Life Extension Magazine®

Issue: Dec 2009

Vitamin D

Antimutagenic potency of chlorophyllin in the Salmonella assay and its correlation with binding constants of mutagen-inhibitor complexes.

Chlorophyllin (CHL) is a water-soluble salt of chlorophyll that exhibits antimutagenic activity in short-term genotoxicity assays and inhibits carcinogen-DNA binding in vivo. The antimutagenic potency of CHL was studied against several structurally related heterocyclic amines using the Salmonella assay. The mutagens included 2-amino-3-methylimidazo[4,5,-f]-quinoline (IQ) and seven related IQ-type compounds, and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and three additional non-IQ-type compounds. No relationship was observed between mutagenic potency (revertants/ng mutagen) and antimutagenic potency when expressed in terms of the CHL dose/plate-inhibiting mutagenicity by 50 percent (I50). However, a correlation was observed between mutagenic potency and the mole ratio of CHL to mutagen giving 50% inhibition (MR50), with most mutagens requiring several hundredfold to several thousandfold molar excess of CHL for inhibition. In spectrophotometric studies, CHL formed noncovalent molecular complexes with the heterocyclic amines, with binding constants in the range 3-13 x 10(3) M-1. Binding constants were inversely correlated with I50 and MR50 values, i.e., with increasing strength of complex formation less CHL/plate and a lower mole ratio of CHL to mutagen was required to inhibit mutagenicity. The results support an inhibitory mechanism in which chlorophylls operate as “interceptor molecules,” interacting with carcinogens and mutagens directly and limiting their bioavailability.

Environ Mol Mutagen. 1993;22(3):164-71

Chlorophyll and chlorophyllin as modifiers of genotoxic effects.

Reports on an inverse relationship between the consumption of fresh vegetables and human gastrointestinal cancer have been followed by screening for the protective activity of a large number of plant extracts, including leafy vegetables. Chlorophyll is ubiquitous in all green plant parts. Chlorophyllins are derivatives of chlorophyll in which the central magnesium atom is replaced by other metals, such as cobalt, copper or iron. An attempt has been made in this article to review the relative efficacy of chlorophyll and chlorophyllin in modifying the genotoxic effects of various known toxicants.

Mutat Res. 1994 Dec;318(3):239-47

Chlorophyllin intervention reduces aflatoxin-DNA adducts in individuals at high risk for liver cancer.

Residents of Qidong, People’s Republic of China, are at high risk for development of hepatocellular carcinoma, in part from consumption of foods contaminated with aflatoxins. Chlorophyllin, a mixture of semisynthetic, water-soluble derivatives of chlorophyll that is used as a food colorant and over-the-counter medicine, has been shown to be an effective inhibitor of aflatoxin hepatocarcinogenesis in animal models by blocking carcinogen bioavailability. In a randomized, double-blind, placebo-controlled chemoprevention trial, we tested whether chlorophyllin could alter the disposition of aflatoxin. One hundred and eighty healthy adults from Qidong were randomly assigned to ingest 100 mg of chlorophyllin or a placebo three times a day for 4 months. The primary endpoint was modulation of levels of aflatoxin-N(7)-guanine adducts in urine samples collected 3 months into the intervention measured by using sequential immunoaffinity chromatography and liquid chromatography-electrospray mass spectrometry. This aflatoxin-DNA adduct excretion product serves as a biomarker of the biologically effective dose of aflatoxin, and elevated levels are associated with increased risk of liver cancer. Adherence to the study protocol was outstanding, and no adverse events were reported. Aflatoxin-N(7)-guanine could be detected in 105 of 169 available samples. Chlorophyllin consumption at each meal led to an overall 55% reduction (P = 0.036) in median urinary levels of this aflatoxin biomarker compared with those taking placebo. Thus, prophylactic interventions with chlorophyllin or supplementation of diets with foods rich in chlorophylls may represent practical means to prevent the development of hepatocellular carcinoma or other environmentally induced cancers.

Proc Natl Acad Sci U S A. 2001 Dec 4;98(25):14601-6

Analysis of the therapeutic effect of sodium copper chlorophyllin tablet in treating 60 cases of leukopenia.

OBJECTIVE: To evaluate the efficacy and safety of sodium copper chlorophyllin (trademarked as “Yebaike Tablet which is abbreviated as YBK in treating leukopenia. METHODS: One hundred and five patients with leukopenia caused by various factors were randomized into 3 groups. The 60 patients in the YBK group took orally YBK Tablets at the dose of 40 mg, three times per day, the 30 patients in the leucogen group were treated with Leucogen Tablets at the dose of 20 mg, three times per day, and the 15 patients in the placebo group were administered with vitamin C tablets 100 mg, three times per day. All the subjects were treated for 1 month. The change of peripheral leucocytes count after treatment and adverse drug reaction that occurred in patients were studied. RESULTS: In the 60 patients treated with YBK, the treatment proved to be markedly effective in 34 cases, effective in 17 and ineffective in 9, the total effective rate being 85%, which was significantly higher than that in the placebo group (26.7%, P < 0.01) and similar to that in the leucogen group (83.3%, P > 0.05). No adverse reaction was found in the treatment course. CONCLUSION: YBK can be used in the treatment of leukopenia caused by various factors, satisfactory in efficacy and safe in use.

Chin J Integr Med. 2005 Dec;11(4):279-82

The importance of carcinogen dose in chemoprevention studies: quantitative interrelationships between, dibenzo[a,l]pyrene dose, chlorophyllin dose, target organ DNA adduct biomarkers and final tumor outcome.

Chlorophyllin (CHL) is a potent antimutagen in vitro, an effective anti-carcinogen in several animal models, and significantly reduced urinary biomarkers of aflatoxin B(1) (AFB(1)) exposure in a human population. Here we report an expanded analysis of CHL chemoprevention using the potent environmental hydrocarbon dibenzo[a,l]pyrene (DBP). A dose-dose matrix design employed over 12,000 rainbow trout to evaluate the interrelationships among dietary carcinogen dose, anti-carcinogen dose, carcinogen-DNA adduct levels at exposure and eventual tumor outcome in two target organs. Included was an evaluation of the pharmaceutical CHL preparation (Derifil), used previously in a study of individuals chronically exposed to AFB(1). CHL was pre-, co- and post-fed at doses of 0-6000 p.p.m. and co-fed with DBP at doses of 0-371.5 p.p.m. for 4 weeks. This protocol generated a total of 21 dose-dose treatment groups, each evaluated with three or more replicates of 100 animals. The DBP-only treatment produced dose-responsive increases in liver and stomach DBP-DNA adducts, whereas increasing CHL co-treatment doses produced successive inhibition in liver (49-83%) and stomach (47-75%) adduct levels at each DBP dose examined. The remaining 8,711 trout were necropsied, 10 months later. DBP treatment alone produced a logit incidence versus log [DBP] dose-response curve in stomach that was linear; CHL co-treatment provided dose-dependent tumor inhibition which ranged from 30 to 68% and was predictable from the adduct response. The Derifil CHL preparation was also found to effectively reduce DNA adduction and final tumor incidence in stomach (as well as liver), with a potency compatible with its total chlorin content. Liver tumor incidence in the DBP-only groups appeared to plateau near 60%. At DBP doses of <or=80 p.p.m., increasing CHL doses generally reduced tumor incidence and multiplicity consistent with early DNA adducts as biomarkers. At 225 p.p.m. DBP, however, very high CHL doses were required to reduce tumor incidence below the 60% plateau. Apparent tumor multiplicity in liver was neither linear nor monotonic with DBP dose, but peaked at 80 p.p.m. DBP and declined at 225 p.p.m., where it was increased by all but one CHL dose. Consequently, the effects of a given CHL dose and the predictivity of DNA adducts as biomarkers were highly dependent on carcinogen dose. These results underscore the critical importance of establishing carcinogen-end point dose-response relationships in chemoprevention studies, and the potential otherwise for misleading interpretations in chemoprevention studies carried out solely at high-carcinogen dose.

Carcinogenesis. 2007 Mar;28(3):611-24

Chlorophyllin significantly reduces benzo[a]pyrene-DNA adduct formation and alters cytochrome P450 1A1 and 1B1 expression and EROD activity in normal human mammary epithelial cells.

We hypothesized that chlorophyllin (CHLN) would reduce benzo[a]pyrene-DNA (BP-DNA) adduct levels. Using normal human mammary epithelial cells (NHMECs) exposed to 4 microM BP for 24 hr in the presence or absence of 5 microM CHLN, we measured BP-DNA adducts by chemiluminescence immunoassay (CIA). The protocol included the following experimental groups: BP alone, BP given simultaneously with CHLN (BP+CHLN) for 24 hr, CHLN given for 24 hr followed by BP for 24 hr (preCHLN, postBP), and CHLN given for 48 hr with BP added for the last 24 hr (preCHLN, postBP+CHLN). Incubation with CHLN decreased BPdG levels in all groups, with 87% inhibition in the preCHLN, postBP+CHLN group. To examine metabolic mechanisms, we monitored expression by Affymetrix microarray (U133A), and found BP-induced up-regulation of CYP1A1 and CYP1B1 expression, as well as up-regulation of groups of interferon-inducible, inflammation and signal transduction genes. Incubation of cells with CHLN and BP in any combination decreased expression of many of these genes. Using reverse transcription real time PCR (RT-PCR) the maximal inhibition of BP-induced gene expression, >85% for CYP1A1 and >70% for CYP1B1, was observed in the preCHLN, postBP+CHLN group. To explore the relationship between transcription and enzyme activity, the ethoxyresorufin-O-deethylase (EROD) assay was used to measure the combined CYP1A1 and CYP1B1 activities. BP exposure caused the EROD levels to double, when compared with the unexposed controls. The CHLN-exposed groups all showed EROD levels similar to the unexposed controls. Therefore, the addition of CHLN to BP-exposed cells reduced BPdG formation and CYP1A1 and CYP1B1 expression, but EROD activity was not significantly reduced.

Environ Mol Mutagen. 2009 Mar;50(2):134-44

Mechanisms of the in vitro antimutagenic action of chlorophyllin against benzo[a]pyrene: studies of enzyme inhibition, molecular complex formation and degradation of the ultimate carcinogen.

Mechanisms of the antimutagenic action of chlorophyllin (CHL) towards benzo[a]pyrene (BP) were studied in vitro. In the Salmonella assay, CHL inhibited the mutagenic activity of BP in the presence of an S9 activation system and was particularly effective against the direct-acting ultimate carcinogen, benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE). Spectral studies indicated that the time-dependent hydrolysis of BPDE to tetrols was augmented in the presence of CHL concentrations on the order of 5 microM. Dose-related inhibition of several cytochrome P450-dependent enzyme activities was observed upon addition of CHL to in vitro incubations. Spectral changes for the interaction between CHL and cytochrome P450 indicated that CHL does not bind to the active site of the enzyme, but exerts its inhibitory effect indirectly. This was achieved by inhibiting NADPH-cytochrome P450 reductase (Ki approximately 120 microM with cytochrome c as substrate), and did not involve lowering of the effective substrate concentration by complex formation with the procarcinogen. It is concluded that the in vitro antimutagenic activity of CHL towards BP involves accelerated degradation of the ultimate carcinogen, with inhibition of carcinogen activation occurring only at high CHL concentrations. The latter mechanism is unlikely to occur in vivo following p.o. administration due to the limited uptake of CHL from the gut, but tissue concentrations may be sufficiently high to cause degradation of BPDE.

Mutat Res. 1994 Jul 16;308(2):191-203

Chlorophyll, chlorophyllin and related tetrapyrroles are significant inducers of mammalian phase 2 cytoprotective genes.

Plant chlorophylls and carotenoids are highly colored, conjugated polyenes that play central roles in photosynthesis. Other porphyrins (tetrapyrroles), such as cytochromes, which are structurally related to chlorophyll, participate in redox reactions in many living systems. An unexpected new property of tetrapyrroles, including tetramethyl coproporphyrin III, tetrabenzoporphine, copper chlorin e4 ethyl ester, and of carotenoids including zeaxanthin and alpha-cryptoxanthin is their ability to induce mammalian phase 2 proteins that protect cells against oxidants and electrophiles. The capacity of these compounds to induce the phase 2 response depends upon their ability or that of their metabolites to react with thiol groups, a property shared with all other classes of phase 2 inducers, which show few other structural similarities. Pseudo second-order rate constants of these inducers are correlated with their potency in inducing the phase 2 enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) in murine hepatoma cells. One of the most potent inducers was isolated from chlorophyllin, a semisynthetic water-soluble chlorophyll derivative. Although chlorophyll itself is low in inducer potency, it may nevertheless account for some of the disease-protective effects attributed to diets rich in green vegetables because it occurs in much higher concentrations in those plants than the widely studied ‘phytochemicals’.

Carcinogenesis. 2005 Jul;26(7):1247-55

The chlorophyllin-induced cell cycle arrest and apoptosis in human breast cancer MCF-7 cells is associated with ERK deactivation and Cyclin D1 depletion.

Targeting the mitogen-activated protein kinases (MAPKs) has been suggested as a novel strategy to treat cancer. Chlorophyllin (CHL) is the sodium-copper salt of chlorophyll derivative and is a commonly used food dye for green coloration; CHL was found previously to retard growth of the human breast carcinoma MCF-7 cells. Extracellular signal-regulated kinases (ERKs) constitute a subfamily of MAPKs, participating in cell survival, proliferation and differentiation. We report here the first evidence that CHL deactivates ERKs to inhibit the breast cancer cell proliferation. The results from flow cytometry showed that 200 microg/ml CHL reduced the phosphorylated and activated ERK-positive cells in different cell cycle phases from the control of >96 to <38% at 24 h of incubation; the ERK deactivations occurred in both dose- and time-dependent manner, so that nearly all ERKs were de-activated by 400 microg/ml CHL at 72 h of treatment. Immunoblot studies, however, illustrated that the levels of total ERKs were not significantly affected by the CHL treatments, suggesting that the phytochemical retards the enzyme activation rather than its expression. Cyclin D1, but not its enzyme Cdk6, was also depleted after the CHL treatments; the depletions were associated with elevations of G0/G1 cells. Apoptosis occurred time-dependently with the ERK deactivations by 400 microg/ml CHL; the apoptotic cells elevated from 2.7-fold of the control level at 24 h, to 4.7-fold at 48 h and to 16.6-fold at 72 h of treatment. Bcl-2 was also depleted at 72 h when there was the most prominent elevation of the apoptotic cells, suggesting that it participates during the exacerbation rather than the initiation phases of the CHL-induced apoptosis. Results from this study support further research on CHL for preventing and treating those tumors with deregulated ERK activations.

Int J Mol Med. 2005 Oct;16(4):735-40

Protection by chlorophyllin and indole-3-carbinol against 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced DNA adducts and colonic aberrant crypts in the F344 rat.

The most abundant heterocyclic amine in fried ground beef, 2-amino-1-methyl-6-pheny-limidazo[4,5-b]pyridine (PhIP), induces colon carcinomas in the male F344 rat. The potential chemopreventive effects of two compounds, namely, the ‘interceptor molecule’ chlorophyllin (CHL) and a modulator of carcinogen activation, indole-3-carbinol (I3C), were examined in a PhIP colon carcinogenesis model. During weeks 3 and 4 of a 16-week study, F344 rats were given PhIP by oral gavage (50 mg/kg body weight, alternating days). Inhibitors were given either before and during PhIP exposure, after PhIP treatment, or continuously for 16 weeks. Treatment of rats with 0.1% CHL in the drinking water inhibited the formation of aberrant crypt foci (ACF) with > or = 4 crypts/focus, from 1.4 +/- 0.9 in controls to 0.7 +/- 0.3 following post-initiation CHL treatment, and to 0.3 +/- 0.5 in rats given CHL continuously for 16 weeks (mean +/- SD; P < 0.05). Potent inhibition of PhIP-induced ACF occurred following initiation, post-initiation and continuous exposure to 0.1% I3C in the diet. Using the initiation protocol, I3C completely inhibited the induction of the ACF with > or = 4 crypts/focus. In a separate experiment, rats were given 0.1% CHL in the drinking water or 0.1% I3C in the diet for 4 weeks. At the end of week 3, animals received 50 mg PhIP/kg body weight by single oral gavage and PhIP-DNA adducts were quantified in the colon and several other tissues by 32P-postlabeling analysis. In addition, the urine and feces were collected to study the effects of inhibitor treatment on PhIP metabolism and excretion. No significant protection against PhIP-DNA adduct formation was detected in the colon after CHL dosing, nor was a consistent pattern of CHL inhibition observed in several other tissues. In contrast, I3C shifted the time-course of adducts in all tissue; compared with controls, adducts were increased by I3C at 6 h but decreased at 24 h and 7 days following PhIP treatment. Analysis of urine metabolites revealed that I3C and CHL decreased the excretion of unmetabolized PhIP and 4’-hydroxy- << PhIP but increased the phase II detoxification products PhIP-4’-O-glucuronide and PhIP-4’- sulfate. In the feces, the elimination of unmetabolized PhIP was increased from 54.5% in controls to approximately 67% in CHL-treated rats and decreased to 28% in rats given I3C (P < 0.05). These results support a protective role for CHL and I3C against PhIP-induced colon carcinogenesis through mechanisms which alter the uptake or metabolism of the carcinogen, and by suppression in the post-initiation phase.

Carcinogenesis. 1995 Dec;16(12):2931-7

Effects of tea and chlorophyllin on the mutagenicity of N-hydroxy-IQ: studies of enzyme inhibition, molecular complex formation, and degradation/scavenging of the active metabolites.

Green tea and black tea inhibit the formation of carcinogen-DNA adducts and colonic aberrant crypts in rats given 2-amino-3-methylimidazo[4, 5-f]quinoline (IQ), a mutagen from cooked meat. The Salmonella mutagenicity assay was used in the present study to test individual constituents of tea as inhibitors of 2-hydroxyamino-3-methylimidazo[4, 5-f]quinoline (N-hydroxy-IQ), a direct-acting metabolite of IQ. Testing of pure compounds at doses relevant to their levels in tea identified epigallocatechin (EGC) and epigalocatechin-3-gallate (EGCG) as the primary antimutagens. Studies of the inhibitory mechanisms established that the rate of degradation of N-hydroxy-IQ under aqueous conditions was not increased significantly in the presence of tea, in contrast to the results obtained with the complexing agent chlorophyllin (CHL), which rapidly degraded the mutagen. Interaction between N-hydroxy-IQ and several tea constituents was detected in spectrophotometric studies, but the binding constants were only on the order of 1 x 10(3) M-1, suggesting that mechanisms other than complex formation might prevail under the conditions of the Salmonella assay. Comparison of the results in two different strains of Salmonella typhimurium, TA98 and TA98/1,8-DNP6, indicated that the antimutagenic activity of EGCG was dependent, at least in part, on a functional O-acetyltransferase activity in the bacteria. These studies suggest that tea constituents inhibit the enzyme(s) which generate the aryl nitrenium ion and directly scavenge the reactive electrophile, whereas CHL complexes with heterocyclic amines and facilitates the degradation of active metabolites.

Environ Mol Mutagen. 1997;30(4):468-74

Antiapoptotic and immunomodulatory effects of chlorophyllin.

Chlorophyllin (CHL) was earlier shown to reduce the level of intracellular ROS and apoptosis induced by ionizing radiation and 2,2’-azobis(2-propionimidinedihydrochloride) (AAPH). In the present studies, the effect of CHL on radiation-induced immunosuppression and modulation of immune responses in mice was examined. Chlorophyllin inhibited the in vitro lymphocyte proliferation induced by concanavalin A (Con A) in a dose dependent manner at doses>or=50 microM. At lower doses (10 microM) CHL significantly inhibited activation induced cell death (AICD) in Con A stimulated spleen cells. Spleen cells obtained from CHL treated mice showed an inhibition of response to Con A depending on dose of CHL and the time after its administration. Spleen cells obtained from CHL treated mice (24 h) showed lower inhibition of response to Con A following in vitro (5 Gy) as well as whole body irradiation (2 Gy). The expression of antiapoptotic genes bcl-2 and bcl-xL was up-regulated in these cells. Chlorophyllin treatment of mice led to splenomegaly and increase in the number of peritoneal exudate cells (PEC). The numbers of T cells, B cells and macrophages in the spleen were also increased. Increased phagocytic activity was seen in PEC obtained from CHL treated mice. Most importantly, CHL administration to mice immunized with sheep red blood cells (SRBC) augmented both humoral and cell-mediated immune responses.

Mol Immunol. 2007 Jan;44(4):347-59

Chlorophyllin as a protector of mitochondrial membranes against gamma-radiation and photosensitization.

Ionizing radiation and photosensitization are highly damaging events and they generate oxygen-derived free radicals as well as excited species. However, the types as well as extent of reactive oxygen species (ROS) differ. They have been linked to various pathological conditions. Hence natural compounds capable of preventing oxidative damage induced by these agents may have potential applications. Chlorophyllin (CHL), the water-soluble analogue of chlorophyll, has been examined for its ability to inhibit membrane damage induced by y-radiation and photosensitization involving methylene blue plus visible light. Using rat liver mitochondria as model systems the mechanisms of damage induced by these two agents as well as its possible prevention by CHL have been examined. The parameters used were lipid peroxidation as assessed by formation of thiobarbituric acid reactive substances (TBARS) and 4-hydroxynonenal (4-HNE), protein oxidation besides glutathione (GSH) and superoxide dismutase (SOD). Peroxidation increases with radiation dose, in the range of 75-600 Gy. A similar observation also was observed with photosensitization, as a function of time. CHL, at a concentration of 10 microM offered a high degree of protection against radiation and photosensitization as indicated by decreased peroxidation, protein oxidation as well as the restoration of GSH and SOD. When compared with the established antioxidants, ascorbic acid and GSH, CHL offered a much higher degree of protection. Pulse radiolysis studies show that this compound has a relatively high rate constant with hydroxyl radical (*OH), a crucial species generated during y-radiation. Hence the studies show that CHL is a potent antioxidant in mitochondrial membranes.

Toxicology. 2000 Nov 30;155(1-3):63-71

Feeding a diet containing a fructooligosaccharide mix can enhance Salmonella vaccine efficacy in mice.

Fructooligosaccharides (FOS) are considered prebiotics because of their ability to promote growth of specific beneficial gut bacteria, such as bifidobacteria. Some studies reported potential immune-modulating properties. The aim of this study was to investigate the effect of FOS:inulin mix on murine response to Salmonella vaccine and evaluate the relevance toward protection against Salmonella infection. Balb/c mice were fed a diet containing 5% FOS:inulin mix or a control diet 1 wk before oral immunization with a suboptimal dose of live attenuated Salmonella typhimurium vaccine. Four weeks after vaccination, mice were infected with LD100 of virulent S. typhimurium. Specific blood Salmonella immunoglobulin G and fecal immunoglobulin A significantly increased in mice fed the diet containing prebiotics compared with control mice 4 wk postimmunization. Peritoneal macrophage phagocytic activity also significantly increased in FOS:inulin-fed mice at 1 wk postimmunization compared with control mice. No detectable effects were observed on the percentage of lymphoid cell subsets in the spleen. However, production of cytokines, interferon-gamma, interleukin-12, and tumor necrosis factor alpha, was numerically increased in spleen cell cultures stimulated with mitogens from FOS:inulin-fed mice 1 and 4 wk postimmunization. Salmonella translocation to lymphoid organs was not affected by feeding FOS:inulin. However, the improved response to Salmonella vaccine was concomitant with an increase in the survival rate of FOS:inulin-fed mice upon challenge with virulent Salmonella. No detectable effects were observed on the composition or the metabolic activity of the microbiota. Overall, the data suggest that a diet supplemented with FOS:inulin mix stimulates mucosal immunity and seems to improve efficacy of an oral vaccine.

J Nutr. 2008 Jan;138(1):123-9

Probiotics and prebiotic galacto-oligosaccharides in the prevention of allergic diseases: a randomized, double-blind, placebo-controlled trial.

BACKGROUND: The increase in allergic diseases is attributed to a relative lack of microbial stimulation of the infantile gut immune system. Probiotics, live health-promoting microbes, might offer such stimulation. OBJECTIVE: We studied the effect of a mixture of 4 probiotic bacterial strains along with prebiotic galacto-oligosaccharides in preventing allergic diseases. METHODS: We randomized 1,223 pregnant women carrying high-risk children to use a probiotic preparation or a placebo for 2 to 4 weeks before delivery. Their infants received the same probiotics plus galacto-oligosaccharides (n = 461) or a placebo (n = 464) for 6 months. At 2 years, we evaluated the cumulative incidence of allergic diseases (food allergy, eczema, asthma, and allergic rhinitis) and IgE sensitization (positive skin prick test response or serum antigen-specific IgE level >0.7 kU/L). Fecal bacteria were analyzed during treatment and at age 2 years. RESULTS: Probiotic treatment compared with placebo showed no effect on the cumulative incidence of allergic diseases but tended to reduce IgE-associated (atopic) diseases (odds ratio [OR], 0.71; 95% CI, 0.50-1.00; P = .052). Probiotic treatment reduced eczema (OR, 0.74; 95% CI, 0.55-0.98; P = .035) and atopic eczema (OR, 0.66; 95% CI, 0.46-0.95; P = .025). Lactobacilli and bifidobacteria more frequently (P < .001) colonized the guts of supplemented infants. CONCLUSION: Probiotic treatment showed no effect on the incidence of all allergic diseases by age 2 years but significantly prevented eczema and especially atopic eczema. The results suggest an inverse association between atopic diseases and colonization of the gut by probiotics. CLINICAL IMPLICATIONS: The prevention of atopic eczema in high-risk infants is possible by modulating the infant’s gut microbiota with probiotics and prebiotics.

J Allergy Clin Immunol. 2007 Jan;119(1):192-8

Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics.

Because the human gut microbiota can play a major role in host health, there is currently some interest in the manipulation of the composition of the gut flora towards a potentially more remedial community. Attempts have been made to increase bacterial groups such as Bifidobacterium and Lactobacillus that are perceived as exerting health-promoting properties. Probiotics, defined as microbial food supplements that beneficially affect the host by improving its intestinal microbial balance, have been used to change the composition of colonic microbiota. However, such changes may be transient, and the implantation of exogenous bacteria therefore becomes limited. In contrast, prebiotics are nondigestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of one or a limited number of bacterial species already resident in the colon, and thus attempt to improve host health. Intake of prebiotics can significantly modulate the colonic microbiota by increasing the number of specific bacteria and thus changing the composition of the microbiota. Nondigestible oligosaccharides in general, and fructooligosaccharides in particular, are prebiotics. They have been shown to stimulate the growth of endogenous bifidobacteria, which, after a short feeding period, become predominant in human feces. Moreover, these prebiotics modulate lipid metabolism, most likely via fermentation products. By combining the rationale of pro- and prebiotics, the concept of synbiotics is proposed to characterize some colonic foods with interesting nutritional properties that make these compounds candidates for classification as health-enhancing functional food ingredients.

J Nutr. 1995 Jun;125(6):1401-12

Perspectives on the role of the human gut microbiota and its modulation by pro- and prebiotics.

One of the most topical areas of human nutrition is the role of the gut in health and disease. Specifically, this involves interactions between the resident microbiota and dietary ingredients that support their activities. Currently, it is accepted that the gut microflora contains pathogenic, benign and beneficial components. Some microbially induced disease states such as acute gastroenteritis and pseudomembranous colitis have a defined aetiological agent(s). Speculation on the role of microbiota components in disorders such as irritable bowel syndrome, bowel cancer, neonatal necrotising enterocolitis and ulcerative colitis are less well defined, but many studies are convincing. It is evident that the gut microflora composition can be altered through diet. Because of their perceived health-promoting status, bifidobacteria and lactobacilli are the commonest targets. Probiotics involve the use of live micro-organisms in food; prebiotics are carbohydrates selectively metabolized by desirable moieties of the indigenous flora; synbiotics combine the two approaches. Dietary intervention of the human gut microbiota is feasible and has been proven as efficacious in volunteer trials. The health bonuses of such approaches offer the potential to manage many gut disorders prophylactically. However, it is imperative that the best methodologies available are applied to this area of nutritional sciences. This will undoubtedly involve a genomic application to the research and is already under way through molecular tracking of microbiota changes to diet in controlled human trials.

Nutr Res Rev. 2000 Dec;13(2):229-54

The effect of ageing with and without non-steroidal anti-inflammatory drugs on gastrointestinal microbiology and immunology.

Elderly individuals are more susceptible to gastrointestinal problems such as constipation than young adults. Furthermore, the common use of non-steroidal anti-inflammatory drugs (NSAID) among the elderly is known to further increase such gastrointestinal ailments. To describe the specific changes in elderly, intestinal microbes, their metabolites and immune markers were measured from faecal samples obtained from fifty-five elderly individuals (aged 68-88 years), using either NSAID or not, and fourteen young adults (aged 21-39 years). The faecal DM content increased with age but was significantly lower among the elderly NSAID users. The microbial metabolism was especially influenced by NSAID use and/or ageing, although fewer changes were observed in the composition of the microbial community, whilst the level of aerobes was increased in the elderly and the level of Clostridium coccoides-Eubacterium rectale reduced in the elderly NSAID users as compared with young adults. An increase in the concentrations of some branched SCFA and l-lactate but a decrease in some major SCFA concentrations were observed. Evidently, the decreased defecation frequency in the elderly directed colonic fermentation toward an unfavourable microbial metabolism but this was partially offset by the use of NSAID. Irrespective of the use of NSAID, the elderly subjects had significantly lower concentrations of faecal PGE2 than the young adults, reflecting possibly a reduced immune response. According to the present study more attention should be paid to the development of dietary products that seek to enhance bowel function, saccharolytic fermentation and immune stimulation in the elderly population.

Br J Nutr. 2008 Jul;100(1):130-7

The inflammatory status of old age can be nurtured from the intestinal environment.

PURPOSE OF REVIEW: Recent studies suggest an association between inflammation status and the presence of chronic disease in the elderly. The review examines publications that address the low level of chronic inflammation and emphasizes how an altered host-microbiota interaction at the gut level could contribute to maintaining a low systemic inflammatory status in the elderly. RECENT FINDINGS: The first population cross-sectional studies with relevant numbers of healthy elderlies show age-related global changes in gut microbiota with a consistent increase in nonpathogenic Gram-negative mainly Enterobacteria and country-specific changes in bifidobacteria. Noninvasive methods have permitted us to detect subclinical intestinal inflammation in the elderly population. Furthermore, few studies report on immune and/or inflammatory response; however, prebiotics, probiotics or synbiotics might improve the inflammatory condition of the elderly. SUMMARY: A better understanding of the mechanisms of host-gut microbiota cross-talk would significantly help in the design of novel nutritional strategies targeting immune reactivity at the mucosal level.

Curr Opin Clin Nutr Metab Care. 2008 Jan;11(1):13-20

Influence of prebiotics on the human immune system (GALT).

Prebiotics have great potential to improve human health in specific intestinal disorders. The knowledge about the influence of prebiotics on the gut-associated lymphoid tissues (GALT) for the improvement of human health is still growing. This paper reviews the latest evidence for the immunity-enhancing effects of prebiotics. Prebiotics, include inulin, fructooligosaccharides, mannosoligosaccharides, and arabinogalactans, are a therapeutic nutritional preparation used for the gut function favoring growth of normal bacterial flora and impedes growth of pathogenic organisms. There is convincing preliminary data to suggest that the consumption of prebiotics can modulate immune parameters in GALT, secondary lymphoid tissues and peripheral circulation. There is increasing evidence that the newly described prebiotics and innovative means of administration can modulate various properties of the immune system, including those of the gut-associated lymphoid tissues (GALT). Authors of recently published patents showed new mechanisms for immuno-modulation, and the ultimate impact on immunological health of prebiotics.

Recent Pat Inflamm Allergy Drug Discov. 2008;2(2):149-53

Cancer prevention by dietary bioactive components that target the immune response.

Dietary bioactive food components that interact with the immune response have considerable potential to reduce the risk of cancer. Reduction of chronic inflammation or its downstream consequences may represent a key mechanism that can be reduced through targeting signal transduction or through antioxidant effects. Major classes of macronutrients provide numerous examples, including amino acids such as glutamine or arginine, lipids such as the omega-3 polyunsaturated fatty acids, DHA or EPA, or novel carbohydrates such as various sources of beta-glucans. Vitamins such as C and E are commonly used as antioxidants, while zinc and selenium are minerals with a wide spectrum of impacts on the immune system. Some of the most potent immunomodulators are phytochemicals such as the polyphenols, EGCG or curcumin, or isothiocyanates such as PEITC. There is accumulating evidence for cancer prevention by probiotics and prebiotics, and these may also affect the immune response. Genomic approaches are becoming increasingly important in characterising potential mechanisms of cancer prevention, optimising the rational selection of dietary bioactive food components, or identifying humans with differing nutrient requirements for cancer protection.

Curr Cancer Drug Targets. 2007 Aug;7(5):459-64

Benefits of a synbiotic formula (Synbiotic 2000Forte) in critically Ill trauma patients: early results of a randomized controlled trial.

BACKGROUND: Since probiotics are considered to exert beneficial health effects by enhancing the host’s immune response, we investigated the benefits of a synbiotics treatment on the rate of infections, systemic inflammatory response syndrome (SIRS), severe sepsis, and mortality in critically ill, mechanically ventilated, multiple trauma patients. Length of stay in the intensive care unit (ICU) and number of days under mechanical ventilation were also evaluated. METHOD: Sixty-five patients were randomized to receive once daily for 15 days a synbiotic formula (Synbiotic 2000Forte, Medipharm, Sweden) or maltodextrin as placebo. The synbiotic preparation consisted of a combination of four probiotics (10(11) CFU each): Pediococcus pentosaceus 5-33:3, Leuconostoc mesenteroides 32-77:1, L. paracasei ssp. paracasei 19; and L. plantarum 2,362; and inulin, oat bran, pectin, and resistant starch as prebiotics. Infections, septic complications, mortality, days under ventilatory support, and days of stay in ICU were recorded. RESULTS: Synbiotic-treated patients exhibited a significantly reduced rate of infections (P = 0.01), SIRS, severe sepsis (P = 0.02), and mortality. Days of stay in the ICU (P = 0.01) and days under mechanical ventilation were also significantly reduced in relation to placebo (P = 0.001). CONCLUSION: The administration of this synbiotic formula in critically ill, mechanically ventilated, multiple trauma patients seems to exert beneficial effects in respect to infection and sepsis rates and to improve the patient’s response, thus reducing the duration of ventilatory support and intensive care treatment.

World J Surg. 2006 Oct;30(10):1848-55

Synbiotic effect of Lactobacillus helveticus M92 and prebiotics on the intestinal microflora and immune system of mice.

The synbiotic effect of the oral treatment of Swiss albino mice with milk-based diets supplemented with Lactobacillus helveticus M92 and various kinds of prebiotics was investigated. Survival, competition, adhesion and colonization, as well as, immunomodulating capability of Lb. helveticus M92, in synbiotic combination, in the gastrointestinal tract (GIT) of mice, were monitored. After the mice were fed with synbiotics, the lactic acid bacteria (LAB) counts in faeces were increased and reduction of enterobacteria and sulphite-reducing clostridia was observed. Similar results were obtained in homogenates of small and large intestine of mice on the 1st and 14th day, after feeding with synbiotics. After the mice were orally given viable Lb. helveticus M92 cells, alone or in combination with prebiotic, the concentration of faecal SIgA and total serum IgA antibodies from all immunized mice were higher compared with the control. The specific humoral immune response was not evoked after oral administration, therefore their synbiotic application is suitable. Among inulin, lactulose and raffinose, Lb. helveticus M92 in combination with inulin, has shown the best synbiotic effect on intestinal and faecal microflora and immune system of mice.

J Dairy Res. 2009 Feb;76(1):98-104

Early dietary intervention with a mixture of prebiotic oligosaccharides reduces the incidence of allergic manifestations and infections during the first two years of life.

A mixture of neutral short-chain galactooligosaccharides (scGOS) and long-chain fructooligosaccharides (lcFOS) has been shown to reduce the incidence of atopic dermatitis (AD) and infectious episodes during the first 6 mo of life. This dual protection occurred through the intervention period. The present study evaluated if these protective effects were lasting beyond the intervention period. In a prospective, randomized, double-blind, placebo-controlled design, healthy term infants with a parental history of atopy were fed either a prebiotic-supplemented (8 g/L scGOS/lcFOS) or placebo-supplemented (8 g/L maltodextrin) hypoallergenic formula during the first 6 mo of life. Following this intervention period, blind follow-up continued until 2 y of life. Primary endpoints were cumulative incidence of allergic manifestations. Secondary endpoints were number of infectious episodes and growth. Of 152 participants, 134 infants (68 in placebo, 66 in intervention group) completed the follow-up. During this period, infants in the scGOS/lcFOS group had significantly lower incidence of allergic manifestations. Cumulative incidences for AD, recurrent wheezing, and allergic urticaria were higher in the placebo group, (27.9, 20.6, and 10.3%, respectively) than in the intervention group (13.6, 7.6, and 1.5%) (P < 0.05). Infants in the scGOS/lcFOS group had fewer episodes of physician-diagnosed overall and upper respiratory tract infections (P < 0.01), fever episodes (P < 0.00001), and fewer antibiotic prescriptions (P < 0.05). Growth was normal and similar in both groups. Early dietary intervention with oligosaccharide prebiotics has a protective effect against both allergic manifestations and infections. The observed dual protection lasting beyond the intervention period suggests that an immune modulating effect through the intestinal flora modification may be the principal mechanism of action.

J Nutr. 2008 Jun;138(6):1091-5

Circulating 25-hydroxyvitamin D levels indicative of vitamin D sufficiency: implications for establishing a new effective dietary intake recommendation for vitamin D.

It has been more than 3 decades since the first assay assessing circulating 25-hydroxyvitamin D [25(OH)D] in human subjects was performed and led to the definition of “normal” nutritional vitamin D status, i.e., vitamin D sufficiency. Sampling human subjects, who appear to be free from disease, and assessing “normal” circulating 25(OH)D levels based on a Gaussian distribution of these values is now considered to be a grossly inaccurate method of identifying the normal range. Several factors contribute to the inaccuracy of this approach, including race, lifestyle habits, sunscreen usage, age, latitude, and inappropriately low dietary intake recommendations for vitamin D. The current adult recommendations for vitamin D, 200-600 IU/d, are very inadequate when one considers that a 10-15 min whole-body exposure to peak summer sun will generate and release up to 20,000 IU vitamin D-3 into the circulation. We are now able to better identify sufficient circulating 25(OH)D levels through the use of specific biomarkers that appropriately increase or decrease with changes in 25(OH)D levels; these include intact parathyroid hormone, calcium absorption, and bone mineral density. Using these functional indicators, several studies have more accurately defined vitamin D deficiency as circulating levels of 25(OH)D < or = 80 nmol or 32 microg/L. Recent studies reveal that current dietary recommendations for adults are not sufficient to maintain circulating 25(OH)D levels at or above this level, especially in pregnancy and lactation.

J Nutr. 2005 Feb;135(2):317-22

Epidemic influenza and vitamin D.

In 1981, R. Edgar Hope-Simpson proposed that a ‘seasonal stimulus’ intimately associated with solar radiation explained the remarkable seasonality of epidemic influenza. Solar radiation triggers robust seasonal vitamin D production in the skin; vitamin D deficiency is common in the winter, and activated vitamin D, 1,25(OH)2D, a steroid hormone, has profound effects on human immunity. 1,25(OH)2D acts as an immune system modulator, preventing excessive expression of inflammatory cytokines and increasing the ‘oxidative burst’ potential of macrophages. Perhaps most importantly, it dramatically stimulates the expression of potent anti-microbial peptides, which exist in neutrophils, monocytes, natural killer cells, and in epithelial cells lining the respiratory tract where they play a major role in protecting the lung from infection. Volunteers inoculated with live attenuated influenza virus are more likely to develop fever and serological evidence of an immune response in the winter. Vitamin D deficiency predisposes children to respiratory infections. Ultraviolet radiation (either from artificial sources or from sunlight) reduces the incidence of viral respiratory infections, as does cod liver oil (which contains vitamin D). An interventional study showed that vitamin D reduces the incidence of respiratory infections in children. We conclude that vitamin D, or lack of it, may be Hope-Simpson’s ‘seasonal stimulus’.

Epidemiol Infect. 2006 Dec;134(6):1129-40

On the epidemiology of influenza.

The epidemiology of influenza swarms with incongruities, incongruities exhaustively detailed by the late British epidemiologist, Edgar Hope-Simpson. He was the first to propose a parsimonious theory explaining why influenza is, as Gregg said, “seemingly unmindful of traditional infectious disease behavioral patterns.” Recent discoveries indicate vitamin D upregulates the endogenous antibiotics of innate immunity and suggest that the incongruities explored by Hope-Simpson may be secondary to the epidemiology of vitamin D deficiency. We identify - and attempt to explain - nine influenza conundrums: (1) Why is influenza both seasonal and ubiquitous and where is the virus between epidemics? (2) Why are the epidemics so explosive? (3) Why do they end so abruptly? (4) What explains the frequent coincidental timing of epidemics in countries of similar latitude? (5) Why is the serial interval obscure? (6) Why is the secondary attack rate so low? (7) Why did epidemics in previous ages spread so rapidly, despite the lack of modern transport? (8) Why does experimental inoculation of seronegative humans fail to cause illness in all the volunteers? (9) Why has influenza mortality of the aged not declined as their vaccination rates increased? We review recent discoveries about vitamin D’s effects on innate immunity, human studies attempting sick-to-well transmission, naturalistic reports of human transmission, studies of serial interval, secondary attack rates, and relevant animal studies. We hypothesize that two factors explain the nine conundrums: vitamin D’s seasonal and population effects on innate immunity, and the presence of a subpopulation of “good infectors.” If true, our revision of Edgar Hope-Simpson’s theory has profound implications for the prevention of influenza.

Virol J. 2008 Feb 25;5:29

Circulating vitamin D3 and 25-hydroxyvitamin D in humans: An important tool to define adequate nutritional vitamin D status.

Circulating 25-hydroxyvitamin D [25(OH)D] is generally considered the means by which we define nutritional vitamin D status. There is much debate, however, with respect to what a healthy minimum level of circulation 25(OH)D should be. Recent data using various biomarkers such as intact parathyroid hormone (PTH), intestinal calcium absorption, and skeletal density measurements suggest this minimum level to be 80 nmol (32 ng/mL). Surprisingly, the relationship between circulating vitamin D(3) and its metabolic product-25(OH)D(3) has not been studied. We investigated this relationship in two separate populations: the first, individuals from Hawaii who received significant sun exposure; the second, subjects from a lactation study who received up to 6,400 IU vitamin D(3)/day for 6 months. Results (1) the relationship between circulating vitamin D(3) and 25(OH)D in both groups was not linear, but appeared saturable and controlled; (2) optimal nutritional vitamin D status appeared to occur when molar ratios of circulating vitamin D(3) and 25(OH)D exceeded 0.3; at this point, the V(max) of the 25-hydroxylase appeared to be achieved. This was achieved when circulating 25(OH)D exceeded 100 nmol. We hypothesize that as humans live today, the 25-hydroxylase operates well below its V(max) because of chronic substrate deficiency, namely vitamin D(3). When humans are sun (or dietary) replete, the vitamin D endocrine system will function in a fashion as do these other steroid synthetic pathways, not limited by substrate. Thus, the relationship between circulating vitamin D and 25(OH)D may represent what “normal” vitamin D status should be.

J Steroid Biochem Mol Biol. 2007 Mar;103(3-5):631-4

Use of vitamin D in clinical practice.

The recent discovery—from a meta-analysis of 18 randomized controlled trials—that supplemental cholecalciferol (vitamin D) significantly reduces all-cause mortality emphasizes the medical, ethical, and legal implications of promptly diagnosing and adequately treating vitamin D deficiency. Not only are such deficiencies common, and probably the rule, vitamin D deficiency is implicated in most of the diseases of civilization. Vitamin D’s final metabolic product is a potent, pleiotropic, repair and maintenance, seco-steroid hormone that targets more than 200 human genes in a wide variety of tissues, meaning it has as many mechanisms of action as genes it targets. One of the most important genes vitamin D up-regulates is for cathelicidin, a naturally occurring broad-spectrum antibiotic. Natural vitamin D levels, those found in humans living in a sun-rich environment, are between 40-70 ng per ml, levels obtained by few modern humans. Assessing serum 25-hydroxy-vitamin D (25(OH)D) is the only way to make the diagnosis and to assure treatment is adequate and safe. Three treatment modalities exist for vitamin D deficiency: sunlight, artificial ultraviolet B (UVB) radiation, and vitamin D3 supplementation. Treatment of vitamin D deficiency in otherwise healthy patients with 2,000-7,000 IU vitamin D per day should be sufficient to maintain year-round 25(OH)D levels between 40-70 ng per mL. In those with serious illnesses associated with vitamin D deficiency, such as cancer, heart disease, multiple sclerosis, diabetes, autism, and a host of other illnesses, doses should be sufficient to maintain year-round 25(OH)D levels between 55-70 ng per mL. Vitamin D-deficient patients with serious illness should not only be supplemented more aggressively than the well, they should have more frequent monitoring of serum 25(OH)D and serum calcium. Vitamin D should always be adjuvant treatment in patients with serious illnesses and never replace standard treatment. Theoretically, pharmacological doses of vitamin D (2,000 IU per kg per day for three days) may produce enough of the naturally occurring antibiotic cathelicidin to cure common viral respiratory infections, such as influenza and the common cold, but such a theory awaits further science.

Altern Med Rev. 2008 Mar;13(1):6-20

Vitamin D: a D-Lightful health perspective.

Sunlight provides most humans with their vitamin D requirement. Adequate vitamin D(3) by synthesis in the skin or from dietary and supplemental sources is essential for bone health throughout life. Vitamin D deficiency is defined as a 25(OH)D concentration <20 ng/mL (50 nmol/L); vitamin D sufficiency as a 25(OH)D >30 ng/mL (75 nmol/L), and insufficiency as 21-29 ng/mL. Vitamin D deficiency and insufficiency has been linked to a wide variety of chronic diseases including common cancers, autoimmune, cardiovascular, and infectious diseases. Healthcare professionals need to be aware of the vitamin D deficiency pandemic. Guidelines for sensible sun exposure and supplemental vitamin D of 800-1000 IU/day are needed.

Nutr Rev. 2008 Oct;66(10 Suppl 2):S182-94

Benefits and requirements of vitamin D for optimal health: a review.

Vitamin D sufficiency is required for optimal health. The conditions with strong evidence for a protective effect of vitamin D include several bone diseases, muscle weakness, more than a dozen types of internal cancers, multiple sclerosis, and type 1 diabetes mellitus. There is also weaker evidence for several other diseases and conditions. There are good reasons that vitamin D sufficiency be maintained during all stages of life, from fetal development to old age. Adequate calcium intake is also recommended. The current vitamin D requirements in the United States are based on protection against bone diseases. These guidelines are being revised upward in light of new findings, especially for soft-tissue health. The consensus of scientific understanding appears to be that vitamin D deficiency is reached for serum 25-hydroxyvitamin D (25OHD) levels less than 20 ng/mL (50 nmol/L), insufficiency in the range from 20-32 ng/mL, and sufficiency in the range from 33-80 ng/mL, with normal in sunny countries 54-90 ng/mL, and excess greater than 100 ng/mL. Solar ultraviolet-B (UVB) irradiation is the primary source of vitamin D for most people. In general, the health benefits accruing from moderate UV irradiation, without erythema or excess tanning, greatly outweigh the health risks, with skin pigmentation (melanin) providing much of the protection. In the absence of adequate solar UVB irradiation due to season, latitude, or lifestyle, vitamin D can be obtained from fortified food, oily fish, vitamin D supplements, and artificial sources of UVB radiation.

Altern Med Rev. 2005 Jun;10(2):94-111

Human cathelicidin antimicrobial peptide (CAMP) gene is a direct target of the vitamin D receptor and is strongly up-regulated in myeloid cells by 1,25-dihydroxyvitamin D3.

The innate immune system of mammals provides a rapid response to repel assaults from numerous infectious agents including bacteria, viruses, fungi, and parasites. A major component of this system is a diverse combination of cationic antimicrobial peptides that include the alpha- and beta-defensins and cathelicidins. In this study, we show that 1,25-dihydroxyvitamin D3 and three of its analogs induced expression of the human cathelicidin antimicrobial peptide (CAMP) gene. This induction was observed in acute myeloid leukemia (AML), immortalized keratinocyte, and colon cancer cell lines, as well as normal human bone marrow (BM) -derived macrophages and fresh BM cells from two normal individuals and one AML patient. The induction occurred via a consensus vitamin D response element (VDRE) in the CAMP promoter that was bound by the vitamin D receptor (VDR). Induction of CAMP in murine cells was not observed and expression of CAMP mRNA in murine VDR-deficient bone marrow was similar to wild-type levels. Comparison of mammalian genomes revealed evolutionary conservation of the VDRE in a short interspersed nuclear element or SINE in the CAMP promoter of primates that was absent in the mouse, rat, and canine genomes. Our findings reveal a novel activity of 1,25-dihydroxyvitamin D3 and the VDR in regulation of primate innate immunity.

FASEB J. 2005 Jul;19(9):1067-77

Antimicrobial peptides in the airway.

The airway provides numerous defense mechanisms to prevent microbial colonization by the large numbers of bacteria and viruses present in ambient air. An important component of this defense is the antimicrobial peptides and proteins present in the airway surface fluid (ASF), the mucin-rich fluid covering the respiratory epithelium. These include larger proteins such as lysozyme and lactoferrin, as well as the cationic defensin and cathelicidin peptides. While some of these peptides, such as human beta-defensin (hBD)-1, are present constitutively, others, including hBD2 and -3 are inducible in response to bacterial recognition by Toll-like receptor-mediated pathways. These peptides can act as microbicides in the ASF, but also exhibit other activities, including potent chemotactic activity for cells of the innate and adaptive immune systems, suggesting they play a complex role in the host defense of the airway. Inhibition of antimicrobial peptide activity or gene expression can result in increased susceptibility to infections. This has been observed with cystic fibrosis (CF), where the CF phenotype leads to reduced antimicrobial capacity of peptides in the airway. Pathogenic virulence factors can inhibit defensin gene expression, as can environmental factors such as air pollution. Such an interference can result in infections by airway-specific pathogens including Bordetella bronchiseptica, Mycobacterium tuberculosis, and influenza virus. Research into the modulation of peptide gene expression in animal models, as well as the optimization of peptide-based therapeutics shows promise for the treatment and prevention of airway infectious diseases.

Curr Top Microbiol Immunol. 2006;306:153-82

Injury enhances TLR2 function and antimicrobial peptide expression through a vitamin D-dependent mechanism.

An essential element of the innate immune response to injury is the capacity to recognize microbial invasion and stimulate production of antimicrobial peptides. We investigated how this process is controlled in the epidermis. Keratinocytes surrounding a wound increased expression of the genes coding for the microbial pattern recognition receptors CD14 and TLR2, complementing an increase in cathelicidin antimicrobial peptide expression. These genes were induced by 1,25(OH)2 vitamin D3 (1,25D3; its active form), suggesting a role for vitamin D3 in this process. How 1,25D3 could participate in the injury response was explained by findings that the levels of CYP27B1, which converts 25OH vitamin D3 (25D3) to active 1,25D3, were increased in wounds and induced in keratinocytes in response to TGF-beta1. Blocking the vitamin D receptor, inhibiting CYP27B1, or limiting 25D3 availability prevented TGF-beta1 from inducing cathelicidin, CD14, or TLR2 in human keratinocytes, while CYP27B1-deficient mice failed to increase CD14 expression following wounding. The functional consequence of these observations was confirmed by demonstrating that 1,25D3 enabled keratinocytes to recognize microbial components through TLR2 and respond by cathelicidin production. Thus, we demonstrate what we believe to be a previously unexpected role for vitamin D3 in innate immunity, enabling keratinocytes to recognize and respond to microbes and to protect wounds against infection.

J Clin Invest. 2007 Mar;117(3):803-11

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