Search Results (405)

Candidatus Rickettsia colombiensis is a new candidate species of Rickettsiae spotted fever group that have been isolated only from ticks. The pathogenicity of Ca. R. colombiensis to human and animals is unknown. This study evaluated the pathogenic potential of Ca. R. colombiensis in Syrian hamsters and assessed the cross-reactivity between Ca. R. colombiensis and other Rickettsia in human and hamster sera. Shell vial technique was employed to isolate Ca. R. colombiensis. Subsequently, five male Syrian hamsters were inoculated intraperitoneally (IP) and five intradermally (ID) with 1 × 10⁶ Vero cells infected with Ca. R. colombiensis. One control hamster was used in each group. The health status was assessed daily, and necropsies were performed. Serum samples were tested by indirect immunofluorescence and tissues were processed by qPCR and histological stains. All Syrian hamsters remained healthy during the trial. No histopathological damages associated with rickettsial infection were observed. No Rickettsial DNA was detected in tissues. Syrian hamsters showed IgG antibody titers ranging from 1:64 to 1:1024. Control hamsters were negative. Regarding human sera, 56% (84/150) had IgG cross-reactivity antibodies against Ca. R. colombiensis. Subsequently, in a selected subset of 30 sera with moderate to high titers, all samples reacted with Ca. R. colombiensis antigen. Under specific conditions of this study, Ca. R. colombiensis did not behave as a highly virulent pathogen in the hamster model, although all infected Syrian hamsters developed IgG antibodies responses. Regarding cross-reactivity, it is possible to serologically diagnose rickettsial infection using Ca. R. colombiensis as an antigen. Full article

Prostate cancer diagnostics have evolved substantially with the integration of multiparametric magnetic resonance imaging (mpMRI), refined prostate-specific antigen (PSA) metrics, and targeted biopsy techniques. While mpMRI has become a central gatekeeper in biopsy decision-making, it is not infallible. Clinically significant prostate cancer may therefore remain undetected, particularly in patients with elevated PSA density, adverse PSA kinetics, or MRI-occult disease. This narrative review synthesizes contemporary evidence on PSA interpretation, mpMRI performance, and biopsy strategy selection, highlighting the limitations of single-parameter approaches. We discuss the diagnostic yield and clinical implications of targeted, systematic, and combined biopsy techniques, emphasizing scenarios in which systematic sampling remains necessary despite negative or equivocal imaging findings. Emerging data support combined targeted and systematic biopsy as the most robust strategy for maximizing the detection of clinically significant disease while limiting overdiagnosis in most biopsy-naive and high-risk patients. By integrating PSA dynamics, prostate volume, imaging findings, and individual risk profiles, a structured, risk-adapted diagnostic pathway can be achieved. The proposed framework is intended as a conceptual, expert-derived clinical aid to support risk-adapted decision-making. It should be interpreted alongside established guidelines, and prospective validation in future studies is warranted. Full article

Background/Objectives: Despite clonal expansion during a primary immune response, or after subsequent antigen encounters, the frequency of memory B cells (B_mem) specific for an antigen remains low, making their detection difficult. However, unlike serum antibodies, which have a short half-life in vivo and thus require continuous replenishment to maintain stable titers, circulating B_mem are long-lived; they preserve immunological preparedness through their ability to rapidly engage in recall responses and differentiate into antibody-secreting cells (ASCs) upon antigen encounter. To this end, development of assays suited for the reliable detection of rare antigen-specific B_mem is critical and can provide insights into an individual’s antigen exposure history and immune status beyond that offered by traditional serum antibody measurements alone. Methods: ImmunoSpot^® has emerged as a suitable technique for the detection of individual antigen-specific B cells through visualizing their antibody-derived secretory footprints. Here, we report the theoretical and practical foundations for detecting rare antigen-specific B_mem in human peripheral blood mononuclear cells (PBMC). Leveraging the unique availability of verifiably naïve vs. antigen-experienced human samples, we used SARS-CoV-2 Spike (S-) and Nucleocapsid (NCAP) antigens to interrogate the presence of B_mem with these respective specificities. Results: While 100% diagnostic accuracy was achieved for both antigens, detection of NCAP-specific B_mem required reducing the lower detection limit of the standard assay. Specifically, this was achieved by testing a total of 2 million PBMC across multiple replicate assay wells and assessing the cumulative number of secretory footprints detected. Conclusion: The protocols described here should facilitate the reliable detection of ASCs present at varying precursor frequencies and serve as guidance for routine immune monitoring of rare B_mem with specificity for any antigen. Full article

Introduction: Radiotracer-based nuclear imaging, including positron emission tomography (PET) and single-photon emission computed tomography (SPECT), can complement conventional cross-sectional imaging in renal cell carcinoma (RCC) by providing biological characterisation of tumour metabolism, angiogenesis, hypoxia, and the tumour microenvironment. While computed tomography (CT) and magnetic resonance imaging (MRI) remain the diagnostic standard, accumulating evidence suggests that selected nuclear imaging techniques may offer incremental value in specific clinical scenarios. Methods: A narrative literature review was performed using PubMed, Embase, and Web of Science to identify preclinical, retrospective, and prospective studies evaluating PET and SPECT radiotracers in localised and metastatic RCC. Priority was given to meta-analyses, multicentre prospective trials, and studies with histopathological correlation. Results: [¹⁸F]fluorodeoxyglucose (FDG) PET/CT demonstrates limited sensitivity for primary renal tumours (pooled sensitivity of approximately 60%) but performs substantially better in metastatic and recurrent disease (pooled sensitivity and specificity of approximately 85–90%), where uptake correlates with tumour grade, progression-free survival, and overall survival. [^99mTc]sestamibi SPECT/CT differentiates oncocytoma and hybrid oncocytic/chromophobe tumours from malignant RCC with pooled sensitivity and specificity of around 85–90%, supporting its role in evaluating indeterminate renal masses rather than staging. Prostate-specific membrane antigen (PSMA) PET/CT shows high detection rates in clear-cell RCC, particularly in metastatic disease, with reported sensitivities of approximately 85–90% and management changes in up to 40–50% of selected cohorts. Carbonic anhydrase IX (CAIX)-targeted PET/CT enables the biologically specific visualisation of clear-cell RCC, achieving sensitivities and specificities in the range of 85–90% in prospective phase II and III trials for primary tumour characterisation. Fibroblast activation protein inhibitor (FAPI) PET/CT demonstrates high tumour-to-background uptake in early RCC studies, but evidence remains preliminary, with small cohorts and recognised non-specific uptake in benign inflammatory and fibrotic conditions. Conclusions: Radiotracer-based nuclear imaging provides complementary, biology-driven insights in RCC that extend beyond anatomical assessment. While most modalities remain adjunctive or investigational and are not recommended for routine use, selective application in carefully chosen clinical scenarios may enhance tumour characterisation, prognostication, and personalised treatment planning. Full article

Most intracellular proteins are degraded by the ubiquitin–proteasome system (UPS), with proteasomes directly hydrolyzing protein substrates. Specific forms of proteasomes (non-constitutive proteasomes), implicated in antigen presentation, cellular homeostasis maintenance and stress response have been described. However, proteasomes were also identified outside cells, where their function remains unclear. Proteasome secretion via extracellular vesicles (EVs) have been reported, though the direct transmission of non-constitutive proteasomes between cells has not been shown. Using genetically modified cells, including a human adenocarcinoma cell line SW620B8-mCherry expressing the β5i subunit of non-constitutive proteasomes fused to the mCherry protein, and a number of techniques, such as differential centrifugation, affinity isolation, unspecific precipitation, NTA and microscopy, EVs containing non-constitutive proteasomes were obtained and characterized. Different cell lines were shown to secrete varying amounts of vesicles containing non-constitutive proteasomes. The content of these proteasomes in EVs was increased after the stimulation of cells with IFN-γ. The interaction of vesicles secreted by SW620B8-mCherry cells with recipient cells was demonstrated. The β5i-mCherry chimera was detected in lysates of different recipient cells following incubation with EVs secreted from SW620B8-mCherry cells. The obtained results highlight the transfer of non-constitutive proteasomes from one cell to another via EVs. Full article

Background: Memory B cells (B_mem) facilitate the generation of renewed and rapid antigen-specific antibody responses long after the initial antigen exposure, at a time when circulating serum antibodies may have declined. As the generation and/or recruitment of B_mem is at the core of most vaccination strategies, the assessment of antigen-specific B_mem is highly informative for forecasting and profiling the elicited B cell immune response. Methods: The two prevalent techniques used to detect antigen-specific B_mem cells at single-cell resolution are probe-based flow cytometry and B cell ImmunoSpot, while the measurement of B cell-derived antibodies in culture supernatants of stimulated B cells offers a semi-quantitative alternative. To the best of our knowledge, a direct side-by-side comparison of these assay systems has not yet been reported using the same starting PBMC material in a blinded fashion to test all three assays simultaneously. Results: These three assay systems were run in parallel to detect SARS-CoV-2 Wuhan-1 strain Spike-specific IgG⁺ B_mem in peripheral blood mononuclear cell (PBMC) samples obtained from well-defined cohorts comprising pre-COVID-19 era “naïve” individuals (negative controls), individuals shortly after recovery from a PCR-verified SARS-CoV-2 infection (positive controls), and a cohort of donor PBMCs isolated in 2024 (the experimental group). Each assay was able to discern Spike-exposed individuals from naïve , with ImmunoSpot suggesting superior sensitivity and specificity. ImmunoSpot and flow cytometry results were closely correlated. Conclusions: The study demonstrates that all three assays are suited for the detection of specific B_mem in antigen-primed individuals when such B_mem occur in the mid- to high-frequency range, and that they broadly concur. Strengths and weaknesses of the three test systems are discussed. Full article

Nanomedicine has now become a transformative platform that enhances the precision and efficacy of immunotherapy approaches and allows customizations like never before when it comes to cancer, as well as autoimmune conditions. Using platforms based on nanoscale, researchers have been able to manipulate immune responses operating across spatial and temporal scales to address key limitations of conventional immunotherapy associated with working with immune response such as immune evasion, systemic toxicity, and poor pharmacokinetics. Sophisticated nanoparticles (such as stimuli-sensitive ones, exosome-mimetic vesicle nanoparticles, and nanoparticles with CRISPR) allow directed immunomodulators, antigens, and gene-editing systems to reach one or more particular immune compartments. The innovations allow reprogramming of immune cells, immune tolerance rejuvenation, and expansion of antitumor immunity without significant off-target effects. Finding applications in integrating the artificial intelligence as well as multi-omics techniques, the process leads to personalization of the nano-immunotherapies based on patient-specific immuno-signatures. The chapter discusses the mechanistic rationale, therapeutic advancement, and the translational opportunities of nanotechnology-based immunotherapies that define them as part of a foundation of future generations of clinical approaches to precision immune modulation in oncology and autoimmune diseases. Full article

Several liquid-, and tissue-based markers are available to guide primary diagnosis-, active surveillance-, and treatment-related decision-making for patients with prostate cancer. Most of these tests can improve the balance of harms and benefits associated with early detection, and aid patient selection for treatment intensification. However, the costs of these tests can make their integration in routine clinical practice challenging. To date, prostate-specific antigen (PSA) is still one of the most well-known and widely utilized tumor markers worldwide, with a unique facility ranging from the diagnosis to the treatment-related follow-up of patients with prostate cancer. Future research efforts are needed to integrate biomarkers and novel imaging techniques, such as prostate magnetic resonance imaging, in the decision-making pathways. Despite the growing body of knowledge and evidence, considerable challenges remain in optimizing risk-stratification, improving patient selection and cost-efficacy in different prostate cancer (PCa)-related settings. 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

Objectives: Pancreatic cystic lesions (PCLs) are increasingly detected due to the widespread use of imaging techniques. The identification of pancreatic mucinous cysts is especially important since these carry a risk of malignant transformation and require follow-up or surgical resection. The aim of this study was to determine the diagnostic yield of the molecular analysis of K-RAS (Kirsten RAt Sarcoma virus) and GNAS (Guanine Nucleotide-binding protein, Alpha Stimulating protein activity) gene mutations in pancreatic cyst fluid (PCF) obtained by endoscopic ultrasound (EUS)-guided fine needle aspiration (FNA). Methods: In this prospective trial, we assessed the sensitivity, specificity, and positive and negative predictive values of K-RAS and GNAS mutation analysis in differentiating mucinous versus non-mucinous cysts and the subsequent impact on decision-making in daily clinical practice. The reference standard used comprised the combination of morphology on cross-sectional imaging and EUS, string sign, cyst fluid cytology, intracystic carcinoembryonic antigen (CEA), and glucose levels, with subsequent correlation of surgical pathology in resected cases. Fluid samples of 47 cysts obtained by EUS-FNA over a 39-month period were analyzed. Mutation analysis of KRAS (exon 2) was performed in all cases, and additionally, GNAS (exon 8) in 28 cases using Sanger sequencing. Results: 33 out of 47 PCLs were classified as mucinous cysts and 14 as non-mucinous cysts defined using conventional standards, including morphological characteristics, string-sign, cytology, cyst fluid testing, and histology in resected cases. Of these 33 mucinous cysts, KRAS mutation was detected in 14 samples. A further 23 mucinous lesions were additionally tested for GNAS mutation, which was detected in 10 of the 23 cysts. A 42.4% sensitivity for KRAS and 43.5% for GNAS mutation analysis was calculated, with a specificity of 92.9% and 100%, respectively, for detecting mucinous lesions. The clinical management was altered through the genetic testing results in one single case. Conclusions: In this cohort, K-RAS and GNAS mutational analysis in cyst fluid did not improve the detection of mucinous pancreatic cysts significantly after conventional testing. However, the method may be useful due to its high specificity in uncertain cases. Full article

Backgroud/Objectives: Canine Visceral Leishmaniasis (CVL), caused by Leishmania infantum, is a significant public health concern due to dogs serving as reservoirs for human infection. An accurate and rapid diagnostic method to distinguish symptomatic and asymptomatic CVL from healthy and vaccinated animals is essential for controlling canine and human disease. Developing innovative antibody detection techniques and exploring new antigens are essential for enhancing CVL testing efficiency. Our study focuses on a multiplex flow cytometry technique to detect Leishmania-specific antibodies in canine serum. This involved conjugating small peptides with carrier proteins or peptide tags, sequences designed to facilitate bead coupling. Methods: A peptide from the L. infantum A2 protein was coupled to beads in three forms: unconjugated, conjugated with BSA, and conjugated with a C-terminal β-alanine–lysine (x4)–cysteine TAG. This TAG was previously designed to enhance peptide solubility, improve binding efficiency, and provide functional groups for covalent attachment to the beads, ensuring stable immobilization in the multiplex assay. Results: Our results suggest that the multiplex approach shows promise as a rapid serological test for CVL, particularly with TAG-conjugated peptides, which optimize bead coupling. However, peptide/BSA conjugation revealed anti-BSA antibodies in samples from healthy and CVL dogs. Conclusions: In conclusion, our findings highlight the potential of multiplex methodologies to enhance CVL diagnostics and caution against using BSA as a bead coupling agent in serological tests for canine samples due to its impact on test specificity and sensitivity. Full article

Background: Neurofilament light chain (NfL) is a well-established biomarker of neuroaxonal damage, detectable in serum through immunoassays. Its potential relevance in psychiatric conditions, including anorexia nervosa (AN), is currently under investigation. This study aims to quantify serum NfL levels in individuals with AN, evaluate their correlation with autoantibodies detection, and critically examine the specificity of NfL as a biomarker in this context. Methods: A total of 100 participants were enrolled, comprising 50 individuals diagnosed with AN and 50 age-matched, normal-weight controls. Serum concentrations of NfL and immunoglobulin G (IgG) antibodies reactive to hypothalamic antigens were measured using validated immunoassay techniques. Results: Serum NfL concentrations were markedly higher in the AN group compared to healthy controls. Interestingly, NfL levels tended to decrease with longer disease duration and with the recovery of body mass index (BMI), indicating a possible association between clinical improvement and reduced neuroaxonal damage. Furthermore, the results confirmed the presence of anti-hypothalamic autoantibodies and revealed a positive correlation between their levels and serum NfL concentrations. Conclusions: Clinical remission in AN appears to be linked to a decrease in both markers neuronal damage and hypothalamic autoimmunity. However, as elevated serum NfL is observed across a spectrum of neurological and psychiatric disorders, its specificity as a biomarker for AN should be further investigated. While NfL may reflect neuroaxonal injury in AN, its interpretation should be contextualized within a broader clinical and immunological framework. Full article

Toll-like receptor 9 (TLR9) and Toll-like receptor 3 (TLR3), which are widely expressed in dendritic cells (DCs), function as key pattern recognition receptors (PRRs) in the immune system. Their primary roles involve specifically detecting pathogen-associated molecular patterns (PAMPs): TLR9 recognizes unmethylated CpG motifs predominantly found in bacterial and viral DNA, while TLR3 identifies viral double-stranded RNA (dsRNA), a molecular signature associated with viral replication. Their specific agonists [CpG ODN (a TLR9 agonist) and poly(I:C) (a TLR3 agonist)] can effectively activate DCs and enhance the expression of immune activation-related molecules. In this study, by establishing a mouse primary dendritic cell model and a glioma-bearing mouse model, and employing techniques such as transcriptome sequencing, we found that combined stimulation with CpG ODN and poly(I:C) significantly enhanced the anti-tumor function of DCs: in vitro, DCs subjected to combined stimulation showed upregulation of anti-tumor-related surface markers, enhanced migratory capacity, and a more effective activation of CD8⁺ T cells; in vivo, a DC vaccine loaded with tumor lysate antigen and stimulated with this combined regimen significantly delayed the progression of glioma in tumor-bearing mice. Further investigation revealed that the underlying mechanism for this enhanced effect may involve TLR9 activation promoting TLR3 upregulation through the p38 MAPK-ATF3 signaling axis. Consequently, we designed a sequential stimulation protocol (first CpG ODN then poly(I:C)), which demonstrated a stronger anti-glioma effect compared to simple combined stimulation. This study provides a new strategy for enhancing the immune efficacy of DC vaccines and has potential significance for promoting the clinical translation of DC vaccines. Full article

Background/Objectives: Satoyoshi syndrome is a rare, autoimmune disorder currently diagnosed based on clinical criteria: painful muscle spasms, diarrhea, and alopecia. Two previous reports showed a specific immunoreactive band in three Satoyoshi syndrome patients using Western blot analysis, with brain homogenate as the antigen source. These findings could be the basis for a future diagnostic test. The aim of our study was to evaluate the efficacy of using SH-SY5Y cell lysate instead of brain homogenate for a potential laboratory test for Satoyoshi syndrome using the Western blot technique. Methods: Western blot analyses were conducted using brain homogenate, SH-SY5Y cell lysates, and differentiated SH-SY5Y cell lysates. Serum samples were obtained from three Satoyoshi syndrome patients, alongside control samples from thirty blood donors and six patients with other neurological conditions. Results: Sera from patients with Satoyoshi syndrome displayed a three-band pattern in the 70–100 kDa range. This pattern was reproducible across all tested antigen sources (brain homogenate, SH-SY5Y lysate, and differentiated SH-SY5Y lysate) but was not observed for the sera from the control groups. The bands were more visible when using either type of SH-SY5Y lysate compared to brain homogenate. No differences were found between the SH-SY5Y lysate and the differentiated SH-SY5Y lysate. Conclusions: Sera from our Satoyoshi syndrome patients showed a specific band pattern that could be used for a future evaluation of Satoyoshi syndrome using Western blot. The use of SH-SY5Y cell lysate vs. brain homogenate as an antigen source may improve visualization and reproducibility of the immunobands and be less costly. Full article

COVID-19, caused by SARS-CoV-2, has created unprecedented global health challenges, necessitating rapid and reliable diagnostic strategies. The spike (S) protein, particularly its S1 subunit, plays a critical role in viral entry, making it a prime biomarker for early detection. In this study, we present a disposable, low-cost, and portable electrochemical biosensor employing specifically optimized aptamers (Optimers) for SARS-CoV-2 S1 recognition. The sensing approach is based on aptamer–protein complex formation in solution, followed by immobilization onto pencil graphite electrodes (PGEs). The key parameters, including aptamer concentration, interaction time, redox probe concentration, and immobilization time, were systematically optimized by performing electrochemical measurement in redox probe solution containing ferri/ferrocyanide using differential pulse voltammetry (DPV) technique.Under optimized conditions, the biosensor achieved an ultralow detection limit of 18.80 ag/mL with a wide linear range (10⁻¹–10⁴ fg/mL) in buffer. Importantly, the sensor exhibited excellent selectivity against hemagglutinin antigen and MERS-CoV-S1 protein, while maintaining high performance in artificial saliva with a detection limit of 14.42 ag/mL. Furthermore, its integration with a smartphone-connected portable potentiostat underscores strong potential for point-of-care use. To our knowledge, this is the first voltammetric biosensor utilizing optimized aptamers (Optimers) specific to SARS-CoV-2 S1 on disposable PGEs, providing a robust and field-deployable platform for early COVID-19 diagnostics. Full article