Search Results (135)

Background/Objectives: Colistin (COL), administered as a prodrug colistimethate sodium (CMS), is commonly used to treat infections caused by multidrug-resistant Gram-negative bacteria in critically ill patients. Given high CMS instability, very complex and variable pharmacokinetics (PK) and high incidence of toxicity, therapeutic drug monitoring (TDM) of active COL might play an important role. This study aimed to develop and validate an accessible immunoassay-based approach for COL monitoring in human serum. Methods: A direct competitive enzyme-linked immunosorbent assay (dcELISA) was developed using polyclonal (pAb) anti-polymyxin antibody alongside a polymyxin B–horseradish peroxidase conjugate. CMS conversion to COL along with serum deproteinization was achieved using 5% trichloroacetic acid (TCA) treatment at 37 °C. Assay accuracy and precision were assessed by spike-and-recovery experiments in healthy volunteer serum. The assay was applied to serum samples from critically ill patients with burns or pneumonia receiving CMS therapy. The reliability of the measurements was confirmed by parallel dcELISA based on a reference monoclonal antibody (mAb) against fragmented polymyxin molecule. Results: Both ELISA formats demonstrated high sensitivity, with limits of detection of 0.053 ng/mL (pAb) and 0.047 ng/mL (mAb). TCA treatment achieved maximal CMS hydrolysis under tested conditions within one hour. Clinical sample analysis showed excellent agreement between the two assays (R² = 0.996), with Bland–Altman analysis revealing a minimal bias of 3.7%. Exploratory PK analysis in burn patients demonstrated increased total drug volume of distribution (45.7–64.9 L) and clearance (8.3–16.3 L/h). Conclusions: This is the first report of ELISA for COL TDM in critically ill patients. The method offers acceptable analytical performance and practical simplicity, with potential to broaden TDM access beyond specialist centers. Full article

Background/Objectives: Ocrelizumab is a humanized monoclonal antibody targeting CD20, approved for the treatment of adult patients with relapsing multiple sclerosis (RMS) and primary progressive multiple sclerosis (PPMS). The neutralizing activity of anti-drug antibodies (ADAs), especially neutralizing ADAs (nADAs) activity, should be examined considering that it can alter pharmacokinetic (PK) and pharmacodynamic (PD) profiles, reduce drug efficacy, and lead to life-threatening adverse events. Methods: This article presents data on the development and validation of an assay for neutralizing anti-drug antibodies (nADA) based on ADCC reporter cells for the analysis of patient sera in the context of ocrelizumab clinical studies. Results: Critical steps and conditions to minimize assay variability were identified. The lower limit of detection was 549.6 ng/mL. The cutoff for nonspecific neutralization was determined as 19.7%. The presence of 0.37–3.0 μg/mL ocrelizumab in a biological sample enables the detection of 1.1–10.0 μg/mL polyclonal anti-ocrelizumab idiotype antibodies, respectively. Conclusions: The developed method can be used for immunogenicity studies of medicinal products containing ocrelizumab. Full article

Delayed reperfusion of an ischemic heart is known to impair the recovery of cardiac function, and the occurrence of intracellular Ca²⁺ overload in the myocardium is considered to play a critical role in the development of ischemia-reperfusion (I/R) injury. Since Ca²⁺/Mg²⁺ ecto-ATPase, which is activated by millimolar concentrations of Ca²⁺ or Mg²⁺, has been shown to serve as a Ca²⁺ gating mechanism for the entry of Ca²⁺ and subsequent development of intracellular Ca²⁺ overload, we investigated the role of depression in Ca²⁺/Mg²⁺ ecto-ATPase activity by polyclonal antibodies against Ca²⁺/Mg²⁺ ecto-ATPase in promoting the recovery of cardiac function in isolated perfused rat hearts upon subjection to I/R injury. Incubation of sarcolemma (SL) membranes with immune serum or purified IgG antibody fraction was found to depress both Ca²⁺-ATPase and Mg²⁺-ATPase activities. Pretreatment of hearts with immune serum or purified antibodies was observed to improve the recovery of cardiac function and depress the SL Ca²⁺/Mg²⁺ ecto-ATPase activities in hearts subjected to I/R injury. A marked increase in myocardial Ca²⁺ content in I/R hearts was also attenuated by immune serum treatment. Furthermore, treatment of cardiomyocytes from normal hearts with immune serum or purified antibodies reduced the ATP-induced increase in intracellular Ca²⁺ concentration. These results suggest that improvement in the recovery of cardiac function in hearts subjected to I/R injury by polyclonal Ca²⁺/Mg²⁺ ecto-ATPase antibodies may be due to the attenuation of intracellular Ca²⁺ overload. Full article

Autoimmune diseases result from a breakdown of immune tolerance influenced by genetic and environmental factors. Regulatory T cells (Tregs) maintain immune homeostasis, while interferon-γ (IFNγ) has context-dependent proinflammatory and regulatory roles. In B10.S mice, mercury-induced autoimmunity (HgIA) emerges within approximately 4 weeks of Hg exposure and is marked by antinucleolar antibody (ANoA) production, polyclonal B-cell activation, and deposition of immune complexes in the kidney. We investigated whether Tregs attenuate HgIA and evaluated IFNγ’s role in this regulation. Female WT and IFNγ^−/− B10.S mice received HgCl₂ or water for 4 weeks until all mice developed ANoA. CD4⁺CD25⁺Foxp3⁺ Tregs or CD4⁺CD25⁻Foxp3⁻ cells were transferred into HgCl₂-exposed WT recipients and monitored for 13 weeks. Compared with Hg-primed non-Tregs, Hg-primed WT Tregs were statistically associated with significantly reduced autoantibody levels, lower IgG1/IgG2a, and significantly decreased glomerular IgG/C3c deposition, suggesting that Hg exposure may modulate Treg function. Conversely, both water- and Hg-primed Tregs and non-Tregs from IFNγ^−/− donors elicited profoundly diminished autoantibody production and renal pathology in recipients. IFNγ^−/− mice lacked fibrillarin-specific responses, highlighting its requirement for HgIA initiation. While non-Treg transfer failed to suppress HgIA, Treg transfer reduced HgIA and highlighted relevance for immune-regulatory therapies, especially where environmental toxicants may drive autoimmune disease. Full article

Our laboratory identified the susceptible allelic variant of equine CXCL16 protein (EqCXCL16S) as an entry receptor for equine arteritis virus (EAV). However, EAV has a broad host cell tropism and infects cells that lack EqCXCL16S. Thus, we hypothesized that EAV interacts with other host cell protein(s) that facilitate EAV infection. A virus overlay protein-binding assay in combination with a Far-Western blot from EAV-susceptible equine pulmonary artery endothelial cells (EECs) and equine dermal fibroblasts (E. Derm) identified a 57 kDa protein, present in the membrane fraction of the protein lysate, as a possible EAV-binding protein. Subsequent LC-MS/MS analysis identified this 57 kDa protein as vimentin. Screening of different mammalian cell lines has shown that only cells expressing vimentin are susceptible to EAV infection. Pre-treatment of EECs with an anti-vimentin polyclonal antibody and Withaferin A partially inhibit EAV infection. Finally, the overexpression of equine vimentin (EqVim) in HEK-293 cells increases their susceptibility to EAV infection. Overall, our data strongly indicate that EAV binds to the host cell protein equine vimentin, which actively participates in EAV infection, potentially serving as an attachment factor. The data suggest that EAV interacts with various host cell proteins to achieve its diverse cell tropism. Full article

The preparation of an antibody to treat snake envenomation requires a large amount of snake venom. In China, only four types of anti-snake venom sera are clinically available, and the production and immunization strategies for clinically approved anti-snake venom sera still mainly rely on detoxified antigens, which is a mature technical route commonly adopted by domestic pharmaceutical enterprises. At present, researchers immunize animals with low doses of certain snake venom toxic components or prokaryotically expressed toxic components to reduce the amount of venom needed, and use prepared antisera for their specific investigation purposes. However, it is unclear if low-dose immunized antibody titers and toxin-neutralizing activities are consistent with those of high-dose detoxified crude venom immunized antibodies. In this study, we developed a method for the preparation of highly effective rabbit polyclonal antisera while saving a large amount of toxin. Rabbit polyclonal antisera prepared by low-dose natural α-bungarotoxin (α-BGT) had strong neutralizing effects on the toxin itself and achieved the same antibody titers as antisera prepared with high doses of detoxified α-BGT. Antigen of A maltose binding protein (MBP) fused with α-BGT (MBP-α-BGT) expressed in prokaryotes had low antibody titer and low neutralizing activity. This study provides an effective dosage selection guide and methods for the preparation of polyclonal antibodies and antiserum for investigation purposes. Full article

Enzyme-Linked-Immunosorbent-Spot (ELISpot) is a highly sensitive technique capable of detecting low-level immune responses, offering critical insights into therapy-induced immune activation. Our mouse interferon-gamma (mIFN-γ) ELISpot assay was originally based on a monoclonal capture antibody and a rabbit polyclonal detection antibody. The objective of our study was to replace the polyclonal detection antibody with a monoclonal alternative, using a llama immune library and phage display technology. A llama was immunized with recombinant mIFN-γ, and an immune VHH library was constructed. The library underwent two rounds of panning using the recombinant antigen. Subsequently, 190 clones were screened by Enzyme-Linked-Immunosorbent Assay (ELISA), yielding 27 specific binders to mIFN-γ. Sequence analysis revealed 24 unique clones grouped into four families based on their CDR3-VH sequences. One representative clone from each family was reformatted as VHH-Human Fragment Crystallizable (VHH-hFc) fusion and produced recombinantly for testing in the ELISpot assay. The purified candidates were evaluated in pairs on native mIFN-γ from mouse splenocytes. Two candidates, H3 and G4, were selected for further trial. Comparative analysis of ELISpot performance showed that G4 is a promising substitute for the original rabbit polyclonal antibody, enhancing the overall performance of the mIFN-γ ELISpot assay. This study highlights the potential of VHH antibodies in ELISpot applications and supports their use as a robust, reproducible alternative to polyclonal antibodies. Full article

Background. Inhalable monoclonal antibodies were explored as therapeutics for respiratory viral infections due to their high specificity, which, however, can become a drawback if virus mutational escape occurs. Serum-derived polyclonal antibodies for prophylaxis reflect the diverse response of the immune system, reducing susceptibility to virus mutations and targeting multiple epitopes. Objectives. The aim of this work was the development of inhalable powders containing serum of rats immunized against SARS-CoV-2. Methods & Results. In a preliminary screening, combinations of sugar and an amino acid outperformed single excipients in terms of retention of protein size and residual moisture content. Four formulations were further developed on neat and albumin-depleted serum: HPβCD/L-leucine in water, HPβCD/L-leucine in phosphate buffer (KP), trehalose/L-leucine in water and HPβCD/glycine in KP. These were subsequently evaluated for aerosol performance and protein stability. All spray-dried formulations afforded respirable particles (MMAD ≤ 5 µm, FPF 70–80%), with L-leucine reducing hygroscopicity and particle aggregation while improving aerosol dispersibility. Conclusions. Albumin did not positively affect aerodynamic properties but provided greater protection of immunoglobulin activity (approximately 80% and 90% in albumin-depleted and neat serum, respectively). Buffer selection had no remarkable impact on the considered parameters. L-leucine with HPβCD offered the best balance of aerodynamic performance and protein stabilization. Full article

Antibiotic stereoisomers as components of medicines are typically characterized by different biological activities. Because pharmaceuticals can include a racemic mixture of stereoisomers, monitoring of all forms is required. One contaminant of food products, antibiotic ofloxacin (OFL), as a chiral compound, has two enantiomers—the biologically active S-isomer and less active R-isomer. In this study, a sensitive immunochromatographic test system for simultaneous enantiospeсific detection of the two OFL isomers was developed for the first time. For this, polyclonal antibodies were produced, and conditions for a double lateral flow immunoassay (LFIA) were selected and optimized so that the cross-reactivity with another enantiomer was negligible. The LFIA was performed in a competitive format with gold nanoparticles as a label for secondary antibodies. The achieved LODs/cutoffs were 0.001/10 and 0.007/30 ng/mL for S-OFL and R-OFL detection, respectively; the assay procedure took only 15 min. A double LFIA was performed to detect S-OFL and R-OFL in milk with minimal sample pretreatment; the recoveries were 85–95%. The developed test system is an effective tool for the selective detection of both isomers of OFL, allowing for the avoidance of false negative results. This immunochromatographic approach can be promising for the control of other optically active food toxicants. Full article

Background: Understanding the immune mechanisms that differentiate protective from pathogenic responses during dengue virus (DENV) infection is critical for effective vaccine development. Objective: To investigate how CD4⁺ T cell depletion alters viral control and the humoral immune response during primary DENV2 infection in a non-human primate (NHP) model. Methods: Rhesus macaques were depleted of CD4⁺ T cells prior to DENV2 infection. Viral kinetics, B cell activation, antibody specificity, and functional outcomes were evaluated longitudinally, including cross-reactivity and antibody-dependent enhancement (ADE) potential. Results: CD4⁺ T cells were essential for early viral clearance and the generation of robust, type-specific neutralizing antibodies. In their absence, animals exhibited early non-specific polyclonal B cell activation, delayed isotype switching, and an expanded repertoire of cross-reactive antibodies to DENV and Zika virus (ZIKV), with diminished neutralizing capacity. CD4-depleted macaques also showed increased ADE potential, particularly against ZIKV, and elevated anti-NS1 IgG titers that persisted one-year post-infection. Conclusion: CD4⁺ T cells play a critical role in orchestrating effective, durable, and type-specific antibody responses during primary DENV infection. Their absence leads to delayed antibody maturation, greater cross-reactivity, and higher ADE potential. These findings emphasize the need for DENV and ZIKV vaccines to include CD4⁺ T cell epitopes that promote high-quality, type-specific antibody responses and minimize ADE risk. Full article

Hepatocellular carcinoma (HCC) is the main leading cause of cancer-related deaths. Treatments for advanced HCC include multikinase inhibitors (Sorafenib or Lenvatinib), with limited response rates and serious side effects, or immunotherapy applicable to a small fraction of patients. Thus, new strategies are needed to improve the management of HCC. We evaluate here the efficacy and safety of XON9, a first-in-class glyco-humanized polyclonal antibody (GH-pAb). Cytotoxic activity of XON9 against Hep3B, Huh7, HepG2 or primary hepatocytes was investigated. Apoptosis, caspase activity, production of reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were evaluated. Efficacy of XON9 was then assessed in vivo in NMRI nude mice, while pharmacokinetics and safety were evaluated in a non-human primate. XON9 showed a potent complement-dependent cytotoxicity (CDC) against Hep3B and Huh7 (EC50 < 10 µg/mL), and to a less extent against HepG2. XON9 induced apoptosis of HCC cells with activation of caspases 8 and 9, increase in ROS and drop in MMP. Overall, in vitro lytic activity of XON9 was superior to that of Sorafenib. In vivo, XON9 significantly reduced tumor progression and outperformed Sorafenib. No toxicity was observed after repeated injections of XON9 in a non-human primate. XON9 represents a promising and selective immunotherapy against refractory HCC. Full article

Low-density lipoprotein receptor-related protein 5 (LRP5) is a multifunctional transmembrane coreceptor that plays a pivotal role in development and disease. Wnt/β-catenin signaling is the primary downstream signaling pathway activated by LRP5. Furthermore, some LRP5 functions are mediated by noncanonical pathways, such as AKT/P21 and TGF-β/Smad signaling. Pathologically, both loss-of-function and gain-of-function mutations in LRP5 produce distinct phenotypes, ranging from osteoporosis-pseudoglioma syndrome to high bone mass disorders. Beyond the skeletal system, LRP5 has emerged as a key regulator of retinal angiogenesis, vascular integrity, renal tubular function, neurodevelopment, and lipid metabolism. Its physiological functions are highlighted by its ability to influence adipocyte differentiation, insulin sensitivity, and neuronal synaptic plasticity. Moreover, LRP5 displays a dual role in development and disease progression. Although it plays a protective role in acute injuries such as myocardial infarction and acute kidney injury, LRP5 also contributes to chronic pathologies such as tubulointerstitial fibrosis, polycystic kidney disease, and atherosclerosis through fibrotic and inflammatory pathways. Recent therapeutic interest has focused on modulating LRP5 activity using agents such as anti-Dickkopf-related protein 1 antibody, sclerostin inhibitors, polyclonal antibodies, CRISPR/Cas9 knockout, and some natural products. This review discusses the current understanding of LRP5's physiological and pathological roles across organ systems and highlights its therapeutic potential, emphasizing the need for targeted approaches considering its context-dependent effects. Full article

Phospholipase A1 (Ves a 1), a major toxin from Vespa affinis venom, poses significant risks to allergic individuals. Nevertheless, the epitope determinants of Ves a 1 have not been characterized. Thus, identifying its linear B-cell epitopes is crucial for understanding envenomation mechanisms. In this study, we predicted and identified B-cell epitopes EP5 and EP6 as potential candidates. EP5 formed an α-helix at the active site of Ves a 1, whereas EP6 adopted an extended loop conformation. Both synthetic peptides were synthesized and evaluated for their inhibitory effects using immune-inhibitory assays with polyclonal antibodies (pAbs) targeting both native (nVes a 1) and recombinant (rVes a 1) forms. The Ves a 1 polyclonal antibodies (pAb-nVes a 1 and pAb-Ves a 1) were produced, and their specificity binding to Ves a 1 was confirmed by Western blot. Next, ELISA inhibition assays showed that EP5 and EP6 significantly blocked pAb binding to both nVes a 1 and rVes a 1. Dot blot and Western blot assays supported these findings, particularly with stronger inhibition toward rVes a 1. Furthermore, enzymatic assays indicated that nVes a 1 and rVes a 1 retained phospholipase activity. Immunoinformatics docking showed that EP5 and EP6 specifically bind to a single-chain variable fragment antibody (scFv) targeting Naja naja PLA2. Molecular analysis revealed similar amino acid interactions to the template, suggesting effective paratope–epitope binding. These results support the potential of EP5 and EP6 for future diagnosis and therapy of V. affinis venom allergy. Full article

Background/Objectives: Radiotherapy (RT) is a mainstay of clinical cancer therapy that causes broad immune responses. The complement system is a pivotal effector mechanism in the innate immune response, but the impact of RT is less well understood. This study investigates the interaction between RT and the complement system as a possible approach to improve immune responses in cancer treatment. Methods: Human solid cancer (lung, prostate, liver, breast cancer), lymphoma, and leukemia cells were irradiated using X-rays and treated with polyclonal antibodies or anti-CD20 monoclonal antibodies, respectively. Chromium release assay was applied to measure cell lysis after radiation with or without complement-activating antibody treatment. The expression of membrane-bound complement regulatory proteins (mCRPs; CD46, CD55, CD59), which confer resistance against complement activation, CD20 expression, apoptosis, and radiation-induced DNA double-strand breaks (γH2AX), was measured by flow cytometry. The radiosensitivity of tumor cells was assessed by colony-forming assay. Results: We demonstrate that RT profoundly impacts complement function by upregulating the expression of membrane-bound complement regulatory proteins (mCRPs) on tumor cells in a dose- and time-dependent manner. Impaired complement-mediated tumor cell lysis could thus potentially contribute to radiotherapeutic resistance. We also observed RT-induced upregulation of CD20 expression on lymphoma and leukemic cells. Notably, complement activation prior to RT proved more effective in inducing RT-dependent early apoptosis compared to post-irradiation treatment. While complement modulation does not significantly alter RT-induced DNA-damage repair mechanisms or intrinsic radiosensitivity in cancer cells, our results suggest that combining RT with complement-based anti-cancer therapy may enhance complement-dependent cytotoxicity (CDC) and apoptosis in tumor cells. Conclusions: This study sheds light on the complex interplay between RT and the complement system, offering insights into potential novel combinatorial therapeutic strategies and a potential sequential structure for certain tumor types. Full article

FOXP3+ regulatory T cells (Tregs) play a central role in the regulation of the immune system. Human Tregs preferentially express a FOXP3 isoform known as delta 2, which lacks exon 2. In addition to FOXP3, Tregs also express isoforms of other Treg-related molecules, such as CTLA-4 and IKZF-2. It is hypothesized that Tregs possess a unique isoform repertoire based on their unique gene and isoform expression profiles, which include FOXP3. Here, we identified a Treg-specific unique isoform repertoire confirmed by long-read high-throughput isoform sequencing called Iso-seq, which is uniquely capable of providing data on genome-wide isoform usage. Notably, while conventional T cells (Tconvs) do not exhibit this pattern, Tregs preferentially express the full-length FOXP3 isoform. Interestingly, the preferential expression of ICOS and PD-L1 upon T-cell receptor (TCR) stimulation was noted in activated Tregs but not in Tconvs or non-activated Tregs. Moreover, using a PD-L1 antibody blockade on Tregs did not diminish FOXP3 expression; however, it significantly reduced the suppressive function. Therefore, Tregs may have a unique isoform repertoire, which becomes pronounced upon polyclonal TCR stimulation. Full article