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Alleviation of Histopathologic Effects of Avian Influenza Virus by a Specific Nutrient Synergy
Abstract and Figures
This study aimed at alleviating the histopathologic effects of avian influenza virus by a specific nutrient synergy present in Epican Forte (R) (EF). The daily administration of EF at the level of 48.8 mg/mL/bird between 7-14 days of age, to birds challenged at 7-days old with H9N2-avian influenza virus, had different impacts on tracheal deciliation, goblet cell degeneration, mucus accumulation, hypertrophy of mucosal layer, and counts of tracheal heterophils and specific heterophils containing inclusion bodies. Other different impacts by EF were observed in histopathology observations of thoracic air sacs and lungs. The 2 most apparent significant reductions in tracheal histopathologic effects (P < 0.05) due to EF administration were the mucus accumulation and the drop in specific heterophils manifesting a presence of inclusion bodies.
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... 1,2 The Nutrient Synergy developed by Dr. Rath Institute in Santa Clara, USA (Epican Forte®) had nine molecules, including amino acids, vitamins, minerals, and a molecule purified from green tea . 3,4 The evaluation of the efficacy of such a developed material against colibacillosis required standardization of a successful E. coli challenge following primary infections, such as those caused by low pathogenic avian influenza viruses, 5 or other primary viruses . 6,7 The purpose of this work was to evaluate the efficacy of a developed material known as Epican forte® in broilers against a standardized high and low dose-E. ...
... Actually, previous research on benefits of NS on the surviving host challenged with the viruses only was documented. 3,4 Certain components of NS, including the green tea extract, vitamin C, selenium, magnesium, and copper, were proven to improve the regeneration of tissues damaged by infection, the healing of tumors, and even the reduction of viral multiplication. 9,10,11 The low E. coli challenge in group 4 treated with NS helped the survivors to gain a significantly higher weight with better feed conversion, compared to the similarly challenged group 2 that was deprived of NS treatment (P<0.05; ...
The objective of this study was to evaluate the efficacy of a Nutrient Synergy (NS; Blend of nine nutrients) in maintaining the performance and alleviating the pathologic effects in broilers challenged with a high and a low dose-E. coli, following a primary challenge with H9N2-avian influenza virus. Six groups of broilers were included (19 birds/group). Each bird in group 1-4 received at an age of 20 d. a primary intratracheal challenge of 2 HA Units of H9N2 virus. At the age of 23 days, birds of groups 1 and 3 received a high dose-E. coli challenge in the right thoracic air sac (1.5x 10 9 cfu/0.5ml/bird), while birds of groups 2 and 4 received-E. coli challenge in the same,I low close route (1.5 x 106 cfu/ml). The initiation of a NS-daily administration, intraesophageally. was according to the manufacturer instructions (Epican Forte (R)) (976 mg/Kg of body weight). The treatment was restricted to birds in groups 3, 4, and 5, effective the age of 17 days and Until 28th day of age. Birds of group 6 were unchallenged and Untreated.However, the average weight and feed conversion at 28 days of age was significantly improved (p<0.05) in the NS-treated group compared to NS-deprived group, with similar low dose-E. coli challenge. The frequency of ocular exudates-sign and diarrhea at 2 days post the E. coli challenge dropped significantly (p<0.05) in the NS-treated groups in comparison to deprived birds, with a similar dose of E. coli challenge. The frequency of diarrhea was kept low at 5d. post-challenge, with the high dose of E. coli in birds treated with NS (P<0.05). The frequencies of the right and left thoracic airsacculitis, and the frequency of pancreatitis were reduced significantly in NS-treated birds with low-dose E. coli in comparison to similarly challenged birds, deprived of NS (p<0.05). However in the high-dose E. coli. challenge groups, the NS treatment lowered only the frequency of abdominal airsacculitis (P<0.05).
... Based on this study and our earlier findings [18,19,27], this combination of plant-derived compounds and micronutrients may constitute a new anti-SARS-CoV-2 strategy by simultaneously affecting multiple aspects of viral life cycle, including viral entry and replication. This strategy was also implemented in our earlier studies, including those of human influenza H1N1, bird flu H1N5, and others, which were based on selecting natural components that simultaneously affect key pathology mechanisms across a wide spectrum of infective agents [28][29][30][31]. ...
Despite vaccine availability, the global spread of COVID-19 continues, largely facilitated by emerging SARS-CoV-2 mutations. Our earlier research documented that a specific combination of plant-derived compounds can inhibit SARS-CoV-2 binding to its ACE2 receptor and controlling key cellular mechanisms of viral infectivity. In this study, we evaluated the efficacy of a defined mixture of plant extracts and micronutrients against original SARS-CoV-2 and its Alpha, Beta, Gamma, Delta, Kappa, and Mu variants. The composition containing vitamin C, N-acetylcysteine, resveratrol, theaflavin, curcumin, quercetin, naringenin, baicalin, and broccoli extract demonstrated a highest efficacy by inhibiting the receptor-binding domain (RBD) binding of SARS-CoV-2 to its cellular ACE2 receptor by 90%. In vitro exposure of test pseudo-typed variants to this formula for 1 h before or simultaneously administrated to human pulmonary cells resulted in up to 60% inhibition in their cellular entry. Additionally, this composition significantly inhibited other cellular mechanisms of viral infectivity, including the activity of viral RdRp, furin, and cathepsin L. These findings demonstrate the efficacy of natural compounds against SARS-CoV-2 including its mutated forms through pleiotropic mechanisms. Our results imply that simultaneous inhibition of multiple mechanisms of viral infection of host cells could be an effective strategy to prevent SARS-CoV-2 infection.
... Such a comprehensive effects of naturally occurring compounds on several mechanisms associated with viral infectivity is not surprising. This strategy was also implemented in our earlier studies, including those of human influenza H1N1, bird flu H1N5, and others, which were based on selecting natural components that simultaneously affect key pathology mechanisms across a wide spectrum of infective agents [30]- [33]. ...
SARS-CoV-2 pandemic remains a challenge to human health and economy worldwide. Previously we have shown that a combination of active plant-derived compounds and plant extracts can dose-dependently inhibit binding of RBD-spike protein SARS-CoV-2 to the ACE2 receptor and its expression on human alveolar epithelial cells. Here we use eGFP-luciferase-SARS-CoV-2 spike protein pseudo-virions and SARS-CoV-2-RdRp, to show if the antiviral effectiveness of this combination of plant-derived compounds and plant extracts expands to other important key mechanisms of SARS-CoV-2 infection. Or results revealed that this combination of five plant-derived compounds inhibited the attachment of the SARS-CoV-2 pseudo-typed particles with lung hACE2/A549 cells. In addition, it down-regulated the activity of key enzymes known to be crucial for the entry of the SARS-CoV-2 virus, such as TMPRSS2, furin and cathepsin L, but not their expression at protein level. This combination did not affect ACE2 binding to and ACE2 enzymatic activity, but modestly decrease cellular expression of neuropilin-1 molecule and significantly inhibited activity of viral RdRp. This study demonstrates inhibitory effects of this combination on key cellular mechanisms of SARS-CoV-2 infection. The findings further support the use of plant-derived compounds as effective health measures against SARS-CoV-2-caused infection.
... [18][19][20] Numerous in vitro, in vivo, and human studies conducted by us and others, involving different viruses, for example, HIV, H1N1, H1N9, and bird flu, confirm the universality of ascorbic acid efficacy in various viral infections and pleiotropic effects achieved from its combination with other natural compounds. [21][22][23] Our findings show that vitamin C has a consistent and significant lowering effect on ACE2 expression exercised at different molecular levels in human alveolar epithelial cells, but also in microvascular endothelial cells-the 2 main cell types affected by the SARS-CoV-2. In microvascular endothelial cells, ascorbic acid could inhibit ACE2 expression at the protein (Western blot) and RNA levels. ...
Background
Angiotensin-converting enzyme II or ACE2 is an integral membrane protein present on many types of cells, including vascular endothelial cells and lung alveolar epithelial cells. This receptor serves as the entry point for SARS-coronaviruses (SARS-CoVs), including a novel coronavirus 2019-nCoV. Limited availability of these receptors can thwart cellular entry of this virus.
Methods
We tested the effects of ascorbic acid (vitamin C) on cellular expression of ACE2 at the protein and RNA levels in human small alveolar epithelial cells and microvascular endothelial cells. In addition, we investigated whether combinations of ascorbic acid with other natural compounds can affect ACE2 expression.
Results
The results show that ascorbic acid itself has moderate but consistent lowering effects on ACE2 expression at the cellular, protein, and RNA levels. Some natural compounds were effective in lowering ACE2 cellular expression, with the highest inhibitory effects observed for baicalin (75%) and theaflavin (50%). Significantly, combinations of these and other test compounds with ascorbic acid further decreased ACE2 expression. The highest impact of ascorbate on ACE2 expression was noted when combined with theaflavin (decrease from 50% to 87%), zinc (decrease from 22% to 62%), and with 10-undecenoic acid (from 18% to 53%). Ascorbic acid showed moderate additional benefits in decreasing ACE2 expression when combined with N-acetylcysteine and baicalin.
Conclusion
Our study provides valuable experimental confirmation of the efficacy of micronutrients in controlling ACE2 expression—the coronavirus cellular “entry” point. It further validates the importance of nutrient interactions in various aspects of cellular metabolism and in considering potential therapeutic applications of nutrient-based approaches. The study shows that ascorbic acid and its combination with some natural compounds could be included in developing preventive and therapeutic approaches toward the current pandemic.
Selenium (Se) deficiency has previously been shown to induce myocarditis in mice infected with a benign strain of coxsackievirus. To determine if Se deficiency would also intensify an infection with influenza virus, Se-deficient and Se-adequate mice were infected with a mild strain of influenza, influenza A/Bangkok/1/79 (H3N2). Infected Se-deficient mice developed much more severe interstitial pneumonitis than did Se-adequate mice. This increase in pathology was associated with significant alterations in mRNA levels for cytokines and chemokines involved in pro-inflammatory responses. These results demonstrate that adequate nutrition is required for protection against viral infection and suggest that nutritional deprivation may be one of many factors that increase the susceptibility of individuals to influenza infection.
Reactive oxygen intermediates (ROI) and cytokines, particularly tumor necrosis factor (TNF) have been implicated in the pathogenesis of influenza. Using a murine model of influenza, we have studied the levels of TNF, interleukin 6 (IL-6) and of superoxide-generating xanthine oxidase (XO). Mice infected intranasally with influenza virus APR/8 had high levels of XO, TNF and IL-6 in the broncoalveolar lavage, as early as 3 d after infection. XO was elevated also in serum and lung tissue. Administration of the antioxidant N-acetylcysteine (NAC,1 g/kg per day, orally) significantly decreased the mortality in infected mice, indicating a role for RO1 in the lethality associated with influenza infection.
Oxidative stress is implicated in the pathogenesis of pulmonary damage during viral infections. In a previous study we observed a significant improvement of survival of influenza-infected mice with NAC, 1g/kg divided in two daily administrations, for 8 days including a pretreatment on day 1 before infection. In order to test NAC in a more realistic model, we studied the effect of combined treatment with NAC and the antiviral drug, ribavirin. Since in the present work we wanted to test a possible synergistic effect by combination of NAC and ribavirin, we used a different NAC's treatment regimen (1 g/kg, once a day for 4 days) that, alone, did not significantly protect mice from death. Mice (12 per group) infected intranasally with a lethal dose of influenza A virus APR/8. NAC was given as a single daily dose of 1000 mg/kg starting from 4 h after infection and until day 4 after infection, in association with ribavirin (100 mg/kg, i.p.). End-point evaluation was 14-day survival. With this schedule survival in infected mice was 17%, it was not significantly changed by NAC (25%). Survival increased to 58% with ribavirin and to 92% (n=12) with a combined treatment with ribavirin and NAC. This suggest that antioxidant therapy can increase survival by either improving the defenses against virus or by protecting from the pathogenesis of lung inflammation.
We investigated a broad spectrum of immunoactive mediators in a mouse model of influenza. ICR mice (4-5 wk old) that were infected with a 10 LD50 dose of influenza A/PR8/34 virus died after 6 days without evidence of bacterial superinfection. Maximal virus titers were reached by day 2 postinfection, whereas the multifocal pneumonia with mononuclear cell infiltration reached its maximum at the end of infection. We measured the cytokines IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-6, IFN-gamma, TNF-alpha, granulocyte (G)/macrophage (M)-CSF, G-CSF, M-CSF, and the lipid mediators leukotriene B4 and platelet-activating factor in the cellfree bronchoalveolar lavage fluid of mice during infection. We found an early increase of IL-1 alpha, IL-1 beta, IL-6, TNF-alpha, GM-CSF, IFN-gamma, and leukotriene B4. Levels of these factors peaked between 36 h and day 3 postinfection, with the exception of IL-6 that remained at elevated levels throughout infection. G-CSF and M-CSF increased slowly and reached a maximum by day 5 postinfection. We were unable to detect IL-2, IL-3, or IL-4. PAF remained at the same level throughout infection. Our results suggest that lung-resident cells, and possibly the alveolar macrophages, participate actively in the onset of the inflammatory response against the invading virus. The inability to detect the T cell products IL-2, IL-3, and IL-4 was unexpected considering the role of T cells in the elimination of the virus in infected mice. Our observation confirms thus earlier findings about the inability of specific T cell clones to elicit an unspecific antiviral effect.
Reactive oxygen and nitrogen metabolites play a complex role in many diseases and in metabolic regulation. Because viruses replicate in living cells, such metabolites influence the growth of viruses in addition to serving as a host defense mechanism. Low levels of reactive oxygen species (ROS) play a role in mitogenic activation, and the early phase of lytic and nonlytic virus infection indeed resembles that of mitogenic cell activation. In addition to these subtle cell-activating effects shared by many viruses, influenza and paramyxoviruses activate a respiratory burst in phagocytic cells. These viruses are toxic when injected in animals. Cells lavaged from the lungs of mice infected with influenza virus are primed for enhanced superoxide generation. Moreover, xanthine oxidase is enhanced and the buffering capacity of small molecular antioxidants is decreased in the lungs, suggesting that infection leads to oxidative stress. The wide array of cytokines produced in the lungs during influenza could contribute to the systemic effects of influenza. Oxidative stress has also been shown in human immunodeficiency virus (HIV) infection in humans. Via activation of NF kappa B, ROS may activate viral replication, but oxidants are believed to contribute also to the loss of CD4 T cells by apoptosis. Antioxidants, together with agents interfering with the harmful effects of cytokines and lipid mediators, may have a role in the treatment of viral diseases. Such agents could not only alleviate disease symptoms but also decrease the long-term effects of chronic oxidative stress, which have been linked to the development of cancer in some viral infections.
It is known that resident peritoneal (RP) cells from BALB/c female mice express a constitutive non-specific antiviral immunity which is progressively reduced during several days of cultivation in vitro. In this report, we have studied the effect of a proline-rich polypeptide (PRP) isolated from ovine colostrum on the kinetics of vesicular stomatitis virus (VSV) replication in freshly isolated and one-day cultured RP cells. The polypeptide was added to the cells immediately after virus adsorption or one day before or after viral infection. Independently on time of PRP addition, an inhibition of VSV replication (virus titres reduced by up to 4 log units) was observed. Occasionally, however, a weak stimulation of VSV replication by PRP (virus titres increased by 1-2 log units) was noticed in RP cells constitutively resistant to the infection.
An ever increasing demand to evaluate the effect of dietary supplements on specific health conditions by use of a "significant scientific" standard has prompted the publication of this study.
To study the effect of megadose Vitamin C in preventing and relieving cold and flu symptoms in a test group compared with a control group.
Prospective, controlled study of students in a technical training facility.
A total of 463 students ranging in age from 18 to 32 years made up the control group. A total of 252 students ranging in age from 18 to 30 years made up the experimental or test group.
Investigators tracked the number of reports of cold and flu symptoms among the 1991 test population of the facility compared with the reports of like symptoms among the 1990 control population. Those in the control population reporting symptoms were treated with pain relievers and decongestants, whereas those in the test population reporting symptoms were treated with hourly doses of 1000 mg of Vitamin C for the first 6 hours and then 3 times daily thereafter. Those not reporting symptoms in the test group were also administered 1000-mg doses 3 times daily.
Overall, reported flu and cold symptoms in the test group decreased 85% compared with the control group after the administration of megadose Vitamin C.
Vitamin C in megadoses administered before or after the appearance of cold and flu symptoms relieved and prevented the symptoms in the test population compared with the control group.
Matrix metalloproteinases (MMPs), vascular endothelial growth factor (VEGF), Ki 67 (proliferative protein) and constituents of ECM play a critical role in angiogenesis, and are crucial in neoplastic invasion and metastasis. Based on the antitumor properties of certain nutrients, we investigated the effect of a diet containing lysine, proline, arginine, ascorbic acid and green tea extract on the growth of tumors induced by implanting human prostate cancer PC-3 cells in athymic nude mice and on the expression of MMPs, VEGF, Ki 67 and fibronectin in these tumors, as well as the production of mucin (by PAS staining).
Male nude mice (n =12) were inoculated with 3x10(6) prostate cancer PC-3 cells and randomly divided into two groups; Group A was fed a regular diet and Group B was fed a regular diet supplemented with 0.5% of the nutrient mixture (NM). Four weeks later, tumors were excised, weighed and processed for histology.
The results showed inhibition of tumor growth in Group B. Histological studies revealed inhibition of MMP-9 and VEGF secretion and mitosis in Group B tissues.
Nutrient supplementation strongly suppressed the growth of tumors without any adverse effects in nude mice, suggesting strong potential as an anticancer agent.
Vitamin C (ascorbate) is essential for hydroxylation of prolyl and lysyl residues in nascent collagen, the failure of which leads to connective tissue lesions of scurvy. Of the pyridinium-type cross-links in mature collagen, pyridinoline requires more hydroxylysyl residues than does deoxypyridinoline. Our study tested the hypothesis that pyridinoline:deoxypyridinoline ratios in urinary degradation products may vary with ascorbate status in man. These ratios were compared between British and Gambian prepubertal boys, mean age 8.3 years, and in Gambian boys between two seasons with contrasting ascorbate availability. The mean cross-links ratio in 216 British boys was 4.36 (SD 0.71), significantly greater (P<0.0001) than in sixty-two Gambian boys: 3.83 (SD 0.52). In the Gambians the cross-links ratio was significantly higher in the dry season (with high ascorbate intake and status) than in the rains (with low intake and status). A 7-week controlled intervention was carried out in Gambian boys during the rainy season (the 'hungry' season, when vitamin C-containing foods are virtually unavailable): 100 mg ascorbate/d was given to one group of thirty-two Gambian boys and placebo to another group. The intervention did not, however, significantly alter the cross-link ratio, possibly because the response time and/or intervention-response delay is >7 weeks. If confirmed, the putative association between ascorbate and collagen cross-link ratios in man could become the basis for a functional test for adequacy of ascorbate status.