Activated lymphocytes are considered to play a pathogenic role in Crohn's disease (CD) and ulcerative colitis (UC), although only a limited fraction of the gut-residing lymphocytes in these diseases ...may be pathogenetically involved, due to active recruitment from the peripheral circulation. Our aim was to characterize in situ preactivated lymphocytes in inflammatory bowel disease mucosa by expansion with interleukin-2.
Flow cytometry was performed on T cells expanded from the colon of patients with CD (7), UC (16), and controls (20), with special reference to T-cell activation markers and adhesion molecules.
In CD a decrease in alpha4beta7 integrin expression was associated with an increase in alphaEbeta7. In UC a similar increase in alphaEbeta7 was observed. Moreover, L-selectin and CD30 were overexpressed on T helper cells in UC versus CD.
These findings indicate different immunopathogenic pathways for CD and UC.
Extracellular vesicles (EVs) are released by all cells into biofluids and hold great promise as reservoirs of disease biomarkers. One of the main challenges in studying EVs is a lack of methods to ...quantify EVs that are sensitive enough and can differentiate EVs from similarly sized lipoproteins and protein aggregates. We demonstrate the use of ultrasensitive, single-molecule array (Simoa) assays for the quantification of EVs using three widely expressed transmembrane proteins: the tetraspanins CD9, CD63, and CD81. Using Simoa to measure these three EV markers, as well as albumin to measure protein contamination, we were able to compare the relative efficiency and purity of several commonly used EV isolation methods in plasma and cerebrospinal fluid (CSF): ultracentrifugation, precipitation, and size exclusion chromatography (SEC). We further used these assays, all on one platform, to improve SEC isolation from plasma and CSF. Our results highlight the utility of quantifying EV proteins using Simoa and provide a rapid framework for comparing and improving EV isolation methods from biofluids.
L1CAM is a transmembrane protein expressed on neurons that was presumed to be found on neuron-derived extracellular vesicles (NDEVs) in human biofluids. We developed a panel of single-molecule array ...assays to evaluate the use of L1CAM for NDEV isolation. We demonstrate that L1CAM is not associated with extracellular vesicles in human plasma or cerebrospinal fluid and therefore recommend against its use as a marker in NDEV isolation protocols.
Extracellular vesicles (EVs) are released by all cells into biofluids such as plasma. The separation of EVs from highly abundant free proteins and similarly sized lipoproteins remains technically ...challenging. We developed a digital ELISA assay based on Single Molecule Array (Simoa) technology for ApoB-100, the protein component of several lipoproteins. Combining this ApoB-100 assay with previously developed Simoa assays for albumin and three tetraspanin proteins found on EVs (Ter-Ovanesyan, Norman et al., 2021), we were able to measure the separation of EVs from both lipoproteins and free proteins. We used these five assays to compare EV separation from lipoproteins using size exclusion chromatography with resins containing different pore sizes. We also developed improved methods for EV isolation based on combining several types of chromatography resins in the same column. We present a simple approach to quantitatively measure the main impurities of EV isolation in plasma and apply this approach to develop novel methods for enriching EVs from human plasma. These methods will enable applications where high-purity EVs are required to both understand EV biology and profile EVs for biomarker discovery.
Alternatives to soil fumigation are needed for soilborne disease control. The aim of this study was to test anaerobic soil disinfestation (ASD) as an alternative to soil fumigation for control of ...critical soilborne pathogens in Californian strawberry production. Controlled environment experiments were conducted at 25 and 15 °C to test different materials as carbon sources for ASD using soil inoculated with Verticillium dahliae. Field trials were conducted in three locations comparing ASD with 20 Mg ha−1 rice bran (RB) against fumigated and untreated controls, steam, mustard seed meal and fish emulsion. In ASD‐treated soils, temperature and extent of anaerobic conditions were critical for control of V. dahliae, but multiple carbon inputs reduced inoculum by 80–100%. In field trials, ASD with RB provided control of a number of pathogens, and in three of four trials produced marketable fruit yields equivalent to fumigation. Little weed control benefit from ASD was found. ASD with RB also induced changes in the soil microbiome that persisted through the growing season. When equivalent yields were obtained, net returns above harvest and treatment costs with ASD RB were 92–96% of those with bed fumigation based on average prices over the previous 5 years. ASD can be a viable alternative for control of some soilborne pathogens. Growers are adopting ASD in California strawberry production, but research to determine optimal soil temperatures, anaerobicity thresholds and carbon sources for effective control of specific pathogens is needed.
Background and aims To assess the impacts of soil microbes and plant genotype on the composition of maize associated bacterial communities. Methods Two genotypes of Brazilian maize were planted ...indoors on sterile sand, a deep underground subsoil, and a nutrient-rich topsoil from the Amazon jungle (terra preta). DNA was extracted from rhizospheres, phyllospheres, and surface sterilized roots for 16S rDNA fingerprinting and next generation sequencing. Results Neither plant genotype nor soil type appeared to influence bacterial diversity in phyllospheres or endospheres. Rhizospheres showed strikingly similar 16S rDNA ordination of both fingerprinting and sequencing data, with soil type driving grouping patterns and genotype having a significant impact only on sterile sand. Rhizospheres grown in non-sterile soils contained greater bacterial diversity than sterile-sand grown ones, however the dominant OTUs (species of Proteobacteria and Bacteroidetes) were found in all rhizospheres suggesting seeds as a common source of inoculum. Rhizospheres of the commercial hybrid appeared to contain less bacterial diversity than the landrace. Conclusions Maize rhizospheres receive diverse bacteria from soil, are influenced by the genotype or treatment of the seed, and are dominated by species of Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes. As many dominant 16S rDNA sequences were observed in rhizospheres grown in both sterile and non-sterile substrate, we conclude that the most common bacterial cells in juvenile maize rhizospheres are seed transmitted.
Sensitive assays are essential for the accurate identification of individuals infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we report a multiplexed assay for the ...fluorescence-based detection of seroconversion in infected individuals from less than 1 µl of blood, and as early as the day of the first positive nucleic acid test after symptom onset. The assay uses dye-encoded antigen-coated beads to quantify the levels of immunoglobulin G (IgG), IgM and IgA antibodies against four SARS-CoV-2 antigens. A logistic regression model trained using samples collected during the pandemic and samples collected from healthy individuals and patients with respiratory infections before the first outbreak of coronavirus disease 2019 (COVID-19) was 99% accurate in the detection of seroconversion in a blinded validation cohort of samples collected before the pandemic and from patients with COVID-19 five or more days after a positive nasopharyngeal test by PCR with reverse transcription. The high-throughput serological profiling of patients with COVID-19 allows for the interrogation of interactions between antibody isotypes and viral proteins, and should help us to understand the heterogeneity of clinical presentations.
Lymph node follicles capture and retain antigens to induce germinal centers and long-lived humoral immunity. However, control over antigen retention has been limited. Here we discovered that antigen ...conjugated to nanoparticle carriers of different sizes impacts the intralymph node transport and specific cell interaction. We found that follicular dendritic cell (FDC) networks determine the intralymph node follicle fate of these nanoparticles by clearing smaller ones (5–15 nm) within 48 h and retaining larger ones (50–100 nm) for over 5 weeks. The 50–100 nm-sized nanoparticles had 175-fold more delivery of antigen at the FDC dendrites, 5-fold enhanced humoral immune responses of germinal center B cell formation, and 5-fold more antigen-specific antibody production over 5–15 nm nanoparticles. Our results show that we can tune humoral immunity by simply manipulating the carrier size design to produce effectiveness of vaccines.
Abstract
Background
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected over 21 million people worldwide since August 16, 2020. Compared to PCR and serology tests, SARS-CoV-2 ...antigen assays are underdeveloped, despite their potential to identify active infection and monitor disease progression.
Methods
We used Single Molecule Array (Simoa) assays to quantitatively detect SARS-CoV-2 spike, S1 subunit, and nucleocapsid antigens in the plasma of patients with coronavirus disease (COVID-19). We studied plasma from 64 patients who were COVID-19 positive, 17 who were COVID-19 negative, and 34 prepandemic patients. Combined with Simoa anti-SARS-CoV-2 serological assays, we quantified changes in 31 SARS-CoV-2 biomarkers in 272 longitudinal plasma samples obtained for 39 patients with COVID-19. Data were analyzed by hierarchical clustering and were compared to longitudinal RT-PCR test results and clinical outcomes.
Results
SARS-CoV-2 S1 and N antigens were detectable in 41 out of 64 COVID-19 positive patients. In these patients, full antigen clearance in plasma was observed a mean ± 95% CI of 5 ± 1 days after seroconversion and nasopharyngeal RT-PCR tests reported positive results for 15 ± 5 days after viral-antigen clearance. Correlation between patients with high concentrations of S1 antigen and ICU admission (77%) and time to intubation (within 1 day) was statistically significant.
Conclusions
The reported SARS-CoV-2 Simoa antigen assay is the first to detect viral antigens in the plasma of patients who were COVID-19 positive to date. These data show that SARS-CoV-2 viral antigens in the blood are associated with disease progression, such as respiratory failure, in COVID-19 cases with severe disease.
Abstract Study question What is the impact of ultrasound guidance performed by a well-trained versus untrained medical personnel on clinical pregnancy rate following embryo transfer (ET)? Summary ...answer The utilization of a trained versus untrained ultrasound operator during ET did not demonstrate an improvement in the clinical pregnancy rate. What is known already ET represents the crucial final step in in vitro fertilization (IVF) cycles. While various aspects of the procedure have been studied for their potential impact on treatment outcomes, the influence of ultrasound operator guidance during ET has not been extensively explored in practice guidelines. Notably, only two previous studies compared trained and untrained medical staff for US guidance during ET and have not shown significant differences in clinical outcomes. Study design, size, duration This retrospective cohort study encompassed all ET procedures conducted in a single university-affiliated IVF unit between February 1st, 2023, and December 1st, 2023. The study compared the clinical pregnancy rate between patients undergoing ET with US guidance by an experienced sonographer versus guidance provided by an untrained medical staff. The study setting allowed for a natural experiment, as the availability of experienced personnel was based on random availability. Participants/materials, setting, methods Experienced sonographers included ultrasound technicians or fertility reproductive medicine senior physicians (referred to as “trained transfers”). Unexperienced sonographers comprised operating room nurses (referred to as “untrained transfers”). Covariates tested as possible confounders included age at oocyte retrieval, Body Mass Index (BMI), treatment indication, endometrial thickness, frozen versus fresh embryo transfer, number of embryos transferred, and embryo developmental stage. Main results and the role of chance A total of 694 embryo transfers were analyzed; 316 performed by trained operators (45.5%) and 378 by untrained personnel (54.4%). Demographic characteristics were comparable, with mean age and BMI not significantly different between untrained (33.3±6.6 years, 26±5.6 kg/m²) and trained transfers (33.5±6.7 years, 25.9±5.4 kg/m², p = 0.7, 0.9). Day of transfer and mean number of embryos transferred were similar (p = 0.4, 0.9). Although clinically sufficient, endometrial thickness differed statistically (9.9mm±0.4 untrained vs. 9.4mm±1.9 trained, p < 0.01). Main diagnosis distribution varied (p = 0.02), with both groups mainly treated for male and unexplained infertility. Proportion of frozen transfers differed significantly (66.9% untrained vs. 81.5% trained, p < 0.01). Regarding the main outcome measure, clinical pregnancy rate, crude rate was similar (35.7% untrained vs. 31.0% trained, p = 0.2). Clinical pregnancy crude rate, the main outcome measure, was similar (35.7% untrained vs. 31.0% trained, p = 0.2). In order to address unevenly distributed variables, a stepwise conditional logistic regression analysis was conducted. The trained operator was not a significant contributor to the clinical pregnancy rate (p = 0.14). Frozen embryo transfer positively correlated with successful implantation (p = 0.01), as did the main diagnosis (p = 0.03). Limitations, reasons for caution The retrospective nature of the study introduces limitations. Confounding variables were addressed through regression modeling. The random availability of trained assistants, independent of the procedure team, mitigates bias. Wider implications of the findings This study contributes evidence to the refinement of the embryo transfer procedure, suggesting that with US guidance, the contribution of a trained sonographer is not a significant factor in the standardized protocol for the transfer. Trial registration number not applicable
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