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As read on the SV Facebook: Baking makes gluten resilient to digestion, the presence of protein does the opposite. The food matrix makes the difference.

In view of the fact that "gluten" is or isn't (literally) in everyone's mouth or tummy, I thought that it may make sense to keep you up-to-date on the latest interesting research in the area of non-celiac gluten sensitivity and celiac disease.

Don't worry I am not going to bother you with the typical Internet bogus about how cancer, obesity and everything else we are or are not suffering from (yet) is triggered by gluten. What I will do, though, is to summarize and discuss the results of two recent studies and one review on the connection between our microbiome and our very individual susceptibility to gluten-related health problems

• Celiac disease and non-celiac gluten sensitivity may be all about the microbiome and begin at the very moment you're born via cesarean section and worsen when you're not breastfed - In a soon-to-be-published review in Nutrients Cenit et al. try to elucidate whether gluten intolerance and celiac disease are consequences or triggers of significant imbalances in the bacterial composition of the human microbiome and how one or the other may eventually come about..
  
  As the authors point out, there are in fact studies which suggest that the early colonization of the infant’s gut in conjunction with environmental factors (e.g., breast-feeding, antibiotics, etc.) could influence the development of our kids' oral tolerance to gluten.
  

  Figure 1: Proposed model for celiac disease (CD) pathogenesis. Specific host genetic makeup and environmental factors could promote the colonization of pathobionts and reduce symbionts, thus leading to dysbiosis. Dysbiosis may contribute to disrupting the immune homeostasis and gut integrity, thereby favoring CD onset and aggravating the pathogenesis (Cenit. 2015).

  In that, the early colonization of the intestinal tract is of particular importance, because it programs a normal or abnormal immune reaction to gluten (and other potential allergens). It is thus no wonder that celiac disease and a whole host of other autoimmune diseases have been linked to a lack of, or an improper early colonization of the intestinal tract and the consequential misprogramming of the immune cells. In that, it has been suggested that the resulting dysbiosis may affect autoimmunity by altering the balance between tolerogenic and inflammatory members of the microbiota and, therefore, the host immune response.
  
  Needless to say that the increased risk of autoimmune diseases is a standalone problem. It is after all not a mere reaction to the bacteria, but a bacterially induced phenomenon that involves the epigentic reprogramming of a whole host of genes. This process is however (unfortunately) so complex that we haven't yet fully understood the individual bacteria-gene and gene-gene interactions. Everyone, who tells you otherwise is lying - probably to sell his snake oil or snake oilish lifestyle advise.
  

  Figure 2: While there's one outlier, 3/4 studies on the effects of breastfeeding when the first gluten containing foods are introduced show significantly reduced risks of developing celiac disease (Akobeng. 2006).

  Among the few things we do know, though, is that breastfeeding and the way it promotes the early colonization of the gut with Bifidobacterium spp. is associated with a reduced risk of gluten intolerance. This is particularly true, if gluten containing foods are introduced while the kids are still breastfed (-52% according to a meta-analysis by Akobeng et al. 2006).

• The fact that these benefits do not apply for every breastfed child may be explained by (epi-)genetic polymorphisms of the mother, such as the altered concentration of several immune markers that have been observed by several researchers in the breast milk of mothers with celiac disease (Olivares. 2014). If that's in fact the case, it's hardly astonishing that the number of celiac patients began to rise when the use of formula peaked and is exploding now that more and more women with celiac disease (or non-celiac gluten sensitivity) are feeding their children with "non-protective" breast milk. We must be careful, though, not to jump to conclusions. There are, after all, as Cenit et al. point out "no robust prospective studies revealing how differences in breast milk composition and intestinal microbiota acquisition and evolution early in life might ultimately protect or contribute to CD onset" (Cenit. 2015).
  
  A similar healthy skepticism is necessary with respect to the link of cesarean sections and an increased susceptibility to gluten sensitivity (Dominguez-Bello. 2010). While it would appear logical to assume that the lack of exposure to the vaginal microbiome may contribute to the previously mentioned misprogramming of the immune system, it would be overtly simplistic to assume that gluten wouldn't be a problem if we were all breastfed and born the natural way.
  

  Figure 3: Increases in risk of full-blown celiac, intestinal inflamation and the presence of markers of celiac disease in the blood in subjects with previous exposure to antibiotics; in all fairness it must be said that the risk increase decreased when individuals who were exposed within the last 24m were excluded - even then the reduced 30% increase was statistically significant and practically relevant, though (Mårild. 2013)

  In conjunction with the indisputable link between the (early) use of antibiotics (Mårild. 2013 | see Figure 3) and the first successful efforts to ameliorate the chronic inflammation in celiac guts with prebiotics, there is yet little doubt that the "right" microbial make-up may be what distinguishes celiacs from patients with non-celiac gluten insensitivity and the still large number of people who don't appear to react to gluten at all.
• Study in healthy subjects, celiacs and their relatives suggests that the way your bacteria metabolize gluten may make you sick - From the first study, or rather review, I've analyzed in this feature article we've already learned that the inability to digest or handle gluten may be transmitted via certain immune factors in the breast milk from mother to child. It is thus particularly interesting that scientists from the Universidad de Léon in Spain who compared the stool of sixteen healthy volunteers on normal diet, eleven healthy volunteers on gluten-free diet (GFD), seventy-one relatives of CD patients on normal diet and sixty-nine relatives on GFD for several proteolytic activities, cultivable bacteria involved in gluten metabolism, SCFA and the amount of gluten found that significant differences in how celiac disease patients metabolized gluten.

• Table 1: Cultivable bacteria involved in gluten metabolism isolated from faeces of healthy subjects, active coeliac disease patients and firstdegree relatives (Caminaro. 2015).

  In contrast to healthy volunteers, their feces showed a significantly higher glutenasic activity (FGA), tryptic activity (FTA), SCFA, but lower levels of faecal gluten. That's interesting, yet counter-intuitive. After all, we've previously thought that one of the main problems of celiac disease and non-celiac gluten sensitivity is that the proteins are not broken down. Rather than that the results of the study at hand suggest that celiacs harbour bacteria that generate immunogenic peptides or pro-inflammatory metabolites which are the actual triggers of the problem (otherwise they'd have to poo out at least as much gluten as the other subjects on the gluten-free diets).
  
  Needless to say that this result does, once more, point toward fecal transplants or prebiotics as potential future treatment options celiac disease and non-celiac gluten sensitivity... with the only problem being: We don't know yet which bacteria we want to support and which to annihilate to solve the problem. If you look at the data in Table 1, though, killing the Clostridium and promoting the Lactobacillus population could be a first step to reinstalling a less celiac-prone gut microbiome.
• More 60% of non-celiac gluten patients don't react to gluten in randomized clinical study - That's certainly an impressive number Zanini et al. report in their latest paper in Alimentary Pharmacology and Therapeutics (Zanani. 2015).
  
  

  Let's hope aspergillus niger does not produce the wrong proteins when breaking down gluten otherwise it would make NCGS worse not better. Thus, further studies are needed.

  In the corresponding study the researchers studied 35 non-CD subjects (31 females) that were on a gluten-free diet (GFD), in a double-blind challenge study. Participants were randomised to receive either gluten-containing flour or gluten-free flour for 10 days, followed by a 2-week washout period and were then crossed over. The main outcome measure was their ability to identify which flour contained gluten. Secondary outcome measures of the study from the University and Spedali Civili of Brescia in Italy were based upon Gastrointestinal Symptoms Rating Scale (GSRS) scores (criteria & results see Figure 4).
  
  In contrast to what the Internet experts are trying to make you believe, only 12 and thus hardly more than 34% of the allegedly highly gluten-intolerant patients were able to tell when they were fed gluten-containing flour based on increases in pain, reflux, indigestion, diarrhoea, and constipation.
  

  Figure 4: Increased severity of symptoms according to whether subjects were able to distinguish whether they were fed a gluten-containing or gluten-free diet (Zanani. 2015).

  Seventeen participants (49%), on the other hand, swore black and blue that they had been fed the gluten-containing flour when they were on the gluten-free diet (and if you look at the data in Figure 4, they even felt worse than those who were actually sensitive ;-).
  
  Now, what's most intriguing about this is that the study proves that there's a non-negligible noocebo effect involved, when it comes to non-celiac gluten sensitivity. One that's powerful enough to have people experience real increases in pain, reflux, indigestion, diarrhoea, and constipation... that tells you something about how infections reading too much bogus on the internet is, right?

References:

• Akobeng, Anthony K., et al. "Effect of breast feeding on risk of coeliac disease: a systematic review and meta-analysis of observational studies." Archives of disease in childhood 91.1 (2006): 39-43.
• Caminero, et al. "Differences in gluten metabolism among healthy volunteers, coeliac disease patients and first-degree relatives." British Journal of Nutrition (2015): Ahead of print.
• Dominguez-Bello, Maria G., et al. "Delivery mode shapes the acquisition and structure of the initial microbiota across multiple body habitats in newborns." Proceedings of the National Academy of Sciences 107.26 (2010): 11971-11975.
• Mårild, Karl, et al. "Antibiotic exposure and the development of coeliac disease: a nationwide case–control study." BMC gastroenterology 13.1 (2013): 109.
• Newnham, Evan D., et al. "Adherence to the gluten‐free diet can achieve the therapeutic goals in almost all patients with coeliac disease: A five‐year longitudinal study from diagnosis." Journal of gastroenterology and hepatology (2015).
• Olivares, Marta, et al. "Human milk composition differs in healthy mothers and mothers with celiac disease." European journal of nutrition 54.1 (2014): 119-128.
• Özkan, T., T. Özeke, and A. Meral. "Gliadin-specific IgA antibodies in breast milk." Journal of international medical research 28.5 (2000): 234-240.
• Zanetti, et al. "Randomised clinical study: gluten challenge induces symptom recurrence in only a minority of patients who meet clinical criteria for non-coeliac gluten sensitivity." Alimentary Pharmacology and Therapeutics (2015): Ahead of print.

2/3 of this can may suffice to make a difference. Whether this difference is (a) relevant or (b) irrelevant is yet as questionable as whether the changes the scientists observed will (i) have a negative (ii) a positive or (iii) no effect.

As a waredownloadsoft user you know that the whole craze about aspartam and sucralose is overblown. You will also know that any potential "pro-insulinogenic" effects occurred only in less than a handful of human studies. If they did, though, they occurred in response to the ingestion of artificial sweeteners and glucose or other insulinogenic carbohydrate sources (learn more). Against that background it's also not surprising that in clinical trials vs. observational bogus, artificial sweeteners have been shown to help people with weight problems lose body fat (learn more).

The one thing about the myth of the bad sweetener that has yet not been completely debunked, though, revolves around their negative effects on the human gut microbiome.
You will remember from my previous post on this topic that - as (unfortunately) usual - all empirically valid data we have is based on rodent trials. In our mammalian cousins, the consumption of artificially sweetened products on top of an obesogenic diet has in fact been shown to have an additional effect on the modulation of the gut bacteria that appears to make turn an already "bad" diet into a nightmare (learn more). It is yet still very questionable, which part of the research can be translated to humans and to which subgroup of the population this would apply. Even if we assume a 1:1 translation from rodent to human, we would after all have to exclude most of you, because none of you will be consuming a hypercaloric, hyper-processed high fat + high carbohydrate diet (at least that's what I'd hope).

If we trust the results of a soon-to-be-published observational study from the George Mason University, though, our gut microbiomes could be in danger - although no one knows for sure.
In their thorough, but small scale (N=31) analysis, Cara L. Frankenfeld and her colleagues analysed the fecal sample of their subjects using Multitag Pyrosequencing. This allowed them to compare the bacterial abundance and bacterial diversityacross consumers and non-consumers of aspartame and acesulfame-K using non-parametric statistics and UniFrac analysis, respectively.

To predict some of the consequences of possible difference in the bacterial make-up Frankenfeld et al. applied a phylogenetic investigation of the communities by reconstruction of unobserved states (PICRUSt) in order to predict mean relative abundance of gene function.

Table 1: There were no sign. differences in BMI, energy intake, total carbohydrate and added sugar intake or the "quality" of the diet (as assessed on the Healthy Eating Index) between AS consumers and non-consumers (Frankenfeld. 2015)

Thus, the results of the gene function analysis must be met with a healthy degree of skepticism, because unlike the bacterial counts and diversity, which was also just estimated based on what "left" the subjects in form of feces, the gene assay is a model-based result... think of it like the weather forecast, one of which studies say it's relatively reliable (Langille. 2013).
Among the seven aspartame consumers and seven acesulfame-K consumers (some consumed both), the researchers did indeed find some significant differences in the bacterial make-up compared to those subjects who had abstained from consuming sweeteners in the four days before the fecal samples were collected. I quote from the FT (Frankenfeld. 2015):

• Bacteriodetes and Firmicutes had the highest median abundances and together accounted for the majority of the bacterial class representation in all individuals. 
• The median Bacteriodetes:Firmcutes ratio did not significantly differ across aspartame non-consumers (0.96, range: 0.15-2.97) and consumers (1.08, range: 0.69-1.87), (median test p-value=0.60). 
• There was no overall visual clustering of individuals by acesulfame-K consumption . 
• Overall bacterial diversity evaluated with UniFrac analysis was different across consumers and non-consumers, but there were no significant differences in relative abundance of gene function across consumers and non-consumers (Figure 3).
• There were no observable difference in the three individuals who consumed both aspartame and acesulfame-K (Supplemental Figure 1). 

So, let's briefly sum this up. In line with the overall hysteria about sweeteners, the changes were more significant in the aspartame group (p<0.01) than they were in the acesulfame-K group (p=0.03), but this could be explained by the simple fact that the subjects consumed significantly more aspartame (one can of diet coke contains 150mg, by the way) than acesulfam-K (5.3 mg/day to 112 mg/day vs. 1.7 mg/day to 33.2 mg/day).

Figure 1: Yes, there were differences in the bacterial make-up of the gut microbiome of the four groups, but no one can tell you what these or the vast individual differences in the groups mean for your health (Frankenfeld. 2015) !

Against that background, the "aspartame is the worst" hypothesis could neither be refuted nor supported based on the study at hand, even if the results would signify overall ill-health effects. With all three potentially important markers remaining unchanged, though, this is not the case:

• the ratio of mostly "bad" gram-negative bacteriodetes and "good" gram positive bacteria remained the same - it's thus hard to argue that the subjects who consumed artificial sweeteners had an unhealthier gut microbiome
• there was no general reduction in gut bacteria, which would indicate a general anti-microbial effect of artificial sweeteners as it occurs with antibiotics - it's thus hard to argue that the anti-microbial effects (which don't exist) of artificial sweeteners would leave you similarly defenseless and open to colonization with "bad" bacteria as antibiotics.
• the gene essays say that despite the differences in the numbers of certain bacteria, the gene expression is the same - it's thus hard to argue that there was an epigenetic effect of artificial sweeteners that precipitates us to obesity or even makes us sick / diabetic / whatever

Probiotics under urgently needed scrutiny - This is the first study to test for antibiotic resistances and to highlight the discrepan- cy between label claims and the actual number of live bacteria in supplements.

There have been plenty of good news about probiotic supplements in the news (including the waredownloadsoft News), lately. One thing that is often forgotten, though, is that the effect of the supplements depends on (a) the exact type of bacteria that are in the pills, (b) the ratio of the different strains and (c) the number of bacteria that are still alive.

Unfortunately, this important truth is rarely mentioned in the edutainment articles on probiotics in the laypress and sales pitches you will find all over the Internet.

Another thing, even you may not have thought about yet is however the potential occurrence of antibiotic resistances among the bazillions of bacteria in your allegedly healthy probiotic supplements.
A group of people who thought of this hitherto overlooked problem are researchers from the  King Abdullah University of Science and Technology in Saudi Arabia and the UCSI University in Malaysia (Wong. 2015). In their recent paper in the scientific journal Nutrition Journal, the international group of researchers are the first to highlight a previously ignored problem i.e. the possibility that certain genes that make bacteria resistant to antibiotics "could transfer to pathogens sharing the same intestinal habitat" - an event that is, as the scientists rightly point out, "conceivable considering the fact that dietary supplements contain high amounts of often heterogeneous populations of probiotics" and could thus "confer pathogens protection against commonly-used drugs" (Wong. 2015).

MRSA in your probiotic supplements? 

Against that background and in view of the numerous reports of antibiotic resistant probiotics in food and biological sources, the antibiogram of probiotics from dietary supplements remains elusive.

Figure 1: Petri dishes from the antibiotic test (top). If the antibiotics still worked on the bacteria in the probiotics, they all should be dead. As you can see (in the graph, as well), that's by no means the case (Wong. 2015).

In fact, Wong et al. are apparently the first researchers to screen five commercially available dietary supplements (the full names were not disclosed) for resistance towards antibiotics of different classes - with somewhat disconcerting results, namely:

• Even probiotics that help weight loss, could transfer antibiotic resistances.

  Probiotics of all batches of products were resistant towards vancomycin.
• Several batches of probiotics from four different brands were also resistant towards streptomycin, aztreonam, gentamycin and / or ciprofloxacin antibiotics (this includes the US and Austrian products, i.e. Cn and Bn, respectively)
• The fifth brand showed a unique resistance towards gentamycin, strepto- mycin and ciprofloxacin antibiotics. 

Now, as previously pointed out, this does not mean that "bad" bacteria which will always be present in your gut, will automatically acquire the same resistances, but the mere fact that it is possible should tell you that the current hype over probiotics as the "go-to supplement" everyone should take is unwarranted, or at least premature.

You're not getting what you're paying for!

The problem with antibiotic resistances is yet not the only intriguing result of Wang's study. The researchers analyses also revealed that you're not just getting more (albeit unwanted) ingredients that you're paying for, they also found a significant discrepancy between the enumerated viable bacteria amounts and the claims of the manufacturers.

Figure 2: Non-strain specific essay that evaluated the number of live bacteria in the products. The products from producers Bi, Bg, and L didn't just contain significantly less living bacteria than the manufacturers claim, the number is even so low that it is absolutely certain that they are 100% useless. The good news may be that the low dose supplements from the Austria(BN) and USA (CN) contained either more or at least roughly the amount of bacteria on the label (Wong. 2015).

In other words, while the scientists claim that you would get more than enough viable bacteria from their product to have a significant impact on your intestinal microbiome, the reality is that many of the good bacteria are dead before you even open the package.
References:

• Wong, Aloysius, et al. "Detection of antibiotic resistance in probiotics of dietary supplements." Nutrition journal 14.1 (2015): 1-6.