Application and Validation of SARS-CoV-2 RBD Neutralizing ELISA Assay.

The establishment of an approach for detecting the anti-severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-receptor-binding domain (RBD) neutralizing antibodies (nAbs) by a safe, easy, and rapid technique without requiring the use of live viruses is essential for facing the coronavirus disease 2019 (COVID-19) pandemic. Depending on competitive enzyme-linked immunosorbent assay (ELISA) methodology, the current study assay was designed to simulate the virus-host interaction using purified SARS-COV-2-RBD from the spike protein and the host cell receptor human angiotensin-converting enzyme 2 protein. The performance of this in-house neutralizing ELISA assay was validated using freshly prepared standards with different known concentrations of the assay. In this regard, a cohort of 50 serum samples from convalescent COVID-19 individuals with different disease severity at different time points post-recovery and a cohort of 50 serum samples from healthy individuals were processed by the in-house developed assay for detecting SARS-CoV-2 nAbs, in comparison with a commercial total anti-SARS-CoV-2 IgG antibody assay as a gold standard. The assay obtained a sensitivity of 88% (95% CI: 75.69-95.47) and a specificity of 92% (95% CI: 80.77- 97.78%). A negative strong correlation was demonstrated in the standard curve between the optical density absorbance and log concentration of the nAbs with a statistical measure of r2 (coefficient of determination) = 0.9539. The SARS-COV-2-RBD neutralizing ELISA assay serves as a high throughput qualitative and quantitative tool that can be applied in most laboratory settings without special biosafety requirements to detect anti-RBD nAbs for seroprevalence, pre-clinical, and clinical evaluation of COVID-19 vaccines efficiency and the rapid selection of convalescent plasma donors for the treatment of COVID-19 patients.

Dynamics of PEGylated-dextran-spermine nanoparticles for gene delivery to leukemic cells.

Leukemic cells are hard-to-transfect cell lines. Many transfection reagents which can provide high gene transfer efficiency in common adherent cell lines are not effective to transfect established blood cell lines or primary leukemic cells. This study aims to examine a new class of cationic polymer non-viral vector, PEGylated-dextran-spermine (PEG-D-SPM), to determine its ability to transfect the leukemic cells. Here, the optimal conditions of the complex preparation (PEG-D-SPM/plasmid DNA (pDNA)) were examined. Different weight-mixing (w/w) ratios of PEG-D-SPM/pDNA complex were prepared to obtain an ideal mixing ratio to protect encapsulated pDNA from DNase degradation and to determine the optimal transfection efficiency of the complex. Strong complexation between polymer and pDNA in agarose gel electrophoresis and protection of pDNA from DNase were detected at ratios from 25 to 15. Highest gene expression was detected at w/w ratio of 18 in HL60 and K562 cells. However, gene expression from both leukemic cell lines was lower than the control MCF-7 cells. The cytotoxicity of PEG-D-SPM/pDNA complex at the most optimal mixing ratios was tested in HL60 and K562 cells using MTS assay and the results showed that the PEG-D-SPM/pDNA complex had no cytotoxic effect on these cell lines. Spherical shape and nano-nature of PEG-D-SPM/pDNA complex at ratio 18 was observed using transmission electron microscopy. As PEG-D-SPM showed modest transfection efficiency in the leukemic cell lines, we conclude that further work is needed to improve the delivery efficiency of the PEG-D-SPM.

Novel phage-based bio-processing of pathogenic Escherichia coli and its biofilms.

To explore new approaches of phage-based bio-process of specifically pathogenic Escherichia coli bacteria in food products within a short period. One hundred and forty highly lytic designed coliphages were used. Escherichia coli naturally contaminated and Enterohemorrhagic Escherichia coli experimentally inoculated samples of lettuce, cabbage, meat, and egg were used. In addition, experimentally produced biofilms of E. coli were tested. A phage concentration of 10(3) PFU/ml was used for food products immersion, and for spraying of food products, 10(5) PFU/ml of a phage cocktail was used by applying a 20-s optimal dipping time in a phage cocktail. Food samples were cut into pieces and were either sprayed with or held in a bag immersed in lambda buffer containing a cocktail of 140 phages. Phage bio-processing was successful in eliminating completely E. coli in all processed samples after 48 h storage at 4°C. Partial elimination of E. coli was observed in earlier storage periods (7 and 18 h) at 24° and 37°C. Moreover, E. coli biofilms were reduced >3 log cycles upon using the current phage bio-processing. The use of a phage cocktail of 140 highly lytic designed phages proved highly effective in suppressing E. coli contaminating food products. Proper decontamination/prevention methods of pathogenic E. coli achieved in this study can replace the current chemically less effective decontamination methods.

The impact of the level of the intestinal short chain Fatty acids in inflammatory bowel disease patients versus healthy subjects.

The aim of this study was to determine the changes of short chain fatty acids (SCFAs) in faeces of inflammatory bowel disease (IBD) patients compared to healthy subjects. SCFAs such as pyruvic, lactic, formic, acetic, propionic, isobutyric and butyric acids were analyzed by using high performance liquid chromatography (HPLC). This study showed that the level of acetic, 162.0 micromol/g wet faeces, butyric, 86.9 micromol/g wet faeces, and propionic acids, 65.6 micromol/g wet faeces, decreased remarkably in IBD faecal samples when compared with that of healthy individuals, 209.7, 176.0, and 93.3 micromol/g wet faeces respectively. On the contrary, lactic and pyruvic acids showed higher levels in faecal samples of IBD than in healthy subjects. In the context of butyric acid level, this study also found that the molar ratio of butyric acid was higher than propionic acid in both faecal samples. This might be due to the high intake of starch from rice among Malaysian population. It was concluded that the level of SCFAs differ remarkably between faecal samples in healthy subjects and that in IBD patients providing evidence that SCFAs more likely play an important role in the pathogenesis of IBD.

Effect of organic acids on Escherichia coli O157:H7 and Staphylococcus aureus contaminated meat.

Appropriate and safe antibacterial agents able to decontaminate meat surfaces have long been big concern of meat industry. In an attempt to manage beef carcass contamination, spray wash treatments utilizing three concentrations (1, 1.5 and 2%) of acetic, lactic, propionic and formic acids were performed to evaluate their efficacy in reducing numbers of Escherichia coli O157:H7 and Staphylococcus aureus on meat tissues. The procured beef pieces of freshly slaughtered animals were decontaminated with hot water and then inoculated with E. coli O157:H7 and S. aureus individually which then were spray washed with organic acids separately. The total plate count of the treated samples showed that the populations of bacteria decreased after being exposed to organic acids. Spray wash of formic acid resulted in the highest reduction of both bacterial species on meat surface. Significantly, higher log reductions were obtained for S. aureus than E. coli O157:H7. It was concluded that organic acids are highly effective in decontaminating meat surfaces and organic acids are shown to be safe, simple, efficient, and cheap modality of meat decontamination which can be highly recommended for industrial scales.

Severity of asthma: the role of CD25+, CD30+, NF-kappaB, and apoptotic markers.

OBJECTIVES: We studied the role of the regulatory T cells CD4+CD25+ (Treg) and activated CD4+CD30+ cells in the pathogenesis of asthma and their association with apoptosis and NF-kappaB in patients with mild intermittent asthma (MA), severe persistent asthma (SA), and healthy volunteers (HV). METHODS: Peripheral blood lymphocytes (PBL) were extracted from asthmatic patients during exacerbations, and CD4+ cells were separated using Dynal beads. Immunostaining of whole PBL for NF-kappaB, Bax, and Bcl-2, and immunostaining of CD4+ cells for CD25+ and CD30+ cells were performed using immunocytochemistry. RESULTS: Treg cells were expressed at higher levels in MA than in HV and SA (P < .05), while CD30+ T cells were expressed at higher levels in both SA and MA than in HV (P < .05), although there was no remarkable difference between SA and MA (P>.05). Levels of NF-kappaB, Bcl-2, and Bcl-2/Bax increased, whereas those of Bax decreased, progressively, from MA to SA (P < .05). NF-kappaB levels correlated directly with the Bcl-2/Bax ratio and with CD4+CD30+ cells in SA and MA, whereas CD4+CD30+ cells correlated inversely with the Bcl-2/Bax ratio. CONCLUSIONS: Unregulated Treg cells probably return inflammatory responses to normal values during exacerbations in MA; however, expression of Treg cells was extensively diminished in SA, leading to probable loss of suppressive control over underlying immune reactions. CD4+CD30+ cells were associated with the pathogenesis of asthma but not with severity. NF-kappaB seems to be the central inflammatory factor in SA, with a remarkable loss of PBL apoptosis, diminished Treg levels, and high CD30+ cell levels that probably induce NF-kappaB, which in turn blocks the proapoptotic potential of CD30 induction itself.

Different inflammatory mechanisms in lungs of severe and mild asthma: crosstalk of NF-kappa-B, TGFbeta1, Bax, Bcl-2, IL-4 and IgE.

OBJECTIVE: To examine differences in the apoptotic, inflammatory, allergic and immunological features in the lungs of adults with asthma. MATERIAL AND METHODS: Thirty-six patients with mild asthma (MA), 16 with severe asthma (SA) and 20 healthy volunteers (HVs) were enrolled. Bronchoalveolar lavage fluid (BALF) was processed into cell-free fluid for enzyme-linked immunosorbent assay detecting soluble TGFbeta1, IL-4 and IgE and BALF lymphocytes for immunocytochemical staining of cellular Bax, Bcl-2 and nuclear factor-Kappa-B (NFkappaB). RESULTS: Cellular NFkappaB expression was higher in SA than in MA and HVs, while extracellular TGFbeta1 was high in both the SA and MA groups but low in the HVs. Bcl-2/Bax ratio was higher in SA than in MA and in MA than in HV groups and correlated significantly with NFkappaB level. Interestingly, the levels of IgE and, to a lesser extent, IL-4 were higher in MA than in SA and both were much higher than in HVs, and were inversely correlated with NFkappaB level in the SA group and with TGFbeta1 level in the MA group. CONCLUSIONS: NFkappaB has a central role in the perpetuation of persistent inflammation in SA and might induce apoptosis via Bcl-2. The SA group appears not associated much with allergen-based IgE and IL-4 reactions as efficiently as in MA. This was supported by the lower levels of IgE and IL-4 in SA compared to MA. TGFbeta1 appears to be associated with asthma pathogenesis, especially allergen-based MA.

The distinctive profile of risk factors of nasopharyngeal carcinoma in comparison with other head and neck cancer types.

BACKGROUND: Nasopharyngeal carcinoma (NPC) and other head and neck cancer (HNCA) types show a great epidemiological variation in different regions of the world. NPC has multifactorial etiology and many interacting risk factors are involved in NPC development mainly Epstein Barr virus (EBV). There is a need to scrutinize the complicated network of risk factors affecting NPC and how far they are different from that of other HNCA types. METHODS: 122 HNCA patients and 100 control subjects were studied in the region of the Middle East. Three types of HNCA were involved in our study, NPC, carcinoma of larynx (CL), and hypopharyngeal carcinoma (HPC). The risk factors studied were the level of EBV serum IgG and IgA antibodies measured by ELISA, age, sex, smoking, alcohol intake, histology, and family history of the disease. RESULTS: EBV serum level of IgG and IgA antibodies was higher in NPC than CL, HPC, and control groups (p < 0.01). NPC was associated with lymphoepithelioma (LE) tumors, males, regular alcohol intake, and regular smoking while CL and HPC were not (p < 0.05). CL and HPC were associated with SCC tumors (p < 0.05). Furthermore, NPC, unlike CL and HPC groups, was not affected by the positive family history of HNCA (p > 0.05). The serum levels of EBV IgG and IgA antibodies were higher in LE tumors, regular smokers, younger patients, and negative family history groups of NPC patients than SCC tumors, non-regular smokers, older patients and positive family history groups respectively (p < 0.05) while this was not found in the regular alcoholics (p > 0.05). CONCLUSION: It was concluded that risk factors of NPC deviate much from that of other HNCA. EBV, smoking, alcohol intake, LE tumors, male patient, and age > 54 years were hot risk factors of NPC while SCC and positive family history of the disease were not. Earlier incidence, smoking, LE tumors, and negative family history of the disease in NPC patients were associated much clearly with EBV. It is proposed that determining the correct risk factors of NPC is vital in assigning the correct risk groups of NPC which helps the early detection and screening of NPC.

Changing survival, memory cell compartment, and T-helper balance of lymphocytes between severe and mild asthma.

BACKGROUND: Asthma is a complicated network of inflammatory reactions. It is classified into mild, moderate, and severe persistent asthma. The success of asthma therapy relies much on understanding the underlying mechanisms of inflammation at each stage of asthma severity. The aim of this study was to explore the differences in apoptotic potential, CD4/CD8 ratio, memory compartment, and T- helper (Th) 1 and 2 profile of peripheral blood lymphocytes (PBL) in patients with mild intermittent asthma and severe persistent asthma during exacerbation periods. RESULTS: Four research lines were investigated and compared among mild asthmatics, severe asthmatics, and healthy groups by applying immunocytochemical staining of PBL. Antiapoptotic and proapoptotic proteins with Bcl-2/Bax ratio, CD4, CD8 markers with CD4+/CD8+ ratio, CD45RO+, CD45RA+ markers with memory/naive ratio (CD45RO+/CD45RA+). Th2/Th1 cytokines balance represented by IL-4/IFN-gamma ratio was measured by enzyme-linked immunosorbent assay (ELISA) for in vitro PBL cytokine synthesis. It was found that Bcl-2/Bax ratio was higher in severe than in mild asthmatics which in turn was higher than in healthy group. And memory/naive ratio of PBL was higher in severe than in mild asthmatics. Moreover, memory cells, CD45RO+ and CD45RO+/CD45RA+ ratio were correlated directly with Bcl-2/Bax, in severe and mild asthma patients. In contrast, CD4+/CD8+ ratio was not changed significantly among healthy group, mild and severe asthmatics. However, CD8+ cells were correlated directly with memory cells, CD45RO+, in severe asthmatics only. Interestingly, the dominant profile of cytokines appeared to change from T helper 2 (Th2) in mild asthmatics to T helper 1 (Th1) in severe asthmatics where the lowest in vitro IL-4/IFN-gamma ratio and highest IFN-gamma were found. CONCLUSION: It was concluded that the underlying mechanisms of inflammation might vary greatly with asthma stage of severity. Mild intermittent asthma is mainly Th2 allergen-oriented reaction during exacerbations with good level of apoptosis making the inflammation as self-limiting, while in severe persistent asthma, the inflammatory reaction mediated mainly by Th1 cytokines with progressive loss of apoptosis leading to longer exacerbations, largely expanded memory cells, CD45RO+, leading to persistent baseline inflammation.