Banking on Breast Milk

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Little Bundles of Joy: Exosomes

For many people, childhood can be summed up in 5 words: peanut butter and jam sandwiches. Unfortunately, many children will never be able to eat one because of an allergy, or they may have to avoid eating one due to allergies their peers have. Peanut allergies have doubled in the last 10 years and effect up to 3% of a population in Western countries. Our bodies have an intricate response to allergens, and it turns out that certain components in breast milk can help with long-term regulation of a child’s reaction and sensitivities to these allergens. Exosomes – small vesicles found in biological fluids like breast milk – provide an infant with key micro RNAs (miRNAs) that can regulate immune responses to allergens, and they express allergen detecting and presenting proteins. In this post, I’m going to focus on one of the immune-related effects that regulatory T cell (Treg) differentiation and function have, and how exosomes regulate this effect by providing certain miRNAs.

Exosomes are small vesicles that bud from many different types of cells, including those involved with allergic responses and intestinal epithelial cells. They contain numerous proteins associated with cell signaling and immune response, and they are packed with all sorts of compounds. Exosomes from breast milk tend to consistently have the same compounds including, but certainly not limited to, 59 immune-related miRNAs and the proteins MHC II, Hsc70, and CD81 (Figure 1). The miRNAs in these exosomes are a form of genetic material similar to DNA, usually consisting of 20-30 nucleotides, but they are not translated into protein. Instead, they exist as various structural forms and interact with other proteins, DNA, or RNA forms to modify cellular activity.

Certain miRNAs, like miR-155 and miR-148a, regulate the differentiation and function of Tregs. T cells are associated with the immunological function of various immune cells, but Tregs are a subset of T cells specifically involved in immunological tolerance and suppression, meaning they can prevent allergies! When Tregs respond to a specific allergen, they become activated and prevent T helper 2 (Th2) cell activity. Th2 activity is very important for cells to elicit an atopic allergic response; that is, a response which is very sensitive to the allergen. The idea is that by preventing Th2 activity, it will be possible to prevent allergic responses to specific allergens, like peanuts.

Back to miR-155 and miR-148a – how do they regulate the differentiation and function of these Tregs? All Treg cells express FoxP3 proteins. FoxP3, the sly fox that it is, is known as the master regulator of Treg cells because it can regulate Treg differentiation and function, so miR-155 takes advantage of FoxP3’s important role. When miR-155 enters the cell, it targets another protein, SOCS1 (suppressor of cytokine signaling 1). Normally, SOCS1 inhibits phosphorylated STAT5 (STAT5-P) and acts as a negative feedback in other pathways. By preventing SOCS1 activity, miR-155 consequently enables STAT5-P to do its job. Just to get an idea of where this pathway is headed, STAT5-P can induce gene expression of specific genes. So, if STAT5-P is active, it will, along with three other proteins, increase FoxP3 expression. Higher levels of FoxP3 has two functions: induce Treg differentiation, and increase miR-155 expression in the cell by binding to the miR-155-encoding gene called bic. The latter point is the most relevant to allergies because increasing miR-155 levels ultimately prevents Th2 hypersensitive allergic immune responses because it blocks two really important transcription factors that are associated with Th2-atopic responses. See Figure 2 for the pathway showing how miR-155 does this.

On the other hand, miR-148a regulates Treg differentiation and function via DNA demethylation to ensure stable FoxP3 expression. The FoxP3 gene has a specific sequence called Treg-specific demethylated region (TSDR) that enables stable FoxP3 expression only when it is demethylated. Of course, there has to be a protein to make life difficult, and it is called DNA methyltransferase (DNMT). This protein methylates TSDR and prevents stable FoxP3 expression. But miR-148a targets and stops DNMT. The end result is demethylation of TSDR. Basically, mir-148a + DNMT = demethylation (yay!) and DNMT = methylation (boo!) (Figure 3). Research has found that people who have atopic diseases have less demethylated TSDR Tregs. When you think about it, this makes sense because it means that in the end they won’t be able to reduce Th2 activity. Melnik et al. speculate that the presence of miRNAs could induce epigenetic modifications that will ultimately lead to Treg stability and prevention of atopic diseases.

The fact that some exosome contents can regulate Tregs is good news, but it’s not really applicable unless the exosomes from milk actually influence the infant’s immune system. In an experiment performed by Admyre et al., they found that peripheral blood mononuclear cell (PBMC – nucleated blood cells) from adult human donors have a higher level of FoxP3 expression after being incubated with breast milk exosome solutions. After observing this, the researchers suggested that human milk does have the potential to influence the infant’s immune system. The fact that milk exosomes can result in higher FoxP3 levels is a definite sign that breast milk has the potential to help reduce allergic responses through lower Th2 activity. Nonetheless, this research is still controversial, and it is unclear exactly what effects breast milk has on allergy development or avoidance.

Assuming that milk exosomes do influence the infant’s immune system, how can they pass through the digestive tract? After all, the digestive tract is a harsh, acidic environment. Gu et al. assessed miRNA stability in an attempt to answer a similar question. They found that breast milk miRNAs were stable after being subjected to harsh environments such as varying temperatures, RNases, and low pH. Knowing that miRNAs are resistant to these harsh conditions, especially low pH, it is promising because it supports the idea that infants take up the miRNAs through the digestive tract. Another recent study by Lässer et al. demonstrated that macrophages are capable of exosome uptake. This makes sense considering macrophages are an important part of the immune system. Still, other studies have shown numerous cells can take up exosomes, so it’s still not entirely clear which cells play a role in breast milk exosome uptake.

The expression of FoxP3 in breast milk-derived exosomes may be influenced by the mother’s atopic status. Melnik et al. suggest that low levels of Foxp3-expressing Tregs in a mother (as a result of allergies) may yield lower miR-155 levels in exosomes. They also suggest that this may have something to do with why atopic mothers pass on atopic allergies to infants more than atopic fathers do. 

There’s still so much to learn about this process that I can’t help but wonder what else these breast milk exosome-derived miRNAs have in store! While there are still a lot of unknowns regarding these miRNAs, it does provide lots of research questions that can be addressed like how these miRNAs interact with proteins, or what other miRNAs can reduce allergic responses. Understanding what factors contribute to allergy development and prevention may, in the future, help reduce the occurrence of some common allergies. Maybe it will someday allow everyone to enjoy the deliciousness that is a PB&J sandwich.

Until next time,

Maria

References:

Melnik BC, John SW, Schmitz G. Milk: an exosomal microRNA transmitter promoting thymic regulatory T cell maturation preventing the development of atopy? Journal of Translational Medicine. 2014; 12(43).

Gu Y,  Li M, Wang T, Liang Y, Zhong Z, Wang X, Zhou Q, Chen L, Lang Q, He Z, Chen X, GonG J, Gao X, Li X, Lv X. Lactation-Related MicroRNA Expression Profiles of Porcine Breast Milk Exosomes. International Journal of Biological Sciences. 2012; 8(1): 118-123.

Lässer C, Alikhani VS, Ekström K, Eldh M, Paredes PT, Bossios A, Sjöstrand M, Gabrielsson S, Lötvall J, Valadi H. Human saliva, plasma and breast milk exosomes contain RNA: uptake by macrophages. Journal of translational Medicine. 2011; 9(9).

Admyre C, Johansson SM, Qazi KR, Filén J-J, Lahesmaa R, Norman M, Neve EPA, Scheynuis A, Gabrielsson S. Are Present in Human Breast Milk Exosomes with Immune Modulatory Features. The Journal of Immunology. 2007; 179: 1969-1978.

Zhou Q, Li M, Wang X, Li Q, Wang T, Zhu Q, Zhou X, Wang X, Gao X, Li X. Immune-related MicroRNAs are Abundant in Breast Milk Exosomes. International Journal of Biological Sciences. 2012; 8: 118-123.

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Your Tum Will Love Colostrum

Colostrum. It’s possibly the most nutrient dense natural milk product out there. If you’re unfamiliar with it, it is the milk produced shortly after birth. It contains many immunological properties, some of which affect the expression of inflammatory and immune genes in intestinal cells. An experiment by Blais et al. studied the effect colostrum has on these inflammatory and immune gene expression after intestinal pig cells were stimulated with E. coli ETEC and Salmonella enterica, two pathogenic bacteria.

The experiment showed that colostrum down-regulates the expression of immune and inflammatory genes and upregulates the expression of wound-healing, cell proliferation and cell migration genes. This response may decrease the negative effects of an immune response that can induce diarrhea and/or inflammatory bowel diseases (IBD). The experiment also showed that colostrum decreases the activity of NF-κB. NF-κB is a transcription factor that responds to pathogens like ETEC and Salmonella and its activation can result in intestinal cell death.

This research has lots of significance in agriculture in which it can help fight off infections in livestock. However, it’s also important in understanding how it may reduce IBD in infants and children. Cheers, Maria Here is the link to the paper: Colostrum whey down-regulates the expression of early and late inflammatory response genes induced by Escherichia coli and Salmonella enterica Typhimurium components in intestinal epithelial cells

http://www.ncbi.nlm.nih.gov/pubmed/25471114

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The Juicy Deets about The Teats

To be, or not to be, that is the question. It’s also a question which bacteria are not given the chance to ask. For years it has been known that breast milk contains secretory antibodies, lipids, proteins, carbohydrates and many other compounds – some of which have not been identified. Even though the protein I’ll be writing about today has not recently been identified, a greater understanding of its function has recently been discovered. In this post, I want to share some really interesting discoveries about an antimicrobial and anti-tumor protein-fatty acid complex found in breast milk.

While Hamlet is defeated by his enemy, there’s a new kind of Hamlet who will not be defeated by his. It is better known as Human α-lactalbumin made lethal to tumor cells. HAMLET’s function is not limited to tumor cells as it has been recently found to act as a single bactericidal compound or one component of a bactericidal combination. HAMELT is an α-lactalbumin protein, a protein involved in lactose production, that is stabilized when it interacts with the human milk lipids oleic or linoleic acid in physiological conditions like an infant’s stomach. It is the most abundant protein in breast milk, but before it interacts with these fatty acids it is in a partially folded, inactive state. As a single bactericidal chemical, HAMLET is particularly effective in preventing growth of the bacteria Streptococcus pneumonia, and somewhat effective against Haemophilus influenza and Moraxella catarrhalis. These three bacteria, especially S. pneumonia, can cause serious infections. In fact, S. pneumonia is the leading cause of death for children under the age of five. As such, it is especially important to be aware of potential antibiotics available to fight off infections. Most antibiotics have a minimum inhibitory concentration (MIC), which is the minimum concentration of an antibiotic required to stop bacterial growth overnight. HAMLET is not as powerful as a typical antibiotic because when it is used alone it requires a higher concentration to inhibit the growth of bacteria compared to the MIC of antibiotics like penicillin, erythromycin and gentamicin. However, this complex is truly a superhero when it is combined with other antibiotics: it reduces the MIC of antibiotics that is needed to inhibit bacterial growth – up to a 300-fold decrease in some cases of antibiotic resistant bacteria! One recent study showed this by combining penicillin, erythromycin, or gentamicin with low levels of HAMLET to demonstrate that the antibiotic-HAMLET combination works better to stop bacterial growth than either the antibiotic or HAMLET alone. By using low levels of HAMLET – specifically, subinhibitory concentrations that were less than the MIC – they showed that HAMLET does not contribute by stopping bacterial growth, but rather by helping the antibiotics stop bacterial growth.

The researchers also showed that specific concentrations of antibiotics and HAMLET could be used to optimize the antibacterial effect of the combination. But it gets better. The specific concentrations of HAMLET and an antibiotic were also shown to sensitize antibiotic-resistant bacteria to the antibiotic-HAMLET combination. In other words, when HAMLET is present, one can use a lower antibiotic concentration to stop bacterial growth even for antibiotic-resistant bacteria compared to the regular antibiotic dosage when the antibiotic acts alone. Way to be, HAMLET!

This may not seem relevant to the development of an infant, but here’s why I think it’s important to know about HAMLET. Many chemicals can be passed on through breast milk to the infant, including antibiotics. While most people taking antibiotics are taking them to combat “bad” bacteria, they may also prevent the growth of the “good” bacteria in a baby’s developing gut micobiome. This is because the presence of both HAMLET and an antibiotic may result in a powerful antibiotic combination that could possibly fight off those good bacteria. Another reason that it’s important to know about is if the baby is given antibiotics to fight off an infection. Perhaps the enhanced antibacterial activity may have an effect on the infant’s recovery.

The tests mentioned above were done outside of the body, but the researches also did tests in live mice. They looked at the effect of the antibiotic-HAMLET combination to kill bacteria growing in the upper part of the throat and biofilms (which grow on liquid-bathed, solid surfaces like teeth). In both situations, the antibiotic-HAMLET combination decreased bacterial growth better than either compound could by itself. If you need a recap of the many ways HAMLET works, look at Figure 1 for a detailed web chart.

Figure 2 – Different combination used to test HAMLET’s antibiotic activity and the effect on the bacteria. 

This protein clearly helps to inhibit bacterial growth, but only to a certain extent. Currently, the mechanism by which the HAMLET-oleic acid complex inhibits bacteria is not entirely known. To learn about HAMLET’s mode of action, the researchers performed a test in which they studied the uptake of antibiotic binding to the bacteria in the presence of low levels of HAMLET. It showed that for some antibiotics, HAMLET increased the bacterial uptake of that antibiotic. Another test showed that HAMLET uses an influx of calcium and kinase activity (which involves a protein adding a phosphate group to a target protein) to increase antibiotic activity. Using HAMLET or certain antibiotics alone typically results in lysis – a response which causes the cell to burst. When HAMLET is combined with antibiotics, however, lysis does not occur and the bacteria dies by another mechanism. If you’re wondering how this is important for breast milk and infants, take note that lysis can result in an immune response like inflammation. If a baby is taking antibiotics and lysis does not occur, maybe it can help remove speed up the infant’s recovery since the baby’s immune system does not respond like it would with cell lysis.

HAMLET is just as interesting if you look at it from an anti-tumor perspective. For several years, studies have shown that HAMLET selectively targets certain types of cancer cells while ignoring healthy cells. Perhaps this function helps keep cellular division in line and ensures tumors do not develop in the rapidly growing baby. How, exactly, they only target malignant cells is unknown, but researchers think it might have to do with an important cell-signaling protein called p53. This protein is highly involved in cell death by apoptosis, which is exactly how HAMLET kills tumor cells. In cancer cells, p53 is often mutated which possibly provides a more favourable binding site for HAMLET. As for the selective targeting, it might have to do with the reductive state of the cancer cells. Because cancer cells grow much faster than healthy cells, they have lower oxygen levels – this means they are reductive. Despite the mechanism being different than that of HAMLET’s antibiotic activity, the result is the same: cell death. See Figure  for the multiple roles HAMLET can play and where it all begins.

Unlike Hamlet who contemplates life versus death, bacteria and tumor cells have no choice in the matter when fighting HAMLET – they will surely die. Considering that α-lactalbumin can be used for lactose production in milk and then recycled to be used as an antibacterial and anti-tumor protein just shows versatile proteins can be! This new research showing the numerous applications of HAMLET’s antibiotic activity are providing us with more information about the great immunological benefits of breast milk.  Until next time, Maria

If you want to read the papers I looked at, here are the links: The human milk protein-lipid complex HAMLET sensitizes bacterial pathogens to traditional antimicrobial agents. http://www.ncbi.nlm.nih.gov/pubmed/22905269 Human alpha-lactalbumin made lethal to tumor cells (HAMLET) kills human glioblastoma cells in brain xenografts by an apoptosis-like mechanism and prolongs survival http://www.ncbi.nlm.nih.gov/pubmed/15026350

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Word art! Read into it for some surprisingly weird and interesting facts about breast milk. 

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Breast milk - as a recovery drink!

We have often heard how chocolate milk is one of the best recovery drinks to have after an intense workout, but has anyone ever heard of drinking breast milk? It has become quite the popular recovery drink to have in the past few years.

Most of the consumers are men who are looking for a healthy, nutritious, and natural energy booster to compensate for nutrients and energy they have lost after a workout. That being said, there is no sound evidence which suggests breast milk has the necessary amount of nutrients for a physically active adult man or woman. For many consumers, this trend started after realizing that adults can enjoy and reap the benefits from breast milk that babies do. But is it worth it? Does it work? It might not be worth the effort since breast milk is specifically tailored to newborns. In fact, it could be dangerous if the milk is obtained from an unknown source and could therefore contain many contaminants. Despite these possible dangers, many remain faithful to its seemingly beneficial effects. This trend was also sparked from the recommendation to drink breast milk while fighting cancer and undergoing chemotherapy. However, this is different than using it as a recovery drink: drinking breast milk during cancer is more understandable as the milk is easy to digest and provides many nutrients that a patient could be missing.

It seems that the overall impression from professionals is that consuming breast milk as a recovery drink has no significant benefits, if any at all. It certainly is not doing any harm, but many are skeptical that the nutrients in breast milk can supply the demand of an adult. Maybe it’s all mind over matter, but considering it can cost up to $2.30/oz. let's hope it does some good!

Maria Take a look at this article from The Washington Post: http://www.washingtonpost.com/news/morning-mix/wp/2014/05/29/the-newest-hottest-workout-recovery-beverage-human-breast-milk/

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Breast milk stem cells may be incorporated into baby - health - 03 November 2014 - New Scientist

Stems cells. They’re pretty neat, and if you haven’t heard of them let me explain. Simply put, they are undifferentiated cells that can develop into any type of cell in the body. That means they can become skin cells, blood cells, liver cells, you name it. They are often referred to as human embryonic stem cells (HESC) in the news. But why is this so important for breast milk? Turns out, breast milk contains maternally derived stem cells that are passed on to the newborn baby. These aren’t the same as HESC, but very similar. The discovery of stem cells in breast milk first came out around 8 years ago, but their function was still unknown. That is until recently. Dr. Foteini Hassiotou and her team have determined that stem cells in mice are passed from mother to offspring and dispersed to a number of different locations in the body. If you’re wondering how this topic fits into the theme of my blog, it is because these stem cells can theoretically develop into immune cells. What’s interesting about the research that is discussed in this article from NewScientist is its application towards our understanding of the function of breast milk components. In more recent years, research has considered the function that immune cells have played in the development of a healthy immune system in a baby as well as any allergies he or she may develop. This new research, however, suggests that the immune system of a baby might also be affected by stem cells. These cells can potentially divide and provide an added advantage to the immune system, since it is, after all, a crucial developmental stage for any infant.

What’s really fascinating is the many applications this research can be used for. It’s not limited to understanding breast milk components, but it can also explore, for example, immunotherapy, stem cell therapy, and tissue regeneration. From the perspective of breast milk, I think it’s safe to say that this new understanding of stem cell function can have lifelong beneficial effects on the nursing baby. As I mentioned in a previous post, I also want to focus on some of the key distinctions between natural breast milk and formula. Perhaps this research means that formula is missing not only key immunological properties (like antibodies), but also stem cells. This article just goes to show us how much there is to learn about breast milk and its benefits.

On that note, don’t forget the wise words of Benjamin Franklin: An ounce of prevention is worth a pound of cure.

So drink up! (Just kidding, stick to cow’s milk, please)

Maria If you have access to the research papers here are the links: Stem cell discover:  http://www.ncbi.nlm.nih.gov/pubmed/17440749 Dr. Hassiotou’s research:  http://www.fasebj.org/content/28/1_Supplement/216.4.short

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All About Me

Hi everyone, welcome to my blog!  I’m Maria, a fourth year biology-chemistry student in my last semester of my undergraduate degree. But I’m certainly more than just my degree; I love playing and listening to music, cooking delicious foods, watching Three’s Company, and volleyball. My interests don’t stop there and, despite my not getting enough exercise as I should and possible coffee addiction, I still have a passion for health and preventative medicine. And what a better time to set your health on the right track than at the get-go with the very best: breast milk!  I travelled to Honduras in 2014 and was amazed at the differences between our cultures! Such a beautiful country with so much culture everywhere. As a Canadian, I’m happy there is so much cultural diversity at home, but I still couldn’t help but notice one major difference. The women in Honduras breast feed their babies whenever and wherever they feel the need, and without hiding them under a blanket. Such freedom! This, as well as my summer job in a hospital, sparked my interest in pro-breastfeeding. Breastfeeding, it seems, has been a hush-hush topic for a while now in our culture and I’m happy that things have been turning around in the past several years! Pro-breastfeeding has come a long way in recent years mostly because of social awareness. But I’m excited to take a deeper look into some of the biochemistry together and dive into some progress and current research happening with regards to breast milk! Recent research has found that breast milk is associated with a healthy immune system in the baby, allergies, and an improved gut flora (the good bacteria in our guts). For this blog I will be focusing on the immunological properties of breast milk, how it has been attempted to be reproduced in formula and what effects these properties might have on allergies. Because everyone deserves peanut butter. Yum. But first, let me ask you what brought you to Banking on Breast Milk? Perhaps you are not one of the 22 other students in my class and you are ordinarily interested in the latest research in breast milk. Perhaps you were intrigued by the title. Perhaps you’re just curious. Whatever the reason, I hope it’s because you are as interested in this topic as I am! It isn’t exactly a topic that applies to everyone all the time, but that doesn’t make it any less important. A good start in life can make all the difference and I truly believe that breast milk is one of the best things a parent can do for their infant.

 I hope you stick around!

Maria

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In my blog, I'm going to be looking at the biochemistry of breast milk, and some recent discoveries about its chemical components and the effect they have on the nursing baby.