A dual-mode aptasensor based on rolling circle amplification enriched G-quadruplex for highly sensitive IFN-γ detection.

BACKGROUND: Aptasensors have been extensively utilized in target detection due to their advantages of high sensitivity and fast response. However, the reliability of the detection results of the single-mode aptasensor cannot be verified in time. Developing efficient detection methods with cross-validation capability is beneficial to achieving highly reliable detection. This study aims to design a colorimetric and fluorescent dual-mode aptasensor by skillfully engineering G-quadruplex assembly and rolling circle amplification for highly reliable IFN-γ detection. RESULTS: The complexes of anti-IFN-γ aptamers and complement sequences (cDNA) were modified on the magnetic beads. In the presence of IFN-γ, the preferential combination of aptamers with IFN-γ resulted in the release of cDNAs. The cDNAs were collected by magnetic separation and then used as primers to trigger rolling circle amplification reaction to generate enriched G-quadruplexes. The G-quadruplex could be utilized to combine with hemin to catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine for colormetric mode or to couple with the fluorogenic dye Thioflavin T for fluorescent mode. The developed dual-mode aptasensor displayed a linear range of 1-10000 pM with a detection limit of 0.406 pM for the colormetric mode and a range of 0.1-10000 pM with a detection limit of 0.037 pM for the fluorescent mode. Further, the designed aptasensor was applied to IFN-γ detection in serum samples and achieved satisfactory recoveries. SIGNIFICANCE: This innovative dual-mode detection strategy skillfully leverages the effective target-binding ability of aptamer, dual-function of the G-quadruplex and the signal amplifying ability of rolling circle amplification. This approach not only provides a reliable testing tool for the detection of IFN-γ, but also promotes the development of multimode sensing platforms.

DNA circuit-based immunoassay for ultrasensitive protein pattern classification.

Cytokines are important immune modulators, and pivotal biomarkers for the diagnostic of various diseases. In standard analytical procedure, each protein is detected individually, using for instance gold standard ELISA protocols or nucleic acid amplification-based immunoassays. In recent years, DNA nanotechnology has been employed for creating sophisticated biomolecular systems that perform neuromorphic computing on molecular inputs, opening the door to concentration pattern recognition for biomedical applications. This work introduces immuno-PUMA (i-PUMA), an isothermal amplification-based immunoassay for ultrasensitive protein detection. The assay couples the convenience of supported format of an ELISA protocol with the computing capabilities of a DNA/enzyme circuit. We demonstrate a limit of detection of 2.1 fM, 8.7 fM and 450 aM for IL12, IL4 and IFNγ cytokines, respectively, outperforming the traditional ELISA format. i-PUMA's versatility extends to molecular computation, allowing the creation of 2-input perceptron-like classifiers for IL12 and IL4, with tunable weight sign and amplitude. Overall, i-PUMA represents a sensitive, low-cost, and versatile immunoassay with potential applications in multimarker-based sample classification, complementing existing molecular profiling techniques.

The effect of infection with Toxoplasma gondii in inducing interferon-gamma in breast cancer patients.

Toxoplasmosis is one of the most widespread zoonotic diseases in the world. Human infection rates range from 10% to 80% in many countries. Female cancer patients receiving chemotherapy are more susceptible to developing acute forms of toxoplasmosis, which can cause brain defects, neurological damage, and encephalitis. The aim of the present study was to investigate the effect of Toxoplasma gondii infection on the induction of interferon-gamma in breast cancer patients from Iraq. This descriptive cross-sectional study was performed on women had breast cancer in Al-Haboubi Teaching Hospital in Nasiriya City-Thi-Qar Province (Iraq) during the period from January to September 2022. Approximately three ml of blood was drawn from all participants and sera were collected. The Sera were then tested for Toxoplasma IgM, IgG, and IFN-γ (Nova Tec Immunodiagnostica GmbH, Germany) using the enzyme-linked immunosorbent assay (ELISA) kits according to the protocols of the manufacturer. Before blood collection, participants completed a printed questionnaire with some demographic information, such as age and place of residence. The total number of positive T. gondii infections from breast cancer patients in the current study was 60 (85.7%). The results of sample analysis by ELISA assay showed that 85.7% and 74.2% of patients were positive for IgG and IgM, respectively. The mean IFN-γ levels in breast cancer patients with toxoplasmosis, without toxoplasmosis, and in the control group were 47.66, 0.00, and 0.57 pg/ ml, respectively. Higher IgG and interferon gamma levels were detected in the group of breast cancer patients with toxoplasmosis than in the group without toxoplasmosis. According to the ELISA findings, T. gondii was the most common parasite in female cancer patients.

The role of vitamin D in amelioration of oral lichen planus and its effect on salivary and tissue IFN-γ level: a randomized clinical trial.

BACKGROUND AND OBJECTIVES: Oral lichen planus (OLP) is a common, prevalent, immune-mediated, inflammatory disease affecting both the skin and oral mucosa and is considered one of the potentially malignant diseases. Since OLP is regarded as an immunologically mediated disease, some studies suggest the use of vitamin D (VD) for its management as it exhibits immune-modulatory, anti-inflammatory, and antimicrobial properties, as well as anti-proliferative, pro-differentiative, and anti-angiogenic effects. VD has demonstrated a suppressive effect on TH1 pro-inflammatory cytokines, including IFN-γ while augmenting the secretion of anti-inflammatory cytokines. At the same time, VD deficiency is a prevalent public issue. Therefore, the present study aimed to investigate the role of VD as an adjunct to steroids in the management of VD-deficient OLP patients as well as its inhibitory effect on IFN-γ through measurement of salivary and tissue IFN-γ levels in OLP patients. METHODS: A total of 40 patients with ulcerative or erythematous OLP, diagnosed according to the World Health Organization's (WHO) modified criteria for OLP, were randomly allocated into one of the two study groups to receive either systemic steroids in addition to VD supplements (Group A) or systemic steroids only (Group B). Blood samples were collected for the measurement of serum VD level (SVDL) using the enzyme-linked immunosorbent assay (ELISA) to involve only patients with VD deficiency or insufficiency (≤ 30 ng/ml). Clinical evaluation of the lesion involved objective signs and subjective symptoms. Also, changes in salivary and tissue INF-γ levels (in pg/mL and pg/mg, respectively) were determined using the ELISA technique. All parameters were measured at baseline and after 4 weeks of treatment. The clinical pharmacy team devised a checklist to record all team interventions. The interventions were categorized into six domains, including drug interactions and/or adverse reactions, medication dose issues, drug selection issues, support with medication history, patient-related concerns, and suggestions for dental medication. RESULTS: After one month of treatment, a significantly greater number of patients in group A showed complete pain relief and resolution of clinical lesions, as well as a greater number of patients showing a reduction in the clinical severity of lesions than in group B (P = 0.005). Also, there was a statistically significant reduction in average VAS pain scores and clinical scores in group A compared to group B after 1 month of treatment (P = 0.001 and 0.002, respectively). Furthermore, there was a statistically significant greater reduction in salivary and tissue IFN-γ levels in group A than in group B (P ≤ 0.001 and 0.029, respectively) after 1 month of treatment. CONCLUSION: Current evidence suggests a significant preventive and therapeutic role for VD as an adjunct to standard therapies indicated for OLP lesions. These protective and therapeutic functions are achieved through the suppressive effect of VD on pro-inflammatory cytokines, particularly IFN-γ. Also, salivary IFN-γ appears to be a valuable prognostic marker for monitoring the progression of OLP. In addition, the inter-professional collaboration between dentists and clinical pharmacists helped to deliver complete, patient-centered primary care and ensured the quality of the medications included in patient kits, thus improving patient treatment and management. Nevertheless, further studies with larger sample sizes, longer follow-ups, and standardized designs may still be needed.

Use of a platform with lens-free shadow imaging technology to monitor natural killer cell activity.

Natural killer (NK) cells are a crucial component of the innate immune system. This study introduces Cellytics NK, a novel platform for rapid and precise measurement of NK cell activity. This platform combines an NK-specific activation stimulator cocktail (ASC) and lens-free shadow imaging technology (LSIT), using optoelectronic components. LSIT captures digital hologram images of resting and ASC-activated NK cells, while an algorithm evaluates cell size and cytoplasmic complexity using shadow parameters. The combined shadow parameter derived from the peak-to-peak distance and width standard deviation rapidly distinguishes active NK cells from inactive NK cells at the single-cell level within 30 s. Here, the feasibility of the system was demonstrated by assessing NK cells from healthy donors and immunocompromised cancer patients, demonstrating a significant difference in the innate immunity index (I3). Cancer patients showed a lower I3 value (161%) than healthy donors (326%). I3 was strongly correlated with NK cell activity measured using various markers such as interferon-gamma, tumor necrosis factor-alpha, perforin, granzyme B, and CD107a. This technology holds promise for advancing immune functional assays, offering rapid and accurate on-site analysis of NK cells, a crucial innate immune cell, with its compact and cost-effective optoelectronic setup, especially in the post-COVID-19 era.

In-vivo and ex-vivo tests for culprit drugs identification in severe cutaneous adverse drugs reactions.

Drug causality assessment in severe cutaneous adverse reactions (SCARs) remains challenging. We investigated the usefulness of in-vivo drug patch tests (PT), ex-vivo interferon (IFN)-γ enzyme-linked immunospot (ELISpot) assay, and lymphocyte transformation test (LTT) in 30 SCARs patients within the past 36 months. Drug PT yielded a 20% positivity rate (n = 6), while IFN-γ ELISpot and LTT showed positive rates of 56.67% (n = 17) and 41.38% (n = 12), respectively. Combining the three tests resulted in an overall positive rate of 66.67% (n = 20) of cases. IFN-γ ELISpot offered additional positivity, especially with oxypurinol. Employing a combined diagnostic approach may enhance the chances of obtaining a positive result.

Multicolor-Assay-on-a-Chip Processed by Robotic Operation (MACpro) with Improved Diagnostic Accuracy for Field-Deployable Detection.

The ability to deploy decentralized laboratories with autonomous and reliable disease diagnosis holds the potential to deliver accessible healthcare services for public safety. While microfluidic technologies provide precise manipulation of small fluid volumes with improved assay performance, their limited automation and versatility confine them to laboratories. Herein, we report the utility of multicolor assay-on-a-chip processed by robotic operation (MACpro), to address this unmet need. The MACpro platform comprises a robot-microfluidic interface and an eye-in-hand module that provides flexible yet stable actions to execute tasks in a programmable manner, such as the precise manipulation of the microfluidic chip along with different paths. Notably, MACpro shows improved detection performance by integrating the microbead-based antibody immobilization with enhanced target recognition and multicolor sensing via Cu2+-catalyzed plasmonic etching of gold nanorods for rapid and sensitive analyte quantification. Using interferon-gamma as an example, we demonstrate that MACpro completes a sample-to-answer immunoassay within 30 min and achieves a 10-fold broader dynamic range and a 10-fold lower detection limit compared to standard enzyme-linked immunosorbent assays (0.66 vs 5.2 pg/mL). MACpro extends the applications beyond traditional laboratories and presents an automated solution to expand diagnostic capacity in diverse settings.

Interferon gamma (IFN-γ)-sensitive TB aptasensor based on novel chitosan-indium nano-kesterite (χtCITS)-labeled DNA aptamer hairpin technology.

There has been increasing interest in the use of biosensors for diagnosis of infectious diseases such as tuberculosis (TB) due to their simplicity, affordability, and potential for point-of-care application. The incorporation of aptamer molecules and nanomaterials in biosensor fabrication explores the advantages of high-binding affinity and low immunogenicity of aptamers as well as the high surface-to-volume ratio of nanomaterials, for increased aptasensor performance. In this work, we employed a novel microwave-synthesized copper indium tin sulfide (CITS) substituted-kesterite nanomaterial, together with a natural biopolymer (chitosan), for signal amplification and increased loading of aptamer molecules. Study of the optical properties of CITS nanomaterials showed strong absorption in the UV region characteristic of kesterite semiconductor nanomaterials. X-ray diffraction analysis confirmed the presence of the kesterite phase with average crystallite size of 6.188 nm. Fabrication of interferon-gamma (IFN-γ) TB aptasensor with a chitosan-CITS nanocomposite (χtCITS) increased the aptasensor's electrochemical properties by 77.5 % and improved aptamer loading by 73.7 %. The aptasensor showed excellent sensitivity to IFN-γ concentrations with limit of detection of 6885 fg/mL (405 fM) and linear range of 850-17000 fg/mL (50 - 1000 fM). The aptasensor also exhibited excellent storage and electrochemical stability, with good selectivity towards IFN-γ and possible real sample application.

Periostin in the relation between periodontal disease and atherosclerotic coronary artery disease: A pilot randomized clinical study.

OBJECTIVE: The aim of this study was to analyze the effects of periodontal treatment on markers of atherosclerotic coronary artery disease and circulating levels of periostin. BACKGROUND: Periostin is necessary for periodontal stability, but it is highly present in atherosclerotic plaques. Treatment of periodontal disease, with low levels of local periostin, is thought to reduce systemic levels of periostin. Thus, this may contribute to cardiovascular health. METHODS: A pilot randomized controlled clinical trial was designed to include patients with severe periodontal disease and history of atherosclerotic coronary artery disease. Samples of gingival crevicular fluid (GCF) and serum were collected before and after periodontal treatment by periodontal surgery or non-surgical therapy. The levels of several markers of inflammation and cardiovascular damage were evaluated including CRP, IFN-γ, IL-1ß, IL-10, MIP-1α, periostin, and TNF-α in GCF and CRP, Fibrinogen, IFN-γ, IL-1ß, IL-6, IL-10, L-Selectin, MIP-1α, Periostin, TNF-α, and vWF in serum. RESULTS: A total of 22 patients with an average of 56 years old were recruited for participating in this study. Twenty of them were male. Most of them (82%) had suffered an acute myocardial event and underwent surgery for placing 1, 2, or 3 stents in the coronary arteries more than 6 months ago but less than 1 year. The treatment of periodontal disease resulted in an overall improvement of all periodontal parameters. Regarding the evaluation of GCF and serum, a significant increase of periostin in the GCF was observed after periodontal surgery. In contrast, although other markers in GCF and serum improved, no significant correlations were found. CONCLUSION: Treatment of periodontal disease through periodontal surgery induces a local and transient increase in the levels of periostin in the gingival crevicular fluid. The effects on systemic markers of inflammation and cardiovascular function have not been confirmed.

[Regulation effect of serum IFN-γ and IL-4 expression on immune response in patients with oral lichen planus].

PURPOSE: To investigate the regulation effect of interferon-γ (IFN-γ) and interleukin-4 (IL-4) in the serum of patients with oral lichen planus (OLP) on immune response. METHODS: A total of 83 OLP patients in Wangfujing Branch of Beijing Stomatological Hospital, Capital Medical University from January 2019 to December 2021 were selected as the experimental group, and another 61 healthy volunteers who came to the hospital during the same period were included as the control group. The expression levels of IFN-γ and IL-4 in the serum and the expression of immune function indexes of the two groups were compared. The relationship between the expression levels of IFN-γ, IL-4 and immune function indicators in the experimental group was analyzed. SPSS 22.0 software package was used to analyze the data. RESULTS: The levels of IFN-γ and IL-4 in the experimental group were significantly higher than those in the control group(P＜0.05). There was no significant difference in the levels of IFN-γ between patients with reticulated OLP and erosive OLP in the experimental group (P＞0.05). The level of IL-4 in patients with erosive OLP in the experimental group was significantly higher than that in patients with reticulated OLP(P＜0.05). The levels of CD3+, CD4, CD16++CD56+ and CD4+/CD8+ in the experimental group were significantly lower than those in the control group(P＜0.05), the levels of CD8+ and CD19+ in the experimental group were significantly higher than those in the control group(P＜0.05). The levels of IgG, IgM and CH50 in the experimental group were significantly higher than those in the control group(P＜0.05), and the levels of IgA, C3 and C4 in the experimental group were significantly lower than those in the control group(P＜0.05). Pearson correlation analysis showed that the level of IFN-γ in the experimental group was negatively correlated with CD16++CD56+ and CD4+/CD8+ (P＜0.05), the level of IL-4 in the experimental group was negatively correlated with CD16++CD56+ and CD4+/CD8+(P＜0.05), and the level of IL-4 in the experimental group was positively correlated with IgG (P＜0.05). CONCLUSIONS: Serum IFN-γ and IL-4 in OLP patients are abnormally high, and IL-4 expression is significantly higher in erosive OLP patients. The cellular and humoral immune functions of OLP patients are disordered and related to the regulation of CD16++CD56+, CD4+/CD8+, and IgG immune responses.

Interferon-γ release assay and mantoux response in infants with tuberculous meningitis in low and intermediate burden countries.

AIM: Until now, the performance of interferon-γ release assay (IGRA) and Mantoux tests remains unclear in infant tuberculous meningitis (TBM). Therefore, a systematic review is performed to evaluate the sensitivity of IGRA and Mantoux tests for the diagnosis of infant TBM in low and intermediate tuberculosis (TB) burden countries, while following PRISMA. METHODS: Several databases, including PubMed, EBSCO, Embase, Scopus, Web of Science, ClinicalTrials.gov, and Cochrane Central Register of Controlled Trials, were searched. Articles describing the results of IGRA or Mantoux tests among infant TBM were included for analysis. Data, such as age, sex, Mantoux test or IGRA, and cerebrospinal fluid (CSF) microbiological examinations (such as acid-fast bacilli (AFB) smear, TB PCR, and TB culture), were extracted from each study. RESULTS: A total of 31 articles were enrolled for further analysis, including 48 cases. The mean age was 9.4 ± 5.8 months and boys accounted for 57.1% of infants (24/42). Mantoux test was positive in 57.4% (27/47) of tested infants and IGRA was positive in 77.8% (7/9) of infants. In addition, among the infants with confirmed TB, 18 (52.9%, 18/34) of them have positive Mantoux responses and 7 (20.0%, 7/35) have positive IGRA results. CONCLUSIONS: In low or intermediate TB burden countries, the Mantoux test has a poor performance for diagnosing TBM among infants, and IGRAs appear to have a moderate sensitivity for the diagnosis of infant TBM.

Stability-indicative and conformation-specific enzyme linked immunosorbent assay for analysis of recombinant human gamma interferon.

Recombinant human interferon gamma (rhIFN-γ) is a promising molecule for the treatment of several diseases. A pair of conformation-specific monoclonal antibodies (mAbs) against rhIFN-γ was selected from generated hybridoma cell lines to design a sensitive, stability-indicative, sandwich-type ELISA. The main assay parameters were optimized by the checkerboard method for the highest signal-to-noise ratio: assay buffer composition, coating buffer pH and composition, coating temperature-incubation time parameters, and coating mAb concentration and conjugate dilution. Detection and quantification limits were estimated between 0.019 and 0.078 ng/mL, respectively, and recovery values were from 92.03% to 98.40%. The coefficient of variation of intra-assay precision parameters ranged from 2.32% to 9.21% while the inter-analyst variation was between 4.70% and 10.63%, supporting the method's repeatability. The ELISA was specific for correctly folded and non-aggregated molecular species, as compared to intrinsic Trp fluorescence (chemical denaturation) and optical density at 340 nm (thermal aggregation), respectively. However, the method was not sensitive to the small C-terminal degradation of full-length rhIFN-γ1-144 (losses of 6-12 amino acid residues) as compared to results with mass spectrometry and gel electrophoresis. ELISA showed good correlation with rhIFN-γ antiviral biological activity. This method was applied to the stability evaluation of rhIFN-γ in physiological buffer at low concentrations using polypropylene and glass vials also in the presence of adsorption protectant excipients. Furthermore, ELISA could be adapted to other applications such as quantification of IFN-γ in serum samples, Mycobacterium tuberculosis diagnosis, etc.

Efecto del estrés crónico y de la masticación sobre los niveles séricos de interleuquina-6 e interferón-gama / Effect of chronic stress and chewing on serum interleukin-6 and interferon-gamma levels

Introducción: el estrés crónico afecta el equilibrio inmunológico alterando los niveles séricos de interleuquina-6 (IL-6) e interferón gama (INF-γ), dicha alteración afecta al sistema nervioso y al comportamiento humano. La masticación adecuada disminuiría dichos efectos. El objetivo del estudio fue determinar el efecto del estrés crónico y de la masticación sobre los niveles séricos de IL-6 e INF-γ. Métodos: experimento donde se emplearon 64 ratones Balb/c de 8 semanas de edad. Se dividieron en 4 tratamientos: Grupo NE: Masticación normal + estrés, Grupo N: masticación normal sin estrés, Grupo DE: Masticación deficiente + estrés, Grupo D: masticación deficiente sin estrés. Mediante test de ELISA se midió IL-6 e IFN-γ alfinal de la 4ta y de la 8va semana de tratamiento. Resultados: tanto la IL-6 como el IFN-γ fueron mayores en el grupo DE (p<0,05) al final de la 4ta semana. Al evaluarlos al término de la 8va semana se observó que en el grupo NE se incrementó la IL-6 respecto al resto de grupos (p<0,0001), y en el grupo DE fue donde se encontró mayor cantidad de IFN-γ (p<0,0001). Conclusión: el estrés crónico y la masticación deficiente incrementan los niveles séricos de IL-6 e IFN-γ. En cambio, la adecuada masticación disminuye el nivel de tales citoquinas al final de la cuarta semana de tratamiento.

Introduction: chronic stress affects the immune balance by altering the serum levels of interleukin-6 (IL-6) and gamma interferon (INF-γ), this alteration affects the nervous system and human behavior. Appropriate chewing would lessen these effects. The aim of this study was to determine the effect of chewing and chronic stress over serum levels of IL-6 and INF-γ. Methods: experiment in which 64 Balb/C mice of 8 weeks of age were used, they were divided into 4 treatments: Group NE: Normal chewing + stress, Group N: normal chewing without stress, Group DE: Chewing poor + stress, Group D: poor chewing without stress. IL-6 and IFN-γ were measured by ELISA after 4 and 8 weeks of treatment. Results: both IL-6 and IFN-γ were higher in the DE group (p < 0,05) at the end of fourth week of treatment. When evaluating the animals at the end of the eighth week of treatment, it was observed that in the NE group, the IL-6 was increased with respect to the rest (p < 0,0001) and the DE group showed more IFN-γ (p < 0,0001). Conclusion: stress and poor chewing increase serum IL-6 and IFN-γ. In contrast, appropriate chewing decreases the effects of stress on the increase of such cytokines at the end of the fourth week of treatment in animals.

Increased interferon-γ levels and risk of severe malaria: a meta-analysis.

Interferon (IFN)-γ contributes to the pathogenesis of severe malaria; however, its mechanism remains unclear. Herein, differences in IFN-γ levels between patients with severe and uncomplicated malaria were evaluated using qualitative and quantitative (meta-analysis) approaches. The systematic review protocol was registered at PROSPERO (ID: CRD42022315213). The searches for relevant studies were performed in five databases, including PubMed, Scopus, Embase, MEDLINE and Web of Science, between 1 January and 10 July 2022. A meta-analysis was conducted to pool the mean difference (MD) of IFN-γ levels between patients with severe malaria and those with uncomplicated malaria using a random-effects model (DerSimonian and Laird method). Overall, qualitative synthesis indicated that most studies (14, 58.3%) reported no statistically significant difference in IFN-γ levels between patients with severe malaria and those with uncomplicated malaria. Meanwhile, remaining studies (9, 37.5%) reported that IFN-γ levels were significantly higher in patients with severe malaria than those in patients with uncomplicated malaria. Only one study (4.17%) reported that IFN-γ levels were significantly lower in patients with severe malaria than those in patients with uncomplicated malaria. The meta-analysis results indicated that patients with severe malaria had higher mean IFN-γ levels than those with uncomplicated malaria (p < 0.001, MD: 13.63 pg/mL, 95% confidence interval: 6.98-20.29 pg/mL, I2: 99.02%, 14 studies/15 study sites, 652 severe cases/1096 uncomplicated cases). In summary, patients with severe malaria exhibited higher IFN-γ levels than those with uncomplicated malaria, although the heterogeneity of the outcomes is yet to be elucidated. To confirm whether alteration in IFN-γ levels of patients with malaria may indicate disease severity and/or poor prognosis, further studies are warranted.

Assessing T-Cell Immunity in Kidney Transplant Recipients with Absent Antibody Production after a 3rd Dose of the mRNA-1273 Vaccine.

The vulnerable population of kidney transplant recipients (KTRs) are low responders to COVID-19 vaccines, so specific immune surveillance is needed. The interferon-gamma (IFN-γ) release assay (IGRA) is effective in assessing T cell-mediated immunity. We assessed SARS-CoV-2-directed T cell responses in KTRs with absent antibody production after a third dose of the mRNA-1273 vaccine, using two different IGRAs. A cohort of 57 KTRs, who were actively followed up, received a third dose of the mRNA-1273 vaccine. After the evaluation of humoral immunity to SARS-CoV-2, 14 seronegative patients were tested with two commercial IGRAs (SD Biosensor and Euroimmun). Out of 14 patients, one and three samples were positive by IGRAs with Euroimmun and SD Biosensor, respectively. The overall agreement between the two assays was 85.7% (κ = 0.444). In addition, multivariate linear regression analysis showed no statistically significant association between the IFN-γ concentration, and the independent variables analyzed (age, gender, years since transplant, total lymphocytes cells/mcl, CD3+ cells/mcl, CD3+ CD4+ cells/mcl, CD3+ CD8+ cells/mcl, CD19+ cells/mcl, CD3-CD16+CD56+ cells/mcl) (p > 0.01). In a vulnerable setting, assessing cellular immune response to complement the humoral response may be advantageous. Since the two commercial IGRAs showed a good agreement on negative samples, the three discordant samples highlight the need for further investigations.

Tuberculosis-specific antigen stimulated and unstimulated interferon-γ for tuberculous meningitis diagnosis: A systematic review and meta-analysis.

OBJECTIVE: Tuberculous meningitis (TBM) is one of the most devastating TB. Accurate identification of TBM is helpful to eliminate TB. Therefore, we assessed the performance of TBAg stimulated IFN-γ (IGRA) and unstimulated IFN-γ in blood and cerebrospinal fluid (CSF) for diagnosing TBM. METHODS: We searched Web of Science, PubMed, Embase and the Cochrane Library databases until March 2022. Bivariate and hierarchical summary receiver operating characteristic models were employed to compute summary estimates for diagnostic accuracy parameters of IGRA and unstimulated IFN-γ in blood and CSF for diagnosing TBM. RESULTS: 28 studies including 1,978 participants and 2,641 samples met the inclusion criteria. The pooled sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic odds ratio (DOR) and area under the curve (AUROC) of blood IGRA were separately as 0.73, 0.83, 4.32, 0.33, 13.22 and 0.86, indicating a good diagnostic accuracy of blood IGRA for detecting TBM. The summary sensitivity, specificity, PLR, NLR, DOR and AUROC of CSF IGRA were separately as 0.77, 0.91, 8.82, 0.25, 34.59 and 0.93, indicating good diagnostic accuracy of CSF IGRA for detecting TBM. The summary sensitivity, specificity, PLR, NLR, DOR and AUROC of CSF IFN-γ were separately as 0.86, 0.92, 10.27, 0.16, 65.26 and 0.95, suggesting CSF IFN-γ provided excellent accuracy for diagnosing TBM. CONCLUSIONS: For differentiating TBM from non-TBM individuals, blood and CSF IGRA are good assays and unstimulated CSF IFN-γ is an auxiliary excellent marker.

Highly accurate multiprotein detection on a digital ELISA platform.

The emerging single-molecule detection platform digital enzyme-linked immunosorbent assay (ELISA) can detect numerous proteins simultaneously at serum concentrations as low as picograms per milliliter. We sought to improve cytokine detection with this platform to aid diagnosis of conditions such as allergy and asthma. We developed a multiple single-molecule detection digital ELISA approach, through the application of encoded magnetic microbeads to simultaneously detect three cytokines in one serum sample. We tested the approach's utility to distinguish asthma-related cytokines in children. Concentrations of interleukin-4 (IL-4) and IL-6 were significantly higher in children with asthma than in healthy controls, while the concentration of interferon-γ (IFN-γ) was significantly lower. Our method has higher accuracy than conventional methods, and our results indicate that the proposed improved high-sensitivity digital ELISA-based diagnosis approach can facilitate early detection and treatment of childhood asthma or related diseases.

Clinical utility of immune function based on IFN-γ monitoring of lymphocyte subsets for parvovirus B19 infection in renal recipients.

BACKGROUND: There should be a heightened index of suspicion for Parvovirus B19 (PVB19)-related anemia in organ transplant recipients. Thus far, there is no consensus or recommendation for clinical routine monitoring methods of PVB19 recipients to allow tailoring of immunosuppression. METHODS: We conducted a retrospective study to evaluate the utility of the function (represented by the abilities to secrete IFN-γ) and numbers of lymphocyte subsets in monitoring PVB19 infections in renal recipients posttransplant. The enrolled 109 patients were split into 2 groups according to whether the recipients had an occurrence of PVB19 infection: 37 (33.94%) recipients developed PVB19 infection and 72 (66.06%) immune-stable recipients. RESULTS: The PVB19 infected group had significantly lower absolute counts and functions of different lymphocyte subsets compared with immune-stable recipients. We showed that the frequencies of IFN-γ + CD4 + T cells, IFN-γ + CD8 + T cells, and IFN-γ + NK cells increased markedly after treatment when compared to the occurrence in patients with timepoint before therapy, especially the percentages of IFN-γ + CD4 + T cells were significantly higher. Receiver operating characteristic (ROC) analysis showed that the optimal infection indicator was IFN-γ + NK cells frequency, with an auROC curve of 0.925. Concomitantly, Cox regression analysis indicated that the post-therapy increasing level of IFN-γ secreting function was significantly predictive of recurrent infections (P < 0.001). CONCLUSIONS: We recommend prospective risk stratification for the high-risk population at risk of early-onset PVB19 infection and its recurrence involves screening strategies of immune-based surveillance with the sensitive IFN-γ + secreting monitoring for antiviral prophylaxis and preemptive therapy goal. Clinical Trial Notation: clinical trial registration number: chiCTR-ROC-17010756.

Interferon-γ detection in point of care diagnostics: Short review.

Interferon (IFN)-γ is a cytokine secreted by immune cells. The elevated levels of IFN-γ are an early indicator of multiple diseases such as tuberculosis and autoimmune diseases. This short review focuses on different sensing methods based on optical, electrochemical, and mechanical principles. We explain how specific biorecognition molecules such as antibodies and aptamers are employed in the sensing methods. We also compare different surface functionalization methods and their details. Although the review gives an overview of only IFN-γ sensing, the same strategies can be applied to sensing other analytes with appropriate modifications.