Production of Disinfective Coating Layer to Facial Masks Supplemented with Camellia sinensis Extract.

Although usefulness of masks for protection against respiratory pathogens, accumulation of pathogens on their surface represents a source of infection spread. Here we prepared a plant extract-based disinfecting layer to be used in coating masks thus inhibiting their capacity to transmit airborne pathogens. To reach this, a polypropylene membrane base was coated with a layer of polyvinyledine difluoride polymer containing 500 µg/ml of Camellia sinensis (Black tea) methanolic extract. Direct inhibitory effects of C. sinensis were initially demonstrated against Staphylococcus aureus (respiratory bacteria), influenza A virus (enveloped virus) and adenovirus 1 (non-enveloped virus) which were directly proportional to both extract concentration and incubation time with the pathogen. This was later confirmed by the capacity of the supplemented membrane with the plant extract to block infectivity of the above mentioned pathogens, recorded % inhibition values were 61, 72 and 50 for S. aureus, influenza and adenovirus, respectively. In addition to the disinfecting capacity of the membrane its hydrophobic nature and pore size (154 nm) prevented penetration of dust particles or water droplets carrying respiratory pathogens. In summary, introducing this layer could protect users from infection and decrease infection risk upon handling contaminated masks surfaces.

Sensitivities of a Rapid Test Versus an ELISA Kit for Detecting Anti-SARS-CoV-2 IgM/IgG in Sera from an Egyptian Cohort.

This study aimed to compare diagnostic sensitivities of a rapid test (Rt) and an ELISA kit for detecting anti-SARS-CoV-2 IgM/IgG in virus-RT-PCR-positive (VPP) and virus-RT-PCR-unchecked (VPU) subjects in an Egyptian cohort during the first wave of SARS-CoV-2 infection. The results revealed higher sensitivity of the Rt for detecting IgM/IgG in the VPP subjects. Both the Rt and ELISA showed identical sensitivities for IgM detection in the VPU subjects. The ELISA was more sensitive for detecting IgG in the VPU subjects. Generally, within both the VPP and the VPU groups, Rt was more sensitive for detecting IgM/IgG among the symptomatic (S) compared to asymptomatic (AS) subjects than ELISA. Within the VPP group, the Rt was more sensitive for detecting both IgM/IgG among the AS subjects than ELISA. In the VPU group, the Rt was more sensitive for detecting IgM among the S subjects than ELISA. The ELISA was more sensitive for detecting IgM/IgG among AS subjects than the Rt. From these results we concluded that, despite the limitation of sample size, this study indicates suitability of the used Rt for detecting anti-SARS-CoV-2 IgM/IgG among S subjects and sheds light on possibility of relying on the used ELISA for IgG detection among AS human subjects.

Individual expression and processing of hepatitis C virus E1/E2 epitopes-based DNA vaccine candidate in healthy humans' peripheral blood mononuclear cells.

Purpose: The development and study of hepatitis C virus (HCV) vaccine candidates' individualized responses are of great importance. Here we report on an HCV DNA vaccine candidate based on selected envelope (E1/E2) epitopes. Besides, we assessed its expression and processing in human peripheral blood mononuclear cells (PBMCs) and in vivo cellular response in mice. Materials and Methods: HCV E1/E2 DNA construct (EC) was designed. The antigen expression of EC was assayed in PBMCs of five HCV-uninfected donors via a real-time quantitative polymerase chain reaction. Serum samples from 20 HCV antibody-positive patients were used to detect each individual PBMCs expressed antigens via enzyme-linked immunosorbent assay. Two groups, five Swiss albino mice each, were immunized with the EC or a control construct. The absolute count of lymph nodes' CD4+ and CD8+ T-lymphocytes was assessed. Results: Donors' PBMCs showed different levels of EC expression, ranging between 0.83-2.61-fold in four donors, while donor-3 showed 34.53-fold expression. The antigens expressed in PBMCs were significantly reactive to the 20 HCV antibody repertoire (all p=0.0001). All showed comparable reactivity except for donor-3 showing the lowest reactivity level. The absolute count % of the CD4+ T-cell significantly increased in four of the five EC-immunized mice compared to the control group (p=0.03). No significant difference in CD8+ T-cells % was observed (p=0.89). Conclusion: The inter-individual variation in antigen expression and processing dominance was evident, showing independence in individuals' antigen expression and reactivity levels to antibodies. The described vaccine candidate might result in a promising natural immune response with a possibility of CD4+ T-cell early priming.

Prevalence of enteric viruses in wastewater in Egypt after the COVID-19 pandemic.

People's hygienic habits greatly affect the spreading rate of enteric viruses. After the COVID-19 pandemic, many people followed announced precautions and improved their hygienic status to protect themselves from SARS-CoV-2 infection. Here, we studied if this indirectly affected the prevalence of enteric viruses in Egypt. A total of 21 samples (one sample per week) were collected from the Zenin wastewater treatment plant (WWTP) through the period between August 2021 and March 2022. Detection of adenovirus, hepatitis A virus (HAV), and rotavirus showed their presence in 66, 14.3, and 9.5% of the collected samples, respectively. Comparing those percentages to previously published data concerned with the detection of the same viruses from the same WWTP or others revealed a remarkable decrease in the prevalence of the three viruses after the COVID-19 pandemic. This allows the conclusion that safety precautions against SARS-CoV-2 lead indirectly to a reduction of adenovirus, HAV, and rotavirus prevalence rates.

Carbohydrates Metabolic Signatures in Immune Cells: Response to Infection.

Introduction: Metabolic reprogramming in immune cells is diverse and distinctive in terms of complexity and flexibility in response to heterogeneous pathogenic stimuli. We studied the carbohydrate metabolic changes in immune cells in different types of infectious diseases. This could help build reasonable strategies when understanding the diagnostics, prognostics, and biological relevance of immune cells under alternative metabolic burdens. Methods: Search and analysis were conducted on published peer-reviewed papers on immune cell metabolism of a single pathogen infection from the four known types (bacteria, fungi, parasites, and viruses). Out of the 131 selected papers based on the PIC algorithm (pathogen type/immune cell/carbohydrate metabolism), 30 explored immune cell metabolic changes in well-studied bacterial infections, 17 were on fungal infections of known medical importance, and 12 and 57 were on parasitic and viral infections, respectively. Results and Discussion: While carbohydrate metabolism in immune cells is signaled by glycolytic shift during a bacterial or viral infection, it is widely evident that effector surface proteins are expressed on the surface of parasites and fungi to modulate metabolism in these cells. Conclusions: Carbohydrate metabolism in immune cells can be categorized according to the pathogen or the disease type. Accordingly, this classification can be used to adopt new strategies in disease diagnosis and treatment.

Addition of Camellia sinensis extract to water to disinfect respiratory viruses accumulated over different surfaces.

New precautions have become part of our daily life since COVID-19 pandemic such as wearing masks, maintaining distance and disinfecting products bought from markets before using them which is exhausting. We aimed to test the inhibitory effect of Camellia sinensis (black tea) water extracts on respiratory viruses and the inhibition of viruses accumulated over different surface types after being soaked in water supplemented with the extracts. Two water extraction methods (extract A: maceration at 80 °C for 30 min and extract B: boiling for 40 min) were applied; extracts were analyzed by high-performance liquid chromatography to detect polyphenolic compounds. Results showed that 200 µg/ml of extract A and 50 µg/ml of extract B in water caused 100% inhibition of influenza A (enveloped virus) virus after 1.5 h and similar results were obtained for adenovirus (non-enveloped virus) but at the same concentration of extract A and at 100 µg/ml of extract B. Different surfaces (aluminum, glass, plastic or carton, vegetables of smooth (tomato) or rough (lemon) surfaces and green leaves) were inoculated with both viruses for 20 min and then soaked in the water supplemented with 200 µg/ml of extract A or 100 µg/ml of extract B for 1.5 h, and this resulted in complete inhibition of both viruses.

TLR3\TLR7 as Differentially Expressed Markers Among Viral, Nonviral, and Autoimmune Diseases in Egyptian Patients.

Toll-like receptors (TLRs) represent the immune link between the innate and the adaptive immune signals against various pathogens. This study aimed to evaluate the TLRs3 and 7 as immune-markers in differentiating between hepatitis C virus (HCV)-infected and -uninfected patients. Also, the use of the TLR3 and TLR7 as immune markers was compared with the prevalent bio and immune markers for autoimmune diseases in HCV-infected or -uninfected patients. The levels of GPT, GOT, B cell activated factors, tumor necrosis factor-alpha (TNF-α), and interleukin (IL)-10 were measured in plasma, while the levels of TLR3 and TLR7 were quantified in lysates of peripheral blood mononuclear cells from healthy donors, HCV-infected patients, nonalcoholic fatty liver (NAFL) patients without autoimmune diseases and with autoimmune diseases (HCV-infected patients with autoimmune diseases [HCV+auto], nonalcoholic fatty liver patients with autoimmune diseases [NAFL+auto]), rheumatoid arthritis (RA), and systemic lupus erythematosus (SLE) patients. The relative expression of TLR3, TLR7, TNF, and IL-10 in cell lysates was assessed against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) by quantitative real time-polymerase chain reaction (qRT-PCR). Results showed that TLRs 3 and 7 levels were significantly higher in SLE, RA, HCV, HCV+auto, and the NAFL patients compared to the normal control. The cell lysates from SLE patients expressed TLR3 at relatively significantly higher mRNA levels compared to normal subjects or other patient groups. The NAFL+auto patients expressed TLR7 at relatively significantly high mRNA levels compared to normal subjects or other patients. The RA patients expressed TLR7 at relatively significantly higher mRNA levels when compared to HCV, HCV+auto, and NAFL+auto patients. Conclusions: At the protein level, TLR7 can differentiate between HCV and NAFL patients. In addition, both TLRs3 and 7 can serve as potent markers in differentiating between NAFL and NAFL+auto.

Glycolytic and immunological alterations in human U937 monocytes in response to H1N1 infection.

We monitored changes that took place in glycolytic enzymes, the pyruvate end product of glycolysis, tumor necrosis factor α (TNFα), and toll-like receptors (TLRs) both at the transcriptional and translational levels upon direct interaction between PR8-H1N1 and the human monocytes U937 in vitro system. U937 were first treated with H1N1 infectious viral particles or phorbol-12-myristate-13-acetate (PMA) or left untreated and later infected with the H1N1 virus. Levels of phosphofructokinase 1 (PFK1) and pyruvate were biochemically quantified. In addition, levels of TNFα, TLR3, and TLR7 were measured by ELISA. The transcriptional profiles of PFKs, inflammatory cytokines, TLR3 and TLR7 were relatively quantified by qRT-PCR. The results generally revealed significant changes in both the transcriptional and translational profiles of the studied biochemical and immunological parameters upon influenza infection in a time-dependent manner. In conclusion, H1N1 infection triggers transcriptional and translational changes in immortalized human monocytes, which might serve as markers for infection subject for further validation for their specificities.

H1N1 Infection Reduces Glucose Level in Human U937 Monocytes Culture.

Infection with influenza A (H1N1) virus contributes significantly to the global burden of acute respiratory diseases. Glucose uptake and metabolic changes are reported in different cell types after infections with different virus types, including influenza A virus. Alteration of glucose metabolism specifically in immune cells has major health consequences. The aim of this study was to monitor glucose concentration in unstimulated and stimulated U937 human monocytes with infectious or heat inactivated H1N1 or Staphylococcus aureus or in nonpathogenically stimulated monocytes with phorbol-12-myristate-13-acetate. Stimulated or unstimulated U937 human monocytes were subjected to H1N1 infection for different time points and the glucose profile in the growth medium was measured post infection. Results showed that regardless to whether the initial stimuli on U937 cells were of pathogen or nonpathogen origins, challenge infection by H1N1 causes a significant reduction of glucose levels 36 h post infection. In conclusion, H1N1 infection has a direct effect on the glucose uptake of U937 cells in vitro. This effect can be related to either H1N1 infection or cell differentiation status that might occur due to the exerted stimuli.

Immune responses to killed reassorted influenza virus supplemented with natural adjuvants.

In this study, we investigated the immunomodulatory effects of a supplemented killed influenza virus (V) by Echinacea purpurea (E) and Nigella sativa (N) extracts and effect of changing the route of immunization from intramuscular (IM) to intraperitoneal (IP). At the 2nd-, 3rd- and 4th-week post-IM immunizations (WPIMI), the supplemented V with N (VN) induced the most significant IgM response unlike N alone. At the 2nd WPIMI, V or VN induced the highest significant IgG levels. At the 2nd-week post-IP immunization (WPIPI), E and VN induced the most significant IgG levels. Both at the 3rd and 4th WPIMI or WPIPI, various treatments induced significant increases in IgG. At the 4th WPIMI, E, V, and V with E (VE) induced significant increases in the CD4+ thymocytes while all IP treatments caused significant increase in their counts. V and VN induced the most significant IM induction of CD8+ thymocytes while their best IP stimulation was induced by N, VE, and VN. At the 4th WPIMI, various treatments caused significant increases in the mesenteric lymph node (MLN) CD4+, CD8+ counts. WPIPI with V or VE caused significant increases in both the CD4+- and CD8+ MLN cells, whereas VN significantly induced CD8+ MLN cells only. WPIPI with various treatments caused significant increases in the B-cell counts and the peak was obtained by VN.

Detection and characterization of hepatitis A virus circulating in Egypt.

Hepatitis A virus (HAV) still poses a considerable problem worldwide. In the current study, hepatitis A virus was recovered from wastewater samples collected from three wastewater treatment plants over one year. Using RT-PCR, HAV was detected in 43 out of 68 samples (63.2%) representing both inlet and outlet. Eleven positive samples were subjected to sequencing targeting the VP1-2A junction region. Phylogenetic analysis revealed that all samples belonged to subgenotype IB with few substitutions at the amino acid level. The complete sequence of one isolate (HAV/Egy/BI-11/2015) showed that the similarity at the amino acid level was not reflected at the nucleotide level. However, the deduced amino acid sequence derived from the complete nucleotide sequence showed distinct substitutions in the 2B, 2C, and 3A regions. Recombination analysis revealed a recombination event between X75215 (subgenotype IA) and AF268396 (subgenotype IB) involving a portion of the 2B nonstructural protein coding region (nucleotides 3757-3868) assuming the herein characterized sequence an actual recombinant. Despite the role of recombination in picornaviruses evolution, its involvement in HAV evolution has rarely been reported, and this may be due to the limited available complete HAV sequences. To our knowledge, this represents the first characterized complete sequence of an Egyptian isolate and the described recombination event provides an important update on the circulating HAV strains in Egypt.

Schistosoma mansoni soluble egg antigens enhance HCV replication in mammalian cells.

BACKGROUND: This work demonstrates successful propagation of HCV in HepG2 and human blood cells as well as viral shedding into their culture media. The influence of Schistosoma mansoni crude soluble egg antigens (SEA) on the rate of viral propagation in both mammalian cells was also monitored. METHODOLOGY: HepG2 cells were inoculated with HCV viremic human sera and some wells were exposed to HCV infection in presence of SEA. Cells were harvested for RT-PCR and Western blotting analysis. HepG2 media was collected for HCV ELISA. Blood samples from HCV-infected humans were cultured in the presence and absence of SEA. Media were collected at different time points post culturing and subjected to HCV ELISA. RESULTS: The ELISA concentration of HCV antigens were generally higher in media of infected HepG2 cells compared to media of control cells at all time intervals post infection. Western blots showed reactivity to immunogenic peptides of different molecular weights in lysate of infected HepG2 cells that were not evidenced in uninfected cells. In presence of SEA, RT-PCR results revealed earlier detection of viral RNA in infected HepG2 cells compared to in absence of such bilharzial antigen. Also, ELISA results revealed higher levels of detected HCV antigens in media of both infected HepG2 and blood cells cocultured with S. mansoni SEA compared to that of cultured infected cells in absence of the parasite antigens. CONCLUSION: HepG2 cells as well as whole blood cultures maintain HCV replication. Furthermore, SEA has the potential to enhance HCV propagation.

Escherichia coli shares T- and B-lymphocyte epitopes with Schistosoma mansoni.

BACKGROUND: In this study, we tested the cross-reaction between crude Escherichia coli antigen (ECA) and 3 crude Schistosoma mansoni antigens. METHODOLOGY: The schistosomal antigens used were cercarial antigen preparation (CAP), soluble worm antigen preparation (SWAP), and soluble egg antigen (SEA). Four groups each of 3 mice received 2 intraperotineal immunizations with the above-mentioned antigens at a two-week interval. The dose of the ECA was 20 microg/100 microl PBS/mouse and that of any of the used schistosomal antigens was 50 microg/100 microl PBS/mouse. IgM and IgG reactivities and cross-reactivities were tested in individual immunized mice sera (IMS) against the above-mentioned antigens by ELISA and Western blotting. The changes in the B, CD4+ and CD8+ -T cells' counts post immunization were recorded. RESULTS: Priming with ECA caused significant increases in IgM (P<0.05) against CAP and SWAP, while both priming and boosting with ECA caused a significant elevation in the IgG only against SWAP. Priming and boosting with ECA or schistosomal antigens caused significant increases in IgM against ECA. Priming with ECA or SWAP caused significant elevation in IgG against ECA. In Western blotting, ECA-IMS recognized 16, 33, 38 and 94 kDa ECA peptides that cross-reacted with CAP-IMS. ECA peptides at 30 and 38 kDa cross-reacted among ECA, SWAP and SEA-IMS. CAP peptides at 40, 71, 85 and 97 kDa cross-reacted with ECA-IMS. A 59 kDa SWAP peptide cross-reacted with ECA. SEA peptides at approximately 55, 96 and 101 kDa cross-reacted with ECA-IMS. Immunization with ECA, CAP, SWAP or SEA caused significant increases in mesentric lymph nodes (MLN)-CD4+, CD8+ -T cells and MLN-B cells. For thymocytes, CD4+ -T cells significantly increased upon immunization with ECA and SWAP while CD8+-T cells significantly increased upon immunization with SWAP. CONCLUSION: It is necessary to include E. coli antigens as controls while establishing schistosomal antigens-based diagnostic tests to ensure the specificity of the detected immune responses. Characterization of the cross-reactive ECA antigens with protective potential against S. mansoni infection remains a future research objective.

Characterization of NS3 protease from an Egyptian HCV genotype 4a isolate.

The role of the NS3 protease in HCV replication was demonstrated by the ability of a protease inhibitor cocktail (10 microg/ml) to abolish the induced cytopathic effect in RAW macrophages upon infection with Egyptian sera. The HCV protease gene was amplified from Egyptian sera by nested PCR and cloned downstream of the CMV promotor in a mammalian expression plasmid, which was then used to transform bacteria. Colonies carrying the gene in the correct orientation were subjected to large-scale plasmid purification followed by sequencing. Phylogenetic comparison of the sequence obtained with published sequences from different genotypes confirmed that our sequence belongs to genotype 4a. Of the other genotypes, the most closely related ones were from genotype 1. Multiple alignments of protease peptides showed that the catalytic triads and binding residues for substrate, Zn2+ and the NS4 cofactor are conserved among different isolates, including ours, and confirmed the closer homology between NS3 of genotypes 4 and 1. The HCV-protease-encoding construct was successfully transcribed in both mammalian cells and mice. Mouse antibodies produced against the protease-encoding-construct detected the 18-kDa enzyme in lysates of cells transfected with the construct by Western blotting, and in the media of infected cells by ELISA.

Synthesis of new 4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine derivatives with an incorporated thiazolidinone moiety and testing their possible serine protease and cercarial elastase inhibitory effects with a possible prospective to block penetration of Schistosoma mansoni cercariae into the mice skin.

5-Substituted 4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine were synthesized by interaction of 4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-sulfonylhydrazide with some aldehydes to give the corresponding Schiff-bases, which after cyclization gave corresponding thiazolidinones. For some of the thiazolidinones, Mannich bases reaction was carried out. All the derivatives were tested for their possible inhibitory effect on Schistosoma mansoni cercarial elastase (CE). Only, N-(4-methylbenzyledine)-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-sulfonylhydrazide was found to have potent inhibitory effect on the CE activity with IC50 = 264 microM. Upon its use as a paint for mice tails before infection with S. mansoni cercariae, the compound formulated in jojoba oil caused a significant reduction (93%; P-value = 0.0002) in the worm burden. IgG & IgM in mice sera were measured by using several S. mansoni antigens by ELISA. Sera from treated infected mice (TIM) 2, 4, and 6 weeks (W) post infection (PI) showed 1.2 folds lower, 1.2 folds higher, 1.7 folds lower IgM reactivity against soluble cercarial antigenic preparation (CAP), respectively, when compared with sera collected from infected untreated mice (IUM). Sera from TIM 2, 4, and 6WPI showed 1.3, 1.6, and 1.7 folds higher IgG reactivity, respectively against CAP than the IgG reactivity from IUM. Sera from TIM 2, 4 and 6WPI showed 1.5, 1.2 folds lower and 1.4 folds higher IgM reactivity, respectively against soluble worm antigenic preparation (SWAP) when compared with sera collected from IUM. Sera from TIM 2, 4, and 6WPI showed 1.4, 1 folds lower and 1 fold higher IgG reactivity, respectivley to SWAP when compared with sera from IUM. Sera from TIM 2, 4, and 6WPI had generaly lower IgM and IgG reactivities against soluble egg antigen (SEA) when compared with sera from IUM.