Abstract: The human skin is constantly exposed to microbial pathogens but infections only rarely occur. Innate cutaneous immunity is a primary system for protection against infection, and antimicrobial peptides (AMPs) expressed in skin are essential defence molecules. The AMPs include molecules such as the defensins that were first characterized for their antimicrobial properties as well as other peptides and proteins first known for their activity as chemokines, enzymes, enzyme inhibitors and neuropeptides. Cathelicidins are unique AMPs that act as defensive and signalling molecules. Two different pathways are involved in this function: cathelicidins have direct antimicrobial activity and they also initiate a host of cellular responses in cytokine release, inflammation and angiogenesis. Several skin diseases are associated with cathelicidin dysfunction. In atopic eczema, for example, cathelicidin expression is suppressed, whereas in rosacea cathelicidin peptides are abnormally processed to forms that induce cutaneous inflammation and a vascular response. In psoriasis cathelicidin peptide converts self-DNA to a potent stimulus in an autoinflammatory cascade. Current studies have unexpectedly identified vitamin D3 as a major factor for the regulation of cathelicidin expression. This finding may provide new strategies in the management of infectious and inflammatory diseases of the skin by targeting control of the expression and function of cathelicidin and other AMPs.

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Abstract: Hormonally active vitamin D(3)-1,25-dihydroxyvitamin D(3) (1,25D3)-acts as a signaling molecule in cutaneous immunity by increasing pattern recognition through Toll-like receptor-2 (TLR2), and increasing the expression and function of antimicrobial peptides. Here we show that the actions of 1,25D3 on keratinocyte innate immune responses are influenced by histone acetylation and require the steroid receptor coactivator 3 (SRC3), which mediates inherent histone acetyltransferase (HAT) activity. SRC3 was detected in the suprabasal and granular layer of the skin, similar to cathelicidin expression. HAT activity was important to keratinocyte cathelicidin expression as the combination of histone deacetylase inhibitors (HDACi) (butyrate or trichostatin A) and 1,25D3 increased cathelicidin and CD14 expression and enhanced the antimicrobial function of keratinocytes against Staphylococcus aureus. This treatment, or activation of TLR2, also directly increased acetylation of histone 4. Small interfering RNA silencing of the vitamin D receptor or SRC3 blocked the induction of cathelicidin and CD14 by 1,25D3. HDACi could not reverse this effect or influence cathelicidin in the absence of 1,25D3, suggesting that both are necessary for function. These studies demonstrate that the epigenetic control of gene transcription by histone acetylation is important for 1,25D3-regulated antimicrobial and TLR function of keratinocytes, essential elements of the innate immune response of the skin.

Abstract: Our skin is constantly challenged by microbes but is rarely infected. Cutaneous production of antimicrobial peptides (AMPs) is a primary system for protection, and expression of some AMPs further increases in response to microbial invasion. Cathelicidins are unique AMPs that protect the skin through 2 distinct pathways: (1) direct antimicrobial activity and (2) initiation of a host response resulting in cytokine release, inflammation, angiogenesis, and reepithelialization. Cathelicidin dysfunction emerges as a central factor in the pathogenesis of several cutaneous diseases, including atopic dermatitis, in which cathelicidin is suppressed; rosacea, in which cathelicidin peptides are abnormally processed to forms that induce inflammation; and psoriasis, in which cathelicidin peptide converts self-DNA to a potent stimulus in an autoinflammatory cascade. Recent work identified vitamin D3 as a major factor involved in the regulation of cathelicidin. Therapies targeting control of cathelicidin and other AMPs might provide new approaches in the management of infectious and inflammatory skin diseases.

Abstract: Keratinocytes produce and secrete antimicrobial peptides which function as endogenous antibiotics and as signaling molecules within the cutaneous innate immune system. Recent studies demonstrate that the antimicrobial peptide cathelicidin LL-37 plays an important role in the pathogenesis of atopic eczema, rosacea and psoriasis. Whereas skin in atopic eczema shows decreased cathelicidin expression which leads to increased susceptibility to superinfection in those patients, overabundant expression of cathelicidin peptide fragments causes inflammation in rosacea. Finally, in psoriasis cathelicidin peptide binds to self DNA which triggers an autoimmune response. These studies demonstrate the role of cathelicidin as a central factor in the pathogenesis of cutaneous inflammation. Therapies targeting cathelicidin expression and function could lead to new treatments for these diseases.

Abstract: Calcineurin inhibitors are potent inhibitors of T-cell-receptor mediated activation of the adaptive immune system. The effects of this class of drug on the innate immune response system are not known. Keratinocytes are essential to innate immunity in skin and rely on toll-like receptors (TLRs) and antimicrobial peptides to appropriately recognize and respond to injury or microbes. In this study we examined the response of cultured human keratinocytes to pimecrolimus. We observed that pimecrolimus enhances distinct expression of cathelicidin, CD14, and human beta-defensin-2 and beta-defensin-3 in response to TLR2/6 ligands. Some of these responses were further enhanced by 1,25 vitamin D3. Pimecrolimus also increased the functional capacity of keratinocytes to inhibit growth of Staphylococcus aureus and decreased TLR2/6-induced expression of IL-10 and IL-1beta. Furthermore, pimecrolimus inhibited nuclear translocation of NFAT and NF-kappaB in keratinocytes. These observations uncover a previously unreported function for pimecrolimus in cutaneous innate host defense.

Abstract: Cathelicidin is strongly expressed in lesional skin in psoriasis and may play an important role as both an antimicrobial peptide and as an autoinflammatory mediator in this chronic skin disease. The mechanism of increased cathelicidin in psoriatic keratinocytes is not known, but recent observations have found that psoriasis has abundant Th17 cells that produce IL-17A and IL-22. We found that human keratinocytes stimulated with supernatants from T cells isolated from lesional psoriatic skin increased expression of cathelicidin when stimulated in the presence of 1,25-dihydroxyvitamin D(3) (1,25D(3)). This increase was signaled through the IL-17RA. In vitro, IL-17A, but not IL-22, enhanced cathelicidin mRNA and peptide expression in keratinocytes dependent on the presence of 1,25D(3). At the same time, coincubation with 1,25D(3) blocked induction of human beta-defensin 2 (HBD2), IL-6, and IL-8, which are other target genes of IL-17A. Act1, an adaptor associated with IL-17RA and essential for IL-17A signaling, mediated cathelicidin induction, as its suppression by small interfering RNA inhibited HBD2 and cathelicidin. Both, 1,25D(3) and IL-17A signaled cathelicidin induction through MEK-ERK. These results suggest that increased IL-17A in psoriatic skin increases cathelicidin through a vitamin D(3)-, Act1-, and MEK-ERK-dependent mechanism. Therapy targeting this cathelicidin-regulating system might be beneficial in patients suffering from psoriasis.

Abstract: The surface of our skin is constantly challenged by a wide variety of microbial pathogens, still cutaneous infections are relatively rare. Within cutaneous innate immunity the production of antimicrobial peptides (AMPs) is a primary system for protection against infection. Many AMPs can be found on the skin, and these include molecules that were discovered for their antimicrobial properties, and other peptides and proteins first known for activity as chemokines, enzymes, enzyme inhibitors and neuropeptides. Cathelicidins were among the first families of AMPs discovered on the skin. They are now known to have two distinct functions; they have direct antimicrobial activity and will initiate a host cellular response resulting in cytokine release, inflammation and angiogenesis. Dysfunction of cathelicidin is relevant in the pathogenesis of several cutaneous diseases including atopic dermatitis where cathelicidin induction is suppressed, rosacea, where cathelicidin peptides are abnormally processed to forms that induce cutaneous inflammation and a vascular response, and psoriasis, where a cathelicidin peptide can convert self-DNA to a potent stimulus of an autoinflammatory cascade. Recent work has unexpectedly identified vitamin D3 as a major factor involved in the regulation of cathelicidin expression. Therapies targeting the vitamin D3 pathway and thereby cathelicidin may provide new treatment modalities in the management of infectious and inflammatory skin diseases.

Abstract: PURPOSE OF REVIEW: To review recently published studies presenting novel and relevant information on antimicrobial peptides in gastrointestinal infections. RECENT FINDINGS: Defensins and cathelicidins are important antimicrobial peptides expressed by the gastrointestinal epithelium. Their localization and regulation have been the focus of current research establishing the relevance of these peptides both in counteracting an attack by pathogens as well as in controlling the endogenous bacterial flora. In the small intestine, Paneth cell alpha-defensins maintain a low level of microorganisms and regulate the composition of the bacterial flora. In contrast, a constitutive beta-defensin can be found in nearly all gastrointestinal tissues. Other relevant beta-defensins as well as human cathelicidin are inducible by inflammation or infections. Thus Helicobacter pylori enhances defensin expression in the gastric mucosa and Campylobacter jejuni and Salmonella provoke a similar response in the colon. Other pathogenic bacteria may suppress the antimicrobial peptide response as an escape strategy. Notably, the therapeutic induction of cathelicidins alleviates experimental shigellosis, suggesting a future role of endogenous antibiotics in medical therapy. SUMMARY: These recent findings together with a better understanding of underlying mechanisms involved in the regulation and biology of antimicrobial peptides will open up new therapeutic avenues to battle infections.

Abstract: 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.

Abstract: Antimicrobial peptides are gene-encoded molecules first discovered for their microbicidal properties but recently shown to have pro- or anti-inflammatory functions. Their role as immune regulators is being expanded with evidence that some antimicrobial peptides stimulate keratinocyte migration, proliferation and cytokine or chemokine production. Poorly named, antimicrobial peptides are multifunctional pillars around which the innate and adaptive immune response has evolved.

Abstract: Second only to cardiovascular diseases, malignant tumors are the most common fatal disease, with malignant neoplasms in the gastrointestinal tract playing an important role. Underlying the most numerous of these malignancies is a complex interaction between genetic and environmental factors. The data relating to the role of environmental factors (for the most part dietary factors) in the development of gastrointestinal tumors derive mainly from, epidemiological research. The current evidence is "convincin" with regard to complex lifestyle patterns, but at most "plausible" when the chemically defined individual substances are considered. Summarizing the potential protective value of dietary factors reveals that the risk of contracting the majority of the gastrointestinal tumors can be reduced by increasing the intake of fruit and vegetables. An additional protective effect is associated with a balanced diet, physical activity, preservation of normal weight, avoidance of smoking, and moderation in the amount of alcohol consumed.

Abstract: In innate immune responses, activation of Toll-like receptors (TLRs) triggers direct antimicrobial activity against intracellular bacteria, which in murine, but not human, monocytes and macrophages is mediated principally by nitric oxide. We report here that TLR activation of human macrophages up-regulated expression of the vitamin D receptor and the vitamin D-1-hydroxylase genes, leading to induction of the antimicrobial peptide cathelicidin and killing of intracellular Mycobacterium tuberculosis. We also observed that sera from African-American individuals, known to have increased susceptibility to tuberculosis, had low 25-hydroxyvitamin D and were inefficient in supporting cathelicidin messenger RNA induction. These data support a link between TLRs and vitamin D-mediated innate immunity and suggest that differences in ability of human populations to produce vitamin D may contribute to susceptibility to microbial infection.

Abstract: Due to its low digestibility in the small intestine, a major fraction of the polyol isomalt reaches the colon. However, little is known about effects on the intestinal microflora. During two 4-week periods in a double-blind, placebo-controlled, cross-over design, nineteen healthy volunteers consumed a controlled basal diet enriched with either 30 g isomalt or 30 g sucrose daily. Stools were collected at the end of each test phase and various microbiological and luminal markers were analysed. Fermentation characteristics of isomalt were also investigated in vitro. Microbiological analyses of faecal samples indicated a shift of the gut flora towards an increase of bifidobacteria following consumption of the isomalt diet compared with the sucrose diet (P<0.05). During the isomalt phase, the activity of bacterial beta-glucosidase decreased (P<0.05) whereas beta-glucuronidase, sulfatase, nitroreductase and urease remained unchanged. Faecal polyamines were not different between test periods with the exception of cadaverine, which showed a trend towards a lower concentration following isomalt (P=0.055). Faecal SCFA, lactate, bile acids, neutral sterols, N, NH3, phenol and p-cresol were not affected by isomalt consumption. In vitro, isomalt was metabolized in several bifidobacteria strains and yielded high butyrate concentrations. Isomalt, which is used widely as a low-glycaemic and low-energy sweetener, has to be considered a prebiotic carbohydrate that might contribute to a healthy luminal environment of the colonic mucosa.

Abstract: BACKGROUND: Inflammatory bowel diseases (IBDs) are characterized by a breakdown of colon epithelial barrier function. Antimicrobial peptides like cathelicidins are molecules of the innate immune system located at epithelial surfaces. Cathelicidins influence microbial growth and inflammation and may play a role in IBD. In this study, the expression of human cathelicidin hCAP18/LL-37 was investigated in the intestinal mucosa from patients suffering from ulcerative colitis or Crohn's disease. METHODS: Biopsy material from colon and ileal mucosa of a total of 89 patients (34 with Crohn's disease, 27 with ulcerative colitis, 28 control patients) was evaluated for cathelicidin expression by real-time reverse-transcriptase polymerase chain reaction and immunohistochemistry. Colon epithelial cells were stimulated in vitro with various cytokines to evaluate mechanisms that influence cathelicidin production. RESULTS: Cathelicidin expression was significantly increased in inflamed and non-inflamed colon mucosa from ulcerative colitis patients compared to non-inflamed control mucosa. In patients with Crohn's disease cathelicidin expression was not changed in inflamed or non-inflamed colon or ileal mucosa independent of NOD2 status. Biopsies evaluated by immunohistochemistry showed epithelial cathelicidin expression in the upper crypt that was diffuse in controls and only basal in IBD patients. Inflammation mediators, alone or in combination with the known cathelicidin inducer butyrate, had no effect on cathelicidin expression in cultured colon cells. CONCLUSIONS: In IBD the colonic expression of human cathelicidin is altered: cathelicidin expression is increased in inflamed and non-inflamed mucosa in patients suffering from ulcerative colitis but not in Crohn's disease. This deficiency may further compromise the antimicrobial barrier in Crohn's disease.

Abstract: Immune defence against microbes depends in part on the production of antimicrobial peptides, a process that occurs in a variety of cell types but is incompletely understood. In this study, the mechanisms responsible for the induction of cathelicidin and beta-defensin antimicrobial peptides were found to be independent and specific to the cell type and stimulus. Vitamin D3 induced cathelicidin expression in keratinocytes and monocytes but not in colonic epithelial cells. Conversely, butyrate induced cathelicidin in colonic epithelia but not in keratinocytes or monocytes. Distinct factors induced beta-defensin expression. In all cell types, vitamin D3 activated the cathelicidin promoter and was dependent on a functional vitamin D responsive element. However, in colonic epithelia butyrate induced cathelicidin expression without increasing promoter activity and vitamin D3 activated the cathelicidin promoter without a subsequent increase in transcript accumulation. Induction of cathelicidin transcript correlated with increased processed mature peptide and enhanced antimicrobial activity against Staphylococcus aureus. However, induction of beta-defensin-2 expression did not alter the innate antimicrobial capacity of cells in culture. These data suggest that antimicrobial peptide expression is regulated in a tissue-specific manner at transcriptional, post-transcriptional and post-translational levels. Furthermore, these data show for the first time that innate antimicrobial activity can be triggered independently of the release of other pro-inflammatory molecules, and suggest strategies for augmenting innate immune defence without increasing inflammation.

Abstract: The effect of regular consumption of the low-digestible and prebiotic isomalt versus the digestible sucrose on gene expression in rectal mucosa was examined in a randomized double-blind crossover trial. Nineteen healthy volunteers received 30 g isomalt per day or 30 g sucrose as part of a controlled diet over two 4-week test periods with a 4-week washout period in between. At the end of each test phase rectal biopsies were obtained. After RNA extraction mucosal gene expression was assayed using GeneChip microarrays. In addition, expression of cathelicidin hCap18/LL37, cellular detoxification enzymes GSTpi, UGT1A1 and CYP3A4, cyclooxygenase 2 and barrier factors MUC2 and ZO-1 were determined by real-time RT-PCR. Microbiological analyses of fecal samples revealed a shift of the gut flora towards an increase of bifidobacteria following consumption of the diet containing isomalt. Isomalt consumption did not affect rectal mucosal gene expression in microarray analyses as compared to sucrose. In addition, the expression of cathelicidin LL37, GSTpi, UGT1A1, CYP3A4, COX-2, MUC2 and ZO-1 was not changed in rectal biopsies. We conclude that gene expression of the human rectal mucosa can reliably be measured in biopsy material taken at endoscopy. Dietary intervention with the low digestible isomalt compared with the digestible sucrose did not affect gene expression in the lining rectal mucosa.

Abstract: The presence of cathelicidin antimicrobial peptides provides an important mechanism for prevention of infection against a wide variety of microbial pathogens. The activity of cathelicidin is controlled by enzymatic processing of the proform (hCAP18 in humans) to a mature peptide (LL-37 in human neutrophils). In this study, elements important to the processing of cathelicidin in the skin were examined. Unique cathelicidin peptides distinct from LL-37 were identified in normal skin. Through the use of selective inhibitors, SELDI-TOF-MS, Western blot, and siRNA, the serine proteases stratum corneum tryptic enzyme (SCTE, kallikrein 5) and stratum corneum chymotryptic protease (SCCE, kallikrein 7) were shown to control activation of the human cathelicidin precursor protein hCAP18 and also influence further processing to smaller peptides with alternate biological activity. The importance of this serine protease activity to antimicrobial activity in vivo was illustrated in SPINK5-deficient mice that lack the serine protease inhibitor LEKTI. Epidermal extracts of these animals show a significant increase in antimicrobial activity compared with controls, and immunoabsorption of cathelicidin diminished antimicrobial activity. These observations demonstrate that the balance of proteolytic activity at an epithelial interface will control innate immune defense.

Abstract: Tight junctions as an epithelial barrier against paracellular diffusion have mainly been investigated on the protein level with particular respect to subcellular localization. In this study, real-time PCR has been established to investigate the influence of protein kinase C (PKC) modulation on the transcription of tight junction elements occludin and ZO-1 in the cell line T84. Activation of PKC by the phorbol ester TPA induced ZO-1 and occludin transcription, whereas PKC inhibition lead to decreased expression levels. Activation of PKC exerted its effect on transcript level directly. PKC signal was partially transduced via MEK1/MEK2 but depended strongly on MAPK independent pathways probably involving nuclear localized PKC, whereas p38 signaling was not implicated. TPA induced loss of function concomitant with a dislocation of ZO-1 and occludin could be prevented by inhibition of MEK1 by PD98059. Overall ZO-1 and occludin seem to be identically regulated in colonic epithelium on the transcript level.

Abstract: The polyol isomalt (Palatinit) is a well established sugar replacer. The impact of regular isomalt consumption on metabolism and parameters of gut function in nineteen healthy volunteers was examined in a randomised, double-blind, cross-over trial with two 4-week test periods. Volunteers received 30 g isomalt or 30 g sucrose daily as part of a controlled diet. In addition to clinical standard diagnostics, biomarkers and parameters currently discussed as risk factors for CHD, diabetes or obesity were analysed. Urine and stool Ca and phosphate excretions were measured. In addition, mean transit time, defecation frequency, stool consistency and weight were determined. Consumption of test products was affirmed by the urinary excretion of mannitol. Blood lipids were comparable in both phases, especially in volunteers with hyperlipidaemia, apart from lower apo A-1 (P=0.03) for all subjects. Remnant-like particles, oxidised LDL, NEFA, fructosamine and leptin were comparable and not influenced by isomalt. Ca and phosphate homeostasis was not affected. Stool frequency was moderately increased in the isomalt phase (P=0.006) without changes in stool consistency and stool water. This suggests that isomalt is well tolerated and that consumption of isomalt does not impair metabolic function or induce hypercalciuria. In addition, the study data indicate that isomalt could be useful in improving bowel function.

Abstract: In vitro studies addressing the primary prevention of colon carcinoma are preferably conducted using normal colonic cells, because these cells are more likely to represent the potential target for prevention in vivo. Established cell lines of normal colonic origin are mostly lacking; however, this is probably due to the difficulties associated with establishment of such cell lines. Cross-contamination with malignant cells is a frequent event, and so any successfully established cell line of normal origin should be scrutinized prior to further investigation. We performed a cytogenetic (spectral karyotyping) and genetic fingerprint (Promega PowerPlex ES multiplex system and Applied Biosystems AmpFlSTR SGM Plus multiplex system) analysis of the putative normal colon epithelial cell line NCOL-1, derived from two different sources (NCOL-1a and 1b). We show that NCOL-1a and 1b are probably derived from the colon carcinoma cell line LoVo, with a matching probability of 99.9995, most probably through cross-contamination. Karyotypes of LoVo and NCOL-1a were identical; NCOL-1b displayed additional marker chromosomes. Our findings highlight the importance of molecular and cytogenetic characterization of established cell lines to avoid drawing misleading conclusions from the original findings.

Abstract: BACKGROUND AND AIMS: On the genetic level colonic carcinogenesis is best described by the adenoma-carcinoma sequence, but it may be modulated by exogenous factors, particularly by dietary factors and chemopreventive agents. The protective effects of exogenous factors are thought to be exerted rather in the early stages of the adenoma-carcinoma sequence. Thus, an in vitro model consisting of cells stemming from an colon adenoma would be desirable. However, establishing such a cell line has proven difficult. MATERIALS AND METHODS: We report the establishment of a colon adenoma cell line. The cells were generated from a colon adenoma and propagated as a stable cell line for more than 40 passages. The cells are microsatellite stable and confirmed to be of epithelial origin by cytokeratin staining. RESULTS: In contrast to commercially available colon cancer cell lines, cytogenetic analysis with spectral karyotype analysis revealed no chromosomal alterations in this adenoma cell line. Incubation with butyrate resulted in a time- and dose-dependent inhibition of proliferation and in an significant increase in cellular differentiation. The cdk inhibitor p21/waf which plays a pivotal role in growth inhibition and differentiation is expressed consecutively in GEKI-2 cells. The expression of cdk1 and cdk2, important regulatory elements in the cell cycle, is downregulated following treatment with butyrate. CONCLUSION: The presented colon adenoma cell line GEKI-2 may prove a versatile tool for further exploring the underlying mechanisms of protective and promoting factors in early colon cancerogenesis.

Abstract: BACKGROUND: Leukocyte recruitment to areas of inflammation depends on Integrin-VCAM/ICAM interaction. Blocking the vascular cell adhesion molecule (VCAM-1) and the intracellular adhesion molecule (ICAM-1) may have therapeutic benefit for the inflammatory component of bowel disease. Notably, the induction of ICAM and VCAM is mediated by a nuclear factor kappaB (NF-kappaB)-dependent mechanism. We investigated whether the anti-inflammatory properties of butyrate are mediated via the modulation of VCAM and ICAM on human endothelial cells. METHODS: VCAM-1 and ICAM-1 expression on human endothelial cells upon tumor necrosis factor-alpha (TNF-alpha) stimulation was assessd by FACS analysis. A monocyte adhesion assay was performed to evaluate the relevance of a modulated CAM-expression. Electrophoretic mobility shift assays were applied to investigate NF-kappaB activation. RESULTS: The observed butyrate-associated inhibition of monocyte adhesion to endothelial cells is associated with an inhibition of NF-kappaB activation in human endothelial cells. In this context, the observed suppression of the TNF-alpha induced VCAM-1 expression is likely to play an essential role. CONCLUSIONS: Butyrate inhibits VCAM-1 mediated leukocyte adhesion to human endothelial cells. This inhibition may contribute to the anti-inflammatory effects of butyrate in patients with distal ulcerative colitis.

Abstract: Histone-deacetylase (HDAC) -inhibitors enhance acetylation of core proteins and this is linked to formation of transcriptionally active chromatin in various cells. In this study, the effect of HDAC inhibitors (butyrate, trichostatin A (TSA)) on the expression of the cathelicidin LL-37 in colon, gastric and hepatocellular cells was investigated. METHODS: LL-37 expression was assessed in colon, gastric and hepatocellular cancer cells after treatment with HDAC-inhibitors. In parallel, histone H4 and HMGN2, a non-histone protein, acetylation was evaluated. In addition, the intracellular signalling pathway MEK-ERK was explored. RESULTS: In contrast to normal colon epithelial cells, gastrointestinal cancer cells lacked LL-37 expression. LL-37 was induced following treatment with HDAC-inhibitors in all investigated cell lines. This induction was time-dependent in butyrate-treated cells while TSA exerted a transient effect. Induction of LL-37 by butyrate was paralleled by acetylation of the histone H4 and the non-histone HMGN2. Again, TSA resulted in transient acetylation. Furthermore, inhibition of MEK-ERK blocked HDAC inhibitor-induced LL-37 expression in colonic and gastric cells. CONCLUSIONS: We have previously shown that butyrate induces LL-37 in colon epithelial cells. In the present study, we demonstrate that cathelicidin expression is modulated by HDAC-inhibitors in various gastrointestinal cells including gastric and hepatocellular cells. This is paralleled by changes in the acetylation of distinct core proteins suggesting a common regulatory mechanism of cathelicidin LL-37 regulation in these cells.

Abstract: BACKGROUND AND AIMS: Short chain fatty acids (SCFA) exert profound effects on the colonic mucosa. In particular, SCFA modulate mucosal immune functions. The antimicrobial cathelicidin LL-37 is expressed by colon epithelial cells. In the present study the effect of SCFA on LL-37 expression was investigated. METHODS: LL-37 expression in vivo was assessed by immunohistochemistry. Real time quantitative reverse transcription-polymerase chain reaction was employed to determine LL-37 expression in colonocytes in vitro after treatment with various cytokines, SCFA, or flavone. LL-37 levels were correlated to cell differentiation which was determined by alkaline phosphatase (AP) activity. In addition, intracellular signalling pathways such as MEK-ERK (mitogen/extracellular signal protein kinase (MEK)/extracellular signal regulated protein kinase (ERK)) and p38/mitogen activated protein (MAP) kinase were explored. RESULTS: In vivo, LL-37 expression in healthy mucosa was restricted to differentiated epithelial cells in human colon and ileum. In colonocytes, increased LL-37 expression associated with cell differentiation was detected in vitro following treatment with butyrate, isobutyrate, propionate, and trichostatin A. Flavone induced LL-37 transcription but did not affect AP activity while cytokines had no effect. To dissect pathways mediating differentiation and LL-37 expression, specific inhibitors were applied. Inhibition of the protein kinase MEK enhanced butyrate induced AP activity while LL-37 expression in colon epithelial cells was blocked. In contrast, inhibition of p38/MAP kinase blocked cell differentiation without inhibiting LL-37 expression. CONCLUSIONS: Expression of the cathelicidin LL-37 in colonocytes and cellular differentiation are separately modulated by SCFA via distinct signalling pathways. These data may provide a rationale for dietary modulation of mucosal defence mechanisms.

Abstract: BACKGROUND: In ulcerative colitis (UC) the activation (i.e. nuclear translocation) of nuclear factor kappa B (NF-kappaB) is an important step in the regulation of cytokines secreted by lamina propria macrophages. Clinical trials suggest anti-inflammatory effects of locally administered butyrate in UC. The potential effects of butyrate on NF-kappaB activation in lamina propria macrophages of UC patients were investigated. METHODS: Eleven patients with distal UC were treated for up to 8 weeks with butyrate at 100 mM (n = 6) or placebo (n = 5) enemas. At entry and after 4 and 8 weeks, clinical status was noted and intestinal inflammation was graded endoscopically and histologically. Double-staining with antibodies against NF-kappaB (p65) and CD68 was employed to detect NF-kappaB and macrophages, respectively. RESULTS: In untreated patients, nuclear translocation of NF-kappaB was detectable in virtually all macrophages. Butyrate treatment for 4 and 8 weeks resulted in a significant reduction in the number of macrophages being positive for nuclear translocated NF-kappaB. In addition, butyrate significantly reduced both the number of neutrophils in crypt and surface epithelia and of the lamina propria lymphocytes/plasma cells. These findings correlated with a significant decrease in the Disease Activity Index (DAI). CONCLUSIONS: The decrease in DAI and mucosal inflammation in butyrate-treated patients is associated with a reduction of NF-kappaB translocation in lamina propria macrophages. Since the inflammatory process in UC is mainly sustained by macrophage-derived cytokines, the known anti-inflammatory effects of butyrate may in part be mediated by an inhibition of NF-kappaB activation in these macrophages.

Abstract: Butyrate, a short chain fatty acid (SCFA), is generated by anaerobic fermentation of undigested carbohydrates within the colon. Butyrate enhances acetylation of core histones, a process directly linked to the formation of active chromatin and gene expression. However, additional chromatin components also contribute to the formation of transcriptionally active chromatin. The high mobility group protein N2 (HMG-N2), a nonhistone protein, is involved in chromatin structure modulation. We examined the effects of butyrate on HMG-N2 expression, hyperacetylation and chromatin binding. HT29 human adenocarcinoma cells were incubated with butyrate. Levels of HMG-N2 mRNA and of total or acetylated HMG-N2 protein were analyzed. Protein dynamics were investigated with transfected cells expressing HMG-N2-EGFP fusion proteins. Treatment of HT29 cells with butyrate led to significant hyperacetylation of HMG-N2. Levels of HMG-N2 protein remained unchanged. Northern blot analysis revealed a significant reduction in HMG-N2 mRNA levels after treatment with butyrate. Analysis of HMG-N2-EGFP transfected HT29 cells demonstrated that butyrate treatment changes the binding properties of HMG-N2-EGFP to chromatin. In addition, butyrate treatment resulted in solubilization of endogenous acetylated HMG-N2 into the supernatant of permeabilized cells. We demonstrate that butyrate treatment is associated with hyperacetylation of HMG-N2 protein in HT29 cells. The modulation of this nonhistone chromatin protein resulted in altered binding properties to chromatin. This may represent an additional step in changing chromatin structure and composition with subsequent consequences for transcription and gene expression. Modulation of nonhistone chromatin proteins, like the ubiquitous HMG-N2 proteins, may be partly responsible for the wide range of butyrate-associated effects.

Abstract: Laboratory and epidemiological studies suggest that butyrate, a metabolic product of microbial fermentation of dietary fibre, and aspirin, a non-steroidal antiphlogistic drug, both reduce the risk of developing colon cancer. Notably, few data exist on potential interactions of these two substances. In this study, the effects of a butyrate-aspirin combination on human colon cancer cells were compared with treatment with aspirin or butyrate alone. Both substances decreased proliferation and induced differentiation and apoptosis. Butyrate reduced mutant p53 expression, whereas aspirin did not affect p53 expression. Butyrate-induced apoptosis correlated with an increase in Bak expression and a decrease in the expression of Bcl-XL. Aspirin had no effect on the investigated apoptosis-controlling factors. The antiproliferative and pro-apoptotic effects of the butyrate-aspirin combination were markedly enhanced. The combination resulted in a stronger decrease in the expression of PCNA and cdk2. Our data suggest that the anticarcinogenic effect of aspirin might effectively be augmented by combination with the short-chain fatty acid butyrate.

Abstract: BACKGROUND: Tumor necrosis factor (TNFalpha)-induced apoptosis is limited by concomitant activation of nuclear factor kappa B (NF-kappaB)-dependent anti-apoptotic genes. Butyrate inhibits NF-kappaB activation so that co-treatment with butyrate effectively enhances TNFalpha-induced apoptosis. In this context, the inhibition of NF-kappaB activation and subsequent modulation of (NF-kappaB)-dependent genes was assessed MATERIALS AND METHODS: Human colon adenocarcinoma cells (SW620) were incubated with TNFalpha and butyrate. Apoptosis was determined by annexin V/propidium iodide staining and flow cytometry. NF-kappaB activation was detected by electrophoretic mobility shift assay. The expression of NF-kappaB-dependent genes was assessed by RNase protection assay (RPA) RESULTS: The TNFalpha/butyrate combination yielded an additive increase in the number of apoptotic cells. NF-kappaB nuclear translocation was successfully inhibited by co-incubation with butyrate. However, the expression pattern of NF-kappaB-dependent genes remained essentially unchanged CONCLUSION: Butyrate enhances TNFalpha-induced apoptosis in the human adenocarcinoma cell line SW620. This additive effect may, at least in part, be mediated by the inhibition of NF-kappaB activation, presumably by impairing the anti-apoptotic properties of NF-kappaB.

Abstract: Butyrate, a short-chain fatty acid, is generated by anaerobic fermentation within the colon. Clinical trials suggest that short-chain fatty acids ameliorate inflammation in ulcerative colitis. Nuclear factor (NF) kappaB, an inducible transcription factor that is activated in inflamed colonic tissue, is sequestered to the cytoplasm by its inhibitory IkappaB proteins. The anti-inflammatory effects of butyrate are associated with an inhibition of NF-kappaB nuclear translocation. To investigate the mechanism of NF-kappaB inhibition we examined the effects of butyrate on IkappaBalpha. Human adenocarcinoma cells (SW480, SW620, and HeLa229) were treated with butyrate for up to 48 h followed by tumor necrosis factor (TNF) alpha stimulation. NF-kappaB was detected by immunofluorescence staining with an antibody against its p65 subunit. Levels of IkappBalpha and phosphorylated IkappaBalpha were determined by western blot. Stimulation with TNFalpha resulted in rapid phosphorylation and degradation of IkappaBalpha followed by NF-kappaB nuclear translocation. Butyrate pretreatment successfully inhibited NF-kappaB activation. Pretreatment of adenocarcinoma cells with butyrate is associated with inhibition of TNFalpha-mediated phosphorylation and degradation of IkappaBalpha and effective blocking of NF-kappaB nuclear translocation. The anti-inflammatory effects of butyrate may at least in part be mediated by an inhibition of IkappaBalpha mediated activation of NF-kappaB.

Abstract: BACKGROUND: Rectal instillation of short-chain fatty acids (SCFA), important nutrients for the colorectal mucosa, has been suggested to be of therapeutic value in distal intestinal inflammation. METHODS: In this study nine patients with Hartmann-closed rectum after colectomy for acute colitis were investigated. In a double-blind crossover trial an enema containing SCFA or a placebo solution was administered twice daily for 3 weeks. Before entry into the protocol, after each treatment period, and 6 weeks after the study period the patients' symptoms were evaluated, rectal endoscopy was performed, histologic samples were scored, and microbiologic analyses were carried out. RESULTS: No significant differences in symptoms, in mucosal inflammation, in histologic scoring, or in microbiologic studies were found between SCFA and placebo periods. Unexpectedly, all but one patient entirely lacked coliform bacteria in the rectum. CONCLUSIONS: In this study SCFA enemas had no beneficial effect on inflammation in excluded rectum in patients earlier submitted to colectomy for colitis. However, a different rectal flora was detected in these patients.