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Effect of Human milk oligosaccharides (HMO) on flu virus and can HMO help against coronavirus?

Cieo® Human milk oligosaccharides (HMO) is made by fermentation can help against respiratory virus like flu virus etc. by increasing immunity. Roger Naik reg. pharmacist believes the same mechanism may help against coronavirus. Human milk oligosaccharides (HMO) can boost human immunity against infection. There is no study done for usefulness of Human milk oligosaccharides (HMO) against coronavirus.

Following research paper is narrated by Roger Naik R. Ph. to simplify without altering original content.

Food and Nutrition Sciences, 2014, 5, 1387-1398

Published Online August 2014 i

Human Milk Oligosaccharides Enhance Innate Immunity to Respiratory Syncytial Virus and Influenza in Vitro

Geralyn Duska-McEwen1, Albert P. Senft2, Teah L. Ruetschilling2,

Edward G. Barrett2, Rachael H. Buck1

1Discovery R & D, Abbott Nutrition, Columbus, USA

2Lovelace Respiratory Research Institute, Albuquerque, USA

Abstract

Human milk oligosaccharides (HMO) contribute to innate immunity by enhancing growth of beneficial bacteria, epithelial cell maturation and mucosal barrier integrity. They have immunomodulatory effects and can block pathogen binding to host cell surface glycans or receptors. We investigated the effects of 2’-fucosyllactose (2’FL), 6’-sialyllactose (6’SL), 3’-sialyllactose (3’SL) and lacto-N-neoTetraose (LNnT) on human respiratory epithelial cell lines or peripheral blood mononuclear cells (PBMCs) following respiratory viral infection in vitro. Expression of cytokines and viral load were monitored in infected cells. These biomarkers of innate immunity were selected since viral load and cytokine levels (IP-10, MIP-1α, IL-6, IL-8, TNF-α) have been correlated with disease severity in respiratory syncytial virus (RSV) and influenza (IAV) virus infection in vivo. 2’FL significantly decreased RSV viral load and cytokines associated with disease severity (IL-6, IL-8, MIP-1α) and inflammation (TNF-α, MCP-1) in airway epithelial cells. LNnT and 6’SL significantly decreased IAV viral load in airway epithelial cells. 6’SL dose-dependently down-regulated IP-10 and TNF-α in RSV infected PBMCs. HMO at or below levels found in breast milk enhance innate immunity to respiratory viruses in vitro and may interact directly with cells to modulate biomarkers of innate immunity.

Keywords

Human Milk Oligosaccharides, Respiratory Syncytial Virus, Influenza Virus, Inflammation, Innate Immunity

1. Introduction

Human milk oligosaccharides (HMO) contribute to innate immunity through several potential paths. HMO support initial development of infant gut microbiota by cultivating colonization with beneficial bacteria. HMO enhance growth [1] [2] and adhesion [3] of probiotic bacteria to gut epithelial cells. Bifidobacteria grown in the presence of HMO stimulate increased expression of tight junction proteins in gut epithelial cells [3] in vitro, suggesting HMO indirectly promote gut barrier integrity. Recently, 2’FL and LNnT were shown to promote gut epithelial cell maturation and barrier function in vitro, suggesting specific HMO directly promote epithelial cell maturation in the small intestine [4].

Some HMO share structural homology with host cell receptors and can block pathogen binding and infection of these host cells by acting as a “receptor decoy” by binding to the pathogen. For example, α-1,2-fucosyllated HMO bind to Campylobacter jejuni and inhibit pathogen binding to intestinal epithelial cells [5]. Other HMO can block pathogens or toxins by binding to the target cell surface receptor, preventing the pathogen/toxin from adhering to the cell. For instance, α-1,2-fucosyllated HMO block binding of the stable toxin from enterotoxigenic Escherichia coli by binding to its target receptor—guanylyl cyclase C [6].

A recent review by Yang and colleagues highlighted several viral surface lectins for glycans found on the surface of human epithelial cells as well as in human milk, suggesting intake of HMO may block or prevent viral infections directly or indirectly [7]. There is evidence that HMO can protect against infection directly by mimicking viral receptors on the cell’s surface to block infection [7]-[9] or by competing with a virus (e.g., human immunodeficiency virus) to bind a C-type lectin receptor (e.g., Dendritic Cell-Specific Intercellular adhesion molecule-3-grabbing non-integrin or DC-SIGN) on a target cell’s surface [10].

The effects of individual HMO on early immune responses in vitro to respiratory viral infection in circulating immune cells or airway epithelial cells have not been studied to date. Respiratory epithelial cells become infected with respiratory syncytial virus (RSV) or influenza (IAV) upon inhalation or inoculation of the respiratory mucosa [11]. Infection then spreads along the respiratory mucosa through cell-to-cell transfer (RSV) [11] [12], viral budding (IAV) [13] and aspiration of nasopharyngeal secretions [12]. Others have shown that RSV viral replication and inflammatory processes occurring in nasal air passages reflect those in lower airways [14] [15].

Since HMO bathe the laryngopharyngeal region during oral consumption, it has been postulated that HMO may reduce pathogen adhesion at the entry of the upper respiratory tract [16]. Cells exposed to HMO in the laryngopharyngeal region would include respiratory mucosal epithelial cells as well as locally resident or transient immune cells (lymphocytes, dendritic cells, monocytes, macrophages, NK cells and M cells) in the palatine and lingual tonsils. HMO have been shown to dampen platelet-neutrophil mediated inflammation [17] [18] and have been shown to be absorbed into the peripheral circulation [19] [20] with potential access through tonsils or the gut mucosa [21] [22]. For these reasons, we investigated the effects of HMO interaction with human respiratory epithelial cells or peripheral blood mononuclear cells (PBMCs) during respiratory viral infection in vitro. We monitored HMO effects on virus load and cytokine expression. These biomarkers of innate immunity were selected since viral load and cytokine levels (IP-10, MIP-1α, IL-6, IL-8, TNF-α) have been correlated with disease severity in respiratory syncytial virus (RSV) [23]-[28] and influenza (IAV) [29]-[32] virus infection in vivo.

2. Materials and Methods

2.1. Biosynthesized Human Milk Oligosaccharides

Purity of oligosaccharides and their endotoxin levels are summarized in Table 1. HMO identity was confirmed by the manufacturer using nuclear magnetic resonance (NMR) to confirm chemical structure, and mass spectrometry (MS) to confirm molecular weight. HMO purity was measured by high performance anion exchange chromatography with pulsed amperometric detection (HPAEC/PAD) using relative peak area comparisons. Moisture content was determined separately using the Karl Fischer method for moisture determination [33]. 3’-Sialyllactose (3’SL), 6’Sialyllactose (6’SL) and 2’Fucosyllactose (2’FL) were all derived from bacterial synthesis. Lacto-N-neoTetraose (LNnT) was synthesized from a yeast fermentation system and purified by crystallization [34]. The endotoxin content was measured in 10 mg HMO/mL reconstitutions using manufacturer’s instructions to perform the end point chromogenic QCL-1000 Limulus Amebocyte Lysate test (Lonza). Briefly, a 50 μL sample of each reconstituted carbohydrate was mixed with an equal volume of the Limulus Amebocyte-Lysate (LAL) and incubated at 37˚C (±1˚C) for 10 minutes. Next 100 μL of substrate solution was mixed with the LAL-sample and incubated at 37˚C (±1˚C) for an additional 6 minutes. The reaction was then stopped with the addition of 100 μL stop reagent and mixed. Endotoxin present is directly proportional to the absorbance read at405 – 410 nm, so the amount of endotoxin present in each sample tested was directly calculated from the assay standard curve.

2.3. Influenza A Virus (IAV)—H1N1

A/New Caledonia/20/99 (H1N1) (NC99), a seasonal influenza strain included in the seasonal influenza vaccine for 7 years, was obtained from the Centers for Disease Control. The stock virus was passaged twice in 10-day old embryonated chicken eggs and then in Madin-Darby Canine Kidney (MDCK) cells prior to infection of lung epithelial cells described herein. Briefly, MDCK cells were infected one day post plating, when the cells were subconfluent. Prior to infection, cells were washed twice with MDCK Influenza Infection Media (Minimum Essential Medium Eagle, 25 mM HEPES, 1% L-glutamine, 1% sodium pyruvate, 0.15% sodium bicarbonate, 1X penicillin/streptomycin and 3 μg/mL TPCK-treated Trypsin). The viral solution was prepared by adding 100 μL of IAV (post passage in chicken eggs) to 24 mL of infection media, and then 12 mL of the viral solution was added to each flask of cells. Cells were incubated at 37˚C and 5% CO2, and 2 days post infection, the MDCK cell monolayer was completely disrupted by viral infection. Flasks were scraped to remove any remaining cells,

Rest of the article can be available by request RCNAIK@GMAIL.COM Roger Naik R. Ph

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