The Unregulated Probiotic Market.

BACKGROUND & AIMS: This narrative review provides an overview of the current regulation of probiotics, with a focus on those used for the dietary management of medical conditions (Medical Foods). FINDINGS: The probiotic market has grown rapidly, both for foods and supplements intended to enhance wellness in healthy individuals, and for preparations for the dietary management of disease. Regulation of probiotics varies between regions. Unless they make specific disease-related health claims, probiotics are regulated as food supplements and regulation is focused on the legitimacy of any claims, rather than efficacy, safety and quality. Many properties of probiotics are strain-specific, and safety and efficacy findings associated to specific formulations should not be generalized to other probiotic products. Manufacturing processes, conditions and ingredients are important determinants of product characteristics and changes to manufacturing are likely to give rise to a product not identical to the "original" in efficacy and safety if proper measures and controls are not taken. Current trademark law and the lack of stringent regulation of probiotic manufacturing mean that the trademark owner can commercialize any formulation under the same brand, even if significantly different from the original. These regulatory deficits may have serious consequences for patients where probiotics are used as part of clinical guideline-recommended management of serious conditions such as inflammatory bowel diseases, and may make doctors liable for prescribing a formulation not previously tested for safety and efficacy. CONCLUSIONS: Current regulation of probiotics is inadequate to protect consumers and doctors, especially when probiotics are aimed at the dietary management of serious conditions.

Pretreatment with the probiotic VSL#3 delays transition from inflammation to dysplasia in a rat model of colitis-associated cancer.

Evidence supports involvement of microflora in the transition of chronic inflammation to neoplasia. We investigated the protective efficacy of the probiotic VSL#3 in a model of colitis-associated colorectal cancer. Chronic colitis was induced in Sprague-Dawley rats by administration of trinitrobenzene sulfonic acid (TNBS), followed 6 wk later by systemic reactivation. To induce colitis-associated dysplasia and cancer, the animals received TNBS (intravenously) twice a week for 10 wk. One group received VSL#3 in drinking water from 1 wk before colitis induction until death. The colons were examined for damage and presence of dysplasia or cancer. Samples were analyzed for cell proliferation and apoptosis, vitamin D receptor (VDR) expression, angiogenic factors, and presence of alkaline sphingomyelinase or phosphatase. Microbial community composition was evaluated by terminal restriction fragment-length polymorphism analysis of the bacterial 16S rRNA gene. None of the probiotic-treated animals developed carcinoma, and no high-grade dysplasia was found in either the proximal or mid colon. In contrast, 29% of the animals in the control group developed carcinoma in one or more regions of the colon. VSL#3-treated animals had significantly less damage than the vehicle treated-controls in all areas of the colon, and this correlated with decreased richness and diversity of the mucosally adherent microbiota. Treatment with the probiotic increased the antiangiogenic factor angiostatin, VDR expression, and alkaline sphingomyelinase. We concluded that pretreatment with the probiotic VSL#3 can attenuate various inflammatory-associated parameters, delaying transition to dysplasia and cancer, thus offering its potential therapeutic use in patients with long-standing colitis.

Diet, but not oral probiotics, effectively reduces urinary oxalate excretion and calcium oxalate supersaturation.

We examined the effect of a controlled diet and two probiotic preparations on urinary oxalate excretion, a risk factor for calcium oxalate kidney stone formation, in patients with mild hyperoxaluria. Patients were randomized to a placebo, a probiotic, or a synbiotic preparation. This tested whether these probiotic preparations can increase oxalate metabolism in the intestine and/or decrease oxalate absorption from the gut. Patients were maintained on a controlled diet to remove the confounding variable of differing oxalate intake from food. Urinary oxalate excretion and calcium oxalate supersaturation on the controlled diet were significantly lower compared with baseline on a free-choice diet. Neither study preparation reduced urinary oxalate excretion nor calcium oxalate supersaturation. Fecal lactobacilli colony counts increased on both preparations, whereas enterococcal and yeast colony counts were increased on the synbiotic. Total urine volume and the excretion of oxalate and calcium were all strong independent determinants of urinary calcium oxalate supersaturation. Hence, dietary oxalate restriction reduced urinary oxalate excretion, but the tested probiotics did not influence urinary oxalate levels in patients on a restricted oxalate diet. However, this study suggests that dietary oxalate restriction is useful for kidney stone prevention.

Comment on: "Search and Selection of Probiotics that Improve Mucositis Symptoms in Oncologic Patients: A Systematic Review. Nutrients 2019, 11, 2322".

I have read the systematic review by Picò et al [...].

Rupture of the spleen after colonoscopy: a life-threatening complication.

During colonoscopy, the risk of injuring the spleen or other viscera except the colon is negligible. We report here a patient in whom spleen rupture did complicate the very early course of colonoscopy, but this remains an extremely rare complication with no more than 50 cases so far described. Diagnosis may be difficult, and the risk of spleen rupture seems to be greatest within 24 hours of colonoscopy. Mechanisms leading to spleen injury in the setting of colonoscopy are unclear; however, direct trauma, colon distension by insufflated air, and the excessive traction on the splenocolic ligament may be involved. Patients with splenomegaly and those with preexisting adhesions are at greater risk for this complication. Patients complaining of persistent abdominal pain after colonoscopy should be closely monitored and aggressively investigated for the suspect of spleen injury and rupture.

VSL#3 probiotic preparation has the capacity to hydrolyze gliadin polypeptides responsible for Celiac Sprue.

The native structure and distribution of gliadin epitopes responsible for Celiac Sprue (CS) may be influenced by cereal food processing. This work was aimed at showing the capacity of probiotic VSL#3 to decrease the toxicity of wheat flour during long-time fermentation. VSL#3 (10(9) cfu/ml) hydrolyzed completely the alpha2-gliadin-derived epitopes 62-75 and 33-mer (750 ppm). Two-dimensional electrophoresis, immunological (R5 antibody) and mass spectrometry analyses showed an almost complete degradation of gliadins during long-time fermentation of wheat flour by VSL#3. Gliadins non-hydrolyzed during fermentation by VSL#3 were subjected to peptic-tryptic (PT) digestion and analyzed by CapLC-ESI-Q-ToF-MS (Capillary Liquid Chromatography-Electrospray Ionization-Quadrupole-Time of Flight-Mass Spectrometry). Search for several epitopes showed the only presence of alpha2-gliadin-fragment 62-75 at a very low concentration (sub-ppm range). Compared to IEC-6 cells exposed to intact gliadins extracted from the chemically acidified dough (control), VSL#3 pre-digested gliadins caused a less pronounced reorganization of the intracellular F-actin which was mirrored by an attenuated effect on intestinal mucosa permeability. The release of zonulin from intestinal epithelial cells treated with gliadins was considerably lower when digested with VSL#3. Agglutination test on K 562 (S) cells showed that the PT-digest of wheat flour treated with VSL#3 increased the Minimal Agglutinating Activity of ca. 100 times. Wheat proteins were extracted from doughs and subjected to PT digestion. Compared to PT-digest from chemically acidified dough, celiac jejunal biopsies exposed to the PT-digest from the dough fermented by VSL#3 did not show an increase of the infiltration of CD3(+) intraepithelial lymphocytes. Proteolytic activity by probiotic VSL#3 may have an importance during food processing to produce pre-digested and tolerated gliadins for increasing the palatability of gluten-free products.

Probiotic preparation has the capacity to hydrolyze proteins responsible for wheat allergy.

This study was aimed at showing the capacity of probiotic VSL#3 to hydrolyze wheat flour allergens. Hydrolysis was investigated either by the use of baker's yeast bread treated with digestive enzymes and VSL#3, an experimental design that mimicked the activity of probiotics during gut colonization, or by the use of VSL#3 as a starter for dough fermentation, an experimental design that mimicked the predigestion of wheat flour proteins during food processing. Albumins, globulins, and gliadins extracted from wheat flour and chemically acidified and started dough and total proteins extracted from breads were analyzed by immunoblotting with pooled sera from patients with an allergy to wheat. Hydrolysis of wheat flour proteins was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis (2DE). Mass spectrometry matrix-assisted laser desorption and ionization-time of flight was used to identify some immunoglobulin E (IgE)-binding proteins. As shown by immunoblotting with sera from allergic patients, several IgE-binding proteins persisted after treatment of baker's yeast bread by pepsin and pancreatin. The signal of all these IgE-binding proteins disappeared after further treatment by VSL#3. As shown by SDS-PAGE and related immunoblotting and 2DE analyses, when VSL#3 was used as a starter for bread making, it caused a marked degradation of wheat proteins, including some IgE-binding proteins such as the putative transcription factor APFI and wheat alpha-amylase inhibitors. Indeed, the IgE-binding profile of the bread manufactured by VSL#3 was largely different from that of baker's yeast bread. The IgE-binding proteins that persisted in the bread made with VSL#3 were completely degraded by pepsin and pancreatin.

Efficacy and Safety of a Multistrain Probiotic Formulation Depends from Manufacturing.

BACKGROUND: Variability in probiotics manufacturing may affect their properties, with potential implications for their efficacy and safety. This is of particular concern with probiotic products destined for use in patients with serious medical conditions, including human immunodeficiency virus (HIV) infection. The purpose of the study was to carry out a series of experiments comparing the properties of the US-made probiotic formulation originally commercialized under the brand name VSL#3®, with those of the Italian-made formulation now commercialized under the same name. The US-made formulation has previously shown beneficial effects at the intestinal and neurological levels in HIV-infected subjects as well as in patients with inflammatory bowel diseases and hepatic encephalopathy. METHODS: Eleven subjects receiving combined antiretroviral therapy for HIV-1 were treated for 6 months with the US-made VSL#3 formulation. At baseline and 6 months, T-cells were analyzed for phenotype and activation markers, and fecal samples were analyzed for bifidobacteria, lactobacilli, and their metabolites. The fecal metabolome was assessed using 1H-NMR spectroscopy. Production of metabolites of interest by bacteria obtained from sachets of the two formulations was compared in vitro and their effects on a rat intestinal epithelial cell line (IEC-6) were assessed. Particular attention was paid to the metabolite 1,3-dihydroxyacetone (DHA). RESULTS: At 6 months, fecal samples showed a significant increase in the specific bacterial genera contained in the probiotic supplement. Immune activation was reduced as shown by a significant reduction in the percentage of CD4+CD38+HLA-DR+ T-cells at 6 months. Fecal concentrations of DHA decreased significantly. In vitro, significant differences in the production and metabolism of DHA were found between bacteria from the US-made and Italian-made formulations: the US-made formulation was able to metabolize DHA whereas the bacteria in the Italian-made formulation were producing DHA. DHA reduced the viability of Streptococcus thermophilus, reduced IEC-6 cell viability in a dose-dependent manner, and also led to a lower rate of repair to scratched IEC-6 cell monolayer. CONCLUSION: Our data, in conjunction with previously published findings, confirm that the new Italian-made formulation of VSL#3® is different from the previous US-made VSL#3 and therefore its efficacy and safety in HIV-infected subjects is still unproven.

Detection of alkaline sphingomyelinase activity in human stool: proposed role as a new diagnostic and prognostic marker of colorectal cancer.

OBJECTIVES: Intestinal alkaline sphingomyelinase, by exerting a major role in dietary sphingomyelin digestion, is responsible for the generation of messengers able to trigger the rapid turnover and apoptosis in intestinal epithelial cells. Markedly reduced mucosal alkaline sphingomyelinase activity has been associated with human colorectal neoplasms. The aim of this study was to analyze the alkaline sphingomyelinase activity in feces from healthy subjects and colorectal adenocarcinoma patients and to correlate it with the enzyme activity in intestinal tissues. MATERIALS AND METHODS: The enzyme activity was measured both in the intestinal samples from 12 healthy controls and 51 patients with colorectal adenocarcinoma (tumoral and paratumoral tissue) and in the fecal samples of 34 healthy subjects and 29 patients with adenocarcinoma. The relation between sphingomyelinase activity and Dukes' stage, cell differentiation degree, age, and gender was also analyzed. RESULTS: Alkaline sphingomyelinase was significantly decreased (P < 0.001; mean reduction >90%) in tumoral intestinal mucosa of patients compared with controls independently of Dukes' stage and tumor differentiation grade. Interestingly, the enzyme activity in histologically normal paratumoral tissues was statistically lower than control samples (P < 0.001). As occurs in neoplastic tissues, a relevant mean reduction (P < 0.0001; almost 90%) of alkaline sphingomyelinase was revealed in stool samples from tumor patients when compared with controls. CONCLUSION: These findings may have implications for cancer biology and perhaps also for the design of clinical test, thus suggesting that the fecal sphingomyelinase activity could really reflect the human intestinal mucosa enzyme level and could represent a new marker for human colorectal adenocarcinoma, mainly taking into account its early appearance in intestinal neoplasms.

Effects of probiotic bacteria (VSL#3) on the polyamine biosynthesis and cell proliferation of normal colonic mucosa of rats.

BACKGROUND: Probiotics seem to possess tumour inhibitory properties, but few studies have investigated their actions on the cell proliferation of normal colonic mucosa. The effects of a probiotic mixture (VSL#3) on polyamine biosynthesis, Ki-67 levels and apoptosis in the normal colon of rats were studied. MATERIALS AND METHODS: For a 4-week period, 20 rats were fed a VSL#3 solution and 20 rats a saline solution. Samples from the colonic mucosa were collected at the end of treatment. Polyamines were detected by HPLC, ornithine decarboxylase activity by a radiometric technique, and apoptosis and Ki-67 by histochemical and immunohistochemical methods. RESULTS: VSL#3 caused a significant decrease in colonic polyamine levels, ornithine decarboxylase activity and Ki-67 compared to controls. A significant increase in the apoptotic index was also observed. CONCLUSION: Probiotics could also reduce proliferation rates in a condition not affected by hyperproliferative or neoplastic growth, when the normal control mechanisms are still completely effective.

Probiotic lactobacilli: an innovative tool to correct the malabsorption syndrome of vegetarians?

Vegetarians may have subtle nutritional deficiencies which have been related to the occurrence of an unrecognized malabsorption syndrome. The excess phytate content in cereals, nuts, legumes and oilseeds which represent the mainstay of their food intake, seems to play a central role in the pathogenesis of this malabsorption syndrome as an inverse relationship has been shown to link the phytate content of the diet with the intestinal absorption of trace minerals and proteins. We postulate that manipulating the endogenous digestive microflora of subjects on a vegetarian diet through administering probiotic lactic bacteria would represent an innovative tool to counteract the occurrence of the malabsorption syndrome dependent on the high phytate content of their diet. Even though there are no data about the composition of endogenous digestive microflora in subjects on a vegetarian diet, we expect that probiotic lactobacilli can interact with or affect distinct yet interrelated components within the intestinal milieu, such as epithelial cells, enteric flora, and/or mucosal immune cells. This would ultimately translate into the correction of the unregulated mechanisms implicated in the altered intestinal absorption of trace metals and proteins commonly seen in vegetarians. Clinical experience with probiotic therapy of patients with inflammatory bowel disease fully agrees with this view. One additional point of interest is that probiotic lactobacilli, and other species of the endogenous digestive microflora as well, are an important source of the enzyme phytase which catalyses the release of phosphate from phytate and hydrolyses the complexes formed by phytate and metal ions or other cations, rendering them more soluble ultimately improving and facilitating their intestinal absorption. The regular intake of probiotic preparation, may represent a cheap and safe tool in order to convert a diet with a low potential for bioavailability of trace minerals and proteins, such as the vegetarian diet, into a diet with a high bioavailability potential. The benefit of such an approach would not be restricted to vegetarians.

L-carnitine reduces lymphocyte apoptosis and oxidant stress in HIV-1-infected subjects treated with zidovudine and didanosine.

Apoptosis is critical to the progression of human immunodeficiency virus-1 (HIV-1) infection. It appears reasonable that antiretroviral therapies may not achieve a full control of the infection in the absence of an impact on apoptosis. We assigned 20 asymptomatic HIV-infected subjects with advanced immunodeficiency to receive either zidovudine (AZT), and didanosine (DDI) or the same regimen plus L-carnitine, a known antiapoptotic drug, for 7 months. Immunologic and virologic parameters were measured at baseline and after 15, 60, 120, and 210 days of treatment. We assessed on each time point the following: (a) the frequency of peripheral blood apoptotic CD4 and CD8 lymphocytes, CD4 and CD8 cells with disrupted mitochondrial membrane potential, and CD4 and CD8 cells undergoing oxidant stress; (b) the expression of the molecular markers of apoptosis Fas and caspase-1; and (c) the expression of p35/cdk-5 regulatory subunit that is involved in regulating cell survival and apoptosis. Absolute CD4 and CD8 counts and plasma viremia were also measured. Apoptotic CD4 and CD8 cells, lymphocytes with disrupted mitochondrial membrane potential, and lymphocytes undergoing oxidant stress were greatly reduced in subjects treated with AZT and DDI plus L-carnitine compared with those who did not receive L-carnitine. Fas and caspase-1 were down-expressed and p35 over-expressed in lymphocytes from patients of the L-carnitine group. No difference was found in CD4 and CD8 counts and viremia between the groups. No toxicity of L-carnitine was recognized. The addition of L-carnitine is safe and allows apoptosis and oxidant stress to be greatly reduced in lymphocytes from subjects treated with AZT and DDI.

Probiotic lactobacilli: a new perspective for the treatment of inflammatory bowel disease.

Inflammatory bowel disease, such as ulcerative colitis and Crohn's disease, results from an interaction between susceptibility genes, the host's bacterial environment, gut barrier defects, and immunological factors. New management approaches have been evolved from advances in our understanding of the pathobiology of this common gut disorder In particular, the therapeutic manipulation of the bacterial microenvironment in the gut seems to offer an innovative tool for the treatment of those patients. Since the gut is a highly sensitizing organ that contributes to the systemic immune response, potent treatments need to be developed to reduce gut inflammation in this disorder. Recent studies have demonstrated that probiotic lactobacilli, and also immunostimulatory DNA sequences from those same bacteria have an important anti-inflammatory potential in this context. Future research should better define among patients with inflammatory bowel disease the various clinical phenotypes with the greatest potential of response to probiotic treatment. Identification of the genes leading to the disease and a rather better understanding of the underlying immunoregulatory abnormalities will be crucial steps to define the different profiles of interaction between endogenous digestive bacterial flora and the immune system in each individual patient. Such advances will probably lead to targeting of effective treatments, including bacteriotherapy with probiotic lactobacilli, to subsets of patients with inflammatory bowel disease.

Modulation of apoptosis and improved redox metabolism with the use of a new antioxidant formula.

Oxidative stress is involved in the pathogenesis of a wide spectrum of diseases, implicating that strategies directed at counterbalancing oxidative processes could have a role in clinical medicine. There is also an evidence that oxidative stress acts as a major determinant of apoptotic cell death. Many studies have reported favourable effects of antioxidant formulas on several parameters of the oxidant-antioxidant balance, but none of them has focused whether antioxidant formulas could modulate apoptosis. We investigated in 20 healthy individuals the effect of supplementation with a formula containing alpha-tocopherol, alpha-lipoic acid, coenzyme Q(10), carnitines, and selenomethionine, on plasma oxidant status and peroxide levels, erythrocyte antioxidant enzymes, lymphocyte apoptosis, and generation of ROS at the mitochondrial level. Control subjects received only carnitines or an incomplete formula with alpha-tocopherol, alpha-lipoic acid, coenzyme Q(10), and selenomethionine. Supplementation with the complete formula resulted in a significant increase in the plasma antioxidant status that was mirrored by a decrease in blood peroxide levels and a reduced generation of ROS at the mitochondrial level. This was associated with a significant decrease in the frequency of peripheral blood lymphocytes, with either CD4 or CD8 phenotype, undergoing apoptosis. Less consistent results were found when either incomplete formula was used. Our study suggests that supplementation with antioxidant formulas can modulate the process of apoptosis under in vivo conditions. The clinical potential of this strategy in the treatment of diseases with an elevated commitment to apoptosis should be explored.

Carnitines and its congeners: a metabolic pathway to the regulation of immune response and inflammation.

Carnitine and its congeners may regulate the immune networks, and their influence on functions of immune cells predominantly or exclusively relies on carnitine-dependent energy production from fatty acids. A reduced pool of carnitines has been demonstrated in either serum or tissues, or both, from patients with a wide spectrum of disorders characterized by unregulated or impaired immune responses ranging from sepsis syndrome to systemic sclerosis, infection with human immunodeficiency virus, and chronic fatigue syndrome. Furthermore, experimental studies have consistently reported that the deranged immune responses and the less efficient inflammation towards infectious organisms associated with aging may be enhanced or modulated by treatment with carnitines. There is also evidence that carnitine deprivation could adversely affect the course of the sepsis syndrome, at least in experimental models, and preliminary studies suggest that carnitine deficiency is ultimately implicated in the pathophysiology of endotoxin-mediated multiple organ failure. Several data indicate that carnitine deficiency is a contributing factor to the progression of infection with human immunodeficiency virus, and carnitine therapy in those patients could counteract the unregulated process of lymphocyte apoptosis and improve CD4 counts. Some case reports have suggested the use of carnitine for the treatment of the severe lactic acidosis that complicates in some patients the use of reverse transcriptase inhibitors.