Thinking beyond Vaccination: Promising Add-On Strategies to Active Immunization and Vaccination in Pandemics-A Mini-Review.

There is little doubt that final victories over pandemics, such as COVID-19, are attributed to herd immunity, either through post-disease convalescence or active immunization of a high percentage of the world's population with vaccines, which demonstrate protection from infection and transmission and are available in large quantities at reasonable prices. However, it is assumable that humans with immune defects or immune suppression, e.g., as a consequence of allograft transplantation, cannot be immunized actively nor produce sufficient immune responses to prevent SARS-CoV-2 infections. These subjects desperately need other strategies, such as sophisticated protection measures and passive immunization. Hypertonic salt solutions attack vulnerable core areas of viruses; i.e., salt denatures surface proteins and thus prohibits virus penetration of somatic cells. It has to be ensured that somatic proteins are not affected by denaturation regarding this unspecific virus protection. Impregnating filtering facepieces with hypertonic salt solutions is a straightforward way to inactivate viruses and other potential pathogens. As a result of the contact of salt crystals on the filtering facepiece, these pathogens become denatured and inactivated almost quantitatively. Such a strategy could be easily applied to fight against the COVID-19 pandemic and other ones that may occur in the future. Another possible tool to fight the COVID-19 pandemic is passive immunization with antibodies against SARS-CoV-2, preferably from human origin. Such antibodies can be harvested from human patients' sera who have successfully survived their SARS-CoV-2 infection. The disadvantage of a rapid decrease in the immunoglobulin titer after the infection ends can be overcome by immortalizing antibody-producing B cells via fusion with, e.g., mouse myeloma cells. The resulting monoclonal antibodies are then of human origin and available in, at least theoretically, unlimited amounts. Finally, dry blood spots are a valuable tool for surveilling a population's immunity. The add-on strategies were selected as examples for immediate, medium and long-term assistance and therefore did not raise any claim to completeness.

Coating with Hypertonic Saline Improves Virus Protection of Filtering Facepiece Manyfold-Benefit of Salt Impregnation in Times of Pandemic.

Recently, as is evident with the COVID-19 pandemic, virus-containing aerosols can rapidly spread worldwide. As a consequence, filtering facepieces (FFP) are essential tools to protect against airborne viral particles. Incorrect donning and doffing of masks and a lack of hand-hygiene cause contagion by the wearers' own hands. This study aimed to prove that hypertonic saline effectively reduces the infectious viral load on treated masks. Therefore, a hypertonic salt solution´s protective effect on surgical masks was investigated, specifically analyzing the infectivity of aerosolized Alphacoronavirus 1 in pigs (Transmissible Gastroenteritis Virus (TGEV)). Uncoated and hypertonic salt pre-coated FFPs were sprayed with TGEV. After drying, a defined part of the mask was rinsed with the medium, and the eluent was used for the infection of a porcine testicular cell line. Additionally, airborne microorganisms´ long-term infectivity of sodium-chloride in phosphate-buffered saline comprising 5% saccharose was investigated. In the results from an initial Median Tissue Culture Infectious Dose, infection rate of TGEV was minimally reduced by untreated FFP. In contrast, this could be reduced by a factor of 104 if FFPs were treated with hypertonic salt solutions. Airborne pathogens did not contaminate the growth medium if salt concentrations exceeded 5%. We conclude that hypertonic saline is a vital tool for anti-virus protection, exponentially improving the impact of FFPs.

Improved protection of filtering facepiece through inactivation of pathogens by hypertonic salt solutions - A possible COVID-19 prevention device.

The filtering facepiece operates through filtration without the ability to kill the viruses. If the filtration might be combined with antiviral agents simultaneously in the masks, this would be much more efficient during the use of these masks and against cross-infection after being discarded. For centuries, sodium chloride (NaCl) contributes to inhibiting pathogens on various occasions. If aerosol with infectious agents reaches the filtering face-piecé surface of the filtering face-piece, coated with hypertonic saline, they become attracted by hygroscopic salt crystals. Proteins and nucleic acids lose their structural integrity and become inactivated concerning their infectious properties. We provide further evidence for cell growth inhibition with hypertonic saline in yeast cells comprising a defending cell wall. Proliferation was inhibited in a concentration-dependent manner, i.e., above 50 g/L, yeast cell proliferation was completely blocked. At a NaCl concentration of 100 g/L, even decomposition of the original inoculated organisms was observed. Therefore, we conclude that hypertonic saline- coated filtering facepiece might strongly reduce the numbers of infectious particles on their surfaces and thus protect mask carriers efficiently from infections.

[Bioavailability of natural versus synthetic B vitamins and their effects on metabolic processes]. / Bioverfügbarkeit eines natürlichen versus eines synthetischen Vitamin-B- Komplexes und deren Auswirkungen auf metabolische Prozesse.

BACKGROUND: Owing to the widespread use of vitamin supplements to prevent and compensate for deficiencies, the equivalence of natural versus synthetic vitamins with respect to their bioavailability and metabolic influence is discussed controversially. METHOD: Thirty healthy female (n=22) and male participants (n=8) were investigated in a randomized, double-blind, cross-over study over a supplementation period of 6 weeks for each condition. The participants received a daily dose of a complex of the 8 natural B vitamins (group N), determined by the natural composition of quinoa seedlings, resp. synthetic B vitamins (group S), both corresponding to about 2.5 times the Recommended Dietary Allowance (RDA) of the national nutrition board. The primary criterion under investigation was changes in the blood levels of the individual B vitamins. Secondary criteria were the influence of both B complexes on homocysteine, antioxidant status, polyphenols, peroxide loading and peroxidase activity. RESULTS: Compared to baseline values, serum levels of all B vitamins measured increased: Vitamins B1 (N +23%; S +27%), B2 (N +14%; S +13%), B6 (N +101%; S +101%), B9 (N +86%; S +153%) and B12 (N +16%) were elevated at the end of the first supplementation period (p < 0.05), while serum levels of vitamins B1, B9 and B12 remained elevated compared to baseline even after the 2-week washout phase. During the second supplementation period, the vitamin concentrations in group N, with the exception of vitamin B1, could be increased once again (p < 0.05). In contrast, in group S only for vitamins B2 and B12 substantial increases (p < 0.05) were found. The influence of B vitamins on metabolic parameters such as homocysteine and polyphenols, which were markedly reduced, was also clearly measurable; however, total antioxidant capacity and peroxidase activity increased. The peroxide concentration remained almost unchanged in both groups. CONCLUSION: This clinical pilot study showed comparable bioavailability for both natural and synthetic B vitamins, with a 2.5-fold concentration of the RDA. Both vitamin B preparations showed a clear influence on metabolic parameters, whereas that of the natural B vitamins tended to have a slightly stronger effect than the synthetic analogues.

A Micromethod for Polyphenol High-Throughput Screening Saves 90 Percent Reagents and Sample Volume.

There is ample evidence that polyphenols are important natural substances with pronounced antioxidative properties. This study aimed to develop a fast and reliable method to determine total polyphenol content (TPC) in foodstuffs and human samples. The microtitration format offers the advantage of low sample volumes in the microlitre range, facilitating high-throughput screening with 40 samples simultaneously. We accordingly adjusted the so-called Folin-Ciocalteu method to a microtitre format (polyphenols microtitre-PPm) with 90% reduction of reagents. The assay was standardized with gallic acid in the range between 0.1 and 3 mM, using a 20 µL sample volume. The intra-assay coefficient of variation (CV) was less than 5%, and inter-assay CV was in the range of 10%. Wavelength was measured at 766 nm after two hours of incubation. This micromethod correlates significantly with both the classical Folin-Ciocalteu method and High-Performance Thin-Layer Chromatography (HPTLC) (r2 = 0.9829). We further observed a significant correlation between PPm and total antioxidants (r2 = 0.918). The highest polyphenol concentrations were obtained for red, blue, and black fruits, vegetables, and juices. Extracts of red grapes could be harvested almost sugar free and might serve as a basis for polyphenol supplementation. Beer, flour, and bread contained polyphenol concentrations sufficient to meet the minimal daily requirement. We conclude that PPm is a sensitive and reliable method that detects polyphenols even in samples diluted 10-fold. The literature strongly recommends further investigations on the effects of polyphenol uptake on human and animal health.

A Randomized Pilot Trial to Evaluate the Bioavailability of Natural versus Synthetic Vitamin B Complexes in Healthy Humans and Their Effects on Homocysteine, Oxidative Stress, and Antioxidant Levels.

The vitamin B complex comprises 8 different water-soluble constituents that humans must sequester from the diet. This pilot study compared natural versus synthetic vitamin B complexes for their bioavailability, accumulation, and their impact on antioxidants, homocysteine levels, and oxidative stress. We conducted a double-blind randomized clinical trial with thirty healthy participants. They were randomly assigned to group N (natural) and group S (synthetic). Vitamin B was ingested daily for 6 weeks in the range of about 2.5 times above the recommended daily allowance. Blood samples were taken at baseline, 1.5 h, 4 h, 7 h (diurnal), 6 w (discontinuation of supplements), and 8 w (washout). Blood levels of thiamine (B1), riboflavin (B2), pyridoxine (B6), folic acid (B9), cobalamin (B12), homocysteine, total antioxidants, peroxidase activity, polyphenols, and total peroxides were determined. Compared to initial values, serum levels of each B vitamin increased at the end of the supplementation period: i.e., B1 (+23% N; +27% S), B2 (+14% N; +13% S), B6 (+101% N; +101% S), B9 (+86% N; +153% S), and B12 (+16% N) (p < 0.05). Homocysteine (-13% N) decreased, while peroxidase activity (+41% S) and antioxidant capacity increased (+26% N). Short-term effects were already observed after 1.5 h for B9 (+238% N; +246% S) and after 4 h for vitamin B2 (+7% N; +8% S), B6 (+59% N; +51% S), and peroxidase activity (+58% N; +58% S). During the washout period, serum levels of B vitamins decreased except for thiamine and peroxidase activity, which increased further. This clinical pilot study revealed comparable bioavailability for both natural and synthetic B vitamins but did not show statistically noticeable differences between groups despite some favourable tendencies within the natural vitamin group, i.e., sustained effects for cobalamin and endogenous peroxidase activity and a decrease in homocysteine and oxidative stress levels.

Cultivation and Immortalization of Human B-Cells Producing a Human Monoclonal IgM Antibody Binding to MDA-LDL: Further Evidence for Formation of Atherogenic MDA-LDL Adducts in Humans In Vivo.

Oxidatively modified low-density lipoprotein (oLDL) is firmly believed to play an important role in the initiation and development of atherosclerosis, and malonic dialdehyde (MDA) is one of the major lipid peroxidation breakdown products involved in this process. In recent decades, antibodies against MDA-LDL have been detected in human and animal sera. In our study, human B-cells from the peripheral blood of a healthy female donor were fused with the SP2/0 mouse myeloma cell line. Antibody-producing hybridomas were detected by MDA-LDL-IgG/IgM enzyme-linked immunosorbent assays (ELISA) and Cu++-oxidized LDL IgG/IgM (oLAb) ELISA. Cells with supernatants emitting positive signals for antibodies were then cloned and after sufficient multiplication frozen and stored under liquid nitrogen. Due to the loss of antibody-producing ability, we established an MDA-LDL-IgM-producing cell line by recloning. This allowed isolation and immortalization of several human B-cells. The human donor had not been immunized with MDA-modified proteins, thus obviously producing MDA-LDL antibodies in vivo. Furthermore, using these antibodies for in vitro experiments, we were able to demonstrate that MDA epitopes are among the epitopes generated during Cu++-LDL oxidation as well. Finally, these antibodies compete in ELISA and cell culture experiments with MDA as a challenging toxin or ligand.

Oxidative stress increases continuously with BMI and age with unfavourable profiles in males.

Oxidative stress is a risk factor for chronic diseases and was previously shown to be independently associated with obesity. The authors investigated the relationship between body mass index (BMI), age and oxidative stress on 2190 subjects undergoing a health care examination. Total antioxidant status (TAS), total peroxides (TOC) and autoantibodies against oxidized LDL (oLAb) were used as oxidative stress biomarkers in addition to serum lipoproteins, bilirubin and uric acid. Gender-specific differences were observed for age, BMI, serum concentrations of bilirubin, low-density lipoprotein (LDL), uric acid and TAS, all of which were higher in males (p < 0.001), while high-density lipoprotein (HDL), HDL/LDL ratio and TOC were higher in females (p < 0.001). Total cholesterol (p < 0.05) and LDL were increased (p < 0.05), while HDL was decreased (p < 0.05) in overweight and obese subjects. This was accompanied by increased uric acid and TAS concentrations. Lowest oLAb titers were detected in obese subjects. In extremely obese subjects, increased TOC and decreased TAS were observed in spite of high uric acid levels. These results demonstrate that oxidative stress increases with increasing BMI and age, as a sequel to an impaired antioxidant status, the consumption of oLAbs, an increase of peroxides and uric acid and a disadvantaged lipid profile.

Oxidative stress: potential of distinct peroxide determination systems.

When reactive oxygen species attack biological structures, peroxides, which are short-lived oxidative intermediates, are generated. We evaluated the potential of two different, commercially available peroxide activity assays (Pox-Act and d-ROMS) to see whether the results were associated with the clinical condition of subjects who were participating in a routine health care program. Furthermore, we determined the total antioxidant status (TAS) and the titer of autoantibodies against oxidized low-density lipoprotein (oLAb) to verify the hydroperoxide measurements. Subjects with medical conditions (hereafter referred to as patients) had significantly increased serum peroxide levels compared to healthy subjects. The d-ROMS kit indicated that 86% of subjects had an increased level of total peroxides. Although the assays had a significant correlation (p<0.001), 34% of the subjects had an increased total peroxide concentration in the Pox-Act assay that was clearly associated with clinical symptoms. Furthermore, the sensitivity of the Pox-Act assay was 35 times higher than that of the d-ROMS kit. In subjects with medical conditions, there was a trend toward a decreased TAS and a slightly increased oLAb titer in comparison to healthy subjects, but this was not statistically significant. The Pox-Act assay seems to be a valuable tool for the determination of total peroxides, while the results from the d-ROMS kit should be considered with caution.