Karl M Stuhlmeier
email: karl-manfred.stuhlmeier@meduniwien.ac.at

Short Term Hyperthermia Prevents the Activation of Mitogen-Activated Protein Kinase p38

Keywords: Balneology, Spatherapy, Hyperthermia, Inflammation, Arthritis

Introduction

A number of in vitro and in vivo models have been employed to study heat shock proteins (HSPs) and the modi operandii that account for the protective effects of stress proteins. Out of such studies, a pattern emerged that linked HSPs, newly synthesized by hyperthermia, fever, or chemical means, to the reduction of a number of proinflammatory genes that are characterized by their dependence on the activation of the transcription factor nuclear factor kappaB (NF-κB) [START_REF] Siebenlist | NF-κB: A Mediator of Pathogen and Stress Responses[END_REF]. Interactions between HSP70 and members of the NF-κB family have been associated with heat shock mediated NF-κB inhibition. It has, for example, been shown that blocking the degradation of the inhibitor kappaB (IκB), a protein that sequesters NF-κB in the cytoplasm, is one of the key events that account for the anti-inflammatory effects of HT [START_REF] Lee | Heat shock protein 70 negatively regulates the heat-shock-induced suppression of the IkappaB/NF-kappaB cascade by facilitating IkappaB kinase renaturation and blocking its further denaturation[END_REF][START_REF] Shanley | Heat shock inhibits phosphorylation of I-kappaBalpha[END_REF].

In general it is rather difficult to change the body core temperature by artificial means without risking undesired effects. One disease that affects many might offer the unique opportunity to utilize HT as a mean to ameliorate inflammation without potentially harmful effects to vital organs in the body core. It seems, that due to their location, the temperature in joints of patients suffering from certain forms of arthritis could be modulated with relative ease. It is for this reason that we initiated studies to evaluate HT in an in-vitro model for its effectiveness in dampening the inflammation associated with rheumatic diseases [START_REF] Markovic | Short-term hyperthermia prevents activation of proinflammatory genes in fibroblast-like synoviocytes by blocking the activation of the transcription factor NF-kappaB[END_REF].

Fibroblast-like synoviocytes (FLS) play an important role in creation and propagation of rheumatoid arthritis and are characterized by their ability to up-regulate numerous genes that take part in inflammatory processes [START_REF] Pap | Fibroblast biology. Role of synovial fibroblasts in the pathogenesis of rheumatoid arthritis[END_REF]. As reported earlier from our laboratory, exposing FLS to HT resulted in significantly reduced mRNA and protein levels of many pro-inflammatory genes that would otherwise be highly upregulated in response to IL-1β [START_REF] Markovic | Short-term hyperthermia prevents activation of proinflammatory genes in fibroblast-like synoviocytes by blocking the activation of the transcription factor NF-kappaB[END_REF]. We further demonstrated by electrophoretic mobility shift assays (EMSA) and western blot experiments that HT prevented IκB degradation and nuclear translocation of NF-κB in this cell type [START_REF] Markovic | Short-term hyperthermia prevents activation of proinflammatory genes in fibroblast-like synoviocytes by blocking the activation of the transcription factor NF-kappaB[END_REF].

It is generally thought that it is the newly synthesized HSPs that account for the majority of protective effects of the stress response induced by HT. However, like us, others also demonstrated discrepancies in the time course of HSP70 and/or HO-1 expression and the inhibition of NF-κB mediated by HT [START_REF] Markovic | Short-term hyperthermia prevents activation of proinflammatory genes in fibroblast-like synoviocytes by blocking the activation of the transcription factor NF-kappaB[END_REF][START_REF] Feinstein | Heat shock protein 70 suppresses astroglial-inducible nitric-oxide synthase expression by decreasing NFkappaB activation[END_REF]. Similar to our results using FLS, it has been reported that IκB degradation in heat shocked macrophages is blocked for up to four hours [START_REF] Grossman | Temporal and mechanistic effects of heat shock on LPS-mediated degradation of IkappaBalpha in macrophages[END_REF]. However, HSP70 protein expression induced by HT in the aforementioned publication (as well as in our experiments) peaked after 7 hours. In addition, in FLS, significantly elevated levels of HSP70 protein can be observed for up to 24 hours, a time at which the effect of HT on IκB/NF-κB has long since subsided [START_REF] Markovic | Short-term hyperthermia prevents activation of proinflammatory genes in fibroblast-like synoviocytes by blocking the activation of the transcription factor NF-kappaB[END_REF][START_REF] Feinstein | Heat shock protein 70 suppresses astroglial-inducible nitric-oxide synthase expression by decreasing NFkappaB activation[END_REF].

Such data, together with relative high HSP70 levels in resting, non-stressed FLS, prompted us to further investigate mechanisms and the involvement of HSPs in antiinflammatory effects of short term HT.

As reported, mammalian cells are able to withstand temperatures of up to 45°C without significant effects on their viability [START_REF] Kroeger | The Heat Shock Transcriptional Response[END_REF]. By and large, HT effects are thought to be due to the activation of a number of heat shock proteins [START_REF] Perdrizet | Heat shock and tissue protection[END_REF][START_REF] Tytell | Heat shock proteins: new keys to the development of cytoprotective therapies[END_REF]. The extent of the heat shock response is thought to be a function of the duration and magnitude of the temperature increase. Furthermore, it is widely assumed that a recovery period at 37°C is essential for the protective effects of HT. Such a period is thought to allow for maximal expression of a number of protective HSPs. However, as mentioned, the heat shock proteins HSP70 and HO-1 are maximally expressed at time points at which the protective effect of HT is seemingly past its peak. In the following, data are presented resulting from an investigation into the very early events mediated by short term hyperthermia. GTA GGC CCA GGT CAT GAG G. Primers, amplification curves, and equations for the quantitation of mRNA for other genes have been reported elsewhere [START_REF] Markovic | Short-term hyperthermia prevents activation of proinflammatory genes in fibroblast-like synoviocytes by blocking the activation of the transcription factor NF-kappaB[END_REF][START_REF] Stuhlmeier | Quinacrine but not chloroquine inhibits PMA induced upregulation of matrix metalloproteinases in leukocytes: quinacrine acts at the transcriptional level through a PLA2-independent mechanism[END_REF][START_REF] Stuhlmeier | Adenovirus-mediated gene transfer of mutated IkappaB kinase and IkappaBalpha reveal NF-kappaB-dependent as well as NF-kappaB-independent pathways of HAS1 activation[END_REF].

Materials and Methods

Reagents

Basal mRNA expression levels in unstimulated FLS were chosen to represent 1X expression of a given gene. Data shown in figure 7B resulted from two independent experiments done in duplicates; all other experiments were done at least three times.

Where statistical analysis was done a P-value 0.05 was considered significant.

Viability of cells was neither affected by the stimuli used, by exposure to hyperthermia, nor by a combination of hyperthermia and stimuli. 1A). However, prior exposure to 41°C is no longer sufficient to block

Results

Effects

IκBα degradation in cells that were given time (20 hours) to maximally express HSPs (figure 1B). Also shown in figure 1A and B is that the temperature had to be increasing to 43°C in order to prevent IκBα degradation even after 20 hours of recovery. This is in accordance with observations made by others, namely, that exposing cells to higher temperatures will result in a more pronounced/prolonged protective effects of HT [START_REF] Kroeger | The Heat Shock Transcriptional Response[END_REF].

Next to HO-1, HSP70 is one of the best studied HSPs and also one that very quickly responds to temperature changes [START_REF] Bach | Heme oxygenase-1: a therapeutic amplification funnel[END_REF][START_REF] Yamashita | Heme oxygenase-1 is essential for and promotes tolerance to transplanted organs[END_REF][START_REF] Yoo | Anti-inflammatory effect of heat shock protein induction is related to stabilization of I kappa B alpha through preventing I kappa B kinase activation in respiratory epithelial cells[END_REF]. HSP70 has many times been shown to be involved in, and is thought to account for the majority of protective effects of, the heat shock response [START_REF] Tytell | Heat shock proteins: new keys to the development of cytoprotective therapies[END_REF]. Nevertheless, we found that in FLS HSP protein levels remain unchanged for at least 40 minutes after HT (4), (data not shown). This led to the assumption that HT might also initiate events that are independent of newly synthesized HSPs. Shown in figure 1C is an experiment that demonstrates that in FLS, IL-1β induced IκBα phosphorylation is an event that starts at 30 seconds and peaks at around 5 minutes.

Experiments like the one shown in figure 1D Such data point at early, HT mediated mechanisms that do not require de novo protein synthesis.

Is protein denaturation involved in HT mediated prevention of Iκ κ κ κB

phosphorylation? Data shown in figure 2 point to mechanisms other than de novo synthesis of HSPs by which HT is able to block NF-κB activation. One mechanism that might be at play is based on the assumption that some proteins could be more sensitive to a moderate increase in temperature than others. For example, a method that is frequently used to selectively inactivate proteins of the complement cascade consists of heating serum to 56°C for 30 minutes. Whether exposing cells to 41°C for 15 minutes is sufficient to denature proteins is less clear. However, a recently published article seems to imply heat induced denaturation/insolubilization as one mechanism by which HT mediates its effects [START_REF] Pittet | Stress-induced inhibition of the NF-kappaB signaling pathway results from the insolubilization of the IkappaB kinase complex following its dissociation from heat shock protein 90[END_REF]. Shown in figure 3 are data that implicate that a method used in A459 cells to demonstrate HT induced protein insolubilization is not suited to prove or disprove such a mechanism in FLS. The reason is that in FLS glycerol (used to prevent protein aggregation) itself exerts profound effects on the IL-1β induced NF-κB pathway. HT prevents transcriptional activation of genes that are regulated by the MAPK p38. Two observations made in this laboratory were used to further test the hypothesis that inhibition of p38 by HT is entirely independent of effects of HT on the NF-κB pathway. We demonstrated earlier that TGF-β activates hyaluronansynthase1 (HAS1) via a NF-κB independent pathway [START_REF] Stuhlmeier | Adenovirus-mediated gene transfer of mutated IkappaB kinase and IkappaBalpha reveal NF-kappaB-dependent as well as NF-kappaB-independent pathways of HAS1 activation[END_REF]. However, TGF-β induced activation of HAS1 in FLS relies on an intact p38 MAPK signaling pathway [START_REF] Stuhlmeier | Differential effect of transforming growth factor beta (TGF-beta) on the genes encoding hyaluronan synthases and utilization of the p38 MAPK pathway in TGF-beta-induced hyaluronan synthase 1 activation[END_REF]. We made use of this phenomenon and show in figure 7 that blocking p38 by short term HT results in highly significant inhibition of TGF-β induced HAS1 transcription. Where indicated, cells were exposed to 41°C for 30 minutes prior to stimulation with TGF-β (2 ng/ml) for either 8 or 14 hours. At both time points analyzed, HAS1 mRNA levels in cells exposed to short term HT were significantly lower than in cells that did not undergo such treatment.

Discussion

Despite many novel treatment options available today, rheumatoid arthritis is still a debilitating disease whose progression can only be slowed but not cured. Inflammation plays an important role in ongoing joint destruction. For this reason most drugs used today aim at interrupting cell to cell communication that is contributing to inflammatory processes. While immunocompetent white blood cells clearly play an important role in arthritis, evidence has been presented that FLS could be of significance in the etiology of rheumatoid arthritis [START_REF] Pap | Fibroblast biology. Role of synovial fibroblasts in the pathogenesis of rheumatoid arthritis[END_REF].

We and others have demonstrated that FLS are not only able to respond to but also express and/or release a number of proinflammatory cytokines, chemokines and adhesion molecules known to be potent mediators of inflammation (4). We demonstrated earlier that short term hyperthermia might offer the appealing opportunity to prevent or reduce inflammation associated, for example, with rheumatoid arthritis by blocking the activation of NF-κB (4). Rheumatoid arthritis seems a disease that offers the rather unique opportunity to make use of short term HT to disrupt or at least diminish inflammatory processes. Due to tight control mechanisms, it is nearly impossible to raise the body core temperature significantly without harming the organism. However, normally the temperature in joints is not only considerably lower than the body core temperature, it also seems much easier to increase the temperature in joints to the degree needed for the initiation of a sustained, protective heat shock response. The temperature in joints is about 32°C and the reportedly needed increase in temperature by 6°C for prolonged protective effects (8) might not pose a serious hurdle for an in vivo implementation of HT as a means to ameliorate inflammation in joints.

While, as previously shown, unwanted gene activation in FLS could indeed be diminished by short term HT, the mechanisms involved seem manifold and still largely unknown. By and large, it is thought that the protective effects of HT are due to the transcription and translation of heat shock proteins that in turn interact with the activation of proinflammatory genes by a multitude of mechanisms [START_REF] Yenari | Antiapoptotic and antiinflammatory mechanisms of heat-shock protein protection[END_REF]. However, time course experiments done in this laboratory pointed at yet another mode of action that accounts for the very early anti-inflammatory effects of HT.

As shown in this manuscript, two very important signaling pathways are blocked by short term HT. Also noteworthy is that, at least in FLS, HT clearly blocks these signaling pathways independently of the need for de novo synthesis of heat shock proteins, as protective effects of HT are detectable long before the appearance of newly synthesized HSPs. Over the years, substantial effort has been put into finding ways to specifically block the NF-κB pathway. As demonstrated again herein, short term HT is a very effective method to prevent events in FLS that would otherwise result in the activation of NF-κB dependent genes.

Of similar importance is the demonstration that short term HT is able to block the activation of p38, a serine kinase that plays a central role in numerous proinflammatory responses [START_REF] Schieven | The biology of p38 kinase: a central role in inflammation[END_REF]. Similar to the effect on NF-κB, HT effects on MAPK p38 do not require de novo protein synthesis. As demonstrated, a mere 10 to 15 minutes exposure to 41°C is sufficient to block IL-1β induced p38 activation for several hours.

The MAPK p38 has increasingly become the focus of researchers aiming at interrupting inflammatory processes. As outlined by Schieven [START_REF] Schieven | The biology of p38 kinase: a central role in inflammation[END_REF] the main reasons are that the MAPK p38 regulates the production of key inflammatory mediators such as TNFα, IL-1β and COX-2 and, in addition, also acts downstream of cytokines such as TNFα. The potential efficacy of p38 inhibition may thus be greater than would be expected from the inhibition of the mediators alone [START_REF] Schieven | The biology of p38 kinase: a central role in inflammation[END_REF].

It was surprising to note the differential effect of short term HT on MAPKs. As shown, IL-1β induced activation of p38 is blocked for several hours. However, HT acts as a potent inducer of ERK and JNK/SAPK phosphorylation in FLS. While the consequences of ERK and JNK/SAPK activation by HT are currently unclear, the relevance of MAPK p38 inhibition for the benefit of arthritis patients seems obvious.

Inhibition of p38 has been hailed as a new approach for treating inflammatory disorders such as rheumatoid arthritis [START_REF] Peifer | New approaches to the treatment of inflammatory disorders small molecule inhibitors of p38 MAP kinase[END_REF]. It has also been shown, that blocking p38 activation will prevent bone destruction [START_REF] Pargellis | Inhibitors of p38 mitogen-activated protein kinase for the treatment of rheumatoid arthritis[END_REF]. In addition, several studies confirmed the central role of p38 in the progression of rheumatic disorders [START_REF] Pargellis | Inhibitors of p38 mitogen-activated protein kinase for the treatment of rheumatoid arthritis[END_REF][START_REF] Zwerina | Activation of p38 MAPK is a key step in tumor necrosis factor-mediated inflammatory bone destruction[END_REF][START_REF] Wei | p38 MAPK as a potential therapeutic target for inflammatory osteolysis[END_REF][START_REF] Mbalaviele | Inhibition of p38 mitogen-activated protein kinase prevents inflammatory bone destruction[END_REF][START_REF] Inoue | Mitogenactivated protein kinase kinase 3 is a pivotal pathway regulating p38 activation in inflammatory arthritis[END_REF]. Because of their promise as a potent treatment option, several of the p38 inhibitors are currently under development or are already in phase I or II clinical trials [START_REF] Pargellis | Inhibitors of p38 mitogen-activated protein kinase for the treatment of rheumatoid arthritis[END_REF].

There are only a limited number of reports analyzing early effects of HT on MAPK. Among these, one group reported activation of p38 by HT [START_REF] Dorion | Activation of the p38 signaling pathway by heat shock involves the dissociation of glutathione S-transferase Mu from Ask1[END_REF]. Whether such differences are due to the cell type used, CCL39 and HeLa versus FLS, or to the fact that cells were exposed to 44°C in their experiments, is currently unclear [START_REF] Dorion | Activation of the p38 signaling pathway by heat shock involves the dissociation of glutathione S-transferase Mu from Ask1[END_REF]. Yet, preliminary data from out laboratory imply that under the conditions used to treat FLS (41°C for 30 minutes), HT seemingly also blocks p38 in cells other than FLS (data not shown). However, other mechanisms could be at work as well, since cell type specific variations in the activation pattern of HSPs by HT have indeed been demonstrated [START_REF] Oehler | Cell typespecific variations in the induction of hsp70 in human leukocytes by feverlike whole body hyperthermia[END_REF].

Then again, the early events leading to the prevention of p38 as well as NF-κB activation in FLS are independent of the need for de novo synthesis of HSPs.

Taken together, data are presented which demonstrate that short term HT is able to prevent independently the activation of two important signaling pathways that are essential for the upregulation of many proinflammatory genes. If these in vitro data could be reproduced in in-vivo experiments and/or extended to other cell types, it would be appealing to speculate that eventually patients could greatly benefit from this simple and inexpensive method to ameliorate inflammation associated with arthritis. More importantly, HT prevents also IκB phosphorylation in FLS whose protein synthesis machinery was blocked.

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  If not stated otherwise, reagents e.g. Interleukin-1β (human recombinant), phorbol myristate acetate PMA) etc. were from Sigma (Sigma, Vienna, Austria). Anti-IκBα and the phosphorylation specific antibodies directed against p-IκB, p-p38, p-JNK/SAPK and p-ERK were from CellSignaling, (CellSignaling, Bedford, MA, USA). The MAPK inhibitor set I and II as well as the MAPK inhibitors SP600125, SB 203580, PD 98059 were from Calbiochem, (Merck, Darmstadt, Germany). Antiα-tubulin was from LabVision (Lab Vision Corporation, Fremont, CA, USA). The anti HSP70 and HO-1 antibodies were from Santa Cruz Biotechnology, (Santa Cruz, CA, USA). Cell culture. A series of human CD90 positive fibroblast-like synoviocytes (FLS), isolated from rheumatoid arthritis patients, were used in these experiments. Cells were either purchased from Dominion Pharmakine (Dominion Pharmakine, Derio, Bizkaia, Spain), or were a gift from Dr. G. Steiner, Medical University Vienna, Austria. FLS were cultured and propagated essentially as described (4). The temperature of the medium was measured and monitored in cell culture dishes that contained the same amount of medium as dishes containing FLS used in experiments. Western blot experiments. SDS-PAGE and Western blot experiments were carried out as described (4). In short, cells were washed twice in ice-cold PBS and subsequently dissolved in SDS sample buffer. Proteins were quantitated, separated, blotted, and stained with indicated antibodies. Proteins were made visible using RenaissancePlus (Perkin Elmer Life Science, Boston, USA) and Kodak BioMax MR films or the chemiluminescence detection device GeneGnome (Syngene, Cambridge, UK). In western blot experiments, lower concentrations of proteins were loaded on separate gels that served as controls for loading and protein transfer (LC). Such blots were stained with an antibody recognizing tubulin, or, like other blots, were stained with Ponceau red. Netherlands), using SYBR green as reporter fluorophore for quantitating mRNA levels (4). To normalize the amount of total RNA present in each reaction, mRNA levels of HPRT and/or actin were used. Results are expressed as relative threshold cycle ( Ctvalues) (Ct values of mRNA levels in stimulated minus Ct values of a given gene in resting cells). Primer sequences used but not reported previously are as follows: IL-1α (forward) ATC AGT ACC TCA CGG CTG CT, IL-1α (reverse) CTT CAT CTT GGG CAG TCA CA, HAS1 (forward) CAA GCC GGA GAG AAG AGA GA, HAS1 (reverse)

  of short term HT on IL-1β β β β induced NF-κ κ κ κB signaling pathways in FLS. As shown by data presented in figure 1A and 1B, in which FLS were exposed to short term HT (39, 40, 41, and 43°C for 30 min.) IκBα degradation, induced by IL-1β (5 ng/ml for 20 minutes), can be affected by HT for at least 20 hours. However, the effectiveness of HT to block the NF-κB activation pathway is more pronounced at earlier time points i.e. when cells are challenged immediately after short term HT rather than after 20 hours at which time HSPs are maximally expressed. If cells are stimulated immediately after HT, 41°C is sufficient to completely block IL-1β induced IκBα degradation (figure

  were done to investigate the time requirements for HT mediated inhibition of the NF-κB pathway. FLS were kept at 37°C or were exposed to 41°C for 5, 10, and 15 minutes. Afterwards, IL-1β was added for 5 minutes. IκBα phosphorylation was therefore chosen to monitor the minimal duration of HT necessary to prevent NF-κB activation. Figure 1D shows that 15 minutes of HT are sufficient to completely block IL-1β induced IκBα phosphorylation. Shown in figure 1C is a representative Western blot experiment depicting IL-1β induced IκB phosphorylation in FLS. Protein synthesis is not essential for HT effects on NF-κ κ κ κB activation. In the following, we show data that further support the hypothesis that short term HT exerts effects that do not depend on a recovery period that allows for the generation of HSPs. FLS were left untreated or were pretreated either with Cycloheximide (figure 2A) and Emetine (figure 2B) -two protein synthesis inhibitors. After 60 minutes pretreatment with these substances, parts of the cells were exposed to 41°C for 30 min. Where indicated, cells were subsequently stimulated with IL-1β for 5 minutes at 37°C. Phosphorylation of ΙκBα was monitored by Western blots. As shown in figure 2, short term HT prevents IL-1β induced IκB phosphorylation even in FLS that were prevented from synthesizing protective heat shock proteins in response to being exposed to 41°C. That the NF-κB signaling cascade in Cycloheximide and Emetine treated cells is intact is demonstrated by data showing phosphorylation of IκBα in protein syntheses inhibitor treated cells exposed to IL-1β (Figure 2, lane "CYC + IL-1β", and lane "EME + IL-1β").

  FLS were incubated with 1M of glycerol for 1 hour and subsequently treated with IL-1β (5 ng/ml for 5 min). Unlike the demonstration that 1M of glycerol will restore the NF-κB pathway in HT treated A459 cells, we found that in FLS, glycerol itself prevents IκB phosphorylation. Shown in figure3Ais a dose curve experiment demonstrating that IL-1β induced IκB phosphorylation is blocked at high concentrations of glycerol. FLS were incubated with glycerol (1 to 1000 mM) for 1 hour. Afterwards, cells were treated with IL-1β for 5 more minutes. As in other Western blot experiments, staining blots with tubulin served as control for protein loading and transfer in addition to staining membranes with Ponceau red. As shown in figure 3B, preincubating FLS with 1M glycerol for one hour, will not restore IL-1β induced IκB phosphorylation in cells that were heat treated. If glycerol indeed prevents protein insolubilization, then data presented herein would exclude protein denaturation as the modus operandi of HT in FLS. However, as also demonstrated in figure 3, glycerol itself is able to prevent IL-1β induced IκB phosphorylation. As shown in figure 3B, lane "GLY + IL-1β", treating FLS with 1M glycerol for one hour will prevent IL-1β induced IκB phosphorylation rather than restore it. Differential effect of short term HT on PMA induced gene activation. To get a better sense of early events affected by HT, PMA induced activation of proinflammatory genes was studied next. One reason for using PMA was that it reportedly does not rely on cell surface receptors for its action(s). Cell surface proteins could be among the first molecules affected by a temperature increase to 41°C. Data presented in figure 4 show that HT effects on PMA induced activation are selective and gene dependent. FLS were left untreated or exposed to HT for 30 minutes. Immediately after HT, PMA (2 ng/ml) was added where indicated. After 4 hours stimulation at 37°C, experiments were terminated by removing medium and dissolving FLS in TRIsol for subsequent mRNA isolation. As shown in figure 4, HT was able to completely prevent PMA induced accumulation of IL-1β mRNA. The effects on IL-1α and COX-2 mRNA levels were, however, less pronounced. Steady state levels of IL-1α mRNA in hyperthermia treated cells are slightly less than 50% of controls and COX-2 mRNA levels were only reduced by approximately 50%. Short term HT has a profound impact on mitogen activated protein kinases (MAPK). The MAPKs p38, JNK/SAPK and ERK take part in the initiation and progression of many inflammatory events. Due to their central position at the intersection of many signaling pathways, MAPK have increasingly become the target of drug interventions aimed at blocking activation of proinflammatory genes (17). To investigate whether short term HT affects MAPK activation was the aim of the following experiments. FLS were left untreated, exposed to HT (41°C, 30 minutes), treated with IL-1β (5 ng/ml) for 5 minutes, or first exposed to HT and subsequently treated with IL-1β. Shown in figure 5A are the somewhat surprising results of a series of such experiments. HT blocked IL-1β induced phosphorylation of the MAPK p38 in FLS that were stimulated with IL-1β. However, in FLS, HT alone resulted in the activation of ERK as well as of JNK/SAPK. For data shown in figure 5A, phosphorylation of p38 was measured at 5 minutes. ERK and JNK phosphorylation patterns were measured 60 minutes after simultaneously treating cells with IL-1β and transferring cells back to 37°C.Time course experiments show that HT induced ERK and JNK/SAPK phosphorylation is detectable within minutes but peaks at about 60 minutes in FLS that were exposed to HT for 30 minutes followed by incubation at 37°C (data not shown). That short term HT can have a long lasting effect on IL-1β induced p38 activation is shown in figure 5B. Where indicated, FLS were exposed to 41°C for 30 minutes and subsequently challenged with IL-1β for 5 minutes, either immediately after HT or after the indicated periods at 37°C. Such Western blot experiments demonstrate that HT not only blocks p38 phosphorylation immediately after HT, but does so even 4 hours after HT. Additional data (not shown) imply that even 7 to 10 hours after HT, IL-1β induced p38 activation is still significantly diminished. Since, as shown in figure 5C, IL-1β induced phosphorylation of p38 peaks roughly 5 minutes after addition of IL-1β, the effect of HT on the activation of this kinase was analyzed at 5 minutes. Non-specific bands (marked with asterisks) and tubulin serve as loading and protein transfer controls. Effects of HT on p38 and the NF-κ κ κ κB signaling pathway are two independent events. From the above data the question arises whether inhibition of p38 by HT ultimately also accounts for the inhibition of the many ways HT is reportedly able to prevent the activation of NF-κB-dependent proinflammatory genes. Although unlikely, we tested whether blocking activation of MAPKs plays any role in the activation of NF-κB in FLS. Cells were preincubated with known inhibitors of p38, JNK/SAPK and ERK. For comparison, FLS were left untreated or exposed to hyperthermia for 30 minutes prior to being stimulated with IL-1β. As shown in figure 6, blocking p38 activation had no effect on IL-1β induced IκBα degradation. As in many experiments before, HT (30 minutes, 41°C) completely blocked IL-1β mediated IκBα degradation. Even more remote is the possibility that other MAPKs play a role in the NF-κB pathway upstream of IL-1β induced IκB degradation. This possibility has, however, also been refuted by experiments demonstrating that in FLS blocking JNK/SAPK and ERK were without any effect on IL-1β mediated IκBα degradation (data not shown).

Figure 1 .

 1 Figure 1. Short term HT prevents NF-κB activation. FLS were exposed to

Figure 2 .

 2 Figure 2. De novo synthesis of HSPs is not essential for HT mediated blockage of

Figure 3 .

 3 Figure 3. Glycerol prevents IL-1β induced IκB phosphorylation. As shown in

Figure 4

 4 Figure4HT affects steady state mRNA levels of proinflammatory genes in PMA treated cells. In FLS, HT (41°C, 30 min.) completely prevents PMA (2 ng/ml for 4 hours)

Figure 5 .

 5 Figure 5. HT blocks IL-1β induced phosphorylation of p38 but activates ERK

Figure 6 .

 6 Figure 6. The MAPK p38 plays no role in IL-1β induced IκBα degradation. Cells

Figure 7 .Figure(s) 6 Figure(s) 7

 767 Figure 7. HT prevents transcriptional activation of p38 dependent genes. Steady state mRNA levels of HAS1 are significantly lower in FLS that were exposed to short


Jutta Zwielehner

Kathrin Liszt

Michael Handschur

Alexander Lapin

Alexander G Haslberger
email: alexander.haslberger@univie.ac.at

G Haslberger

Combined PCR-DGGE fingerprinting and quantitative-PCR

Keywords: microbiota, PCR-DGGE, qPCR, Bacteroides, bifidobacteria, Clostridium, 19 feces, ageing 20 21 APS ammonium-persulfate 23

subsets within the genera Bacteroides, bifidobacteria and Clostridium cluster IV. The 32 institutionalized elderly harbored significantly higher numbers of Bacteroides cells 33 than control (28.5 % ± 8.6%; 21.4 % ± 7.7%; p=0.016) but contained less 34 bifidobacteria (1.3 %0.9, 2.7 % ± 3.2%, p=0.026) and Clostridium cluster IV (26.9% 35  11.7%, 36.36%  11.26%, p=0.036). The elderly also displayed less total Bacteria 36 diversity and less diversity with the Clostridium cluster IV (p<0.016) and Bacteroides. 37 It is estimated that between 60-80 % of the microbial diversity resident within the 48 human gastro-intestinal tract (GI) has yet to be cultivated [START_REF] Suau | Direct analysis of genes encoding 16S rRNA from complex communities 550 reveals many novel molecular species within the human gut[END_REF]. 49 However, the use of culture-independent molecular methods has enabled 50 researchers to identify important characteristics of this community. We now know that 51 the composition of GI microbiota is highly distinct between individuals (Dethlefsen et 52 al. 2006). Despite this variability, some general features are apparent. There appear 53 to be 'core species' present in a majority of humans which are quite resilient to 54 external influences [START_REF] Dore | Gut microbiota in health and disease[END_REF]. In addition to this group, there are also 'passengers' 55 or transients, sometimes in great numbers, sometimes below detection limit (Favier 56 et al. 2003). However, the majority of sequences seem to be unique to some 57 individual and not one single phylotype may be present in all humans (Turnbaugh et 58 al. 2009). A number of factors have been identified which influence community 59 composition. The type of bacteria introduced to the environment is determined by 60 those associated with food sources [START_REF] Duncan | Cultivable bacterial diversity from the 483 human colon[END_REF][START_REF] Pryde | The 538 microbiology of butyrate formation in the human colon[END_REF]. The 61 nature and composition of non-digestible carbohydrates in a diet can further stimulate 62 different types of bacteria [START_REF] Kolida | Prebiotic capacity of inulin-type fructans[END_REF], whilst hereditary dispositions 63 and personal gut environmental factors may account for unique personal 64 characteristics [START_REF] Eckburg | The role of microbes in Crohn's disease[END_REF]. 65

Correlations between the GI microbiota and diseases such as allergenicity (Shreiner 66 et al. 2008), inflammatory bowel diseases [START_REF] Sokol | 546 Specificities of the fecal microbiota in inflammatory bowel disease[END_REF]) and individual 67 dispositions such as obesity [START_REF] Zhang | 572 Human gut microbiota in obesity and after gastric bypass[END_REF] ) have been discussed. 68

The colon harbors butyrate-producing species of several genera such as Clostridium, high levels of butyrate in vitro are those related to Faecalibacterium prausnitzii, an 71 ubiquitous member of Clostridium cluster IV [START_REF] Pryde | The 538 microbiology of butyrate formation in the human colon[END_REF]. Bacteroidetes 72 species have been reported to show high variations between individuals, although 73 Bacteroides thetaiotaomicron is found in all human beings [START_REF] Layton | 503 Development of Bacteroides 16S rRNA gene TaqMan-based real-time PCR assays 504 for estimation of total, human, and bovine fecal pollution in water[END_REF]. Both 74 Bacteroidetes and Clostridium cluster IV are known to be involved in beneficial 75 functions, including nutrient absorption, production of short chain fatty acids (SCFAs) 76 and epithelial cell maturation and maintenance [START_REF] Woodmansey | Intestinal bacteria and ageing[END_REF]. 77

Another important subgroup of the human GI microbiota is the bifidobacteria. 78

Stimulation of these bacteria has previously been shown after prebiotic intervention 79 with inulin and fructo-oligosaccharides [START_REF] Kolida | Prebiotic capacity of inulin-type fructans[END_REF]. Furthermore, they 80 have been shown to be involved in prevention of atopic disease (Ouwehand et al. 81 2007), obesity and insulin resistance via enhanced barrier function of the gut 82 epithelium (Cani et al. 2007). 83

With ageing, a decrease in beneficial organisms such as Lactobacilli and 84 bifidobacteria, amongst other anaerobes, and an increase in the number of 85 facultative anaerobes [START_REF] Guigoz | The inflammatory status of old age can 492 be nurtured from the intestinal environment[END_REF]) have been reported. Population cross-86 sectional studies with relevant numbers of healthy elderly also show age-related 87 changes in GI microbiota. These include a consistent global increase in 88 nonpathogenic Gram-negative bacteria (mainly Enterobacteria), as well as country-89 specific changes in bifidobacteria [START_REF] Guigoz | The inflammatory status of old age can 492 be nurtured from the intestinal environment[END_REF]. This, along with a general 90 reduction in species diversity within most bacterial groups, changes to diet and 91 altered digestive physiologies such as intestinal transit time, may result in increased 92 putrefaction in the colon and a greater susceptibility to disease. The aged gut is 93 characterized by increased proteolytic activity, decreased amylolytic activity and 94 reduced levels of SCFA [START_REF] Woodmansey | Intestinal bacteria and ageing[END_REF]. Ageing is associated with reduced levels of prostaglandins such as PGE 2 and PGF 2  as shown in specimens of 96 stomach and duodenum biopsies [START_REF] Tiihonen | The 553 effect of ageing with and without non-steroidal anti-inflammatory drugs on 554 gastrointestinal microbiology and immunology[END_REF]. Subclinical intestinal 97 inflammation in elderly populations has been detected and is believed to contribute to 98 impaired immune functions, the underlying cause of mortality beyond 75-80 years of 99 age [START_REF] Guigoz | The inflammatory status of old age can 492 be nurtured from the intestinal environment[END_REF]. 100

Analyses of individual dispositions associated with changes of the microbiota should 101 consider quantitative and qualitative aspects of gut community structure. Analysis of 102 stool samples can target changes in colonic microbiota, since feces are 103 representative of inter-individual differences [START_REF] Turnbaugh | The human microbiome project[END_REF]. 104

The aim of this work was to investigate shifts in GI microbiota associated with aging, 105 by comparing institutionalized elderly with young healthy volunteers. To analyze 106 changes in total bacterial community composition, along with specific compositional 107 changes within the Bacteroidetes, bifidobacteria and Clostridia cluster IV, we used 108 the polymerase chain reaction (PCR) based community fingerprinting method 109

Denaturing Gradient Gel Electrophoresis. The resolution of this method allows for the 110 characterization of the dominant members of a targeted microbial community. 111

Further, we used quantitative-PCR (q-PCR) to determine the relative load of 112 Bacteriodetes, bifidobacteria and Clostridia cluster IV groups within our samples. 113 Thus, use of these methods in combination allowed the characterization of both 114 along with the cultured type strains, to generate appropriate reference markers for 169 DGGE analysis. Amplifications were carried out using primer pair 27f [START_REF] Edwards | Isolation and 489 direct complete nucleotide determination of entire genes. Characterization of a gene 490 coding for 16S ribosomal RNA[END_REF]) and sg-Clep-R [START_REF] Matsuki | Use of 16S 507 rRNA gene-targeted group-specific primers for real-time PCR analysis of 508 predominant bacteria in human feces[END_REF], which are specific for members of the 171 Clostridium cluster IV, and 32f and 708r [START_REF] Bernhard | A PCR assay To discriminate human and 476 ruminant feces on the basis of host differences in Bacteroides-Prevotella genes 477 encoding 16S rRNA[END_REF] which are 172 specific for members of the Bacteroides. Amplified products were cloned into a p-173 GEM Easy Vector (Promega) following the instructions of the manufacturer. Clone 174 libraries were screened as previously described [START_REF] Schabereiter-Gurtner | An advanced 541 molecular strategy to identify bacterial communities on art objects[END_REF]. 175 The institutionalized elderly of this study did not consume any whole grain products 272 at all but received supplements with soluble fiber (Benfiber, Novartis). 273 274

TaqMan-quantification 275

TaqMan assays were set up quantifying bacterial sub-populations as percentage of 276 the total bacterial DNA. We detected no cross-reactivity of group-specific primers and 277 probes with non-target strains. Test-retest variations were between 2.7% and 5.2%, 278 values after relative quantification varied by less than 4%. The sensitivity was 279 corresponding to a 100 000 fold dilution of DNA from feces, that is 20 copies of 16S 280 rRNA gene per reaction. 281

The elderly harbored only 69%21.6% of the total bacterial load in their faeces 282 compared to control (figure 1). Bacteroides were found to represent a larger 283 percentage than bifidobacteria in all samples (figure 1) and the institutionalized 284 elderly harbored significantly more Bacteroides than young volunteers (p=0.016). 285

Although relative levels of bifidobacteria were highly variable among samples, the 286 differences between young and elderly probands were statistically significant 287 (p=0.026). Furthermore, elderly citizens had significantly less members of Clostridium 288 cluster IV (figure 1) in their fecal microbiota than young volunteers (p=0.036). 289 290 DGGE bandpattern analysis 291 DGGE fingerprinting with primer pair 341GC-518, which amplified the total microbial 292 community, showed high inter-individual variations (figure 2). The mean numbers of 293 bands per individual were 16.6 ± 3 bands for institutionalized elderly and 20 ± 3 frequently in elderly probands. Shannon and Simpson indices of diversity were 296 significantly lower for elderly than control (figure 3). The highly diverse dataset was 297 subjected to principal component analysis (PCA). Extraction of underlying 298 components within the dataset indicated grouping of samples according to their 299 variance (figure 4) along the first two principal components. Cluster analysis could 300 separate the fingerprints of young and elderly (figure 5). Jackknife analysis 301 suggested that the dominant bacteria of the elderly microbiota are a subpopulation of 302 the microbiota of young individuals: Predictability of groupings was 100% for young 303 and only 13.33% for elderly. 304 305 Bifidobacteria 306

An average of thirteen bands were observed in individual DGGE bandpatterns 307 obtained with the primer pair specific for bifidobacteria. Two bands with the same 308 melting behaviour as the type strains B.longum DSM 20219 T and B.pseudolongum 309 DSM 20099 T in our marker were abundant in the majority of probands. PCA was 310 performed and the first two PC's explained 23.64% of variance (figure 4). Although 311 cluster analysis showed high similarity of young and elderly bandpatterns, jackknife 312 testing demonstrated greater similarity of bandpattern for control than for elderly 313 (expressed in predictability of groupings: 91.67% for control and 35.72% for elderly). 314 PCA supported these results and could not separate young and elderly according to 315 variances in the dominant bifidobacteria of their microbiota. 316 317 Bacteroides 318 DGGE fingerprints obtained with the Bacteroides specific primers contained an 319 average of 7.6 ± 2.5 bands for the elderly, whilst young individuals averaged 9.5 ±3 bands. Diversity indices (figure 3) showed a tendency to be lower for elderly than for 321 control. PCA results suggest a tendency for less Bacteroides diversity with ageing 322 (figure 4). Elderly subjects grouped along the first principal component. Clustering of 323

Pearson correlations could separate young and elderly individuals according to 324 DGGE fingerprinting. Jackknife analysis using average similarities could predict 325 groupings of young bandpattern with 76.92% reliability and 75% for elderly. 326 327 Clostridium cluster IV 328 DGGE fingerprints obtained with the Clostridium cluster IV specific primers yielded 329 an average of 10 ±3 .5 bands per elderly individual and 13 ± 3 bands per young 330 individual. Diversity indices (figure 2) were significantly (p=0.02) lower for aged 331 citizens than for the young. One band that occurred more frequently in the young 332 than in the elderly had the same melting characteristics as a band in our marker. This 333 band was generated from a clone from our library related to the genus 334 Faecalibacterium. This clone had the highest similarity (99.2%) to an uncultured 335 bacterium EF403698 and also displayed 96.6% similarity to Faecalibacterium 336 prausnitzii T . Three more bands occurred more frequently in the young than in elderly. 337

These bands were identified from our clone libraries as relating to the genera 338 Ruminococcus (clone had 95.9% similarity to Ruminococcus bromii T , 99% similarity 339 to uncultured AJ408987 from human colon) and Subdoligranulum (clones had 94.7%/ 340 97.5% similarity to Subdoligranulum variabile T , and 97.9%/ 98% similarity to 341 uncultured bacterium DQ793301). 342 PCA (figure 4) indicated separation of young and elderly according to their 343 Clostridium cluster IV DGGE fingerprints along PC1.

Clustering and Jackknife analysis did not result in distinct clustering of elderly and 345 young individuals. Clostridium cluster IV representatives in the elderly microbiota are 346 most likely to be a subset of the species present in young individuals: Only in 35.71% 347 of resamplings elderly bandpattern grouped with each other, whereas Jackknife 348 value for young individuals was 100%. 349 350 DISCUSSION 351 Metagenomic analysis of the human GI microbiota is presently the subject of large 352 research consortia and has already substantiated the concepts of a 'core 353 microbiome' and inter-individual variations [START_REF] Turnbaugh | The human microbiome project[END_REF]). However, the 354 collection of data on 'reference microbiota' is far from complete and thus no definition 355 of a healthy microbiota is available yet. Important information comes from analyses 356 addressing the abundance and diversity of specific bacterial populations with 357 relevance to disease, diet or probiotic intervention. We used a combined molecular 358 approach to compare patterns in several target GI microbial groups between the 359 institutionalized elderly and young healthy volunteers. The community fingerprinting 360 method PCR-DGGE was used to compare the diversity present in total Bacteria and 361 also specifically within the Clostridium cluster IV, bifidobacteria and Bacteroides, 362 whilst q-PCR was used to quantify the relative population abundance of these same 363 bacterial groups in all samples. 364

Our results demonstrate some significant shifts in patterns in GI microbiota between 365 our study groups. Faeces from the institutionalized elderly had less total Bacterial 366 abundance and lower total Bacterial diversity than that from the young subjects, 367 However, samples from the elderly displayed an increase in the relative abundance 368 of Bacteroides, although this group tended to display less diversity than Bacteroides in the young. The relative abundance of the bifidobacteria and Clostridium cluster IV 370 were significantly higher in the young, and the Clostridium cluster IV also displayed 371 greater diversity in the young. Furthermore, cluster analysis revealed that for all 372 microbial groups analysed, the members of the GI microbiota in the elderly could be 373 considered a subset of that present in the young. 374

The results of our bifidobacteria analysis are in agreement with previous studies 375 which have identified a reduction and loss of diversity of bifidobacteria associated 376 with aging [START_REF] Woodmansey | Intestinal bacteria and ageing[END_REF][START_REF] Hopkins | Variation in human intestinal 499 microbiota with age[END_REF]) or hospitalization of aged 377 citizens [START_REF] Bartosch | Characterization of 472 bacterial communities in feces from healthy elderly volunteers and hospitalized 473 elderly patients by using real-time PCR and effects of antibiotic treatment on the fecal 474 microbiota[END_REF]. Our study population of elderly was supplemented 378 with soluble fiber. This prebiotic intervention alone was apparently not able to 379 antagonize ageing-related changes in the bifidobacteria. In this respect Ouwehand et 380 al. (2008) recently reported that supplementation with lactitol and a probiotic 381 Lactobacillus led to an increase in bifidobacteria in the microbiota of elderly. 382 [START_REF] Bartosch | Characterization of 472 bacterial communities in feces from healthy elderly volunteers and hospitalized 473 elderly patients by using real-time PCR and effects of antibiotic treatment on the fecal 474 microbiota[END_REF] also reported a marked reduction in the abundance of 383 Faecalibacterium prausnitzii, a member of the Clostridium cluster IV which decreased 384 in our aged group. However several studies [START_REF] Hopkins | Variation in human intestinal 499 microbiota with age[END_REF], Woodmansey et al. 385 2007[START_REF] Bartosch | Characterization of 472 bacterial communities in feces from healthy elderly volunteers and hospitalized 473 elderly patients by using real-time PCR and effects of antibiotic treatment on the fecal 474 microbiota[END_REF]) have reported a decrease in the relative abundance of 386 the Bacteroides, whereas our aged study population displayed a relative increase in 387 abundance of this group. Like us, all the previous studies reported a decrease in 388 Bacteroides diversity. Increased levels of Bacteroides have been found in individuals 389 with infectious colitis [START_REF] Sokol | 546 Specificities of the fecal microbiota in inflammatory bowel disease[END_REF]. Discrepancies in changes of the Bacteroides 390 abundance might be due to country-specific differences in this bacterial subgroup as 391 indicated by [START_REF] Mueller | 516 Differences in fecal microbiota in different European study populations in relation to 517 age, gender, and country: a cross-sectional study[END_REF]. [START_REF] Mueller | 516 Differences in fecal microbiota in different European study populations in relation to 517 age, gender, and country: a cross-sectional study[END_REF] also observed gender effects 392 within Bacteroides, with levels being generally higher in males than in females. Our 393 study populations were gender balanced with volunteers being 55% females in the group of elderly and 50% in the young. The results presented here do not support 395 gender differences in Bacteroides abundance. Reductions in amylolytic activity 396 observed in a healthy elderly population have been correlated with the occurrence 397 and diversity of Bacteroides [START_REF] Woodmansey | Intestinal bacteria and ageing[END_REF]. 398 399 Reduced numbers of Bacteria in the fecal content of elderly reflect the physiological 400 alterations associated with ageing. These include prolonged colonic transit time and 401 reduced dietary energy requirement and food uptake [START_REF] Morley | The aging gut: physiology[END_REF]. Further 402 reductions in the relative abundance of important sub-populations such as 403

  Conclusion: Despite high individual variations, our analyses indicate the composition 38 of microbiota in the elderly comprises a less diverse subset of young healthy 39 microbiota. 40 Significance and Impact of the Study: A better understanding of the individual 41 composition of the human microbiota and the effects of ageing might result in the 42 development of specifically targeted supplementation for elderly citizens in order to 43 support healthy ageing. 44 INTRODUCTION 46 47

  institutionalized elderly aged 86 ± 8 years, BMI 21.75  5.08, from a 119 geriatric department in Vienna and 17 students from Vienna joined the study. Probands were interviewed following a questionnaire assessing: age; gender; body 121 length and weight; individual health status, including chronic or acute diseases and 122 blood lipid levels; and life-style aspects, such as physical activity and dietary habits. 123 Five percent of geriatric patients suffered from manifest diabetes mellitus type 2. Ten 124 patients were bed-ridden and seven mobile. Causes for loss of mobility were 125 Parkinson's disease, dementia and osteoporosis. Nursing staff reported the 126 application of NSAIDs (non-steroidal anti-inflammatory drugs) on demand. 127 Seventeen young healthy volunteers were aged 24 ± 2.5 years, BMI 22.68  3.41 128 and their dietary habits were typical for Central Europe. 129 Study populations were gender balanced, with 55% females in the group of elderly 130 and 50% in the young group. Only non-pregnant probands with no diagnosed gastro-131 intestinal disease and no antibiotic or chemotherapeutic treatment three months prior 132 to sampling were included in the study. All probands agreed to participate in the 133 study and gave their informed consent. 134 135 Sampling and DNA extraction from Stools and Type Strains 136 From each proband, three stool samples were taken within the course of a week and 137 immediately stored at -70°C. Portions of the three samples from each patient were 138 pooled, a 200 mg aliquot was treated twice for 45 sec in a bead-beater (Mini-139 Beadbeater-8) and DNA extracted with the QIAamp® DNA Stool Mini Kit (QIAGEN) 140 following the manufacturer's protocol and then immediately stored at -20°C. 141 Type strains known to be associated with GI microbiota were grown, and DNA 142 extracted, for use as part of the markers in DGGE analysis (see below). Type strains 143 L. casei DSM 20011 T , L. delbrueckii subsp. lactis DSM 20072 T , Bacteroides fragilis 144 DSM 2151 T , Bacteroides thetaiotaomicron DSM 2079 T , Bifidobacterium longum DSM20219 T , B.longum DSM 20211, B.pseudolongum DSM 20099 and 146 B.thermophilus DSM 20210 were anaerobically cultivated on blood agar, E.coli IMBH 147 252/07 and clones were aerobically cultivated on LB-agar (liquid broth medium). The 148 biomass was resuspended in sterile phosphate buffered saline (1x PBS from 10x 149 PBS Rotistock, ROTH). Tenfold dilutions from these suspensions in sterile 1x PBS 150 were plated in duplicate on blood agar or LB-agar, colony forming units (CFU/ml) 151 were counted and DNA extracted from serial dilutions. DNA was extracted from 152 clones using the Wizard® Plus.SV Minpreps DNA Purification System (Promega). 153 The DNA of all Gram-negative bacteria was extracted with the DNA Mini Kit 154 (QIAGEN), for Gram-positive bacteria, the FastDNA Spin Kit for Soil (MP-155 Biomedicals) was used following the instructions of the manufacturer. 156 157 Polymerase Chain Reaction (PCR) 158 PCR was used to amplify 16S ribosomal RNA gene sequences from type strains and 159 Bacteria in stool samples for use in DGGE analysis and manufacture of clone 160 libraries (see below). All reactions were carried out using a ready-to-use mastermix 161 (Promega) with 1.5mM MgCl 2 . Bovine serum albumin (10mg/ml, Fermentas) was 162 added to a final concentration of 400g/ml, primer concentration in the reaction 163 volume was 0.5µM. Amplifications were carried out in a Robocycler (Stratagene). 164 165 Clone libraries 166 Clone libraries were constructed from stool samples to identify dominant members of 167 the Clostridium cluster IV and the Bacteroidetes. Selected clones were then used, 168

  Clone inserts were sequenced by 'DNA confidence' (Vienna). Nucleotide sequences 176 were corrected for vector and primer sequences in CodonCode Aligner 177 (www.codoncode.com) and taxonomically identified by comparison to previously 178 published sequences using the online tools of the ribosomal database project 179 (http://rdp.cme.msu.edu/). 180 181 DGGE 182 PCR amplifications of 16S rRNA gene fragments from total DNA extracted from stool 183 samples were carried out using the specific primer sets outlined in Table 2. 184 Separate DGGE gels were run to analyse samples for patterns in a) total Bacteria, b) 185 bifidobacteria, c) Clostridium cluster IV and d) Bacteroides. DGGE gels were 186 prepared as described previously (Muyzer and Smalla 1998) with a linear gradient of 187 25-65% for Bacteria (general), 30-65% for bifidobacteria, 20-50% for Bacteroides and 188 30-50% for Clostridium cluster IV, using a gradient mixer (Hoefer SG 30) and a 189 peristaltic pump. We generated reference markers appropriate for each set of DGGE 190 analyses (i.e. a different reference marker was used for each of a,b,c and d above) to 191 enable meaningful comparisons across multiple gels and to provide putative 192 identification of some bands. These reference markers contained fragments of 16S 193 rRNA genes from cultured bacteria and clones generated from fecal material (as 194 described above). Each marker was loaded in triplicate on each gel to allow gel-to-gel comparison. The reference marker for DGGE fingerprinting of general Bacteria 196 consisted of E.coli IMBH 252/07, Enterococcus faecium DSM 20477 T , Bacteroides 197 thetaiotaomicron DSM 2079 T , Bifidobacterium longum DSM 20219 T , Clostridium 198 perfringens (laboratory isolate), clone BT11 (98.8% similarity with Bacteroides 199 uniformis JCM 5828 T ), clone BT17 (96.0% similarity with Bacteroides vulgatus T and 200 bacterium LY88 previously identified in human feces (Eckburg et al. 2005)) and clone 201 CL16 (98.4% similarity with an uncultured bacterium from human feces (Turnbaugh 202 et al. 2006) and 96.7% similarity with Faecalibacterium prausnitzii T ). The reference 203 marker for bifidobacteria specific DGGE analysis consisted of Enterococcus faecium 204 DSM 20477 T , E.coli IMBH 252/07, Bifidobacterium longum DSM 20219 T , B. longum 205 DSM 20211, B. thermophilus DSM 20210 T and B. pseudolongum DSM 20099 T . The 206 reference marker for Bacteroides specific DGGE analysis was composed of 207 Bacteroides thetaiotaomicron DSM 2079 T , fecal clones Bt 17 and Bt11 and 208 Enterococcus faecium DSM 20477 T . The reference marker for Clostridium cluster IV 209 DGGE analysis was constructed from 7 clones all representing previously uncultured 210 species from cluster IV: Similarities with type strains from this cluster were 81.8%, 211 96.6%, 96.7% and 97.8% similarity with Faecalibacterium prausnitzii T , 92.1% and 212 92.9% similarity with Eubacterium desmolans T and 95.9% similarity with 213 Subdoligranulum variabile T . 214 215 TaqMan RTQ-PCR 216 The TaqMan-assay was carried out in a Rotorgene 3000 (Corbett Life Science) in 217 duplicate in a volume of 10 µl containing 5 µl TaqMan SensiMix DNA Kit (Quantace), 218 1 µl of each primer and probe (final concentrations table 1) and 2 µl of the 100-fold 219 dilution of the template. All probes were labeled with 6-FAM at the 5' end and carried a BHQ-1 quencher at the 3' end. TaqMan Probe (Clep-P) for Clostridium cluster IV 221 was designed with CLC DNA Workbench (www.clcbio.com). Analysis with 222 ProbeMatch (rdp.9.58) indicated that Clep-P binds to all members of Clostridium 223 cluster IV. The PCR program for bifidobacteria, Bacteroides and universal bacteria 224 consisted of denaturing at 95°C for 3min and 45 cycles of 95°/60°C for 15/ 45 sec. 225 The amplification program for clostridium cluster IV was: denaturation at 95° for 5 min 226 and 45 cycles at 95°/ 55°C for 30/ 45 sec. 227 DNA of Bacteroides thetaiotaomicron T and Bifidobacterium longum T , clone CL16 and 228 one fecal sample were used to construct standard curves for comparison of PCR 229 reaction efficiencies among different experiments and enumeration of all bacterial 230groups. Reaction efficiency (E) was estimated using the slope of the standard curve 231 and the formula E=10 (-1/slope) -1 as described elsewhere[START_REF] Penders | 534 Quantification of Bifidobacterium spp., Escherichia coli and Clostridium difficile in faecal samples of breast-fed and formula-fed infants by real-time PCR[END_REF]. 232 Quantification was done using standard curves obtained from known concentrations 233 of organisms containing the respective amplicons for each set of primers. The 234 percentage of bacterial group rRNA gene copies in relation to total rRNA gene copies 235 (relative abundance) was calculated for each individual, and the mean was 236 determined for each subject group. Relative quantification (% of bacteria) was 237 performed using Rotor-Gene 3000 calculation software (Corbett operator manual) 238 and Excel. Cross reactivity with non-target strains was tested using the Probe-Match 239 tool at the RDP website and using the strains mentioned above. 240 241 Statistical analysis. 242 Food frequency data were analyzed based on Chi-square approximation as 243 implemented in SPSS. P values <0.05 were considered significant. Band comparison 244 tables were analyzed with principal component analysis (PCA) using the default settings in 'R-software environment for statistical computing' (www.r-project.org) until 246 100% variance was explained. Transformed data were plot in a bi-plot as a function 247 of the first two principal components. Clustering was applied to DGGE fingerprinting 248 data as implemented in the GelComparII environment (www.applied-maths.com). 249 Clustering was performed based on Dice coefficient as well as based on Pearson 250 correlation. Those methods are supplementary to each other, clustering after Dice 251 takes band positions into account, whereas Pearson correlation based clustering 252analyzes the densitometric curves of each fingerprint. UPGMA dendrograms were 253 generated and Jackknife analysis was performed using average similarities and 100 254 resamplings. Jackknife analysis is a leave-one-out method that tests the reliability of 255 the clustering similar to bootstrap analysis. Shannon and Simpson's diversity index 256 were calculated on binary band information (presence-absence) with the default 257 settings implemented in the 'vegan' package in 'R'. Shannon index is defined as H = -258  p i ln p i , where p i is the proportional abundance of species i. For Simpson's index D, 259 this is 1- ((p i )^2), where p i is the relative frequency of the i-th species. Prior to 260 application of Student's t-test for diversity indices and qPCR data, equality of 261 variances of the three datasets was tested using the F-test as implemented in participant's dietary habits indicated similar consumption patterns of 268 fruits, vegetables and milk products in both groups. Young volunteers stated 269 significantly less frequent (Chi 2 Test; p<0.04) consumption of meat than elderly probands and regular consumption of whole grain products several times a week. 271

Figure 1 :Figure 2 :Figure 3 :Figure 4 :Figure 5 :

 12345 Figures 440

Eubacterium and Fusobacterium[START_REF] Pryde | The 538 microbiology of butyrate formation in the human colon[END_REF]. Among strains that produce

bands in control. Two bands, highlighted in figure2, occurred significantly less