Background Individuals recovering from COVID-19 are known to have antibodies against the Spike and other structural proteins. Antibodies against Spike have been shown to display viral neutralization. However, not all antibodies against Spike have neutralizing ability although they may be cross-reactive. There is a need for easy-to-use SARS-CoV-2 neutralizing assays for the determination of virus-neutralizing activity in sera of individuals. Here we describe a PCR-based micro‐neutralization assay that can be used to evaluate the viral neutralization titers of serum from SARS-CoV-2 infected individuals. Methods The SARS-CoV-2 strain used was isolated from a nasopharyngeal specimen of a COVID-19 case. The limiting dilution method was used to obtain a 50% tissue culture infective dose (TCID50) of Vero cells. For the micro‐neutralization assay, 19 serum samples, with positive IgG titers against Spike Receptor-Binding Domain (RBD) were tested. After 24 hours, infected cells were inspected for the presence of a cytopathic effect, lysed and RNA RT-PCR conducted for SARS-CoV-2. PCR target Ct values were used to calculate percent neutralization/inhibition of SARS-CoV-2. Results Out of 19 samples, 13 samples gave 100% neutralization at all dilutions, 1 sample showed neutralization at the first dilution, 4 samples showed neutralization at lower dilutions, while one sample did not demonstrate any neutralization. The RBD ODs and neutralization potential percentages were found to be positively correlated. Conclusion We describe a rapid RT-PCR-based SARS-CoV-2 microneutralization assay for the detection of neutralizing antibodies. This can effectively be used to test the antiviral activity of serum antibodies for the investigation of both disease-driven and vaccine-induced responses.

The differences between XDR-TB and drug-susceptible isolates suggest that the increased expression levels of MTB efflux pump genes may contribute to drug resistance in extensively drug-resistant tuberculosis. Future studies are needed to determine whether combining efflux pump inhibitors to antitubercular drugs would be effective to treat resistant tuberculosis.

Introduction Tuberculosis (TB) infections and latent Mycobacterium tuberculosis (MTB) infection (LTBi) remain prevalent globally. Type 2 diabetes mellitus (DM) worsens TB outcomes but the immune mechanisms that cause this are not yet clear. We investigated a role of suppressor of cytokine signaling molecules (SOCS1 and SOC3) in regulating host cytokine responses in the diabetic host infected with MTB. Materials and Methods We studied peripheral blood cells from health endemic controls (EC), LTBi cases, diabetics with and without LTBi and TB patients. Mycobacterial antigen-stimulated cytokine secretion was determined using the Th1/Th2 11 plex cytokine assay. Antigen-induced gene expression of IFNγ, TNFα, IL6 and SOCS3 was determined by reverse-transcription PCR. Results Purified protein derivative (PPD) antigen stimulation induced higher levels of, IL-6, IL-2, TNFα and GM-CSF levels in DM-LTB as compared with EC and LTB cases. IL-13 levels were raised in DM-LTB cases as compared with DM cases. PPD-induced IFNγ and IL-6 transcripts were raised in DM-LTBi as compared with EC. TNFα mRNA levels were raised in DM-LTBi as compared with LTBi. SOCS3 mRNA levels were reduced in DM-LTBi as compared with LTBi. SOCS3 transcripts were higher in LTBi as compared with EC and TB groups. Discussion We found co-occurrence of LTBi with DM to be associated with an increased release of proinflammatory IL-6, IL-2 and TNF-α but reduced SOCS3 mRNA levels. SOCS3 protects against MTB infection therefore, reduced levels in DM-LTB may be contribute to progression from LTBi to active TB in individuals infected with MTB.M. tuberculosis infection in diabetics is associated with increased inflammatory cytokine and decreased Suppressor of cytokine signaling (SOCS)-3 responses

Introduction: The immune system plays a pivotal role in identification and clearance of tumor cells, but can also be counterproductive and enhance tumor progression. Presence of neutrophils in solid tumors, including the sixth most common cancer globally - head and neck squamous cell carcinomas (HNSCC), is associated with poor prognosis. IL-8 and GM-CSF, whose secretion is regulated by NF-κB, are pro-inflammatory cytokines that promote extravasation of neutrophils into tissue and play an important role in the proliferation, invasion, and metastasis of HNSCC. The onset and progression of HNSCC is also associated with genetic alterations that affect several cellular signaling pathways such as transforming growth factor-β (TGFβ) pathway. TGF-β is responsible for polarization of neutrophils towards anti-tumor (N1) versus pro-tumor (N2) phenotype. Objective: The role of neutrophils in HNSCC and the possible molecular mechanisms of pro-tumor neutrophil recruitment is being investigated. Methods and Results: IL-8 and GM-CSF released at various time points by FaDu (ATCC® HTB-43™), SCC-9 (ATCC® CRL-1629™) and Cal-27 (ATCC® CRL-2095™) was measured by ELISA. Conditioned media from FaDu and Cal-27 cells exhibited consistent expression of IL-8 (115 - 117pg/mL and 103 - 110pg/ml, respectively) at 6 - 72 hours. SCC-9 conditioned media exhibited sustained levels of IL-8 expression (103 - 110pg/ml) at 6 - 24 hours that then declined at 48 - 72 hours. GM-CSF was not significantly expressed in the FaDu conditioned media; even with increasing cell density, the mean expression remained 0.142pg/ml. SCC-9 and Cal-27 expressed GM-CSF at 6 - 72 hours (16 - 20pg/mL and ~15pg/ml, respectively). In all cell lines, IL-8 expression decreased in a dose and time dependent manner after treatment with the NF-κB inhibitor, BAY 11-7082, while effect on GM-CSF expression was negligible. Expression of phosphorylated IκBα in FaDu and SCC-9 cell lysates decreased in a time and dose dependent manner, indicating inhibition of the NF-κB pathway by BAY11-7082. Expression of phosphorylated NF-κB also decreased as compared to control in these cells. In nuclear extracts, BAY 11-7082 dose-dependent alteration in NF-κB expression was observed in FaDu and Cal-27 cells at earlier time points. Conditioned media from SCC-9 and Cal-27 cells also exhibited sustained baseline expression of TGF- β (~30pg/ml) over 6 - 72 hours. Conclusion: HNSCC cells derived from tongue and hypopharyngeal squamous cell carcinoma release pro-inflammatory cytokines. The NF-κB signaling pathway appears to be involved in the release of IL-8, a cytokine that is in turn likely to be involved in recruitment of neutrophils to the cancer site that may be polarized towards the N2 pro-tumor phenotype by TGF-β secreted by HNSCC cells. Citation Format: Hina Shakeel, Fareena Bilwani, Kiran Iqbal, Kulsoom Ghias. NF-κB-mediated pro-inflammatory cytokine release and neutrophil recruitment in head and neck squamous cell carcinoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3152.