Acute-phase Serum Research Articles

Effect of guselkumab on serum biomarkers in patients with active psoriatic arthritis and inadequate response to tumor necrosis factor inhibitors: results from the COSMOS phase 3b study

BackgroundGuselkumab is a selective interleukin (IL)-23 inhibitor targeting the IL-23p19 subunit. In the phase 3b COSMOS trial, guselkumab demonstrated efficacy in treating participants with active psoriatic arthritis (PsA) and inadequate response (IR; lack of efficacy or intolerance) to tumor necrosis factor inhibitors (TNFi).MethodsAdults with active PsA (≥ 3 swollen joints, ≥ 3 tender joints) and IR to one or two TNFi (TNFi-IR) were randomized 2:1 to guselkumab at Weeks 0, 4, then every 8 weeks (Q8W) or placebo➔guselkumab Q8W at Week 24 with possible early escape at Week 16. Levels of serum cytokines, including interferon γ (IFNγ), IL-10, and tumor necrosis factor α (TNFα); T helper 17 (Th17) effector cytokines IL-17A, IL-17F, and IL-22; and acute phase proteins C-reactive protein (CRP), IL-6, and serum amyloid A (SAA), were assessed and compared with demographically matched healthy controls; guselkumab pharmacodynamics through Week 24 were also assessed. Associations between baseline biomarker levels and 1) baseline disease activity (28-joint disease activity score using CRP [DAS28-CRP], psoriasis area and severity index [PASI], and % body surface area [BSA] affected by psoriasis) and 2) clinical response (including ≥ 20% improvement in American College of Rheumatology criteria [ACR20] response) at Week 24 were assessed.ResultsBaseline serum levels of IL-6, IL-10, IL-17A, IL-17F, IL-22, TNFα, and IFNγ were significantly higher in COSMOS TNFi-IR participants than in healthy controls. Baseline IL-6, CRP, and SAA levels were associated with baseline DAS28-CRP. IL-17A and IL-17F levels were associated with baseline PASI score and psoriasis BSA. Baseline swollen or tender joint counts did not associate with baseline biomarker levels. At Week 24, significant decreases from baseline in CRP, SAA, IL-17A, IL-17F, and IL-22 levels were seen in guselkumab-, but not placebo-, treated participants. IL-17F and IL-22 levels at Week 24 in guselkumab-treated participants did not significantly differ from those of healthy controls. Guselkumab-treated participants achieving ACR20 response at Week 24 exhibited higher baseline IL-22 and IFNγ levels versus nonresponders.ConclusionsResults from COSMOS participants with active, TNFi-IR PsA suggest guselkumab reduces levels of effector cytokines associated with the IL-23/IL-17 pathway, including those associated with baseline arthritis and skin disease activity.Trial registrationClinicalTrials.gov: NCT03796858.

Cross-Linking Mass Spectrometry Uncovers Interactions Between High-Density Lipoproteins and the SARS-CoV-2 Spike Glycoprotein

High-density lipoprotein (HDL) levels are reduced in patients with coronavirus disease 2019 (COVID-19), and the extent of this reduction is associated with poor clinical outcomes. While lipoproteins are known to play a key role during the life cycle of the hepatitis C virus, their influence on coronavirus (CoV) infections is poorly understood. In this study, we utilize cross-linking mass spectrometry (XL-MS) to determine circulating protein interactors of the severe acute respiratory syndrome (SARS)-CoV-2 spike glycoprotein. XL-MS of plasma isolated from patients with COVID-19 uncovered HDL protein interaction networks, dominated by acute-phase serum amyloid proteins, whereby serum amyloid A2 was shown to bind to apolipoprotein (Apo) D. XL-MS on isolated HDL confirmed ApoD to interact with SARS-CoV-2 spike but not SARS-CoV-1 spike. Other direct interactions of SARS-CoV-2 spike upon HDL included ApoA1 and ApoC3. The interaction between ApoD and spike was further validated in cells using immunoprecipitation-MS, which uncovered a novel interaction between both ApoD and spike with membrane-associated progesterone receptor component 1. Mechanistically, XL-MS coupled with data-driven structural modeling determined that ApoD may interact within the receptor-binding domain of the spike. However, ApoD overexpression in multiple cell-based assays had no effect upon viral replication or infectivity. Thus, SARS-CoV-2 spike can bind to apolipoproteins on HDL, but these interactions do not appear to alter infectivity.

Antibody signatures in hospitalized hand, foot and mouth disease patients with acute enterovirus A71 infection.

Enterovirus A71 (EV-A71) infection is a major cause of severe hand, foot and mouth disease (HFMD) in young children. The characteristics of EV-A71 neutralizing antibodies in HFMD patients are not well understood. In this study, we identified and cloned EV-A71-neutralizing antibodies by single cell RNA and B cell receptor sequencing of peripheral blood mononuclear cells. From 145 plasmablasts, we identified two IgG1 monoclonal antibodies (mAbs) and six IgM mAbs that neutralized EV-A71. Four of the IgM mAbs harbor germline variable sequences and neutralize EV-A71 potently. Two genetically similar IgM antibodies from two patients have recurrent heavy chain variable domain gene usage and similar complementarity-determining region 3 sequences. We mapped the residues of EV-A71 critical for neutralization through selection of virus variants resistant to antibody neutralization in the presence of neutralizing mAbs. The residues critical for neutralization are conserved among EV-A71 genotypes. Epitopes for the two genetically similar antibodies overlap with the SCARB2 binding site of EV-A71. We used escape variants to measure the epitope-specific antibody response in acute phase serum samples from EV-A71 infected HFMD patients. We found that these epitopes are immunogenic and contributed to the neutralizing antibody response against the virus. Our findings advance understanding of antibody response to EV-A71 infection in young children and have translational potential: the IgM mAbs could potentially be used for prevention or treatment of EV-A71 infections.

Paradoxically Increased Inflammation in Immunosuppressed Individuals with COVID-19

Abstract Dysregulated inflammation is central to the morbidity and mortality associated with COVID-19. Treatment with dexamethasone and IL-6 blockade can be life-saving in patients stratified for moderate - severe disease. Patients with suppressed immunity are often excluded from immune blockade therapy due to the assumption that additional suppression of an impaired immune system will be detrimental to viral clearance. We hypothesised that patients with suppressed immune systems would be slower to clear viral infection, leading to increased damage and therefore, paradoxically, a more intense acute phase response to SARS-CoV-2 infection. This was tested by a sub-group analysis of the Coronavirus Immune Response and Clinical Outcomes (CIRCO) cohort, an observational study of acute COVID-19 in Greater Manchester, UK. Patients were included if they were treated with dexamethasone and had a research blood sample retrieved within 48 hours of admission, and excluded if they were treated with IL-6 blockade or antiviral therapy prior to research sampling. Acute phase serum cytokine levels were compared between eight immunosuppressed and 12 immunocompetent patients positive for SARS-CoV-2. In support of our hypothesis, we found that immunosuppressed individuals had higher levels of the inflammatory cytokines IP-10 (p=0.03) and MCP-1 (p=0.01) compared to immunocompetent patients matched for COVID-19 severity. This suggests that immunosuppressed patients may benefit as much as immunocompetent individuals from systemic treatments to dampen inflammation in COVID-19, and current recommendations of excluding these patients from treatment solely on the basis of immune competence may need to be re-evaluated.

Gastrointestinal permeability and inflammatory status of preweaning dairy calves in response to decreasing the ratio of n-6 to n-3 fatty acid in milk replacer

The ratio of n-6 to n-3 fatty acid (FA) is between 2 and 10 times higher in milk replacer (MR) than in whole milk, which may promote inflammation and compromise the integrity of the intestinal epithelium. To evaluate how decreasing the n-6:n-3 FA ratio of MR affects gastrointestinal (GIT) permeability and inflammatory status, 30 dairy calves (2.8 ± 1.06 d of age; mean ± standard deviation) were randomly assigned to be fed an MR with an n-6:n-3 FA ratio of 40:1 (CON; 29.3% crude fat of DM; n = 15) or 6.5:1 (n-3; 29.1% crude fat of DM; n = 15). Calves were fed 7.0 L/d in 2 meals. Calves were weighed and fecal consistency was analyzed weekly. On d 22, calves were administered Cr-EDTA, lactulose, and d-mannitol to assess GIT permeability. Blood and total urine were sequentially collected for 6 and 24 h, respectively, and analyzed for marker content. Whole blood collected 4 h after the meal was subjected to an ex vivo lipopolysaccharide (LPS) challenge to evaluate cytokine secretion from blood cells. Calves were euthanized on d 25 for collection of intestinal tissue samples. Tissue samples were processed to assess FA composition by gas chromatography, histomorphology by bright-field microscopy, and gene expression of tight junction proteins, lipid metabolism enzymes, and immune molecules by real-time quantitative PCR. Data were analyzed using PROC GLIMMIX in SAS (version 9.4, SAS Institute Inc.). Growth performance and fecal consistency were unaffected. Calves fed MR with a lower ratio of n-6 to n-3 FA had 2-fold higher n-3 FA contents and 2-fold lower ratios of n-6 to n-3 FA in proximal jejunum and ileum tissues. Total urinary recovery (0-24 h relative to marker administration) and plasma concentrations of the markers were unaffected. Expression of TJP1 tended to be higher in proximal jejunum tissue and lower in ileum tissue of n-3 calves. The expression of TLR4 and TNFA tended to be higher and CD14 was higher in ileum tissue of n-3 calves. Plasma concentrations of interleukin-4 were decreased in response to the ex vivo LPS challenge in n-3 calves. Histomorphology and GIT permeability were largely unaffected by treatment. Furthermore, the inclusion of linseed and algae oil may promote inflammation, as suggested by greater concentrations of the acute-phase proteins haptoglobin and serum amyloid A postprandially, demonstrating that fat sources should be evaluated for their suitability for MR formulations. Understanding how MR composition affects dairy calf health may improve nutritional strategies on farm.

Serum biomarkers and anti-flavivirus antibodies at presentation as indicators of severe dengue.

Dengue is the most common vector-borne viral disease worldwide. Most cases are mild, but some evolve into severe dengue (SD), with high lethality. Therefore, it is important to identify biomarkers of severe disease to improve outcomes and judiciously utilize resources. One hundred forty-five confirmed dengue cases (median age, 42; range <1-91 years), enrolled from February 2018 to March 2020, were selected from an ongoing study of suspected arboviral infections in metropolitan Asunción, Paraguay. Cases included dengue virus types 1, 2, and 4, and severity was categorized according to the 2009 World Health Organization guidelines. Testing for anti-dengue virus IgM and IgG and serum biomarkers (lipopolysaccharide binding protein and chymase) was performed on acute-phase sera in plate-based ELISAs; in addition, a multiplex ELISA platform was used to measure anti-dengue virus and anti-Zika virus IgM and IgG. Complete blood counts and chemistries were performed at the discretion of the care team. Age, gender, and pre-existing comorbidities were associated with SD vs. dengue with/without warning signs in logistic regression with odds ratios (ORs) of 1.07 (per year; 95% confidence interval, 1.03, 1.11), 0.20 (female; 0.05,0.77), and 2.09 (presence; 1.26, 3.48) respectively. In binary logistic regression, for every unit increase in anti-DENV IgG in the multiplex platform, odds of SD increased by 2.54 (1.19-5.42). Platelet count, lymphocyte percent, and elevated chymase were associated with SD in a combined logistic regression model with ORs of 0.99 (1,000/μL; 0.98,0.999), 0.92 (%; 0.86,0.98), and 1.17 (mg/mL; 1.03,1.33) respectively. Multiple, readily available factors were associated with SD in this population. These findings will aid in the early detection of potentially severe dengue cases and inform the development of new prognostics for use in acute-phase and serial samples from dengue cases.

Reference Intervals and Clinical Utility of Acute Phase Proteins and Serum Proteins Electrophoresis in the Hamadryas Baboon (Papio hamadryas)

Measurements of specific acute phase proteins (APP) and protein electrophoresis (EPH) fractions have been widely used to better assess the health of species under managed care across numerous taxa. To date, APP assays have not been validated in the hamadryas baboon (Papio hamadryas), and reference intervals have not yet been established. This information is critical for the interpretation of APP and EPH measurements used in the diagnosis of inflammatory diseases during routine veterinary care of this species. To obtain this information, banked serum samples from hamadryas baboons of various age, sex, and health status, under managed care at the North Carolina Zoo, were analyzed. A small pilot study found significantly higher serum amyloid A (SAA) and C-reactive protein (CRP) but not haptoglobin in baboons with acute inflammation compared to healthy counterparts, so these two APPs were investigated further. Reagents for serum amyloid A (SAA) and C-reactive protein (CRP) were validated, although differences in CRP reagents were observed. Based on the results of this study, SAA and CRP were defined as major APPs that were significantly increased in baboons with active inflammation or infection compared to healthy conspecifics. Baboons with acute inflammation additionally had significantly higher gamma globulins compared to healthy baboons. Although mean albumin concentrations were lower in baboons with acute inflammation, the difference from healthy baboons was not statistically significant. This study identifies SAA, CRP, and EPH as useful tools in the diagnosis of inflammatory disease in the hamadryas baboon and establishes reference intervals to aid in the future veterinary care of this species.

Analysis of cross-reactivity among flaviviruses using sera of patients with dengue showed the importance of neutralization tests with paired serum samples for the correct interpretations of serological test results for dengue

Dengue is a febrile illness caused by the dengue virus (DENV) that belongs to the genus Flavivirus in the family Flaviviridae. Cross-reactivity between flaviviruses poses a challenge while interpreting serological test results. In the present study, the cross-reactivity of sera of the patients with dengue, who traveled from Japan to DENV-endemic countries, was analyzed by using an enzyme-linked immunosorbent assay (ELISA) and neutralization test (NT). Sixteen serum samples were collected from patients with dengue and were tested for: i) IgM antibodies against Zika virus (ZIKV), West Nile virus (WNV), Japanese encephalitis virus (JEV), and tick-borne encephalitis virus (TBEV) using IgM ELISA, ii) IgG antibody against TBEV using IgG ELISA, and iii) neutralizing antibody against ZIKV, WNV, TBEV, and JEV. Among the 16 samples tested using ELISA, seven samples were IgM-positive for at least one of the other flaviviruses, and nine samples were IgG-positive for TBEV. Neutralizing antibody titers (NATs) against ZIKV, WNV, and TBEV were one-fourth or lower than those against the causative DENV in all samples. The NATs against JEV were one-fourth or lower than those against the causative DENV in six convalescent-phase serum sample among the seven convalescent-phase serum samples. The NAT against DENV of the residual one convalescent-phase serum was similar to that against JEV and that against JEV of its relevant acute-phase serum sample. These results showed that NTs with paired serum samples are important to correctly interpret the serological test results for DENV.

Evaluation of blood serum iron concentration as an alternative biomarker for inflammation in dairy cows.

This study aimed to clarify the relationship between acute phase protein (APP) concentrations and serum Fe concentrations to determine whether serum iron (Fe) can be clinically applied as a substitute for APPs in cows. One hundred five Holstein-Friesian breed lactating dairy cows were enrolled in this study. Cows with inflammatory diseases were 16 subclinical, and 15 severe mastitis cows, in addition to 15 mild and 16 severe sole ulcer cows. The plasma haptoglobin (HPT), alpha-1 acid glycoprotein (AGP), SAA, serum Fe levels, and other biochemical parameters in the cows were measured. The two-sample t-tests and multiple logistic regression analysis were used to compare the control and inflammatory disease groups. ROC analysis was used to evaluate the ability to diagnose inflammation disease. From the results, the proposed diagnostic cutoff value for plasma SAA and serum Fe concentrations to identify dairy cows with inflammatory diseases based on analyses of ROC curves were set at > 3.65mg/l and < 120.50µg/dl, respectively. Therefore, instead of using expensive inflammatory markers to evaluate the inflammatory state at the first treatment day for inflammatory diseases in cow, it shows the useful for screening with serum Fe concentration that can be measured easily and inexpensively as alternative inflammatory biomarkers.

Diagnostic Usefulness of Varicella Zoster Virus-Specific Immunoglobulin (Ig) A and IgG in Patients With Herpes Zoster.

Whether varicella zoster virus (VZV) antibody titer could discriminate patients with herpes zoster (HZ) from healthy controls (HCs) is unclear. We evaluated the diagnostic usefulness of VZV-specific immunoglobulin A (IgA) and immunoglobulin G (IgG) antibodies in patients with confirmed HZ. Study subjects comprised patients with confirmed HZ by salivary VZV DNA positivity and control age- and sex-matched HCs. Saliva was collected and concurrent blood samples were obtained on the first visit day (acute phase) and after 4 weeks (convalescent phase) from 44 HZ patients. All 44 healthy volunteers provided blood and saliva samples once. The median VZV IgA titers in acute-phase (5.2 mIU/mL, P < 0.001) and convalescent-phase (15.8 mIU/mL, P < 0.001) serum samples from HZ patients were significantly higher than those in HCs (1.35 mIU/mL). VZV IgA positivity was detected in about 20% of acute phase serum and convalescent-phase serum of HZ patients. The median VZV IgG antibody titers of HZ patients during acute (1,471.0 mIU/mL, P < 0.001) and convalescent (4,934.7 mIU/mL, P < 0.001) phases were significantly higher than the median titer reported for HCs (591.6 mIU/mL). A four-fold or higher increase in VZV IgG antibody titer was observed in 36.4% of HZ patients. VZV IgA positivity or four-fold or higher increase in VZV IgG antibody titers were not detected in a satisfactory proportion of HZ-infected patients. However, the titer of VZV IgA or IgG antibody particularly in convalescent-phase sera may discriminate HZ patients from HCs.

Evaluation of Nucleoprotein-Based Enzyme-Linked Immunosorbent Assay for Serodiagnosis of Acute Crimean–Congo Hemorrhagic Fever Virus Infections in a Turkish Population

Background: Crimean-Congo hemorrhagic fever virus (CCHFV) causes a highly contagious tick-borne disease with high case-fatality rates in humans. It is circulating not only in many Asian and African countries, but also spreading to and within Europe. To cope better with future outbreaks of Crimean-Congo hemorrhagic fever (CCHF), the WHO has prioritized the need for the development and validation of CCHF diagnostics, including serological assays. In this study, we evaluated the performance of the new EUROIMMUN anti-CCHFV IgM and IgG enzyme-linked immunosorbent assays (ELISAs). Materials and Methods: Both ELISAs were compared to the Vector-Best VectoCrimean-CHF-IgM and -IgG ELISAs using the EUROIMMUN CCHFV Mosaic 2 IgM and IgG indirect immunofluorescence assays (IFA) as reference. Forty-nine acute-phase serum samples from patients with CCHFV infection confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and/or anti-CCHFV IgM IFA positivity were used to determine assay sensitivity. The assessment of specificity was based on sera from 30 control patients, 30 healthy blood donors, and 29 patients with hantavirus or sandfly fever virus infections. All samples originated from Turkey. Results: Sensitivity of the EUROIMMUN ELISAs (IgM 98.0%, IgG 47.1%) exceeded that of the Vector-Best ELISAs (IgM 95.9%, IgG 35.3%). Specificity of the EUROIMMUN ELISA IgM (86.4%) was slightly higher compared with the Vector-Best ELISA IgM (84.7%), while specificity for IgG was 100% for both assays. Qualitative agreement between the EUROIMMUN and Vector-Best ELISAs was substantial for detecting anti-CCHFV IgM (84.1%, ĸ = 0.673) and IgG (94.9%, ĸ = 0.791), whereas the quantitative results indicated a very strong positive correlation (IgM: r = 0.868, IgG: r = 0.913). Conclusion: The new EUROIMMUN anti-CCHFV ELISAs are standardized and easy-to-use tools that reliably support the identification of acute CCHF cases, and thus suitable for laboratories involved in on-site outbreak support.

Invasive Streptococcal Infection Can Lead to the Generation of Cross-Strain Opsonic Antibodies.

The human pathogen Streptococcus pyogenes causes substantial morbidity and mortality. It is unclear if antibodies developed after infections with this pathogen are opsonic and if they are strain specific or more broadly protective. Here, we quantified the opsonic-antibody response following invasive S. pyogenes infection. Four patients with S. pyogenes bacteremia between 2018 and 2020 at Skåne University Hospital in Lund, Sweden, were prospectively enrolled. Acute- and convalescent-phase sera were obtained, and the S. pyogenes isolates were genome sequenced (emm118, emm85, and two emm1 isolates). Quantitative antibody binding and phagocytosis assays were used to evaluate isolate-dependent opsonic antibody function in response to infection. Antibody binding increased modestly against the infecting isolate and across emm types in convalescent- compared to acute-phase sera for all patients. For two patients, phagocytosis increased in convalescent-phase serum both for the infecting isolate and across types. The increase was only across types for one patient, and one had no improvement. No correlation to the clinical outcomes was observed. Invasive S. pyogenes infections result in a modestly increased antibody binding with differential opsonic capacity, both nonfunctional binding and broadly opsonic binding across types. These findings question the dogma that an invasive infection should lead to a strong type-specific antibody increase rather than a more modest but broadly reactive response, as seen in these patients. Furthermore, our results indicate that an increase in antibody titers might not be indicative of an opsonic response and highlight the importance of evaluating antibody function in S. pyogenes infections. IMPORTANCE The bacterium Streptococcus pyogenes is a common cause of both mild and severe human diseases resulting in substantial morbidity and mortality each year. No vaccines are available, and our understanding of the antibody response to this human pathogen is still incomplete. Here, we carefully analyzed the opsonic antibody response following invasive infection in four patients. Unexpectedly, the patients did not always generate opsonic antibodies against the specific infecting strain. Instead, we found that some patients could generate cross-opsonic antibodies, leading to phagocytosis of bacteria across strains. The emergence of cross-opsonic antibodies is likely important for long-term immunity against S. pyogenes. Our findings question the dogma that mostly strain-specific immunity is developed after infection and add to our overall understanding of how immunity to S. pyogenes can evolve.

Borrelia burgdorferi specific serum and cerebrospinal fluid antibodies in Lyme neuroborreliosis

We used definite Lyme neuroborreliosis (LNB) adult patient acute and convalescent phase serum (n = 63 and 61, respectively) and cerebrospinal fluid (CSF; acute n = 63, 3 weeks timepoint n = 41) samples to characterize Borrelia burgdorferi specific antibody responses in patient subgroups categorized by demographics, infection manifestation and phase, infecting B. burgdorferi genospecies, received antibiotic treatments, and treatment outcome. B. burgdorferi antibodies were analyzed using 4 different assays incorporating a large array of antigens. We observed that B. burgdorferi specific serum antibodies show a universal, antigen independent declining trend after antibiotic treatment of LNB at 1 year. Antibodies declined similarly among women and men over time, and the decline was independent of patient age. The antibody responses were independent of the predominant LNB manifestation, treatment received by the patient, infecting B. burgdorferi genospecies, or the subjective improvement experienced by the patients. Finally, the antibody specificities in CSF reflected the specificities observed in serum samples.

Comparison of Neutralizing Antibody Response Kinetics in Patients with Hand, Foot, and Mouth Disease Caused by Coxsackievirus A16 or Enterovirus A71: A Longitudinal Cohort Study of Chinese Children, 2017-2019.

Hand, foot, and mouth disease (HFMD), which is mainly caused by coxsackievirus A16 (CVA16) or enterovirus A71 (EV-A71), poses a serious threat to children's health. However, the long-term dynamics of the neutralizing Ab (NAb) response and ideal paired-serum sampling time for serological diagnosis of CVA16-infected HFMD patients were unclear. In this study, 336 CVA16 and 253 EV-A71 PCR-positive HFMD inpatients were enrolled and provided 452 and 495 sera, respectively, for NAb detection. Random-intercept modeling with B-spline was conducted to characterize NAb response kinetics. The NAb titer of CVA16 infection patients was estimated to increase from negative (2.1, 95% confidence interval [CI]: 1.4-3.3) on the day of onset to a peak of 304.8 (95% CI: 233.4-398.3) on day 21 and then remained >64 until 26 mo after onset. However, the NAb response level of EV-A71-infected HFMD patients was much higher than that of CVA16-infected HFMD patients throughout. The geometric mean titer was significantly higher in severe EV-A71-infected patients than in mild patients, with a 2.0-fold (95% CI: 1.4-3.2) increase. When a 4-fold rise in titer was used as the criterion for serological diagnosis of CVA16 and EV-A71 infection, acute-phase serum needs to be collected at 0-5 d, and the corresponding convalescent serum should be respectively collected at 17.4 (95% CI: 9.6-27.4) and 24.4 d (95% CI: 15.3-38.3) after onset, respectively. In conclusion, both CVA16 and EV-A71 infection induce a persistent humoral immune response but have different NAb response levels and paired-serum sampling times for serological diagnosis. Clinical severity can affect the anti-EV-A71 NAb response.

Evaluation of biochemical markers in the blood plasma of rats exposed to chronic administration of a mixture of metal nanoparticles

The aim of this study was to determine the effects of a specific mixture of metal nanoparticles (Ag, Cu, Fe and MnО2) orally administered to rats, on biochemical blood markers as indicators of chronic toxicity. Animals (Wistar line rats (n=20)) in experimental groups were given a solution of a mixture of metal salts or a mixture of nanoparticles with food for 90 days. On days 15, 30, 60 and 90 of the experiment, five rats from each group were anaesthetised with CO2 and decapitated, and blood samples were collected for biochemical studies and spectrophotometric measurement. The toxic effect of the chronic administration of the mixture at a dose of 4.0 mg/kg of body weight in white rats caused partial immunosuppression expressed as hypoproteinaemia, excessive formation of circulating immune complexes and acute phase serum mucoid proteins (P&amp;lt;0.05), and cytolytic damage to hepatocyte membranes. Levels of enzyme activity (AST, GGT, ALT and AP) were significantly elevated (P&amp;lt;0.05). It has been shown that the origin of the toxic effect is due to oxidative stress, which slowed lipoperoxidation along with the elevation of the level of carboxylated proteins, depleting the antioxidant defence resources of the organism, as seen by a decrease in the level of catalase and total antioxidant activity (P&amp;lt;0.05). No such effects were observed at the dose of 0.3 mg/kg as there were no significant negative impacts on the biomarkers. Based on this data, the biochemical markers studied may be suitable for use in pre-clinical in vivo toxicological assessment of metal nanoparticle-candidates for pharmaceuticals.

Simple and Economical Extraction of Viral RNA and Storage at Ambient Temperature.

ABSTRACTRNA extraction is essential for the molecular detection of common viral pathogens. However, available extraction methods and the need for ultra-cold storage limit molecular testing in resource-constrained settings. Herein, we describe the development of an economical RNA Extraction and Storage (RNAES) protocol that eliminates requirements for instrumentation, expensive materials, and preserved cold chain. Through an iterative process, we optimized viral lysis and RNA binding to and elution from glass fiber membranes included in simple RNAES packets. Efficient viral lysis was achieved with a nontoxic buffer containing sucrose, KCl, proteinase K, and carrier RNA. Viral RNA binding to glass fiber membranes was concentration dependent across seven orders of magnitude (4.0–10.0 log10 copies/μL) and significantly increased with an acidic arginine binding buffer. For the clinical evaluation, 36 dengue virus (DENV)-positive serum samples were extracted in duplicate with the optimized RNAES protocol and once in an EMAG instrument (bioMérieux). DENV RNA was successfully extracted from 71/72 replicates (98.6%) in the RNAES protocol, and real-time RT-PCR cycle threshold (CT) values correlated between extraction methods. DENV RNA, extracted from clinical samples, was stable when stored on dried RNAES membranes at ambient temperature for up to 35 days, with median eluate RNA concentration decreasing by 0.18 and 0.29 log10 copies/μL between day 0 and days 7 and 35, respectively. At a cost of $0.08/sample, RNAES packets address key limitations to available protocols and may increase capacity for molecular detection of RNA viruses.IMPORTANCE RNA extraction methods and ultra-cold storage requirements limit molecular testing for common viruses. We developed a simple, flexible, and economical method that simultaneously addresses these limitations. At $0.08/sample, the new RNA Extraction and Storage (RNAES) protocol successfully extracted viral RNA from acute-phase sera and provided stable, ambient-temperature RNA storage for 35 days. Using this approach, we expect to improve RNA virus detection and outbreak response in resource-constrained settings.

Revisiting misfolding propensity of serum amyloid A1: Special focus on the signal peptide region

AA amyloidosis is the result of overproduction and aberrant processing of acute-phase serum amyloid A1 (SAA1) by hepatocytes. Proteolytic cleavage of SAA1 is believed to play a central role in AA amyloid formation. The SAA1 protein undergoes a cleavage of 18 residues consisting of the signal peptide at the N-terminal region. To better understand the mechanism behind systemic amyloidosis in the SAA1 protein, we studied the misfolding propensity of the signal peptide region. We first examined the signal peptide amino acid SAA derived from different animal species. A library of 16 peptides was designed to evaluate the propensity of aggregation. The amyloidogenic potential of each SAA1 signal peptide homolog was assessed using in silico Tango program, thioflavin T (ThT) fluorescence, transmission electron microscopy (TEM), and seeding with misfolded human SAA1 signal peptide. After 7 days of incubation, most of the SAA1 signal peptide fragments had the propensity to form fibrils at a concentration of 100 μM in 50 mM Tris buffer at 37 °C by TEM. All peptides were able to generate fibrils at a higher concentration, i.e 500 μM in 25 mM Tris buffer with 50% HFIP, by ThT. All SAA1 signal synthetic peptides designed from the different animal species had the propensity to misfold and form fibrils, particularly in species with low occurrence of systemic amyloidosis. The human SAA1 signal peptide region was capable to seed the SAA1 1–25 and 32–47 peptide regions. Characterizing fibrillar conformations are relevant for seeding intact and/or fragmented SAA, which may contribute, to the mechanism of protein misfolding. This research signifies the importance of the signal peptide region and its possible contribution to the misfolding of aggregation-prone proteins.

Long-Term Stability of Blood Serum Biomarkers in Traumatic Brain Injury: A Feasibility Study.

Few studies on traumatic brain injury (TBI) have investigated the stability of blood serum biomarkers after long-term storage at low temperatures. In the current feasibility study we analyzed acute phase serum samples from patients with mild TBI as well as patients with moderate and severe TBI that were collected more than 10 years ago (old samples). We were particularly interested in mild TBI, because injury effects are more subtle in this category as compared to moderate-severe TBI. Therefore, the primary objective was to find out whether several biomarkers were still detectable for these patients. Additionally, we examined whether biomarker levels varied as a function of injury severity. For comparison, we also analyzed samples from an ongoing mTBI cohort (new samples) and healthy controls. Samples were treated with care and were not being subjected to freeze-thaw cycles. We measured concentrations of interleukins (IL6 and 10) and brain specific markers (total tau, UCH-L1, GFAP, and NF-L). No significant differences in biomarker concentrations were found between old and new mild TBI samples. For IL6, IL10, and UCH-L1 higher concentrations were found in moderate and severe TBI as compared to mild TBI. In conclusion, our study shows that long-term storage does not rule out the detection of meaningful biomarker concentrations in patients with TBI, although further research by other laboratories is warranted.

Acute-phase Serum Cytokine Levels and Correlation with Clinical Outcomes in Children and Adults with Primary and Secondary Dengue Virus Infection in Myanmar between 2017 and 2019.

The dengue virus (DENV) has been endemic in Myanmar since 1970, causing outbreaks every 2–3 years. DENV infection symptoms range from mild fever to lethal hemorrhage. Clinical biomarkers must be identified to facilitate patient risk stratification in the early stages of infection. We analyzed 45 cytokines and other factors in serum samples from the acute phase of DENV infection (within 3–5 days of symptom onset) from 167 patients in Yangon, Myanmar, between 2017 and 2019. All of the patients tested positive for serum DENV nonstructural protein 1 antigen (NS1 Ag); 78.4% and 62.9% were positive for immunoglobulin M (IgM) and G (IgG), respectively; and 18.0%, 19.8%, and 11.9% tested positive for serotypes 1, 3, and 4, respectively. Although the DENV-4 viral load was significantly higher than those of DENV-1 or DENV-3, disease severity was not associated with viral load or serotype. Significant correlations were identified between disease severity and CCL5, SCF, PDGF-BB, IL-10, and TNF-α levels; between NS1 Ag and SCF, CCL5, IFN-α, IL-1α, and IL-22 levels; between thrombocytopenia and IL-2, TNF-α, VEGF-D, and IL-6 levels; and between primary or secondary infection and IL-2, IL-6, IL-31, IL-12p70, and MIP-1β levels. These circulating factors may represent leading signatures in acute DENV infections, reflecting the clinical outcomes in the dengue endemic region, Myanmar.

Misfolding Propensity of SAA1 Fragments: Special Focus on the Signal Peptide Region

AA amyloid is the most common form of systemic amyloidosis reported in human medicine and across animal species. AA amyloidosis is the result of over‐production by hepatocytes and aberrant processing of acute‐phase serum amyloid A1 (SAA1). Proteolytic cleavage of SAA1 is believed to play a central role in AA amyloid formation. The SAA1 protein is of 122 amino acids (aa) and undergoes a first cleavage of 18 residues consisting of the signal peptide at the N‐terminal region. The aggregation propensity of the signal peptide region has never been studied. To better understand the mechanism behind systemic amyloidosis in the SAA1 protein, we studied the misfolding propensity of the signal peptide region considering that SAA1 is going through further cleavage processes. The goal of this research was to determine the propensity of the SAA1 signal peptide region to generate fibrils. We examined the signal peptide amino acid sequence of serum amyloid A1 (SAA1) derived from different animal species. A library of the 25 peptides was designed to study the fibril formation using in silico analysis and in vitro assays. The amyloidogenic potential of each SAA1 signal peptide homolog was assessed using thioflavin T fluorescence assays, Congo red binding assays, and transmission electron microscopy. After 7 days of incubation, 22 SAA1 signal peptide fragments had the propensity to form fibrils at a concentration of 500 µM with 50% hexafluoroisopropanol at 37 ºC. The signal peptide region might be important in contributing to the misfolding of aggregation‐prone proteins. Characterizing these conformations are relevant as they may be involved in seeding intact and/or fragmented SAA1, contributing to mechanism of misfolding. This information could be important for continuation studies in the understanding of misfolding disorders.