Search Results (909)

Vector-borne protozoal infections—including babesiosis, theileriosis, hepatozoonosis, trypanosomosis, and leishmaniosis—impose a substantial burden on livestock and companion animal health worldwide and carry important zoonotic and public health implications. Accurate diagnosis is essential yet challenging, given the diversity of parasite genera, their markedly different tissue tropisms, and the uneven distribution of diagnostic resources across veterinary settings. This review provides an integrated overview of the principal diagnostic approaches available, structured around the biological logic that guides test selection in practice. Microscopic examination remains the first-line method; its strengths and limitations are discussed for intraerythrocytic parasites (Plasmodium spp., Babesia spp., Theileria spp., Cytauxzoon spp.—the latter two with additional extra-erythrocytic schizont stages in leukocytes and tissue macrophages, respectively), leukocyte-associated forms (Hepatozoon spp.), extracellular trypanosomes, and tissue-stage parasites, including emerging applications of artificial intelligence. Serological methods—enzyme-linked immunosorbent assay (ELISA), indirect fluorescence antibody test (IFAT), and point-of-care lateral flow assays—are evaluated for their role in exposure detection, population screening, and international trade certification, with attention to cross-reactivity and the active-versus-past-infection distinction. Molecular diagnostics, encompassing conventional PCR, qPCR, droplet digital PCR, isothermal amplification, and next-generation sequencing, are reviewed with respect to target selection, sensitivity, and point-of-care applicability. Finally, diagnostic challenges are contextualised within a One Health framework, highlighting the fragmentation of veterinary surveillance and the need for integrated, cross-sector approaches to detect emerging threats. Full article

This study aimed to express the canine parvovirus (CPV) VP2 protein prokaryotically and develop an indirect ELISA for detecting CPV-specific antibodies in canine serum. The VP2 gene from a laboratory-isolated CPV strain was amplified and cloned into the pET-28a vector. Following prokaryotic expression optimization, the recombinant protein was purified via Ni-NTA affinity chromatography and validated using Western blotting. An indirect ELISA was established utilizing the purified VP2 as the coating antigen, with optimal parameters determined by checkerboard titration. A 1773 bp VP2 fragment was amplified. Optimal expression of the 64.8 kDa recombinant VP2 was achieved with 2 mmol/L isopropyl β-D-thiogalactoside (IPTG) at 32 °C for 8 h. For the indirect ELISA, the optimal antigen coating concentration was 2 μg/mL, alongside primary (canine serum) and secondary antibody dilutions of 1:320 and 1:4000, respectively. The diagnostic cut-off optical density at 450 nm (OD₄₅₀) threshold was established at ≥0.2066, and the analytical sensitivity reached a serum dilution of 1:5120. Compared with the hemagglutination inhibition (HI) assay using 192 clinical serum samples, the ELISA showed a diagnostic sensitivity of 85.94%, a diagnostic specificity of 88.28%, and an overall agreement rate of 87.50%. The mean intra-assay and inter-assay coefficients of variation were 4.39% and 3.02%, respectively. These findings indicate that the recombinant VP2-based indirect ELISA showed good analytical sensitivity, reproducibility, and diagnostic agreement with the HI assay for detecting CPV-specific antibodies in canine serum under the tested conditions, although broader cross-reactivity validation is still required. Full article

Background: Traumatic brain injury (TBI) affects millions of individuals annually and remains a major global cause of neurological disability and death. Tau protein hyperphosphorylation, particularly in its cis conformation, is a major pathological hallmark contributing to neurodegeneration following TBI. Single-chain variable fragments (scFvs), despite their diagnostic potential, suffer from rapid renal clearance and short circulation half-lives, which limit their in vivo performance. PEGylation is therefore employed to prolong systemic circulation and improve the pharmacokinetic behavior of scFvs, enabling more effective brain retention and target engagement. Methods: In this study, we utilized a previously validated anti-cis p-tau scFv antibody fragment, radiolabeled with technetium-99m tricarbonyl (^99mTc(CO)₃), as a diagnostic tracer to detect tau pathology in TBI rat models. The antibody was conjugated with polyethylene glycol (PEG, 20 kDa); PEGylation efficiency was determined by quantifying the products on SDS-PAGE, and the products were subsequently radiolabeled. Results: Radiochemical purity (RCP) was ~95.4% for the non-PEGylated tracer (^99mTc-AININ20) and ~92.7% for the PEGylated form (^99mTc-AININ20-PEG), with both showing >90% radiochemical purity consistently. Upon systemic administration, PEGylated scFv was able to cross the blood–brain barrier (BBB) and selectively accumulated in injured regions, as confirmed by single-photon emission computed tomography (SPECT) imaging. Both PEGylated and non-PEGylated scFv tracers showed significantly higher brain uptake in TBI rats compared to healthy controls (p < 0.0001). At 24 h, the PEGylated form exhibited a significantly higher brain signal than the non-PEGylated version (p < 0.0001), indicating improved tracer retention. Biodistribution analysis at 2 h post-injection showed significantly reduced renal clearance for the PEGylated tracer and increased hepatic uptake compared to the non-PEGylated form. At 24 h, in vivo imaging confirmed sustained brain retention, highlighting improved pharmacokinetics and imaging potential. Conclusions: These results support PEGylated scFv as a promising SPECT imaging agent for early detection of tauopathy in TBI, offering enhanced brain retention and improved pharmacokinetics. Full article

Saposin-like protein 2 (SAP2) exhibits strong immunogenicity as an antigen for immunodiagnosis in ruminant and human fasciolosis. Most available immunodiagnostic test kits are based on polyclonal and monoclonal antibodies against antigens from Fasciola spp. Previous studies demonstrated that polyclonal and monoclonal antibodies against SAP2 showed high specificity and could effectively detect Fasciola spp. infections at an early stage. However, polyclonal antibodies are extremely difficult to produce, and quality control is not possible during production; the procedure also involves considerable financial investment. To overcome these problems, we developed a single-chain variable fragment (scFv) to control quality in each production cycle and reduce the cost of manufacturing immunodiagnostic kits. Our objectives were to produce and characterize an scFv that binds the SAP2 from the liver fluke Fasciola gigantica. We constructed the scFv by genetic engineering: we cloned immunoglobulin genes and linked them with flexible polypeptide linkers composed of repeating glycine and serine residues. We selected an scFv with high affinity for binding SAP2 using the phage-display technique and produced it using a prokaryotic expression system. The scFv was characterized via in silico and in vitro methods to confirm its specificity for SAP2, including IMGT/V-QUEST, IMGT/Collier-de-Perles, HADDOCK 2.4, ELISA, immunoblotting, and immunohistochemistry. The scFv was successfully produced and purified using Ni-NTA affinity chromatography. The purified scFvFgSAP2 was approximately 27 kDa, as confirmed by SDS-PAGE and immunoblot analysis. An indirect ELISA and immunoblotting indicated that scFvFgSAP2 had strong reactivity with F. gigantica compared to other parasite species. Moreover, immunolocalization of scFvFgSAP2 confirmed that it binds specifically to natural SAP2 in the cecal epithelium cells of F. gigantica. Therefore, this scFv targeting SAP2 is an effective material and can be used to develop immunodiagnostic procedures. Full article

Background/Objectives: The GMZ2.6c malaria vaccine candidate is a multi-stage P. falciparum chimeric protein that contains a fragment of the sexual stage Pfs48/45-6c protein genetically fused to GMZ2, which is an asexual stage vaccine construct consisting of conserved domains of Glutamate-Rich Protein (GLURP) and Merozoite Surface Protein-3 (MSP-3). Previous studies showed that GMZ2.6c is widely recognized by antibodies from individuals living in endemic areas of Brazil and that levels of anti-GMZ2.6c increase with malaria exposure and may contribute to immunity against the parasite. As cell-mediated responses are crucial for parasite control and protection, identifying antigens that elicit antigen-specific T cell recall in naturally exposed populations is the key to vaccine development. This study aimed to evaluate the cellular immune response against GMZ2.6c and its components (MSP-3, GLURP, and Pfs48/45) and to identify promiscuous T cell epitopes in individuals exposed to malaria in the Brazilian Amazon, considering the impact of active P. falciparum infection on antigen-specific T cell recall. Methods: This study was carried out using peripheral blood mononuclear cells (PBMCs) from individuals with active P. falciparum infection (PFI) and non-infected individuals exposed to malaria (NI) from Cruzeiro do Sul and Mâncio Lima, Acre State, and Guajará, Amazonas State. The PBMCs were stimulated with GMZ2.6c and its components, and cellular activation, CD4⁺ and CD8⁺ memory T cell subsets, and cytokine production were evaluated by flow cytometry. IFN-γ-secreting T cells were quantified by ELISpot using predicted T cell epitopes. Results: The individuals infected by P. falciparum displayed more CD8⁺ T cell activation in response to MSP-3 and Pfs48/45 and an increase in CD4⁺ T_CM cells and a reduction in CD4⁺ T_EM cells following stimulation with Pfs48/45 and GMZ2.6c. The PBMCs from both groups showed elevated production of IL-6 and TNF after stimulation with GMZ2.6c, MSP-3, and Pfs48/45, but only the non-infected individuals had high levels of IL-10. T cell epitope prediction identified sequences within MSP-3, GLURP, and Pfs48/45 that elicited IFN-γ responses in both the non-infected and P. falciparum-infected individuals. Conclusions: Individuals exhibit cellular immune responses to MSP-3 and Pfs48/45 that are recalled following GMZ2.6c stimulation. P. falciparum infection may modulate immune response, inducing a prominent pro-inflammatory response. Conversely, in the absence of the parasite, the individuals displayed balanced Th1/Th2 cytokine production. Several promiscuous T cell epitopes were able to recall IFN-γ responses. Further studies are needed to fully ascertain the potential of GMZ2.6c as a protective candidate vaccine against malaria. Full article

Mitochondria are essential organelles that perform irreplaceable functions in neurons. The degeneration of neurons in Alzheimer’s disease (AD) is associated with mitochondrial damage, and Tau pathology represents a significant pathogenic factor in AD. However, the relationship between Tau and mitochondrial dysfunction during neuronal degeneration remains unclear. In this study, we investigated the effects and mechanisms by which extracellular Tau aggregates induce neuronal mitochondrial damage and dysfunction. The results showed that extracellular Tau aggregates lead to structural damage of mitochondria in SH-SY5Y cells and disrupt mitochondrial homeostasis. Extracellular Tau aggregates can also cause mitochondrial oxidative stress and inhibit oxidative phosphorylation in SH-SY5Y cells. Concurrently, extracellular Tau aggregates promote neuronal death through an increase in cytochrome C, mtDNA leakage and activation of the cGAS/STING pathway. We also explored the effects of a single-chain variable fragment antibody (scFv T1) and found that scFv T1 alleviated mitochondrial damage and dysfunction by inhibiting the formation of Tau aggregates. These findings suggest that targeting Tau pathology may be crucial to address neuronal mitochondrial impairment and that reduction of the toxicity associated with extracellular Tau aggregates could help slow Tau pathology progression. Full article

EmCRT is a calreticulin secreted by Echinococcus multilocularis during its infection in host, playing an important role in evading host immune attack as a survival strategy. Our previous study has demonstrated that recombinant EmCRT (rEmCRT) was able to bind to C1q and lectin to interfere with host classical and lectin complement activation pathway, respectively. However, the C1q-binding site on EmCRT and the associated immune evasion mechanism remain unknown. In this study, the C1q-binding site on EmCRT was determined through molecular docking analysis and fragment expression to be localized to the S-domain (EmCRT-S) between Lys¹⁴⁰ at the N-domain and Gln²⁹² at the P-domain. The recombinant EmCRT-S protein (rEmCRT-S) was subsequently expressed in bacteria. Functional analysis confirmed that rEmCRT-S was able to bind to human C1q and inhibit C1q-initiated complement activation at the similar level to the full-length rEmCRT, resulting in the reduction in C4b/C3b deposition and antibody-sensitized sheep red blood cell hemolysis. Furthermore, rEmCRT-S binding to C1q suppressed THP-1-derived macrophage chemotaxis and ROS generation. Given that the identified functional domain EmCRT-S provides similar complement regulatory functions to the full-length EmCRT, this domain is a more feasible and practical target for vaccine development against E. multilocularis infection or for inflammatory and autoimmune diseases. Full article

Background: Transferrin receptor protein 1 (TfR1) plays a central role in cellular iron uptake and is frequently overexpressed in malignant tumor cells, rendering it an attractive target for tumor-directed therapy and drug delivery. Methods: A fully human single-chain variable fragment (scFv) antibody targeting TfR1, termed T8scFv, was isolated from a human scFv phage display library through three rounds of stringent biopanning and subsequently reformatted into a full-length IgG1 antibody (T8IgG1). Binding kinetics were characterized using Octet biolayer interferometry (BLI), while cellular binding and internalization were assessed by flow cytometry and immunofluorescence microscopy, respectively. T8IgG1 was further conjugated to DT3C, a recombinant truncated diphtheria toxin fusion protein, to evaluate its internalization-dependent cytotoxicity in vitro. Results: T8scFv exhibited nanomolar affinity for TfR1 (K_D = 214 ± 1 nM), which was substantially enhanced following conversion to the IgG1 format (T8IgG1, K_D = 18.5 ± 0.1 nM). T8IgG1 specifically recognized TfR1 on the surface of tumor cells and underwent efficient TfR1-mediated internalization. The T8IgG1-DT3C complex significantly reduced cell viability and induced apoptosis in K562 cells in vitro. Conclusions: These findings indicate that T8IgG1 is a moderate-affinity, internalizing anti-TfR1 antibody and highlight its potential as a promising candidate for TfR1-based targeted antitumor drug delivery systems. Full article

Background: Respiratory syncytial virus (RSV) causes severe lung infections in infants and the elderly. The conserved central domain (CCD) of the RSV G protein is a key antigenic fragment for inducing protective antibodies. In this study, we used the hepatitis B surface antigen (HBsAg) as a platform to present this RSV G CCD fragment. Methods: We first sequenced and compared several HBsAg genotypes from clinical samples and selected one as an expression candidate for further development. The RSV G CCD was then inserted into the selected candidate to generate a recombinant expression construct. Subviral particles (SVPs) were produced using both CHO cells and yeast expression systems. Particle assembly was examined using electron microscopy. Finally, the safety and immunogenicity of the recombinant vaccine were evaluated in mice. Results: We successfully identified HBsAg38 as a potential recombinant vaccine expression candidate due to its abundant expression and secretion. The RSV G CCD fragment was inserted into the candidate and efficiently expressed in both CHO cells and yeast. The expressed protein was effectively secreted and formed uniform, spherical particles. The resulting vaccine candidate was safe for mice, causing no detectable weight loss or organ damage. Immunization with the recombinant SVPs elicited antibody responses against both HBsAg and the RSV G CCD. Upon intranasal RSV challenge, vaccinated mice exhibited markedly reduced RSV F protein and mRNA levels in lung tissues compared to PBS controls, with the yeast-derived SVP group showing the most pronounced reduction. Histopathological analysis further revealed that immunized mice had significantly less alveolar destruction and inflammatory cell infiltration than the control group, confirming that the vaccine conferred effective protection against RSV-induced lung pathology. Conclusions: We successfully developed a novel antigen-displaying HBsAg platform for generating vaccines targeting multiple pathogens. The RSV G CCD-expressing HBsAg induced a strong antibody response and provided effective protection against RSV infection. This platform offers a promising new approach for the development of next-generation vaccines. Full article

Background/Objectives: FcγRIIIA presents a single nucleotide polymorphism at position 158 (V/F), which affects its binding affinity to the fragment crystallizable (Fc) of antibodies (Abs). In the presence of immune complexes, FcγRIIIA can mediate the inflammatory signaling, severity of bone disease, and osteoclastogenic activity. Based on this functional relevance, we hypothesized that the FcγRIIIA F158V polymorphism may influence the clinical presentation of multiple myeloma (MM). Methods: FcγRIIIA F158V genotyping was performed on genomic DNA extracted from peripheral blood samples of patients affected by MM or asymptomatic conditions named MGUS and SMM. We compared the allele frequency of FcγRIIIA-F158V polymorphism in 72 MM, 42 MGUS and 31 SMM and evaluated the association with clinical features and occurrence of high-risk chromosome abnormalities. Targeted NGS mutation analysis was performed on genomic DNA isolated from purified CD138⁺ bone marrow plasma cells (BMPCs) of 41 patients, to evaluate the association between somatic mutations and the FcγRIIIA F158V genotype. Results: the FcγRIIIA-158 V/V homozygous genotype was associated with high-risk cytogenetics, anemia, high beta-2 microglobulin levels, and more than 10 osteolytic lesions. V/V homozygous genotype was significantly associated with at least one mutation in RAS pathway genes (N-RAS, K-RAS or B-RAF). In the immune microenvironment, patients carrying the V/V homozygous genotype had a higher percentage of CD14⁺CD16⁺⁺ non-conventional inflammatory monocytes than the V/F or FF genotype. Conclusions: Our study contributes to a better understanding of the interactions between genetic variants, tumor microenvironment, and therapeutic response in plasma cell dyscrasias, to identify molecular biomarkers for precision medicine in MM, MGUS and SMM. Full article

Background: Despite its high vaccination coverage, pertussis remains a public health concern due to waning vaccine-induced immunity and the emergence of pertactin (Prn)-negative strains. Nevertheless, anti-Prn antibodies and memory B cells elicited by vaccinations may contribute to long-term immunity and protection against Prn-positive strains. While most vaccination studies focus on serum antibodies, data on memory B cells remain limited. Methods: In this study, we implemented a flow cytometry-based approach to characterize Prn-specific B-cell fluctuations following Tdap booster vaccination in five healthy adults. Total and Prn- and tetanus toxoid fragment C (TTC)-specific plasma cells and memory B cells were analyzed at baseline and at 7, 14, 21, and 90 days post-vaccination using Prn Klickmers^® and TTC tetramers. Following this, cellular responses were correlated with antigen-specific serum IgG and IgA levels. Results: Prn-specific and TTC-specific memory B cells increased on days 14 and 7 post-vaccination, respectively, accompanied by a phenotypic shift from IgMD+ to IgG+ cells. Clear expansions of total as well as Prn- and TTC-specific plasma cells occurred on day 7. These plasma cells primarily comprised IgG+, but an increase in Prn-specific IgA+ plasma cells was also observed. The numbers of Prn-specific IgG+ memory B cells on day 7 post-vaccination correlated weakly with serum anti-Prn IgG levels at later time points. Conclusion: To our knowledge, this is the first study to use flow cytometry to evaluate Prn-specific B-cell responses and report their fluctuations over time following vaccination. These findings support the potential of this method to complement serological assays and improve our understanding of vaccine-induced immunity. Full article

Background: Protein A resins are indispensable for monoclonal antibody (mAb) production, yet their condition and performance are traditionally assessed using indirect or qualitative methods. In this study, the multi-attribute method (MAM), previously applied to therapeutic protein characterization, is systematically adapted for the first time as a unified liquid chromatography–mass spectrometry (LC-MS) platform for Protein A resin analysis. Method: Four Cytiva Protein A resins, MabSelect™, MabSelect SuRe™, MabSelect SuRe™ LX, and MabSelect™ PrismA, were evaluated by MAM for resin identity, Protein A ligand integrity, fouling by impurities, and cleaning performance. Results: MAM enables resin-specific peptide fingerprinting and quantitative monitoring of Protein A ligand post-translational modifications (PTMs), including deamidation, isomerization, and fragmentation induced by repeated clean-in-place (CIP) cycles. Comparative analysis of virgin and used resins revealed ligand degradation and fouling despite engineered alkaline stability, with MabSelect™ showing the greatest susceptibility. Importantly, residual monoclonal antibodies (mAbs) and host cell proteins (HCPs) were directly detected and quantified from the resin matrix, providing a molecular-level assessment of resin cleaning effectiveness not achievable with conventional approaches. Conclusions: This work establishes MAM as a novel, sensitive, and comprehensive strategy for Protein A resin lifecycle management, delivering actionable insight for resin selection, cleaning optimization, and downstream process development. Full article

The Golgi complex undergoes dynamic remodeling during the cell cycle, as ribbon unlinking in G2 is required for proper mitotic progression. Failure to fragment the ribbon leads to G2 arrest, whereas forced mitotic entry with intact ribbons results in multipolar spindle formation. Phosphorylation of the Golgi matrix protein GRASP65 at serine 277 (S277) in rat (S274 in human) by JNK2 is essential for ribbon unlinking, but its upstream regulation has remained unclear. Here, we generated and validated a phospho-specific antibody recognizing human GRASP65 phosphorylated at S274, enabling accurate detection of this modification. Using this tool, we identify protein kinase D2 (PKD2) as a critical upstream regulator required for GRASP65 phosphorylation and Golgi unlinking. GRASP65-S274 phosphorylation increases during G2 and is markedly reduced upon PKD2 inhibition or depletion, resulting in decreased Golgi unlinking and delayed G2/M transition. Conversely, PKD2-activating stimuli, including phorbol esters and nocodazole, enhance GRASP65 phosphorylation in a PKD2-dependent manner. These findings define PKD2 as a key regulator of the JNK2–GRASP65 signaling axis controlling Golgi disassembly at the G2/M transition. Moreover, the phospho-specific GRASP65 antibody described here provides a valuable tool to dissect upstream signaling mechanisms and to identify the initial triggers driving Golgi unlinking at G2 entry. Full article

Trogocytosis is the process of engulfment of a portion of a cell’s membrane by another cell. This process is characterized by the transfer of membrane fragments and proteins between adjacent cells without their complete fusion or phagocytosis, which distinguishes it from classical cellular uptake pathways. In the immune system, the initiating signal for trogocytosis is antigen presentation or the interaction of the Fc receptor with an antibody bound to the cell. During trogocytosis, T cells transfer not only the MHC molecule with the antigenic peptide, but also the costimulatory molecules CD80, CD86, OX-40 and others. As a result of trogocytosis, cells can transfer various surface molecules, acquire new immunological properties, and modulate each other’s activity. This review examines the basic mechanisms of trogocytosis, the involvement of T2-mediated immunity components in trogocytosis, and its possible role in allergies. Full article

Autoantibodies targeting α6β4 integrin have been identified in individual patients with mucous membrane pemphigoid (MMP). Reactivity against α6 integrin has been associated with oral lesions, while anti-β4 integrin reactivity has been linked to ocular involvement. However, the pathogenic effects of these antibodies have not been fully elucidated. Here, we investigated the pathogenic potential of anti-α6 and anti-β4 integrin IgG both in vitro and in vivo. Immune complexes of anti-α6 and anti-β4 integrin induced the release of reactive oxygen species from normal human leukocytes and stimulated CXCL2 secretion in cultured murine C5N keratinocytes. In vivo, repeated injections of IgG against a recombinant fragment of β4 integrin into C57BL/6 mice led to palpebral conjunctival swelling and mild oral lesions. The latter was observed following injection of IgG against a recombinant fragment of α6 integrin. Histopathological analysis revealed subepithelial inflammatory infiltrates without evidence of split formation. Direct immunofluorescence microscopy showed linear deposits of IgG at the basement membrane zone in most tissues, whereas C3 deposition was largely absent. This lack of complement activation was corroborated by a complement fixation assay, which confirmed that IgG against α6 and β4 integrin failed to induce C3 deposition in normal murine conjunctivae, buccal mucosa, or skin. Collectively, these findings indicate that IgG autoantibodies against α6 and β4 integrin exhibit pathogenic activity in vitro and induce mild disease in vivo, possibly due in part to relatively inefficient complement activation in this model. Full article