Search Results (91)

Despite the prevalence of fucosylated IgG in plasma, specific IgGs with low core fucosylation sporadically emerge in response to virus infections and blood cell alloantigens. This low fucosylation of IgG is implicated in the pathogenesis of SARS-CoV-2 and dengue infections. In COVID-19, the presence of IgGs with low core fucosylation (afucosylated IgGs) targeting spike protein predicts disease progression to a severe form and actively mediates this progression. This study reveals that SARS-CoV-2 infection of megakaryocytes promotes the generation of pathogenic afucosylated anti-spike IgGs, leading to outcomes, such as pulmonary vascular thrombosis, acute lung injury, and mortality in FcγRIIa-transgenic mice. Platelets from mice injected with virus-infected human megakaryocytes express significant activation biomarkers, indicating a direct link between the immune response and platelet activation. Mice injected with virus-infected human megakaryocytes demonstrate an elevated rate of thrombus formation induced by FeCl₃ (4%) and a reduction in bleeding time, emphasizing the intricate interplay of viral infection, immune response, and hemostatic complications. Treatment with inhibitors targeting FcγRIIa, serotonin, or complement anaphylatoxins of mice injected with spike-expressing MKs successfully prevents observed platelet activation, thrombus formation, and bleeding abnormalities, offering potential therapeutic strategies for managing severe outcomes associated with afucosylated IgGs in COVID-19 and related disorders. Full article

The evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its impact on public health continue to demand attention as the virus continues to evolve, demonstrating a remarkable ability to adapt to diverse selective pressures including immune responses, therapeutic treatments, and prophylactic interventions. The SARS-CoV-2 variant landscape remains dynamic, with new subvariants continuously emerging, many harboring spike protein mutations linked to immune evasion. In this study, we characterized a panel of live SARS-CoV-2 strains, including those key subvariants implicated in recent waves of infection. Our findings revealed a significant variability in mutation patterns in the spike protein across the strains analyzed. Commercial antibodies and human convalescent plasma (HCoP) samples from unvaccinated donors were ineffective in neutralizing the most recent Omicron subvariants, particularly after the emergence of JN.1 subvariant. Using human airway epithelial cells derived from healthy bronchiolar tissue (hBAEC), we established both monoinfections and coinfections involving SARS-CoV-2, Influenza A virus H1N1 (IFAV_H1N1) and Respiratory Syncytial Virus (RSV). Assessments were conducted to compare viral infectivity and the production and release of immune mediators in the apical and basolateral compartments. Notably, Omicron KP.3.1.1 subvariant induced a more pronounced cytopathic effect in hBAEC compared to its parental strain JN.1 and even surpassed the impact observed with the ancestral wild-type virus (WA1/2020, Washington strain). Furthermore, the coinfection of KP.3.1.1 subvariant with IFAV_H1N1 or RSV did not attenuate SARS-CoV-2 infectivity; instead, it significantly exacerbated the pathogenic synergy in the lung epithelium. Our study demonstrated that pro-inflammatory cytokines IL-6, IFN-β, and IL-10 were upregulated in hBAEC following SARS-CoV-2 monoinfection with recent Omicron subvariants as well as during coinfection with IFAV_H1N1 and RSV. Taken together, our findings offer new insights into the immune evasion strategies and pathogenic potential of evolving SARS-CoV-2 Omicron subvariants, as well as their interactions with other respiratory viruses, carrying important implications for therapeutic development and public health preparedness. Full article

Fluorescent brighteners (FBs) are a class of chemicals extensively used in industrial and consumer products. Their environmental occurrences and potential health risks have raised significant concerns. However, the lack of analytical methods for FBs in human samples has hindered the accurate assessment of internal exposure levels. Addressing this gap, this study developed and validated a novel method for the simultaneous determination of 13 FBs at trace levels in human plasma using solid-phase extraction combined with HPLC-MS/MS. The method employed EMR-Lipid SPE columns, which can selectively adsorb phospholipids for plasma sample pre-treatment. Detection was achieved through positive ion electrospray ionization (ESI) in multiple reaction monitoring (MRM) modes. The results showed that all 13 FBs exhibited good linearity within their respective ranges, with correlation coefficients (R²) greater than 0.992. The method quantitation limits (MQLs) of the FBs ranged from 0.012 to 0.348 ng/mL, and the spiked recovery rates ranged from 61% to 98%. The method was successfully applied to analyze 10 adult plasma samples, detecting 10 FBs with total concentrations ranging from 0.221 to 0.684 ng/mL. This study provides a reliable analytical method for determining FBs in human plasma, providing a basis for further research on human internal exposure to FBs and associated health risk assessments. Full article

A novel material composed of Au@SiO₂-(3-Aminopropyl Triethoxysilane) (Au@SiO₂-APTES) was successfully synthesised using the sol–gel method, and was used to modify glassy carbon electrodes. Its effectiveness as a molecular recognition element is evaluated in the design of an electrochemical sensor for the precise detection of dopamine. The Au@SiO₂-APTES composite was analysed using Fourier transform infrared spectroscopy, scanning electron microscopy, energy-dispersive X-ray spectroscopy, and X-ray diffraction. Elemental analysis verified the presence of oxygen, silicon, and gold, with atomic percentages of around 77.19%, 21.12%, and 1.65%, respectively. The corresponding elemental mapping for Au@SiO₂-APTES composite showed that the spatial distribution of all the elements was fairly homogeneous throughout the composite, indicating that the Au NPs are embedded in the silica structures. Traces of dopamine were detected by differential pulse voltammetry with a low limit of detection (S/N = 3) and quantification (S/N = 10) of 1.4 × 10⁻⁸ molL⁻¹ and 4.7 × 10⁻⁸ molL⁻¹, respectively. The Au@SiO₂-APTES composite had two linear ranges: from 4.7 × 10⁻⁸ to 1 × 10⁻⁷ molL⁻¹ and 1.25 × 10⁻⁷ to 8.75 × 10⁻⁷ molL⁻¹. Moreover, the sensor showed outstanding selectivity even in the presence of various potential interfering species. It also demonstrated good reusability and signal recovery when tested in human urine and plasma samples spiked with different dopamine concentrations. The electrochemical sensor, constructed using this novel composite material, shows great promise in the selective and sensitive detection of dopamine in the biological matrix. These results underscore the sensor’s capability for practical application in analysing real-world samples. Full article

The simultaneous determination of arsenic and mercury species in human urine is critical for clinical diagnostics and therapeutic monitoring because it reduces the costs, time, and consumption of samples. This study proposes a method of utilizing high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS) for the concurrent analysis of arsenic and mercury species in urine samples. The separation was performed using a Hepu AR 5 μm C₁₈ (250 × 4.6 mm) column, employing a mobile phase composed of 0.1% L-cysteine, 5 mM NH₄H₂PO₄, and 4 mM tetrabutylammonium hydroxide (TBAH). The samples were diluted with water, and matrix interference was reduced through the application of a kinetic energy discrimination (KED) mode. Calibration curves for four arsenic species and three mercury species exhibited strong linearity within the range of 1–20 μg L⁻¹, with correlation coefficients (r) above 0.999. The limits of detection (LOD) ranged from 0.030 to 0.086 μg L⁻¹, while the limits of quantitation (LOQ) were observed to range from 0.10 to 0.29 μg L⁻¹. The spiking recoveries for all species varied from 87.0% to 110.3%, and the intra-day and inter-day relative standard deviations (RSD) were determined to be 1.1–6.0%, and 0.8–9.2%, respectively. These results indicate that the developed method achieves high precision, accuracy, and suitability for clinical applications, offering valuable insights for the diagnosis and treatment of heavy metal exposure. Full article

Stroke affects approximately 12 million individuals annually, necessitating swift diagnosis to avert fatal outcomes. Current hospital imaging protocols often delay treatment, underscoring the need for portable diagnostic solutions. We have investigated silver nanostars (AgNS) incubated with human plasma, deposited on a simple aluminum foil substrate, and utilizing Surface-Enhanced Raman Spectroscopy (SERS) combined with machine learning (ML) to provide a proof-of-concept for rapid differentiation of stroke types. These are the seminal steps for the development of low-cost pre-hospital diagnostics at point-of-care, with potential for improving patient outcomes. The proposed SERS assay aims to classify plasma from stroke patients, differentiating hemorrhagic from ischemic stroke. Silver nanostars were incubated with plasma and spiked with glial fibrillary acidic protein (GFAP), a biomarker elevated in hemorrhagic stroke. SERS spectra were analyzed using ML to distinguish between hemorrhagic and ischemic stroke, mimicked by different concentrations of GFAP. Key innovations include optimized AgNS–plasma incubates formation, controlled plasma-to-AgNS ratios, and a low-cost aluminum foil substrate, enabling results within 15 min. Differential analysis revealed stroke-specific protein profiles, while ML improved classification accuracy through ensemble modeling and feature engineering. The integrated ML model achieved rapid and precise stroke predictions within seconds, demonstrating the assay’s potential for immediate clinical decision-making. Full article

Despite the success of combination antiretroviral therapy (cART) to suppress HIV replication, HIV persists in a long-lived reservoir that can give rise to rebounding viremia upon cART cessation. The translationally active reservoir consists of HIV-infected cells that continue to produce viral proteins even in the presence of cART. These active reservoir cells are implicated in the resultant viremia upon cART cessation and likely contribute to chronic immune activation in people living with HIV (PLWH) on cART. Methodologies to quantify the active reservoir are needed. Here, an automated immunocytochemistry (ICC) assay coupled with computational image analysis to detect and quantify intracellular Gag capsid protein (CA) is described (CA-ICC). For this purpose, fixed cells were deposited on microscopy slides by the cytospin technique and stained with antibodies against CA by an automated stainer, followed by slide digitization. Nuclear staining was used to count the number of cells in the specimen, and the chromogenic signal was quantified to determine the percentage of CA-positive cells. In comparative analyses, digital ELISA, qPCR, and flow cytometry were used to validate CA-ICC. The specificity and sensitivity of CA-ICC were assessed by staining a cell line that expresses CA (MOLT IIIB) alongside a control cell line (Jurkat) devoid of this marker, as well as peripheral blood mononuclear cells (PBMCs) from HIV seronegative donors before or after ex vivo infection with an HIV laboratory strain. The sensitivity of CA-ICC was further assayed by spiking MOLT IIIB cells into uninfected Jurkat cells in limiting dilutions. In those analyses, CA-ICC could detect down to 10 CA-positive cells per million with a sensitivity superior to flow cytometry. To demonstrate the application of CA-ICC in pre-clinical research, bulk PBMCs obtained from mouse and non-human primate animal models were stained to detect HIV CA and SIV p27, respectively. The level of intracellular CA quantified by CA-ICC in PBMCs obtained from animal models was associated with plasma viral loads and cell-associated CA measured by qPCR and ELISA, respectively. The application of CA-ICC to evaluate the activity of small-molecule targeted activator of cell-kill (TACK) in clinical specimens is presented. Overall, CA-ICC offers a simple imaging method for specific and sensitive detection of CA-positive cells in bulk cell preparations. Full article

Anticoagulants can complicate the interpretation of routine and specialised coagulation assays. Several methodologies have been developed to minimise or eliminate anticoagulant-associated interferences; however, no ‘universal methodology’ that encompasses different anticoagulant classes is currently available. Ciraparantag is a promising reversal agent that can bind both direct oral anticoagulants (DOACs) and heparin-like anticoagulants. As such, we aimed to investigate whether ciraparantag could be employed as a ‘universal’ anticoagulant chelator in vitro. Human plasma was spiked with ascending concentrations of ciraparantag, with or without DOACs or heparin, and assayed for routine coagulation parameters. Ciraparantag had minimal effects on coagulation testing when added to human plasma at concentrations similar to pharmacokinetic maxima; however, ciraparantag did not remove DOAC- or heparin-associated activities in vitro, which was likely due to the preferential chelation of anionic substances in the coagulation reagents. In contrast, DOAC-Stop™, a commercial activated charcoal-based adsorbent, efficiently removed both DOAC- and ciraparantag-associated interferences. In conclusion, although ciraparantag is not effective as a ‘universal’ anticoagulant chelator in vitro, we report that activated charcoal-based adsorbents may be clinically useful in situations where laboratory investigations are complicated by the presence of DOACs and/or ciraparantag. Full article

Consistent with the biochemistry of coronaviruses as well established over decades, SARS-CoV-2 makes its initial attachment to host cells through the binding of its spike protein (SP) to sialylated glycans (containing the monosaccharide sialic acid) on the cell surface. The virus can then slide over and enter via ACE2. SARS-CoV-2 SP attaches particularly tightly to the trillions of red blood cells (RBCs), platelets and endothelial cells in the human body, each cell very densely coated with sialic acid surface molecules but having no ACE2 or minimal ACE2. These interlaced attachments trigger the blood cell aggregation, microvascular occlusion and vascular damage that underlie the hypoxia, blood clotting and related morbidities of severe COVID-19. Notably, the two human betacoronaviruses that express a sialic acid-cleaving enzyme are benign, while the other three—SARS, SARS-CoV-2 and MERS—are virulent. RBC aggregation experimentally induced in several animal species using an injected polysaccharide caused most of the same morbidities of severe COVID-19. This glycan biochemistry is key to disentangling controversies that have arisen over the efficacy of certain generic COVID-19 treatment agents and the safety of SP-based COVID-19 vaccines. More broadly, disregard for the active physiological role of RBCs yields unreliable or erroneous reporting of pharmacokinetic parameters as routinely obtained for most drugs and other bioactive agents using detection in plasma, with whole-blood levels being up to 30-fold higher. Appreciation of the active role of RBCs can elucidate the microvascular underpinnings of other health conditions, including cardiovascular disease, and therapeutic opportunities to address them. Full article

Hepatitis E virus (HEV) causes acute hepatitis in humans, which can progress to chronicity in immunosuppressed individuals. Almost all reported HEV infections are caused by Paslahepevirus balayani genotypes 1–4. The structural ORF2 protein is the major antigen detected in the blood of HEV-infected individuals. ELISA assays to detect IgM antibodies to HEV are the first-line diagnostic tests; however, they showed variable performance with frequently discordant results. A qualitative HEV antigen (ORF2) ELISA is currently available for research use. Here, we report a novel quantitative sandwich ELISA to measure HEV ORF2 protein in 3 matrix types. An optimal pair of capture and detection antibodies was selected among 12 unique combinations tested. A sandwich ELISA protocol was developed using these mAbs and biotin–streptavidin technology. The protocol was further optimized to quantify ORF2 antigen in different matrices by interpolating from a standard curve with a linear range of 3.17 to 50.8 femtomoles/mL. Using this method, ORF2 protein was detected in the cell culture medium of Huh7 cells as early as 2–3 days after transfection with HEV genome RNA and in a medium of human hepatocytes infected with HEV. ORF2 antigen was readily detected in the first 2 weeks post-HEV infection in gerbil sera. In immunosuppressed gerbils, ORF2 was detected up to 6 weeks, and the levels were significantly higher between 3 and 6 weeks post-infection. HEV ORF2 antigen levels showed a strong positive correlation with HEV RNA levels in both cell culture medium and gerbil sera. Our novel sandwich ELISA detected at least 7.3 femtomoles/mL ORF2 protein in human plasma spiked with cell culture propagated HEV and detected ORF2 protein in human plasma samples that tested positive for HEV RNA but negative for anti-HEV antibodies. Further, the assay was nonreactive, with negative human plasma, and HBV or HCV-positive human plasma demonstrating specificity. Overall, our ORF2 antigen ELISA will be useful for quantifying ORF2 antigen in cell culture medium, gerbil serum, and human plasma. Further studies are warranted to evaluate its utility in HEV clinical diagnosis. Full article

The surface spike (S) glycoprotein mediates cell entry of SARS-CoV-2 into the host through fusion at the plasma membrane or endocytosis. Omicron lineages/sublineages have acquired extensive mutations in S to gain transmissibility advantages and altered antigenicity. The fusogenicity, antigenicity, and evasion of Omicron subvariants have been extensively investigated at unprecedented speed to align with the mutation rate of S. Cells that overexpress receptors/cofactors are mostly used as hosts to amplify infection sensitivity to tested variants. However, systematic cell entry comparisons of most prior dominant Omicron subvariants using human lung epithelium cells are yet to be well-studied. Here, with human bronchial epithelium BEAS-2B cells as the host, we compared single-round virus-to-cell entry and cell-to-cell fusion of Omicron BA.1, BA.5, BQ.1.1, CH.1.1, XBB.1.5, and XBB.1.16 based upon split NanoLuc fusion readout assays and the S-pseudotyped lentivirus system. Virus-to-cell entry of tested S variants exhibited cell-type dependence. The parental Omicron BA.1 required more time to develop full entry to HEK293T-ACE2-TMPRSS2 than BEAS-2B cells. Compared to unchanged P681, S-cleavage constructs of P681H/R did not have any noticeable advantages in cell entry. Omicron BA.1 and its descendants entered BEAS-2B cells more efficiently than D614G, and it was slightly less or comparable to that of Delta. Serine protease-pretreated Omicron subvariants enhanced virus-to-cell entry in a dose-dependent manner, suggesting fusion at the plasma membrane persists as a productive cell entry route. Spike-mediated cell-to-cell fusion and total S1/S2 processing of Omicron descendants were similar. Our results indicate no obvious entry or fusion advantages of recent Omicron descendants over preceding variants since Delta, thus supporting immune evasion conferred by antigenicity shifts due to altered S sequences as probably the primary viral fitness driver. Full article

Toscana virus (TOSV), a sandfly-borne virus, is an important etiological agent in human acute meningitis and meningoencephalitis in the Mediterranean area during the summer. However, the actual number of TOSV infections is underestimated. Laboratory confirmation is necessary because TOSV infection has overlapping clinical features with other neuro-invasive viral infections. Nowadays, the reference test for direct diagnosis in the acute phase of TOSV infection is the PCR based method for detecting TOSV in cerebrospinal fluid and/or plasma, serum, or blood. Although poorly employed, urine is another helpful biological matrix for TOSV detection. Urine is a matrix rich in PCR inhibitors that affect PCR efficiency; consequently, false negatives could be generated. To investigate the potential effect of urine PCR inhibitors on TOSV detection, we compared undiluted and diluted urine using 10-fold series of spiked TOSV. The results showed a significant improvement in TOSV detection performance in diluted urine (1 TCID₅₀ vs. 1 × 10⁴ TCID₅₀ limit of detection and 101.35% vs. 129.62% efficiency, respectively, in diluted and undiluted urine). In conclusion, our data provide preliminary important insights into the use of diluted urine to limit the impact of the inhibitory effects of urine on the detection of TOSV in RT-PCR-based approaches. Full article

Reinforced by this study, New York City has one of the cleanest water systems in the world. Medgar Evers College (MEC) serves 7000 students/1050 faculty/staff. Given that: (1) students/faculty/staff spend 20–30% of their daily time there; (2) potable water sources must abide by the EPA’s maximum contamination levels (MCLs); and (3) a detrimental impact on human health arises from violations to EPA’s water quality mandates, we quantified the abundance of 27 heavy metals (96 samples, N = 3) using MEC as a case study. Water was collected from all potable water sources, following EPA protocols for sample-matrix preparation, collection, and wet-chemical analysis. Linear polyethylene containers/caps were used to prevent sample contamination while the water samples were spiked with HNO₃ (aq) for preservation. Heavy metal concentrations were quantified using New Jersey’s Meadowlands Environmental Research Institute’s Inductively Coupled Plasma-Mass Spectrometer (ICP-MS, Agilent 7700X) in no gas, and He flow modes. Ninety-five percent of sample concentration relative standard deviations (RSDs) reveal four distinct regions: (1) where one mode is more precise than the other, and sample data exhibit very good to excellent precision, RSD ≤ 15%; (2) despite being at low concentrations, measurements exhibit good to excellent precision, RSD ≤ 20%; (3) species concentrations ≥0.1 ppb very good to excellent precision is shown, RSD ≤ 15%; and (4) species at concentrations ≤ 10⁻³ ppb display fair to very poor precision, RSD ≥ 30%. All heavy metals complied with their respective EPA MCLs (except Fe). Over 90% of Fe sample concentrations were enhanced by up to about 30×. Two samples exhibited [Pb] = 13.7 (No gas mode, RSD = 3.32%) and 14.8 ppb (He mode, RSD = 0.75%), which is close to the EPA Primary MCL, 15 ppb. Based on EPA/WHO end-member equations, we estimate a 1/103 to 1/108 chance of cancer attainment from long-term exposure to the range of concentrations of heavy metals measured in this study. Full article

Follicle-stimulating hormone (FSH) regulates the development, growth, pubertal maturation and reproductive processes of the human body. The determination of serous FSH concentration is significant as an alternative to testicular biopsy in the case of boys suffering from cryptorchidism after orchidopexy, and as a means of determining the menopausal stage in women. The aim of this investigation is to develop a specific array surface plasmon resonance imaging (SPRi) biosensor for the determination of FSH in body liquids such as blood plasma, obtaining sufficient sensitivity to determine FSH at levels characteristic for that hormone in blood plasma, without any signal enhancement. The biosensor consists of a mouse monoclonal anti-FSH antibody attached to the gold surface of a chip via a cysteamine linker. Its linear response range is from 0.08 mIU mL⁻¹ (LOQ) to 20 mIU mL⁻¹, and well covers most of the range of FSH activities found in blood without dilution. The precision of measurement is between 3.2% and 13.1% for model samples, and between 3.7% and 5.6% for spiked plasma samples. Recoveries are in the range from 94% to 108%. The biosensor has good selectivity, and is validated by comparison with ECLE, with good agreement of the results Full article

Three years into the COVID-19 pandemic, mass vaccination campaigns have largely controlled the disease burden but have not prevented virus circulation. Unfortunately, many immunocompromised patients have failed to mount protective immune responses after repeated vaccinations, and liver transplant recipients are no exception. Across different solid organ transplant populations, the plasma levels of Torquetenovirus (TTV), an orphan and ubiquitous human virus under control of the immune system, have been shown to predict the antibody response after COVID-19 vaccinations. We show here a single-institution experience with TTV viremia in 134 liver transplant recipients at their first or third dose. We found that TTV viremia before the first and third vaccine doses predicts serum anti-SARS-CoV-2 Spike receptor-binding domain (RBD) IgG levels measured 2–4 weeks after the second or third dose. Pre-vaccine TTV loads were also associated with peripheral blood anti-SARS-CoV-2 cell-mediated immunity but not with serum SARS-CoV-2 neutralizing antibody titers. Full article