Spot Assay Research Articles

Biological control of Pseudomonas aeruginosa (ATCC-27853) by isolated lytic bacteriophage

Background Pseudomonas aeruginosa is a common bacterial species; it can grow in all fields, even in distilled water. One of the obvious characteristics of P. aeruginosa species is its low antibiotic susceptibility (antibiotic-multi-resistant species); this reflects the urgent need for alternative strategies. Unconventionally, we try to use an alternative destructive antibacterial agent, phage. phage application in our environmental fields (engineering relevance) is a promising safe avenue for replacing chemical biocides. Objective To isolate a lytic phage as a bio-control agent for Pseudomonas aeruginosa (ATCC-27853), evaluate the ability of the isolated phage to inhibit or suppress the growth of the target bacteria. We study phage stability while performing biological and physical characterization, optimizing phage action against bacteria. Materials and methods We use an activated, purified young culture of Pseudomonas aeruginosa (ATCC-27853) as the target bacteria to isolate a specific lytic bacteriophage from a mixture of Enriched sewage and marine water through a sequence of experiments: a Broth clearing assay using nutrient broth and a spot assay test by using nutrient agar medium, both for the detection of phage presence and its lytic action. For the purification of phage, the plaque assay test was performed in many successive subcultures by using the serial dilution method on a semisolid medium. The high-titer lysate obtained from the confluent plates was investigated by TEM to determine its morphology and taxonomy. We performed biological characterization of the isolated phage: one-step growth curve, Phage adsorption rate, host range (using nutrient double layer agar), as well as a bacterial challenge test (using nutrient broth, based on the optical densities). Also, we performed physical characterization of the phage to determine phage Stability, pH, thermal stability, and the influence of UV and organic solvents on the lytic action of the phage. Results and conclusion Pyocyanin and pyoverdin pigments (both fluorescent under UV illumination) are secreted by P. aeruginosa (ATCC-27853), also their ability to thrive at 42°C. After spotting P. aeruginosa plates with enriched sewage filtrate, the results indicated lytic area (+ve). Plaques have a pin tip and are translucent. The lytic profile of bacteria after phage infection revealed that the bacteria was reduced to a MOI of 10 in 4 h. However, the multiplicity of infection(MOI) of 1 and 0.01, respectively, was longer (10, 22/h). The electron microscopy revealed that the phage is a member of the podoviridae family, with a noncontractile short tail (18 nm) and a polyhedral (heptagonal) head (45 nm) in diameter and a phage length of 63 nm. The adsorption rate of the phage was greatest at 20 min. Our goal with the phage application is to replace harmful chemical biocides with lytic bacteriophages in various environmental systems (Phage for Biological Control). Phage bio-control treatment thus holds promise for eliminating the requirement for hazardous chemical biocides.

Personalized neoantigen vaccine and pembrolizumab in advanced hepatocellular carcinoma: a phase 1/2 trial

Programmed cell death protein 1 (PD-1) inhibitors have modest efficacy as a monotherapy in hepatocellular carcinoma (HCC). A personalized therapeutic cancer vaccine (PTCV) may enhance responses to PD-1 inhibitors through the induction of tumor-specific immunity. We present results from a single-arm, open-label, phase 1/2 study of a DNA plasmid PTCV (GNOS-PV02) encoding up to 40 neoantigens coadministered with plasmid-encoded interleukin-12 plus pembrolizumab in patients with advanced HCC previously treated with a multityrosine kinase inhibitor. Safety and immunogenicity were assessed as primary endpoints, and treatment efficacy and feasibility were evaluated as secondary endpoints. The most common treatment-related adverse events were injection-site reactions, observed in 15 of 36 (41.6%) patients. No dose-limiting toxicities or treatment-related grade ≥3 events were observed. The objective response rate (modified intention-to-treat) per Response Evaluation Criteria in Solid Tumors 1.1 was 30.6% (11 of 36 patients), with 8.3% (3 of 36) of patients achieving a complete response. Clinical responses were associated with the number of neoantigens encoded in the vaccine. Neoantigen-specific T cell responses were confirmed in 19 of 22 (86.4%) evaluable patients by enzyme-linked immunosorbent spot assays. Multiparametric cellular profiling revealed active, proliferative and cytolytic vaccine-specific CD4+ and CD8+ effector T cells. T cell receptor β-chain (TCRβ) bulk sequencing results demonstrated vaccination-enriched T cell clone expansion and tumor infiltration. Single-cell analysis revealed posttreatment T cell clonal expansion of cytotoxic T cell phenotypes. TCR complementarity-determining region cloning of expanded T cell clones in the tumors following vaccination confirmed reactivity against vaccine-encoded neoantigens. Our results support the PTCV’s mechanism of action based on the induction of antitumor T cells and show that a PTCV plus pembrolizumab has clinical activity in advanced HCC. ClinicalTrials.gov identifier: NCT04251117.

Abstract 5006: Discovery of immunogenic neo-peptides from actionable RNA fusions for developing cancer vaccines

Abstract The global surge in cancer cases, with a staggering 20 million new instances reported in 2023 alone, underscores an urgent demand for advanced and reliable cancer therapeutics. Immunotherapies centered around neoantigens have emerged as a promising avenue for enhancing treatment efficacy and have become a focal point in cancer research. We hypothesize that the tumor cells present a rich neoantigenic repertoire and the immunogenic neoantigens arising from the junctions of proteins translated from chimeric RNAs present viable targets for peptide and mRNA vaccines, offering a promising approach to impede tumor progression. To validate our hypothesis, we acquired a dataset of RNA fusions detected through RNAseq of tissue extracted from patient-derived xenograft (PDX) models established by XenoSTART, a global non clinical oncology contract research organization. This comprehensive dataset encompassed 985 PDX models across 27 cancer types, revealing three fusions associated with the neuregulin-1 (NRG1) gene. NRG1 gene fusions have been identified as a clinically actionable target for multiple solid tumors as they result in ErbB-mediated pathway activation. CD74-NRG1 has emerged as the most frequently reported NRG1 fusion across many cancers such as pancreatic ductal adenocarcinoma, triple negative breast cancer and ovarian cancer. The CD74-NRG1 fusion was identified in two PDX models harboring lung cancer (ST2891 and ST3204) and in an ovarian cancer model (ST088). The lung cancer PDXs displayed a notable abundance of fusion junction-crossing reads. We generated the CD74 (Exon 1-6) -NRG1 (Exon 6-12) fusion transcript model, where the first 6 exons of CD74 fused to exon 6 of NRG1 leading to an in-frame fusion. Validation of this fusion in PDX tissue was accomplished through PCR and Sanger sequencing of the PCR product. We assessed the affinity of the 9-mer neo peptide sequences formed by the translated junction of CD74-NRG1 to Major Histocompatibility Complex (MHC) Class I molecules using the in-silico prediction pipelines MHCNuggets and MixMHCPred-2 and found HLA-A*68:23 to be the MHC allele with highest binding affinity. HLA-Arena was used to investigate the binding affinity between the peptides and the MHC molecules via molecular docking, which revealed 4 peptides to be strong binders to HLA-A*68:23.The immunogenicity of these neoantigenic peptides to HLA-A*68:23 matched Peripheral Blood Mononuclear Cells (PBMCs) will be assessed by the IFN-γ Enzyme Linked Immunosorbent Spot (ELISpot) assay. To elucidate the specific T-cell clonotypes responding to the neo peptides, the 10X Genomics single-cell 5’ Gene Expression Assay, coupled with T Cell Receptor mapping, will be employed. The objective of the study is to establish a robust combinatorial therapeutic strategy leveraging peptide and mRNA vaccine technology to support an effective framework for personalized cancer treatment. Citation Format: Sakuni Rankothgedera, Micah Castillo, Shiyanth Thevasagayampillai, Cole Woody, Aaranyah Kandasamy, Armando Diaz, Michael Wick, Preethi Gunaratne. Discovery of immunogenic neo-peptides from actionable RNA fusions for developing cancer vaccines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5006.

Prevalence and characteristics of ertapenem-mono-resistant isolates among carbapenem-resistant Enterobacterales in China

ABSTRACT Carbapenem-resistant Enterobacterales (CRE), specifically those resistant to only ertapenem among carbapenems (ETP-mono-resistant), are increasingly reported, while the optimal therapy options remain uncertain. To investigate the prevalence and characteristics of ETP-mono-resistant CRE, CRE strains were systematically collected from 102 hospitals across China between 2018 and 2021. A 1:1 randomized matching study was conducted with ETP-mono-resistant strains to meropenem- and/or imipenem-resistant (MEM/IPM-resistant) strains. Antimicrobial susceptibility testing, whole-genome sequencing, carbapenem-hydrolysing activity and the expression of carbapenemase genes were determined. In total, 18.8% of CRE strains were ETP-mono-resistant, with relatively low ertapenem MIC values. ETP-mono-resistant strains exhibited enhanced susceptibility to β-lactams, β-lactam/β-lactamase inhibitor combinations, levofloxacin, fosfomycin, amikacin and polymyxin than MEM/IPM-resistant strains (P < 0.05). Phylogenetic analysis revealed high genetic diversity among ETP-mono-resistant strains. Extended-spectrum β-lactamases (ESBLs) and/or AmpC, as well as porin mutations, were identified as potential major mechanisms mediating ETP-mono-resistance, while the presence of carbapenemases was found to be the key factor distinguishing the carbapenem-resistant phenotypes between the two groups (P < 0.001). Compared with the MEM/IPM-resistant group, limited carbapenemase-producing CRE (CP-CRE) strains in the ETP-mono-resistant group showed a significantly lower prevalence of ESBLs and porin mutations, along with reduced expression of carbapenemase. Remarkably, spot assays combined with modified carbapenem inactivation method indicated that ETP-mono-resistant CP-CRE isolates grew at meropenem concentrations eightfold above their corresponding MIC values, accompanied by rapidly enhanced carbapenem-hydrolysing ability. These findings illustrate that ETP-mono-resistant CRE strains are relatively prevalent and that caution should be exercised when using meropenem alone for treatment. The detection of carbapenemase should be prioritized.

A mechanism study on the synergistic effects of rifapentine and fluconazole against fluconazole-resistant Candida albicans in vitro

Candida albicans (C. albicans) is one of the most common clinical isolates of systemic fungal infection. Long-term and inappropriate use of antifungal drugs can cause fungal resistance, which poses a great challenge to the clinical treatment of fungal infections. The combination of antifungal drugs and non-antifungal drugs to overcome the problem of fungal resistance has become a research hotspot in recent years. Our previous study found that the combination of rifapentine (RFT) and fluconazole (FLC) has a significant synergistic against FLC-resistant C. albicans. The present study aimed to further verify the synergistic effect between FLC and RFT against the FLC-resistant C. albicans 100, and explore the underlying mechanism. The growth curve and spot assay test not only showed the synergistic effect of FLC and RFT on FLC-resistant C. albicans in vitro but exhibited a dose-dependent effect on RFT, indicating that RFT may play a principal role in the synergic effect of the two drugs. Flow cytometry showed that the combined use of RFT and FLC arrested cells in the G2/M phase, inhibiting the normal division and proliferation of FLC-resistant C. albicans. Transmission electron microscopy (TEM) demonstrated that FLC at a low concentration could still cause a certain degree of damage to the cell membrane in the FLC-resistant C. albicans, as represented by irregular morphologic changes and some defects observed in the cell membrane. When FLC was used in combination with RFT, the nuclear membrane was dissolved and the nucleus was condensed into a mass. Detection of the intracellular drug concentration of fungi revealed that the intracellular concentration of RFT was 31–195 fold that of RFT alone when it was concomitantly used with FLC. This indicated that FLC could significantly increase the concentration of RFT in cells, which may be due to the damage caused to the fungal cell membrane by FLC. In short, the present study revealed a synergistic mechanism in the combined use of RFT and FLC, which may provide a novel strategy for the clinical treatment of FLC-resistant C. albicans.

Biocontrol of Escherichia coli O157:H7 by Enterobacter asburiae AEB30 on intact cantaloupe melons.

Escherichia coli O157:H7 causes >73,000 foodborne illnesses in the United States annually, many of which have been associated with fresh ready-to-eat produce including cantaloupe melons. In this study, we created a produce-associated bacterial (PAB) library containing >7500 isolates and screened them for the ability to inhibit the growth of E. coli O157:H7 using an invitro fluorescence-based growth assay. One isolate, identified by 16S and whole-genome sequence analysis as Enterobacter asburiae, was able to inhibit the growth of E. coli by ~30-fold invitro and produced zones of inhibition between 13 and 21 mm against 12 E. coli outbreak strains in an agar spot assay. We demonstrated that E. asburiae AEB30 was able to grow, persist and inhibit the growth of E. coli on cantaloupe melons under simulated pre- and post-harvest conditions. Analysis of the E. asburiae AEB30 genome revealed an operon encoding a contact-dependent growth inhibition (CDI) system that when mutated resulted in the loss of E. coli growth inhibition. These data suggest that E. asburiae AEB30 is a potential biocontrol agent to prevent E. coli contamination of cantaloupe melons in both pre- and post-harvest environments and that its mode of action is via a CDI system.

Exploring the Antifungal Activity of Various Natural Extracts in a Sustainable Saccharomyces cerevisiae Model Using Cell Viability, Spot Assay, and Turbidometric Microbial Assays

Saccharomyces cerevisiae is a sustainable yeast with many applications in the food industry. Here, we study the use of a Saccharomyces cerevisiae model composed of three different industrial strains (a wine, a beer and a baker’s strain) to assess the antifungal activity of three organic plant-based extracts (Hypericum perforatum 10% w/w, Pistacia lentiscus var. Chia 20% w/w and Rosmarinus officinalis 6% w/w). Three different methods were employed (agar disc diffusion, spot assay, and growth curve analysis). Only the Rosmarinus officinalis extract (6% w/w) exhibited inhibitory activity against all the tested yeast strains in the agar disc diffusion method. In the spot assay, all extracts and their carrier oils (sunflower oil and caprylic triglyceride) exerted similar mild antifungal activity. In the growth curve analysis, all extracts significantly lowered the growth rate of the yeasts, but this was not observed for the carrier oils. The results highlighted that it is important to consider more than one method for testing the antimicrobial activity of different compounds. The three yeast strains exhibited differences in their susceptibility to pharmaceutical antifungals, and the beer and baker’s yeasts were resistant to itraconazole. Moreover, polyphenols were detected in all natural extracts which may be linked to their antifungal activity. Our results suggest that we might consider multiple use of these natural extracts in the food industry as food additives or even preservatives to delay food spoilage.

Development of an Enzyme-Linked Immunosorbent Spot Assay for the Assessment of Adeno-Associated Virus Peptides to Examine Immune Safety.

Adeno-associated virus (AAV)-based gene therapies have shown promise as novel treatments for rare genetic disorders such as hemophilia A and spinal muscular atrophy. However, cellular immune responses mediated by cytotoxic (CD8+) and helper (CD4+) T cells may target vector-transduced cells as well as healthy immune cells, impacting safety and efficacy. In this study, we describe the optimization and reproducibility of interferon-γ (IFNγ)-based and interleukin-2 (IL-2)-based enzyme-linked immunosorbent spot (ELISpot) assays for measuring T cell responses against AAV peptide antigens. For method optimization, peripheral blood mononuclear cells (PBMCs) were isolated from healthy human donors and stimulated with commercially available major histocompatibility complex (MHC) class I or II-specific peptides as positive controls. Peptide pools were designed from published AAV8 and AAV9 capsid protein sequences and then used to assess the presence of AAV-specific T cell responses. Our results demonstrate a measurable increase in IFNγ and IL-2-producing cells after AAV peptide presentation. Furthermore, there was an observed difference in the magnitude and specificity of response to peptide pools based on AAV serotype and donor. Finally, using individual peptides, we identified a region of the AAV9 capsid protein that can elicit an immunogenic response. This work shows the applicability of ELISpot in assessing anti-AAV immune responses and provides insight into how novel recombinant AAV vectors could be designed to reduce immunogenic potential.

Solanum lycopersicum (Tomato)-Derived Nanovesicles Accelerate Wound Healing by Eliciting the Migration of Keratinocytes and Fibroblasts.

Plant-derived nanovesicles have been considered interesting in medicine for their breakthrough biological effects, including those relevant to wound healing. However, tomato-derived nanovesicles (TDNVs) have not been studied for their effects on wound closure yet. TDNVs were isolated from Solanum lycopersicum (var. Piccadilly) ripe tomatoes by ultracentrifugation. Extract (collected during the isolation procedure) and NVs (pellet) were characterized by transmission electron microscopy and laser Doppler electrophoresis. Wound healing in the presence of Extract or NVs was analyzed by a scratch assay with monocultures of human keratinocytes (HUKE) or NIH-3T3 mouse fibroblasts. Cell proliferation and migration were studied by MTT and agarose spot assay, respectively. The vesicles in the Extract and NV samples were nanosized with a similar mean diameter of 115 nm and 130 nm, respectively. Both Extract and NVs had already accelerated wound closure of injured HUKE and NIH-3T3 monocultures by 6 h post-injury. Although neither sample exerted a cytotoxic effect on HUKE and NIH-3T3 fibroblasts, they did not augment cell proliferation. NVs and the Extract increased cell migration of both cell types. NVs from tomatoes may accelerate wound healing by increasing keratinocyte and fibroblast migration. These results indicate the potential therapeutic usefulness of TDNVs in the treatment of chronic or hard-to-heal ulcers.

A novel multicolor fluorescent spot assay for the functional assessment of chimeric antigen receptor (CAR) T-cell products

Background aimsChimeric antigen receptor (CAR) T-cell (CAR-T) therapies have revolutionized the treatment of B-cell lymphomas. Unfortunately, relapses after CD19-targeted CAR-T are relatively common and, therefore, there is a critical need for assays able to assess the function and potency of CAR-T products pre-infusion, which will hopefully help to optimize CAR-T therapies. We developed a novel multicolor fluorescent spot assay (MFSA) for the functional assessment of CAR-T products on a single-cell level, combining the numerical assessment of CAR-T products with their functional characterization. MethodsWe first used a standard single-cell interferon (IFN)-γ enzyme-linked immune absorbent spot assay to measure CD19-targeted CAR-T responses to CD19-coated beads. We then developed, optimized and validated an MFSA that simultaneously measures the secretion of combinations of different cytokines on a single CAR-T level. ResultsWe identified IFN-γ/tumor necrosis factor-α/granzyme B as the most relevant cytokine combination, and we used our novel MFSA to functionally and numerically characterize two clinical-grade CAR-T products. ConclusionsIn conclusion, we have developed a novel assay for the quantitative and functional potency assessment of CAR-T products. Our optimized MFSA is cost-effective, easy to perform, reliable, can be performed overnight, allowing for a fast delivery of the product to the patient, and requires relatively minimal maintenance and training. The clinical value of our novel assay will be assessed in studies correlating the pre-infusion assessment of CAR-T products with the patients’ outcome in a prospective fashion.

In-silico and in-vitro evaluation of antifungal bioactive compounds from Streptomyces sp. strain 130 against Aspergillus flavus

Streptomyces spp. are considered excellent reservoirs of natural bioactive compounds. The study evaluated the bioactive potential of secondary metabolites from Streptomyces sp. strain 130 through PKS-I and NRPS gene-clusters screening. GC-MS analysis was done for metabolic profiling of bioactive compounds from strain 130 in the next set of experiments. Identified antifungal compounds underwent ADMET analyses to screen their toxicity. All compounds’ molecular docking was done with the structural gene products of the aflatoxin biosynthetic pathway of Aspergillus flavus. MD simulations were utilized to evaluate the stability of protein-ligand complexes under physiological conditions. Based on the in-silico studies, compound 2,4-di-tert butyl-phenol (DTBP) was selected for in-vitro studies against Aspergillus flavus. Simultaneously, bioactive compounds were extracted from strain 130 in two different solvents (ethyl-acetate and methanol) and used for similar assays. The MIC value of DTBP was found to be 314 µg/mL, whereas in ethyl-acetate extract and methanol-extract, it was 250 and 350 µg/mL, respectively. A mycelium growth assay was done to analyze the effect of compounds/extracts on the mycelium formation of Aspergillus flavus. In agar diffusion assay, zone of inhibitions in DTBP, ethyl-acetate extract, and methanol extract were observed with diameters of 11.3, 13.3, and 7.6 mm, respectively. In the growth curve assay, treated samples have delayed the growth of fungi, which signified that the compounds have a fungistatic nature. Spot assay has determined the fungal sensitivity to a sub-minimum inhibitory concentration of antifungal compounds. The study’s results suggested that DTBP can be exploited for antifungal-drug development. Communicated by Ramaswamy H. Sarma

Characterization of Levan Fructan Produced by a Gluconobacter japonicus Strain Isolated from a Sugarcane Processing Facility.

During raw sugarcane processing, a significant portion of lost sucrose is attributable to microbial degradation. Sucrose consumption by many bacteria is also linked to the production of exopolysaccharides (EPS) such as dextrans and fructans. These resulting EPS cause operational challenges during raw sugar manufacturing. Here, we report the characterization of EPS from a fructan-forming Gluconobacter japonicus bacterium that we previously isolated from a Louisiana sugarcane factory. The genome sequencing revealed the presence of two encoded levansucrase genes, lsrA and lsrB. One levansucrase, LsrB, was detected in the secreted protein fraction of G. japonicus LASM12 by QTOF LC-MS. The spotting assays indicated that G. japonicus produces EPS using sucrose and raffinose as substrates. The G. japonicus EPS correlated with levan fructan commercial standards by 1H-NMR, and with the characteristic carbohydrate fingerprint region for FTIR spectra, confirming that the G. japonicus EPS is levan fructan. The glycosyl composition and glycosyl linkage analysis revealed a linear β-2,6-fructofuranosyl polysaccharide with occasional (5.7%) β-2,1-fructofuranosyl branches. The gel permeation chromatography of the levan fructan EPS showed two main peaks at 4.5 kDa and 8 kDa and a very minor peak at 500 kDa. G. japonicus was identified as a producer of levan fructan. These findings will be useful for future studies aimed at evaluating the impact of levan fructans on sugar crop processing, which have been historically underestimated in industry.

Utility of the Interferon-Gamma Enzyme-Linked Immunosorbent Spot Assay to Predict Risk of Cytomegalovirus Infection in Kidney Transplant Recipients.

Non-specific interferon-gamma (IFN-γ) enzyme-linked immunosorbent (ELISpot) responses after solid organ transplant (SOT) and their relationship with cytomegalovirus (CMV) reactivation have hardly been investigated. Adult kidney transplant (KT) recipients underwent measurement of IFN-γ-producing T cells using the ELISpot assay before and 1 month after transplantation. Data for CMV infection episodes were collected. Risk factors for post-transplant CMV infection, based on IFN-γ responses, were analyzed using a Cox proportional hazards model. A total of 93 KT recipients were enrolled in the study and 84 evaluable participants remained at 1 month post KT. Thirty-three (39%) recipients developed subsequent CMV infection within 6 months post-transplant. At 1-month post-transplant, IFN-γ-producing T cells with <250 spot-forming units (SFUs)/2.5 × 105 peripheral blood mononuclear cells (PBMCs) were significantly associated with CMV infection (HR 3.1, 95% CI 1.4-7.1, p = 0.007). On multivariable analysis, posttransplant IFN-γ-producing T cells with <250 SFUs/2.5 × 105 PBMCs remained independently associated with CMV infection (HR 3.1, 95% CI 1.2-7.8, p = 0.019). Conclusions: KT recipients with low IFN-γ-producing T cells measured by the ELISpot assay are more likely to develop CMV infection after transplantation. Therefore, measurement of nonspecific cell-mediated immunity ELISpot responses could potentially stratify recipients at risk of CMV infection (Thai Clinical Trials Registry, TCTR20210216004).

Limonene synergistically augments fluconazole susceptibility in clinical Candida isolates from cleft lip and palate patients.

Cleft lip and palate (CLP) patients are prone to Candida infections (oral thrush) mainly due to poor oral hygiene, repetitive surgeries, and orthodontic procedures. This study was undertaken to evaluate the antifungal efficacy of limonene against clinical Candida isolates from CLP patients. The antifungal efficacy of limonene was studied alone and in combination with fluconazole (FLC) against six standards, twenty nine FLC sensitive, and three FLC resistant clinical strains using broth dilution, checkerboard microdilution, agar disk diffusion, growth curves, and spot assays. This nontoxic monoterpene gave low minimum inhibitory concentration (MIC) values of 300-375 µg/mL and 500-520 µg/mL for FLC susceptible and FLC resistant strains, respectively. It showed synergistic interaction with FLC in all clinical and standard Candida strains (fractional inhibitory concentration (FIC) index ≤0.5). Significant chemosensitization of FLC was observed even against resistant clinical isolates. Complete suppression of fungal growth was observed when using combinations. Negligible toxicity, easy availability, and potent antifungal properties suggest that limonene and FLC combinations in appropriate doses can make excellent antifungal mouthwashes during CLP treatment pre and post surgery. Impending in vivo studies are needed to validate the present data.

Assessing Antigen-Specific T Cell Responses Through IFN-γ Enzyme-Linked Immune Absorbent Spot (ELISpot).

T cells are instrumental in protecting the host against invading pathogens and the development of cancer. To do so, they produce effector molecules such as granzymes, interleukins, interferons, and perforin. For the development and immunomonitoring of therapeutic applications such as cell-based therapies and vaccines, assessing T cell effector function is paramount. This can be achieved through various methods, such as 51Cr release assays, flow cytometry, and enzyme-linked immune absorbent spot (ELISpot) assays. For T cell ELISpots, plates are coated with antibodies directed against the effector molecule of interest (e.g., IFN-g). Subsequently, peripheral blood mononuclear cells (PBMCs) or isolated T cells are cultured on the plate together with stimuli of choice, and the production of effector molecules is visualized via labeled detection antibodies. For clinical studies, ELISpot is currently the gold standard to determine antigen-specific T cell frequencies. In contrast to 51Cr release assays, ELISpot allows for the exact enumeration of responding T cells, and compared to flow cytometry, ELISpot is more cost-effective and high throughput. Here, we optimize and describe, in a step-by-step fashion, how to perform a controlled IFN-γ ELISpot experiment to determine the frequency of responding or antigen-specific T cells in healthy human volunteers. Of note, this protocol can also be employed to assess the frequency of antigen-specific T cells induced in, e.g., vaccination studies or present in cellular products.

Development and Evaluation of a Promising Biomarker for Diagnosis of Latent and Active Tuberculosis Infection

Abstract Background Diagnosing latent tuberculosis (TB) infection (LTBI) and active TB (ATB) is crucial for preventing disease progression and transmission. However, current diagnostic tests have limitations in terms of accuracy and sensitivity, making it challenging to diagnose these different infection states. Therefore, this study intends to develop a promising biomarker for LTBI and ATB diagnosis to overcome the limitations of the current diagnostic tests. Methods We developed a novel multiepitope-based diagnostic biomarker (MEBDB) from LTBI region of differentiation antigens using bioinformatics and immunoinformatics. Immune responses induced by MEBDM were detected using enzyme-linked immunosorbent spot and cytometric bead assays. This study was conducted from April 2022 to December 2022 in the Senior Department of Tuberculosis at the 8th Medical Center of PLA General Hospital, China. Blood samples were collected from participants with ATB, individuals with LTBI, and healthy controls (HCs). The diagnostic efficacy of MEBDB was evaluated using receiver operating characteristic curves. Results A novel MEBDB, designated as CP19128P, was generated. CP19128P comprises 19 helper T lymphocyte epitopes, 12 cytotoxic T lymphocyte epitopes, and 8 B-cell epitopes. In silico simulations demonstrated that CP19128P possesses strong affinity for Toll-like receptors and elicits robust innate and adaptive immune responses. CP19128P generated significantly higher levels of tumor necrosis factor (TNF-α), interleukin 4 (IL-4), and IL-10 in ATB patients (n = 7) and LTBI (n = 8) individuals compared with HCs (n = 62) (P &lt; 0.001). Moreover, CP19128P-induced specific cytokines could be used to discriminate LTBI and ATB from healthy subjects with high sensitivity and specificity. Combining IL-2 with IL-4 or TNF-α could differentiate LTBI from HCs (the area under the receiver operating characteristic curve [AUC], 0.976 [95% confidence interval [CI], 0.934–1.000] or 0.986 [0.956–1.000]), whereas combining IL-4 with IL-17A or TNF-α could differentiate ATB from HCs (AUC, 0.887 [0.782–0.993] or 0.984 [0.958–1.000]). Conclusions Our study revealed that CP19128P is a potential MEBDB for the diagnosis of LTBI and ATB. Our findings suggest a promising strategy for developing novel, accurate, and sensitive diagnostic biomarkers and identifying new targets for TB diagnosis and management.

ELISpots for Detecting Antigen-Specific Memory B Cells in Infection or Autoimmunity.

Enzyme-Linked Immunosorbent Spot assay (ELISpot) is an immunoassay used to quantify individual protein-specific secreting cells. Proteins secreted by cells cultured in ELISpot plates (96- or 8-well format) bind to a specific antigen bound to a PVDF membrane at the bottom of the well. A detection antibody followed by an enzymatic reaction is used to identify secreted protein bound to the membrane coated antigen. This reaction results in distinct "spots" on the membrane corresponding to individual protein secreting cells. While the design is similar to an ELISA, ELISpots quantify the number and relative amount of secreted protein on a single cell level, as opposed to an ELISA that reveals the concentration of secreted proteins from a population of cells. The sensitivity, robustness, and diversity of different antigens used by ELISpots have led to an array of research applications such as measuring cytokines from cytotoxic T cells in cancer and quantifying antibody specificity from B cells following vaccinations. Improvements have been made to assays measuring cytokines and antibodies on a single cell basis, such as intracellular flow cytometry. Yet the ability of an ELISpot to evaluate the quantity and quality of protein secretion on an individual cell basis remains unmatched. Here, we describe the use of a modified ELISpot assay to detect antigen-specific memory B cells in the setting of a viral infection and autoimmunity.

Automated identification and segmentation of urine spots based on deep-learning.

Micturition serves an essential physiological function that allows the body to eliminate metabolic wastes and maintain water-electrolyte balance. The urine spot assay (VSA), as a simple and economical assay, has been widely used in the study of micturition behavior in rodents. However, the traditional VSA method relies on manual judgment, introduces subjective errors, faces difficulty in obtaining appearance time of each urine spot, and struggles with quantitative analysis of overlapping spots. To address these challenges, we developed a deep learning-based approach for the automatic identification and segmentation of urine spots. Our system employs a target detection network to efficiently detect each urine spot and utilizes an instance segmentation network to achieve precise segmentation of overlapping urine spots. Compared with the traditional VSA method, our system achieves automated detection of urine spot area of micturition in rodents, greatly reducing subjective errors. It accurately determines the urination time of each spot and effectively quantifies the overlapping spots. This study enables high-throughput and precise urine spot detection, providing important technical support for the analysis of urination behavior and the study of the neural mechanism underlying urination.

Impact of intravascular hemolysis on functional and molecular alterations in the urinary bladder: implications for an overactive bladder in sickle cell disease.

Patients with sickle cell disease (SCD) display an overactive bladder (OAB). Intravascular hemolysis in SCD is associated with various severe SCD complications. However, no experimental studies have evaluated the effect of intravascular hemolysis on bladder function. This study aimed to assess the effects of intravascular hemolysis on the micturition process and the contractile mechanisms of the detrusor smooth muscle (DSM) in a mouse model with phenylhydrazine (PHZ)-induced hemolysis; furthermore, it aimed to investigate the role of intravascular hemolysis in the dysfunction of nitric oxide (NO) signaling and in increasing oxidative stress in the bladder. Mice underwent a void spot assay, and DSM contractions were evaluated in organ baths. The PHZ group exhibited increased urinary frequency and increased void volumes. DSM contractile responses to carbachol, KCl, α-β-methylene-ATP, and EFS were increased in the PHZ group. Protein expression of phosphorylated endothelial NO synthase (eNOS) (Ser-1177), phosphorylated neuronal NO synthase (nNOS) (Ser-1417), and phosphorylated vasodilator-stimulated phosphoprotein (VASP) (Ser-239) decreased in the bladder of the PHZ group. Protein expression of oxidative stress markers, NOX-2, 3-NT, and 4-HNE, increased in the bladder of the PHZ group. Our study shows that intravascular hemolysis promotes voiding dysfunction correlated with alterations in the NO signaling pathway in the bladder, as evidenced by reduced levels of p-eNOS (Ser-1177), nNOS (Ser-1417), and p-VASP (Ser-239). The study also showed that intravascular hemolysis increases oxidative stress in the bladder. Our study indicates that intravascular hemolysis promotes an OAB phenotype similar to those observed in patients and mice with SCD.

Prevalence of glucose-6-phosphate dehydrogenase (G6PD) deficiency among the fulminant hepatitis A virus infection patients.

Hepatitis A is a widespread viral infection with significant public health implications. Assessing glucose 6-phosphate dehydrogenase (G6PD) deficiency in hepatitis A patients is essential for various reasons, including prognosis, disease severity evaluation, encephalopathy risk identification, tailored management, and advancing scientific understanding. This study aimed to investigate the prevalence and clinical implications of G6PD impairment in individuals with fulminant hepatitis A. A cross-sectional descriptive analysis was conducted, involving hospitalized patients with fulminant hepatitis A. Demographic data, prevalence rates, and clinical findings were recorded in a database. The diagnosis of hepatitis A infection was confirmed using an anti-HAV IgM antibody test, and G6PD enzyme activity was measured with a fluorescent spot assay. Out of 81 patients with hepatitis A, 57 (70.4%) were males, and 24 (29.5%) were females, with an average age of 24.6 years. Dark yellow urine and anorexia were the most common clinical symptoms. Notably, 30 (37%) patients lacked G6PD. The group with G6PD deficiency showed significantly higher rates of encephalopathy and mortality (P<0.01), along with elevated bilirubin (P=0.00), abnormal coagulation parameters, and low hemoglobin levels (P=0.00). In light of these findings, the present study proposes the implementation of routine G6PD level assessments and the evaluation of other relevant markers in regions where hepatitis A is endemic. Furthermore, the study underscores the need for vigilant monitoring of hemolysis and encephalopathy in affected patients to optimize clinical management and reduce morbidity and mortality associated with this condition.