Synthesis and Immunological Evaluation of a Single Molecular Construct MUC1 Vaccine Containing l-Rhamnose Repeating Units.

A rhamnose targeting strategy for generating effective anticancer vaccines was successful in our previous studies. We showed that by utilizing natural anti-rhamnose antibodies, a rhamnose-containing vaccine can be targeted to antigen-presenting cells, such as dendritic cells. In this case, rhamnose (Rha) was linked directly to the liposomes bearing the antigen. However, in the current approach, we conjugated a multivalent Tri-Rha ligand with the antigen itself, making it a single component vaccine construct, unlike the previous two-component vaccine construct where Rha cholesterol and Mucin1 (MUC1) antigen were both linked separately to the liposomes. Synthesis required the development of a linker for coupling of the Rha-Ser residues. We compared those two systems in a mouse model and found increased production of anti-MUC1 antibodies and more primed antigen-specific CD4+ T cells in both of the targeted approaches when compared to the control group, suggesting that this one-component vaccine construct could be a potential design used in our MUC1 targeting mechanisms.

A Synthetic MUC1 Glycopeptide Bearing ßGalNAc-Thr as a Tn Antigen Isomer Induces the Production of Antibodies against Tumor Cells.

This study presents the synthesis of the novel protected O-glycosylated amino acid derivatives 1 and 2, containing ßGalNAc-SerOBn and ßGalNAc-ThrOBn units, respectively, as mimetics of the natural Tn antigen (αGalNAc-Ser/Thr), along with the solid-phase assembly of the glycopeptides NHAcSer-Ala-Pro-Asp-Thr[αGalNAc]-Arg-Pro-Ala-Pro-Gly-BSA (3-BSA) and NHAcSer-Ala-Pro-Asp-Thr[ßGalNAc]-Arg-Pro-Ala-Pro-Gly-BSA (4-BSA), bearing αGalNAc-Thr or ßGalNAc-Thr units, respectively, as mimetics of MUC1 tumor mucin glycoproteins. According to ELISA tests, immunizations of mice with ßGalNAc-glycopeptide 4-BSA induced higher sera titers (1:320 000) than immunizations with αGalNAc-glycopeptide 3-BSA (1:40 000). Likewise, flow cytometry assays showed higher capacity of the obtained anti-glycopeptide 4-BSA antibodies to recognize MCF-7 tumor cells. Cross-recognition between immunopurified anti-ßGalNAc antibodies and αGalNAc-glycopeptide and vice versa was also verified. Lastly, molecular dynamics simulations and surface plasmon resonance (SPR) showed that ßGalNAc-glycopeptide 4 can interact with a model antitumor monoclonal antibody (SM3). Taken together, these data highlight the improved immunogenicity of the unnatural glycopeptide 4-BSA, bearing ßGalNAc-Thr as Tn antigen isomer.

KL-6 regulated the expression of HGF, collagen and myofibroblast differentiation.

OBJECTIVES: KL-6 is a pulmonary epithelial-derived mucin which is secreted mainly by type II alveolar epithelial cells. The level of KL-6 in serum is closely correlated to the clinical activity of various interstitial lung diseases (ILD) and acts as a prognostic factor for ILD patients. Previous studies have showed that KL-6 promoted chemotaxis, proliferation and inhibited the apoptosis of human lung fibroblasts. However, the underlying mechanism remains unknown. In this study, we investigated the function of KL-6 on the expression of hepatocyte growth factor (HGF), transforming growth factor-ß1 (TGF-ß1), collagen and α-smooth muscle actin(α-SMA) in human embryonic lung fibroblasts cell line MRC-5. METHODS: Human embryonic lung fibroblasts were cultured in Eagle's minimum essential medium. The cells plated in 6-well plates was cultured in serum-free medium at 37°C in 5% CO2 and challenged with recombinant KL-6 at a final concentration of 0, 10, 20, 40 ng/mL. Five micrograms of total RNA template were transcripted to cDNA by using AMV (Avian Myeloblastosis Virus) reverse transcriptase and random 9 mers as the first-strand primer. Synthesized cDNA was used in PCR experiments. The expression of TGF-ß1 and HGF in cell culture supernatants was measured using ELISA kit. Cells incubated with KL-6 for 72h were collected for flow-cytometry analysis. The analysis was done using a Beckman counter device. RESULTS: It was found that KL-6 up-regulated the expression of collagen type I and III in a dose-dependent manner. However, the addition of KL-6 significantly inhibited the production of HGF. As regard to the biological function, KL-6 induced myofibroblast differentiation confirmed by the elevated expression of a-SMA. CONCLUSIONS: KL-6 is one of the key molecules involved in epithelial-mesenchymal interactions and might contribute to the fibrosis in ILD.

Human milk mucin 1 and mucin 4 inhibit Salmonella enterica serovar Typhimurium invasion of human intestinal epithelial cells in vitro.

Many human milk glycans inhibit pathogen binding to host receptors and their consumption by infants is associated with reduced risk of disease. Salmonella infection is more frequent among infants than among the general population, but the incidence is lower in breast-fed babies, suggesting that human milk could contain components that inhibit Salmonella. This study aimed to test whether human milk per se inhibits Salmonella invasion of human intestinal epithelial cells in vitro and, if so, to identify the milk components responsible for inhibition. Salmonella enterica serovar Typhimurium SL1344 (SL1344) invasion of FHs 74 Int and Caco-2 cells were the models of human intestinal epithelium infection. Internalization of fluorescein-5-isothiocyanate-labeled SL1344 into intestinal cells was measured by flow cytometry to quantify infection. Human milk and its fractions inhibited infection; the inhibitory activity localized to the high molecular weight glycans. Mucin 1 and mucin 4 were isolated to homogeneity. At 150 µg/L, a typical concentration in milk, human milk mucin 1 and mucin 4 inhibited SL1344 invasion of both target cell types. These mucins inhibited SL1344 invasion of epithelial cells in a dose-dependent manner. Thus, mucins may prove useful as a basis for developing novel oral prophylactic and therapeutic agents that inhibit infant diseases caused by Salmonella and related pathogens.

Cancer-associated MUC1 mucin inhibits human T-cell proliferation, which is reversible by IL-2.

A number of adenocarcinomas abundantly express and secrete underglycosylated MUC1 mucin. Underglycosylation exposes tandem repeat peptide sequences on cancer-associated MUC1 mucin that are normally cryptic. High levels of MUC1 mucin are correlated with a poor prognosis and immunosuppression in adenocarcinoma patients. In this report we show that cancer-associated MUC1 mucin, affinity-purified from ascites fluids of cancer patients, and synthetic tandem repeats of MUC1 mucin core peptide can suppress human T-cell proliferative responses. This MUC1 mucin-induced suppression of T-cell responses can be reversed by the addition of exogenous IL-2 or anti-CD28 monoclonal antibody. These results are consistent with other studies showing that lymphocytes present in the vicinity of tumor cells are anergic and can be reactivated with exogenous interleukin-2. Overcoming MUC1 mucin-induced immunosuppression with IL-2 combined with active specific immunotherapy might be an effective immunotherapeutic strategy against human adenocarcinomas.

Mucin1 relieves acute lung injury by inhibiting inflammation and oxidative stress.

Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) is a kind of diffuse inflammatory injury caused by various factors, characterized by respiratory distress and progressive hypoxemia. It is a common clinical critical illness. The aim of this study was to investigate the effect and mechanism of the Mucin1 (MUC1) gene and its recombinant protein on lipopolysaccharide (LPS)-induced ALI/ARDS. We cultured human alveolar epithelial cell line (BEAS-2B) and used MUC1 overexpression lentivirus to detect the effect of MUC1 gene on BEAS-2B cells. In addition, we used LPS to induce ALI/ARDS in C57/BL6 mice and use hematoxylin and eosin (H&E) staining to verify the effect of their modeling. Recombinant MUC1 protein was injected subcutaneously into mice. We examined the effect of MUC1 on ALI/ARDS in mice by detecting the expression of inflammatory factors and oxidative stress molecules in mouse lung tissue, bronchoalveolar lavage fluid (BALF) and serum. Overexpression of MUC1 effectively ameliorated LPS-induced damage to BEAS-2B cells. Results of H&E staining indicate that LPS successfully induced ALI/ARDS in mice and MUC1 attenuated lung injury. MUC1 also reduced the expression of inflammatory factors (IL-1ß, TNF-α, IL-6 and IL-8) and oxidative stress levels in mice. In addition, LPS results in an increase in the activity of the TLR4/NF-κB signaling pathway in mice, whereas MUC1 decreased the expression of the TLR4/NF-κB signaling pathway. MUC1 inhibited the activity of TLR4/NF-κB signaling pathway and reduced the level of inflammation and oxidative stress in lung tissue of ALI mice.

Mucin-1 conjugated polyamidoamine-based nanoparticles for image-guided delivery of gefitinib to breast cancer.

PAMAM dendrimers (PAMs) are a group of polymeric macromolecules with distinctive physicochemical features, which can make them multifunctional theranostic nanoparticles (NPs). This study was designed to examine the impact of mucin-1 aptamer-conjugated NPs which were engineered using PAM for image-guided delivery of gefitinib (GEF) in the breast cancer cells/tumor. For this, PAMAM was conjugated with diethylenetriaminepentaacetic acid (DTPA) and modified with PEG2000 to prepare a multi-functionalized NPs. Subsequently, GEF was loaded onto the DTPA-PAM-PEG NPs, which were then armed with MUC-1 aptamer to form the DTPA-PAM-PEG/GEF@MUC-1 nanosystem. Finally, aptamer-conjugated NPs were radiolabeled by gallium-67 as an imaging agent to construct image-guided nanoplatforms. The prepared NPs were characterized by different techniques. The kinetic release models of gefitinib from radiolabeled NPs offer the sustained-release mechanism of the encapsulated drug for over 7 days. In vitro evaluation showed higher cytotoxicity and enhanced uptake of the mucin-grafted NPs in MCF-7 cells. Nuclear medicine imaging and in vivo investigations revealed significant accumulation of 67Ga-DTPA-PAM-PEG/GEF@MUC-1 in the tumor site of the animal models. These data suggest that the engineered NPs are a promising image-guided nanosystem for mucin-expressing breast cells/tumors with the assistance of nuclear medicine.

ERK and PI3K regulate different aspects of the epithelial to mesenchymal transition of mammary tumor cells induced by truncated MUC1.

Epithelial to mesenchymal transition (EMT) integrates changes to cell morphology and signaling pathways resulting from modifications to the cell's transcriptional response. Different combinations of stimuli ignite this process in the contexts of development or tumor progression. The human MUC1 gene encodes multiple alternatively spliced forms of a polymorphic oncoprotein that is aberrantly expressed in epithelial malignancies. MUC1 is endowed with various signaling modules and has the potential to mediate proliferative and morphological changes characteristic of the progression of epithelial tumors. The tyrosine-rich cytoplasmic domain and the heavily glycosylated extracellular domain both play a role in MUC1-mediated signal transduction. However, the attribution of function to specific domains of MUC1 is difficult due to the concomitant presence of multiple forms of the protein, which stem from alternative splicing and proteolytic cleavage. Here we show that DA3 mouse mammary tumor cells stably transfected with a truncated genomic fragment of human MUC1 undergo EMT. In their EMT, these cells demonstrate altered [i] morphology, [ii] signaling pathways and [iii] expression of epithelial and mesenchymal markers. Similarly to well characterized human breast cancer cell lines, cells transfected with truncated MUC1 show an ERK-dependent increased spreading on fibronectin, and a PI3K-dependent enhancement of their proliferative rate.

One-Step Self-Assembly of Multifunctional DNA Nanohydrogels: An Enhanced and Harmless Strategy for Guiding Combined Antitumor Therapy.

DNA nanostructure-based drug delivery system (DDS) has become an advanced therapeutic strategy for cancer because of its unsurpassed editability, intrinsic biodegradability, and tunable multifunctionality. An intelligent DNA nanosystem integrating targeting, immunostimulation, and chemotherapy was constructed based on unmethylated cytosine-phosphate-guanine oligonucleotides (CpG ODNs) DNA nanohydrogels (CpG-MUC1-hydrogel). By facile one-step self-assembly, the cross-shaped DNAs (C-DNAs) assembled from pH-responsive I-motif sequences and targeted MUC1 aptamer-immunoadjuvant CpG-fused sequences (CpG-MUC1) were integrated into DNA nanohydrogels with controllable size by the hybridization of DNA linkers. Subsequently, DOX was successively intercalated into the base pairs of CpG-MUC1-hydrogel, resulting in CpG-MUC1-hydrogel/Dox that would disassemble and release DOX and CpGs at acidic conditions. After MUC1-mediated internalization, CpG-MUC1-hydrogel/Dox dissociated in the endo/lysosomes and induced favorable apoptosis of tumor cells. Afterward, liberated CpGs triggered vast cytokine secretion from immune cells which elicited potent immune response against malignancy. Notably, CpG-MUC1-hydrogel induced an apoptosis effect on MCF-7 cells via significantly increasing the Bax/Bcl2 ratios and a higher level of tumor necrosis factor (TNF-α) on RAW264.7 cells than naked CpGs. Our results demonstrated that self-assembled CpG-MUC1-hydrogel represented an attractive DDS for precise delivery, potent immunostimulating activity, and considerable combination efficiency with few adverse effects, which is expected to make breakthroughs in clinical translation.

A unique mucin immunoenhancing peptide with antitumor properties.

Implantation of DA-3 mammary tumor cells into BALB/c mice results in tumor growth, metastatic lesions, and death. These cells were transfected with genes encoding for either the transmembrane (DA-3/TM) or secreted (DA-3/sec) form of human mucin 1 (MUC1). Although the gene for the secreted form lacks the transmembrane and cytoplasmic domains, the 5' sequences of these mucins are identical; however, the gene for the secreted mucin isoform ends with a sequence encoding for a unique 11 amino acid peptide. The DA-3/TM or DA-3 cells transfected with the neomycin vector only (DA-3/neo) have the same in vivo growth characteristics as the parent cell line. In contrast, DA-3/sec cells fail to grow when implanted in immunocompetent BALB/c animals. DA-3/sec cells implanted in nude mice resulted in tumor development verifying the tumorigenic potential of these cells. Pre-exposure of BALB/c mice to DA-3/sec cells afforded protection against challenge with DA-3/TM or DA-3/neo mammary tumors and the unrelated tumors K7, an osteosarcoma, and RENCA, a renal cell carcinoma. Partial protection against subsequent tumor challenges was also achieved by substituting the 11 amino acid peptide found only in the secreted MUC1 isoform, for the live DA-3/sec cells. Notably, the efficacy of this peptide is not strain restricted because it also retarded the growth of Lewis lung carcinoma cells in C57 BL/6 mice. These findings reveal that a unique peptide present in the secreted MUC1 has immunoenhancing properties and may be a potential agent for use in immunotherapy.

Mucin 1 and poly I:C activates dendritic cells and effectively eradicates pituitary tumors as a prophylactic vaccine.

Pituitary tumors are the most common type of cancer within the central nervous system. In the present study, the expression levels of mucin 1 (Muc1) were examined in invasive and non­invasive pituitary tumor samples, and the results of immunohistochemical staining and Western blot analysis demonstrated marked positive expression of Muc1. In addition, Muc1 + polyinosinic:polycytidylic acid (poly I:C) was found to stimulate the expression levels of the surface molecules cluster of differentiation (CD)40, CD83 and CD80, and HLA­DRm and decreased the expression of CD14 in the dendritic cells, determined using fluorescence­activated cell sorting. The secretions of interleukin (IL)­6, tumor necrosis factor­α and IL­1ß cytokines were also significantly induced, in a dose­dependent manner. In in vivo experiments, a higher percentage of CD3+CD4+ T lymphocytes was detected, and the levels of interferon­Î³ and IL­2 in the splenocytes were also upregulated. Furthermore, the combination treatment of Muc1 with poly I:C increased anti­Muc1 IgM and anti­Muc1 IgG titers, and altered the balance of IgG2a and IgG1, all of which increased the T helper (Th)1 polarized immune response. Thus, the tumor antigen, Muc1, with poly I:C may produce potent protective effects, which polarize immune responses towards Th1, and elicit antitumor immunity to inhibit the progression of pituitary tumors.

Detection of breast cancer from blood through analysis of lymphocyte fluorescent intensity using MUC1 antigen.

BACKGROUND: Sensitive and reliable early diagnostic markers for breast cancer (BC) are still unavailable today. In this work, we proposed a new complementary method for detection of BC. This method is based on an observation that lymphocytes re-exposed in vitro to antigenic stimulation express cytoplasmic changes. METHODS: In the new protocol, we recorded changes in the fluorescence intensity of light emitted from lymphocytes obtained from females with and without BC after stimulation with MUC1 antigen utilized flow cytometry. RESULTS: Out of 55 BC patients tested, 46 were correctly diagnosed. Of 73 controls, 55 were correctly identified as healthy subjects. The sensitivity of the test was 84 %; the specificity was 75 %. CONCLUSION: These results suggest a potentially valuable method for detection of BC. The clinical importance of this procedure relies on the ability to screen populations for BC with widely available flow cytometry by a relatively fast, accurate, and economical procedure. Another potential benefit would be identification of candidates for vaccination as a primary or secondary preventive measure.

Subcellular localization of MUC1 recognized by a monoclonal antibody MY.1E12 correlates with postsurgical prognosis in differentiated-type gastric carcinomas of stage II and III.

Because of the complex nature of the diverse histologic grade in gastric carcinomas a useful biomarker should be provided to scale the aggressiveness of the disease and to determine surgical strategy, especially for advanced carcinomas. Our previous study of MUC1 in gallbladder carcinoma using mAb MY.1E12 has revealed the stromal localization of MUC1 adjacent to the carcinoma was correlated with poor prognosis. In gastric carcinoma the biological significance of the localization of MUC1 recognized by mAb MY.1E12 has not been fully investigated. We performed immunohistochemical analysis to determine the correlations with the localization of mAb MY.1E12-reactive-MUC1 (MY.1E12-MUC1) and clinicopathological findings. A total of 91 consecutive patients with stage II, IIIA or IIIB gastric carcinoma after curative resection were reviewed retrospectively. The localization of MY.1E12-MUC1 was classified as negative, apical, cytoplasmic or stromal type based on the predominant subcellular localization. Immunostaining of MY.1E12-MUC1 was recognized in 84% of the 55 cases of differentiated-type carcinoma and in 53% of the 36 cases of undifferentiated-type carcinoma (P<0.01). In differentiated-type carcinoma, the proportion of stromal-type dominant localization of MY.1E12-MUC1 was increased at the deepest invading sites. Postsurgical liver metastasis was seen in 11 (30%) of 37 cases showing stromal or cytoplasmic-type localization-dominant group and in 1 (6%) of 18 cases showing apical-type localization-dominant group or negative staining group (P<0.05). The postsurgical survival was significantly poorer in the former group than in the latter (P=0.004). In differentiated-type gastric carcinoma, the presence of the cytoplasmic- or stromal-type localization of MY.1E12-MUC1 at the deepest invading sites correlates with aggressiveness of the disease, such as the tendency to form liver metastasis. This phenotype may serve as a unique biological feature associated with the malignant behavior of differentiated-type gastric carcinomas.

Purified human breast milk MUC1 and MUC4 inhibit human immunodeficiency virus.

BACKGROUND: The HIV-AIDS pandemic is prevalent in sub-Saharan Africa. Breastfeeding is a risk factor, with transmission from mother to child being as high as 40%. OBJECTIVES: To determine the antiviral activity of crude breast milk and its purified mucins MUC1 and MUC4 against HIV-1 in patients who were HIV positive compared to those who were not. METHODS: Twenty-one human milk samples were taken from both groups. Breast milk mucins were purified by density-gradient ultracentrifugation in caesium chloride and analyzed by SDS-PAGE, Western blotting and amino acid content. The inhibition of the virus by crude milk and purified mucin was assayed by an in vitro HIV-1 p24 assay. RESULTS: SDS-PAGE for purified mucin showed several high-molecular-weight bands for the HIV-negative group and prominently stained single bands on the stacking gel with faintly periodic acid Schiff-positive glycoprotein bands observed in some cases in the running gel for the HIV-positive mucins. Western blot analysis identified the mucins in both groups to be MUC1 and MUC4. Both mucins showed more intensity on Western blotting for the HIV-positive group. There was no difference in the content of serine, threonine and proline of purified mucins for both groups. HIV-1 was not inhibited by crude breast milk from normal (13/14 samples) and infected individuals (19/19 samples). Fifteen of 20 and 16/18 samples of purified mucin from the uninfected and HIV-positive groups, respectively, inhibited the virus. CONCLUSIONS: Crude breast milk does not inhibit HIV-1, whilst purified mucins do in an in vitro assay.

The role of salivary proteins in the mechanism of astringency.

Understanding astringency has focused on the interaction of tannins with the salivary proline-rich proteins (PRPs), although it remains unclear if other astringents precipitate the PRPs or how this interaction relates to sensory perceptions of astringency. We used 2 approaches to compare how distinct classes of astringent compounds interacted with the salivary PRPs and mucins. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis, we evaluated protein patterns and characterized the salivary proteins present in the supernatants and pellets of pooled saliva assayed with tannin, alum, and hydrochloric acid solutions. Tannins and alum precipitated many of the PRPs, but acid did not. Mucins were precipitated by both the acid and alum, but not by the tannins. From our research, it appears that the precipitation of salivary proteins may be involved in the mechanism of astringency, but the precipitation of PRPs is not requisite for the development of astringency. We also measured mucin and deoxyribonucleic acid content of expectorated solutions of astringents that panelists swished in their mouths to determine if astringency was associated with a loss of oral lubricating films.

Specific decidual CD14(+) cells hamper cognate NK cell proliferation and cytolytic mediator expression after mucin 1 treatment in vitro.

Mucin 1 (MUC1) forms a glycocalyx on the surface of decidual epithelial cells that needs to be removed for successful embryo attachment. We investigated whether MUC1 affects human early pregnancy decidual CD14(+) cells and their interactions with cognate decidual natural killer (NK) cells. FITC-dextran internalisation, surface and intracellular antigen levels, and proliferation of CD14(+) and/or CD56(+) cells were analysed by flow cytometry. Magnetic separation was used to purify CD56(+) and CD14(+) cells. Uncultured CD14(+) cells expressed a negligible percentage of CD1a and CD83 molecules. They expressed lower levels of CD16, and higher levels of endocytic mannose receptors (MR), dendritic cell-specific intercellular adhesion molecule grabbing non-integrin (DC-SIGN), proinflammatory chemokine CC receptor 5 (CCR5), and CD163 receptor, than their peripheral blood counterparts. Lipopolysaccharide stimulation did not affect FITC-dextran internalisation in CD14(+) cells. MUC1 bound and internalised, in a dose-dependent manner, the carbohydrate recognition domain of MR, increasing the decoy IL-1 receptor type II and decreasing IL-15 expression in CD14(+) cells. In the presence of MUC1-treated macrophages, the expression levels of the proliferation and cytotoxic mediators (perforin, Fas ligand and TNF-related activation-induced ligand or TRAIL) was attenuated, while that of the anti-inflammatory chemokine CCL17 was increased, in NK cells compared with untreated macrophages. In conclusion, MUC1 supports the alternative activation of tissue-specific CD14(+) cells, and may restrict proliferation of NK cells and regulate their content of cytotoxic mediators. Based on the experiments with first-trimester decidual cells in vitro, we conclude that removing MUC1 from decidual tissue might help control trophoblast invasion by NK cells.

Inhibition of human immunodeficiency virus type 1 activity by purified human breast milk mucin (MUC1) in an inhibition assay.

It has been reported that breast-feeding is responsible for approximately 40% of the HIV transmissions from HIV-positive mothers to children. Human breast milk, however, is known to contain numerous biologically active components which protect breast-fed infants against bacteria, viruses, and toxins. The purpose of this study was to purify and characterize breast milk mucin and to determine its anti-HIV-1 activity in an HIV inhibition assay. Sepharose CL-4B column chromatography and caesium chloride isopycnic density gradient purification were used to isolate and purify the mucin. Following Western blotting and amino acid analysis, an HIV-1 inhibition assay was carried out to determine the anti-HIV-1 activity of crude breast milk and purified milk mucin (MUC1) by incubating them with HIV-1 prior to infection of the human T lymphoblastoid cell line (CEM SS cells). SDS-PAGE analysis of the mucin, together with its amino acid composition and Western blotting, suggested that this purified mucin from human breast milk was MUC1. The HIV inhibition assay revealed that while the purified milk mucin (MUC1) inhibited the HIV-1 activity by approximately 97%, there was no inhibition of the HIV-1 activity by crude breast milk. Although the reason for this is not clear, it is likely that because the MUC1 in crude milk is enclosed by fat globules, there may not be any physical contact between the mucin and the virus in the crude breast milk. Thus, there is a need to free the mucin from the fat globules for it to be effective against the virus.