Magnesium sensing via LFA-1 regulates CD8+ T cell effector function.

The relevance of extracellular magnesium in cellular immunity remains largely unknown. Here, we show that the co-stimulatory cell-surface molecule LFA-1 requires magnesium to adopt its active conformation on CD8+ T cells, thereby augmenting calcium flux, signal transduction, metabolic reprogramming, immune synapse formation, and, as a consequence, specific cytotoxicity. Accordingly, magnesium-sufficiency sensed via LFA-1 translated to the superior performance of pathogen- and tumor-specific T cells, enhanced effectiveness of bi-specific T cell engaging antibodies, and improved CAR T cell function. Clinically, low serum magnesium levels were associated with more rapid disease progression and shorter overall survival in CAR T cell and immune checkpoint antibody-treated patients. LFA-1 thus directly incorporates information on the composition of the microenvironment as a determinant of outside-in signaling activity. These findings conceptually link co-stimulation and nutrient sensing and point to the magnesium-LFA-1 axis as a therapeutically amenable biologic system.

Diapedesis-Induced Integrin Signaling via LFA-1 Facilitates Tissue Immunity by Inducing Intrinsic Complement C3 Expression in Immune Cells.

Intrinsic complement C3 activity is integral to human T helper type 1 (Th1) and cytotoxic T cell responses. Increased or decreased intracellular C3 results in autoimmunity and infections, respectively. The mechanisms regulating intracellular C3 expression remain undefined. We identified complement, including C3, as among the most significantly enriched biological pathway in tissue-occupying cells. We generated C3-reporter mice and confirmed that C3 expression was a defining feature of tissue-immune cells, including T cells and monocytes, occurred during transendothelial diapedesis, and depended on integrin lymphocyte-function-associated antigen 1 (LFA-1) signals. Immune cells from patients with leukocyte adhesion deficiency type 1 (LAD-1) had reduced C3 transcripts and diminished effector activities, which could be rescued proportionally by intracellular C3 provision. Conversely, increased C3 expression by T cells from arthritis patients correlated with disease severity. Our study defines integrins as key controllers of intracellular complement, demonstrates that perturbations in the LFA-1-C3-axis contribute to primary immunodeficiency, and identifies intracellular C3 as biomarker of severity in autoimmunity.

Eomes-Dependent Loss of the Co-activating Receptor CD226 Restrains CD8+ T Cell Anti-tumor Functions and Limits the Efficacy of Cancer Immunotherapy.

CD8+ T cells within the tumor microenvironment (TME) are exposed to various signals that ultimately determine functional outcomes. Here, we examined the role of the co-activating receptor CD226 (DNAM-1) in CD8+ T cell function. The absence of CD226 expression identified a subset of dysfunctional CD8+ T cells present in peripheral blood of healthy individuals. These cells exhibited reduced LFA-1 activation, altered TCR signaling, and a distinct transcriptomic program upon stimulation. CD226neg CD8+ T cells accumulated in human and mouse tumors of diverse origin through an antigen-specific mechanism involving the transcriptional regulator Eomesodermin (Eomes). Despite similar expression of co-inhibitory receptors, CD8+ tumor-infiltrating lymphocyte failed to respond to anti-PD-1 in the absence of CD226. Immune checkpoint blockade efficacy was hampered in Cd226-/- mice. Anti-CD137 (4-1BB) agonists also stimulated Eomes-dependent CD226 loss that limited the anti-tumor efficacy of this treatment. Thus, CD226 loss restrains CD8+ T cell function and limits the efficacy of cancer immunotherapy.

How integrin phosphorylations regulate cell adhesion and signaling.

Cell adhesion is essential for the formation of organs, cellular migration, and interaction with target cells and the extracellular matrix. Integrins are large protein α/ß-chain heterodimers and form a major family of cell adhesion molecules. Recent research has dramatically increased our knowledge of how integrin phosphorylations regulate integrin activity. Phosphorylations determine the signaling complexes formed on the cytoplasmic tails, regulating downstream signaling. α-Chain phosphorylation is necessary for inducing ß-chain phosphorylation in LFA-1, and the crosstalk from one integrin to another activating or inactivating its function is in part mediated by phosphorylation of ß-chains. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus receptor angiotensin-converting enzyme 2 (ACE2) and possible integrin coreceptors may crosstalk and induce a phosphorylation switch and autophagy.

Extracellular ISG15 Signals Cytokine Secretion through the LFA-1 Integrin Receptor.

ISG15 is a ubiquitin-like protein that functions in innate immunity both as an intracellular protein modifier and as an extracellular signaling molecule that stimulates IFN-γ secretion. The extracellular function, important for resistance to mycobacterial disease, has remained biochemically uncharacterized. We have established an NK-92 cell-based assay for IFN-γ release, identified residues critical for ISG15 signaling, and identified the cell surface receptor as LFA-1 (CD11a/CD18; αLß2 integrin). LFA-1 inhibition blocked IFN-γ secretion, splenocytes from CD11a-/- mice did not respond to ISG15, and ISG15 bound directly to the αI domain of CD11a in vitro. ISG15 also enhanced secretion of IL-10, indicating a broader role for ISG15 in cytokine signaling. ISG15 engagement of LFA-1 led to the activation of SRC family kinases (SFKs) and SFK inhibition blocked cytokine secretion. These findings establish the molecular basis of the extracellular function of ISG15 and the initial outside-in signaling events that drive ISG15-dependent cytokine secretion.

Carba PBP: a novel penicillin-binding protein-based lateral flow assay for rapid phenotypic detection of carbapenemase-producing Enterobacterales.

Rapid phenotypic detection assays, including Carba NP and its variants, are widely applied for clinical diagnosis of carbapenemase-producing Enterobacterales (CPE). However, these tests are based on the acidification of the pH indicator during carbapenem hydrolysis, which limits test sensitivity and speed, especially for the detection of CPE producing low-activity carbapenem (e.g., OXA-48 variants). Herein, we developed a novel rapid and sensitive CPE detection method (Carba PBP) that could measure substrate (meropenem) consumption based on penicillin-binding protein (PBP). Meropenem-specific PBP was used to develop a competitive lateral flow assay (LFA) for meropenem identification. For the detection of carbapenemase activity, meropenem concentration was optimized using a checkerboard assay. The performance of Carba PBP was evaluated and compared with that of Carba NP using a panel of 94 clinical strains characterized by whole-genome sequencing and carbapenem susceptibility test. The limit of detection of PBP-based LFA for meropenem identification was 7 ng mL-1. Using 10 ng mL-1 meropenem as the substrate, Carba PBP and Carba NP could detect 10 ng mL-1 carbapenemase within 25 min and 1,280 ng mL-1 CPE in 2 h, respectively. The sensitivity and specificity were 100% (75/75) and 100% (19/19) for Carba PBP and 85.3% (64/75) and 100% (19/19) for Carba NP, respectively. When compared with Carba NP, Carba PBP showed superior performance in detecting all the tested CPE strains (including OXA-48-like variants) within 25 min and presented two orders of magnitude higher analytical sensitivity, demonstrating potential for clinical diagnosis of CPE. IMPORTANCE This study successfully achieved the goal of carbapenemase activity detection with both high sensitivity and convenience, offering a convenient lateral flow assay for clinical diagnosis of carbapenemase-producing Enterobacterales.

Development of a triplex RT-RAA-LFA assay for the rapid differential diagnosis of porcine epidemic diarrhea virus, porcine deltacoronavirus and transmissible gastroenteritis virus.

Porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV) and transmissible gastroenteritis virus (TGEV) are three clinically common coronaviruses causing diarrhea in pigs, with indistinguishable clinical signs and pathological changes. Rapid, portable and reliable differential diagnosis of these three pathogens is crucial for the prompt implementation of appropriate control measures. In this study, we developed a triplex nucleic acid assay that combines reverse transcription recombinase-aided amplification (RT-RAA) with lateral flow assay (LFA) by targeting the most conserved genomic region in the ORF1b genes of PEDV, PDCoV and TGEV. The entire detection process of the triplex RT-RAA-LFA assay included 10-min nucleic acid amplification at 42 °C and 5-min visual LFA readout at room temperature. The assay could specifically differentiate PEDV, PDCoV and TGEV without cross-reaction with any other major swine pathogens. Sensitivity analysis showed that the triplex RT-RAA-LFA assay was able to detect the viral RNA extracted from the spiked fecal samples with the minimum of 1 × 100 TCID50 PEDV, 1 × 104 TCID50 PDCoV, and 1 × 102 TCID50 TGEV per reaction, respectively. Further analysis showed that the 95 % detection limit (LOD) of triplex RT-RAA-LFA for PEDV, PDCoV, and TGEV were 22, 478, and 205 copies of recombinant plasmids per reaction, respectively. The diagnostic performance of triplex RT-RAA-LFA was compared with that of PEDV, PDCoV and TGEV respective commercial real-time RT-PCR kits by testing 114 clinical rectal swab samples in parallel. The total diagnostic coincidence rates of triplex RT-RAA-LFA with real-time RT-PCR kits of PEDV, PDCoV and TGEV were 100 %, 99.1 % and 99.1 %, respectively, and their Kappa values were 1.00, 0.958 and 0.936, respectively. Collectively, the RT-RAA-LFA assay is a powerful tool for the rapid, portable, visual, and synchronous differential diagnosis of PEDV, PDCoV, and TGEV.

LFA-1 in T cell priming, differentiation, and effector functions.

The integrin LFA-1 is crucial for T cell entry into mammalian lymph nodes and tissues, and for promoting interactions with antigen-presenting cells (APCs). However, it is increasingly evident that LFA-1 has additional key roles beyond the mere support of adhesion between T cells, the endothelium, and/or APCs. These include roles in homotypic T cell-T cell (T-T) communication, the induction of intracellular complement activity underlying Th1 effector cell polarization, and the support of long-lasting T cell memory. Here, we briefly summarize current knowledge of LFA-1 biology, discuss novel cytoskeletal regulators of LFA-1 functions, and review new aspects of LFA-1 mechanobiology that are relevant to its function in immunological synapses and in specific pathologies arising from LFA-1 dysregulation.

Conjugation of visual enhancers in lateral flow immunoassay for rapid forensic analysis: A critical review.

During crime scene investigations, numerous traces are secured and may be used as evidence for the evaluation of source and/or activity level propositions. The rapid chemical analysis of a biological trace enables the identification of body fluids and can provide significant donor profiling information, including age, sex, drug abuse, and lifestyle. Such information can be used to provide new leads, exclude from, or restrict the list of possible suspects during the investigative phase. This paper reviews the state-of-the-art labelling techniques to identify the most suitable visual enhancer to be implemented in a lateral flow immunoassay setup for the purpose of trace identification and/or donor profiling. Upon comparison, and with reference to the strengths and limitations of each label, the simplistic one-step analysis of noncompetitive lateral flow immunoassay (LFA) together with the implementation of carbon nanoparticles (CNPs) as visual enhancers is proposed for a sensitive, accurate, and reproducible in situ trace analysis. This approach is versatile and stable over different environmental conditions and external stimuli. The findings of the present comparative analysis may have important implications for future forensic practice. The selection of an appropriate enhancer is crucial for a well-designed LFA that can be implemented at the crime scene for a time- and cost-efficient investigation.

Performance of the IMMY® sona Aspergillus lateral flow assay for the detection of galactomannan in tracheal aspirate samples from Brazilian patients with COVID-19-associated pulmonary aspergillosis: Cross-sectional and systematic review of literature.

During the COVID-19 pandemic, many patients in intensive care units (ICUs) were affected by invasive fungal infections, including aspergillosis, contributing to a high mortality rate. Diagnosing proven COVID-19-associated pulmonary aspergillosis (CAPA) requires clinical and radiological evaluations, along with laboratory testing of bronchoalveolar lavage samples or lung biopsies. However, these procedures and equipment are often inaccessible in developing countries or regions with limited resources, including Brazil. Consequently, alternative diagnostic methods, such as measuring Aspergillus galactomannan (GM) in tracheal aspirate (TA), have been explored for CAPA diagnosis. Nonetheless, research on the efficacy of TA-based diagnostic tests is limited. This study aimed to assess the performance of the IMMY® Sona Aspergillus lateral flow assay (LFA) for GM detection in TA samples from 60 ICU patients with suspected CAPA at two tertiary hospitals in Campo Grande, Brazil. The ELISA method (Platelia Aspergillus AG, Bio-Rad®) was used to detect Aspergillus GM in TA samples, serving as the microbiological criterion and reference test. Fifteen patients (12.4%) were identified as having possible CAPA. The overall accuracy of LFA was 94%, and the tests demonstrated an agreement of 93.1% (Cohen's kappa of 0.83). Based on our findings, the LFA for Aspergillus GM detection in TA samples exhibited excellent performance, proving to be a valuable diagnostic tool for potential CAPA. In a systematic review, two studies were included, and the meta-analysis revealed pooled estimates provided a sensitivity of 86% (95% CI, 80%-91%) and specificity of 93% (95% CI, 86%-97%). The diagnostic odds ratio (DOR) for identification of Aspergillus using LFA was 103.38 (95% CI, 38.03-281.03). Despite its lower sensitivity compared to our study, the LFA appears to be a promising diagnostic option for CAPA, particularly in suspected cases that have not received antifungal therapy. This enables timely antifungal treatment and could reduce mortality rates in regions where bronchoscopy is unavailable or limited.

The relationship between lung CT features and serum cryptococcal antigen titers in localized pulmonary cryptococcosis patients.

BACKGROUND: To explore the associations of computed tomography (CT) image features with the serum cryptococcal antigen (CrAg) titers measured by the lateral flow assay (LFA) in localized pulmonary cryptococcosis patients. METHODS: A retrospective analysis of patients with pathologically confirmed pulmonary cryptococcosis admitted to the First Affiliated Hospital of Xiamen University from January 2016 to December 2022 was performed. Clinical data, CT results, serum CrAg-LFA test results, and follow-up data were collected and analyzed. RESULTS: A total of 107 patients with localized pulmonary cryptococcosis were included, of which 31 had a single lesion in chest CT and the other 76 had multiple lesions. The positivity rate was (94.74% vs 64.52%) and titers of serum CrAg-LFA (1.77 ± 0.87 vs 0.91 ± 0.98) in the multiple lesion group were higher than those in the single lesion group, respectively. Multivariate linear regression analysis showed that the serum CrAg titers were positively associated with the number of lesions (ß, 0.08; 95% CI, 0.05 to 0.12) and the lesion size (ß, 0.40; 95% CI, 0.31 to 0.50) after adjusting other covariates. The serum CrAg-LFA titers of 60 pulmonary cryptococcosis patients showed a decreasing trend with the reduction in pulmonary lesion size after effective therapy. CONCLUSION: In pulmonary cryptococcosis patients, the number and size of lung lesions are positively correlated with the titers of the serum CrAg-LFA test. The CrAg-LFA test could be a useful tool for the diagnosis, severity assessment, and therapeutic monitoring of localized pulmonary cryptococcosis patients.

Integrin activation enables rapid detection of functional Vδ1+ and Vδ2+ γδ T cells.

Conformational change of the ß2 integrin lymphocyte function-associated antigen 1 (LFA-1) is an early marker of T cell activation. A protocol using the mAb clone m24 recognizing the active, extended high-affinity conformation has been previously described for the assessment of functional CD4+ and CD8+ T cells in response to MHC-peptide stimulation. We investigated the applicability of the m24 mAb to detect the activation of γδ T cells in response to different soluble and immobilized stimuli. m24 mAb staining was associated with the expression of cytokines and was detectable as early as 10 min after stimulation, but with different kinetics depending on the nature of the stimulus. Hence, we conclude that this assay is suitable for the detection of functional γδ T cells and allows the assessment of activation more rapidly than alternative methods such as cytokine detection. Intracellular staining, protein trafficking inhibitors, or prior knowledge of the stimulating moiety recognized are no longer required for monitoring γδ T cell activation.

Development of a rapid lateral flow assay for detection of anti-coccidioidal antibodies.

Coccidioides spp. are dimorphic fungi that are capable of infecting human and non-human mammals and can cause diverse manifestations of coccidioidomycosis or Valley fever (VF). In combination with clinical symptoms and radiographic findings, antibody-based diagnostic tests are often used to diagnose and monitor patients with VF. Chitinase 1 (CTS1) has previously been identified as the seroreactive antigen used in these diagnostic assays to detect anticoccidial IgG. Here, an indirect enzyme-linked immunosorbent assay to detect IgG to CTS1 demonstrated 165 of 178 (92.7%) patients with a positive result by immunodiffusion (ID) and/or complement fixation (CF) had antibodies to the single antigen CTS1. We then developed a rapid antibody lateral flow assay (LFA) to detect anti-CTS1 antibodies. Out of 143 samples tested, the LFA showed 92.9% positive percent agreement [95% confidence interval (CI), 84.3%-96.9%] and 97.7% negative percent agreement (95% CI, 87.9%-99.6%) with ID and CF assays. Serum or plasma from canines, macaques, and dolphins was also tested by the CTS1 LFA. Test line densities of the CTS1 LFA correlated in a linear manner with the reported CF and ID titers for human and non-human samples, respectively. This 10-min point-of-care test for the rapid detection of anti-coccidioidal antibodies could help to inform healthcare providers in real-time, potentially improving the efficiency of healthcare delivery.

Magnesium enhances the graft-versus-tumor effect of donor lymphocytic infusion on hematologic malignancies.

Donor lymphocyte infusion (DLI) cures relapsed hematologic malignancies after allogeneic hematopoietic stem cell transplantation through the graft-versus-tumor (GVT) effect. Although the important role of magnesium in enhancing immunity has been mentioned in studies, limited clinical data have explored how magnesium affects the efficacy of DLI. Besides, although laboratory data demonstrate that magnesium can enhance CD8+ T cells effector function, whether magnesium regulates the tumor killing effect of peripheral blood mononuclear cells (PBMCs) remains to be explored. Here, for the retrospective study, we collected clinical data of relapsed patients receiving DLI and explored the relationship between different serum magnesium levels and patient outcomes. For in vitro studies, we investigated the effect of magnesium on the cytotoxicity of DLI cells which were PBMCs and preliminarily explored the mechanism. Eighty-one patients were enrolled in this study. It was found that the high post-DLI magnesium level was significantly associated with a higher incidence of complete remission (CR) or partial remission (CR/PR) and a higher possibility of survival. The magnesium level after DLI was an independent risk factor of overall survival. In vitro studies proved that increased magnesium enhanced the cytotoxic function of PBMCs on hematologic malignancies. Besides, magnesium modulated LFA-1 headpiece opening. When blocking the integrin-ligand interaction between LFA-1 and ICAM-1, the regulation effect of magnesium on PBMCs was weakened. Therefore, it was possible that magnesium regulated PBMCs effector function by stimulating LFA-1. These results show that serum magnesium levels affect immunological responses mediated by donor lymphocytes in hematologic malignancies.

LFA-1 knockout inhibited the tumor growth and is correlated with treg cells.

Cancer immunotherapy has been proven to be clinically effective in multiple types of cancers. Lymphocyte function-associated antigen 1 (LFA-1), a member of the integrin family of adhesion molecules, is expressed mainly on αß T cells. LFA-1 is associated with tumor immune responses, but its exact mechanism remains unknown. Here, two kinds of mice tumor model of LFA-1 knockout (LFA-1-/-) mice bearing subcutaneous tumor and Apc Min/+;LFA-1-/- mice were used to confirm that LFA-1 knockout resulted in inhibition of tumor growth. Furthermore, it also demonstrated that the numbers of regulatory T cells (Treg cells) in the spleen, blood, mesenteric lymph nodes were decreased in LFA-1-/- mice, and the numbers of Treg cells in mesenteric lymph nodes were also decreased in Apc Min/+;LFA-1-/- mice compared with Apc Min/+ mice. LFA-1 inhibitor (BIRT377) was administered to subcutaneous tumor-bearing LFA-1+/+ mice, and the results showed that the tumor growth was inhibited and the number of Treg cells was reduced. The analysis of TIMER tumor database indicated that LFA-1 expression is positively associated with Treg cells and TNM stage. Conclusively, this suggests that LFA-1 knockout would inhibit tumor growth and is correlated with Treg cells. LFA-1 may be one potential target for cancer immunotherapy. Video Abstract.

A review of rapid food safety testing: using lateral flow assay platform to detect foodborne pathogens.

The detrimental impact of foodborne pathogens on human health makes food safety a major concern at all levels of production. Conventional methods to detect foodborne pathogens, such as live culture, high-performance liquid chromatography, and molecular techniques, are relatively tedious, time-consuming, laborious, and expensive, which hinders their use for on-site applications. Recurrent outbreaks of foodborne illness have heightened the demand for rapid and simple technologies for detection of foodborne pathogens. Recently, Lateral flow assays (LFA) have drawn attention because of their ability to detect pathogens rapidly, cheaply, and on-site. Here, we reviewed the latest developments in LFAs to detect various foodborne pathogens in food samples, giving special attention to how reporters and labels have improved LFA performance. We also discussed different approaches to improve LFA sensitivity and specificity. Most importantly, due to the lack of studies on LFAs for the detection of viral foodborne pathogens in food samples, we summarized our recent research on developing LFAs for the detection of viral foodborne pathogens. Finally, we highlighted the main challenges for further development of LFA platforms. In summary, with continuing improvements, LFAs may soon offer excellent performance at point-of-care that is competitive with laboratory techniques while retaining a rapid format.

Self-directed molecular diagnostics (SdMDx) system for COVID-19 via one-pot processing.

Rapid, sensitive, and specific detection of the severe acute respiratory syndrome coronavirus (SARS-CoV)- 2 during early infection is pivotal in controlling the spread and pathological progression of Coronavirus Disease 2019 (COVID-19). Thus, highly accurate, affordable, and scalable point-of-care (POC) diagnostic technologies are necessary. Herein, we developed a rapid and efficient self-directed molecular diagnostic (SdMDx) system for SARS-CoV-2. This system combines the sample preparation step, including virus enrichment and extraction processes, which involve dimethyl suberimidate dihydrochloride and diatomaceous earth functionalized with 3-aminopropyl(diethoxy)methylsilane, and the detection step using loop-mediated isothermal amplification-lateral flow assay (LAMP-LFA). Using the SdMDx system, SARS-CoV-2 could be detected within 47 min by hand without the need for any larger instruments. The SdMDx system enabled detection as low as 0.05 PFU in the culture fluid of SARS-CoV-2-infected VeroE6 cells. We validated the accuracy of the SdMDx system on 38 clinical nasopharyngeal specimens. The clinical utility of the SdMDx system for targeting the S gene of SARS-CoV-2 showed 94.4% sensitivity and 100% specificity. This system is more sensitive than antigen and antibody assays, and it minimizes the use of complicated processes and reduces contamination risks. Accordingly, we demonstrated that the SdMDx system enables a rapid, accurate, simple, efficient, and inexpensive detection of SARS-CoV-2 at home, in emergency facilities, and in low-resource sites as a pre-screening platform and POC testing through self-operation and self-diagnosis.

Fluorophore-encapsulated nanobeads for on-site, rapid, and sensitive lateral flow assay.

Since December 2019, the rapid and sensitive detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a priority for public health. Although the lateral flow assay (LFA) sensor has emerged as a rapid and on-site SARS-CoV-2 detection technique, the conventional approach of using gold nanoparticles for the signaling probe had limitations in increasing the sensitivity of the sensor. Herein, our newly suggested methodology to improve the performance of the LFA system could amplify the sensor signal with a facile fabrication method by concentrating fluorescent organic molecules. A large Stokes shift fluorophore (single benzene) was encapsulated into polystyrene nanobeads to enhance the fluorescence intensity of the probe for LFA sensor, which was detected on the test line with a longpass filter under ultraviolet light irradiation. This approach provides comparatively high sensitivity with the limit of detection of 1 ng mL-1 for the SARS-CoV-2 spike protein and a fast detection process, which takes less than 20 min. Furthermore, our sensor showed higher performance than gold nanoparticle-based commercial rapid diagnostics test kits in clinical tests, proving that this approach is more suitable and reliable for the sensitive and rapid detection of viruses, bacteria, and other hazardous materials.

Proteomic analysis in primary T cells reveals IL-7 alters T cell receptor thresholding via CYTIP/cytohesin/LFA-1 localisation and activation.

The ability of the cellular immune system to discriminate self from foreign antigens depends on the appropriate calibration of the T cell receptor (TCR) signalling threshold. The lymphocyte homeostatic cytokine interleukin 7 (IL-7) is known to affect TCR thresholding, but the molecular mechanism is not fully elucidated. A better understanding of this process is highly relevant in the context of autoimmune disease therapy and cancer immunotherapy. We sought to characterise the early signalling events attributable to IL-7 priming; in particular, the altered phosphorylation of signal transduction proteins and their molecular localisation to the TCR. By integrating high-resolution proximity- phospho-proteomic and imaging approaches using primary T cells, rather than engineered cell lines or an in vitro expanded T cell population, we uncovered transduction events previously not linked to IL-7. We show that IL-7 leads to dephosphorylation of cytohesin interacting protein (CYTIP) at a hitherto undescribed phosphorylation site (pThr280) and alters the co-localisation of cytohesin-1 with the TCR and LFA-1 integrin. These results show that IL-7, acting via CYTIP and cytohesin-1, may impact TCR activation thresholds by enhancing the co-clustering of TCR and LFA-1 integrin.

Comparative assessment of homemade ELISA and lateral flow assay (LFA)in the rapid, specific and sensitive detection of SARS-CoV-2 anti-nucleocapsid protein in sera of Egyptian patients.

Several diagnostic measures have been employed to precisely detect the SARS-CoV-2 viral infection using viral antigens, nucleic acids, and other serological approaches. The sensitivity and specificity of the serological tests remain a challenging need. Here, we describe the detection of human anti-SARS-CoV-2 IgG and IgM antibodies qualitatively through two optimized in-house ELISA and lateral flow immunoassay. Both approaches are based on the prokaryotic expression of 50 kDa SARS-CoV-2 recombinant nucleocapsid protein. This SARS-CoV-2rN-6×His was used either to coat ELISA plates or to be conjugated to gold nanoparticles followed by colorimetric detection of bound human IgG or IgM. In the LFA, we show the optimization of nanoparticle size, protein-binding capacity, membrane treatment, and finally testing the potential capacity of using either the optimized ELISA or LFA in detecting antibodies raised against viral infection. Assessment of both methods was carried out using human sera-positive and negative SARS-CoV-2 antibodies. The ELISA and LFA tests showed 86%, 96.5% sensitivity, 92%, 93.75% specificity, 97%, 98.2% PPV, and 64%, 88.2% NPV, respectively. In conclusion, both approaches were able to successfully detect human antibodies against SARS-CoV-2 nucleocapsid protein. The importance of both protocols cannot be overstated in the detection and diagnosis of viral infections, especially in developing countries.