Clearly, several approaches may be taken to enhance DC tolerogenecity. We previously Ruxolitinib clinical trial demonstrated that genetic modification of immature DCs

with inhibitory cytokines such as IL-10, TGF-β or soluble TNF receptor could inhibit pathogenesis of autoimmune diseases or prolong allograft survival 14–16. In addition, exosomes derived from IL-10-treated or IL-4 gene-modified DCs could also suppress inflammation and attenuate progression of autoimmune diseases 17, 18. In spite of the existing findings, new approaches to enhance DC tolerogenecity or utilizing new subsets of tolerogenic/suppressive/regulatory DCs for the control of inflammation and autoimmune diseases with increased efficacy and identifying the underlying mechanisms remains a hot topic in immunology. Ligation of Fc receptors for IgG (FcγRs) by immune complexes selleckchem (IC) may trigger two opposing signals, activating or inhibitory, depending on the FcγR subtypes 19. Three FcγR subtypes are currently known: FcγRI, FcγRIIa and FcγRIII that trigger cell activation by the presence

of an immunoreceptor tyrosine-based activation motif in their cytoplasmic fragments 19, and FcγRIIb that deliveries inhibitory signal through its intracellular domain containing an immunoreceptor tyrosine-based inhibition motif 20. Accumulating evidences have shown that inhibitory FcγRIIb is important for the maintenance of peripheral tolerance, oxyclozanide and FcγRIIb deficiency is associated with the pathogenesis of experimental autoimmune models and of systemic lupus erythematosus (SLE) 21, 22. SLE is characterized by high levels of autoantibodies in circulation. Tissue deposition of IC plays a major role in the pathogenesis of inflammatory injuries in SLE. Therefore, enhancement

of FcγRIIb expression and function may be useful to the prevention and treatment of inflammatory autoimmune diseases such as SLE. Disorders of DC subsets and functions are associated with the pathogenesis of SLE. High level of type I IFN from overactivated plasmocytoid DCs (pDCs) are also involved in the pathogenesis of SLE 23. SLE patients have significantly decreased expression of BDCA2, a negative regulator of TLR9-dependent activation and, accordingly, have markedly elevated IFN-α levels 24. Furthermore, the decrease in myeloid DCs with a tolerizing phenotype was reported in SLE patients 25. Given these lines of evidence, it is plausible that intervention of DC function may be another approach for the treatment of SLE.

, but with the two structures repeatedly alternating every 2 min. Under these circumstances, there was no evidence of learning either of the two syllable statistics, presumably because the 2-min exposure was insufficient to “tag” the fact that there were two structures. However, when each structure was spoken by a different talker or voice, this tagging was obvious and now subjects learned both syllable statistics. Thus, as in Gebhart et al., when there is a strong cue that indicates the presence

of two different contexts, XAV-939 molecular weight learners are quite adept at keeping track of two separate sets of statistics that describe the two underlying structures. This notion of context is crucial not only for the efficacy and efficiency of learning, but also for the propensity to generalize. Consider a situation in which a naïve learner is attempting to understand a corpus of environmental input. Even if the learner has a stationarity bias, there are a variety of contextual cues that are very obvious (e.g., time of the day as indicated by sunlight versus darkness or when a given parent is present versus a preschool teacher). How does the learner decide which of Y-27632 in vivo these contextual cues is relevant—leading

to the inference that there is a new structure to be learned—and which contextual cues should be ignored because they are uncorrelated with a change in structure? As noted by Qian, Jaeger, and Aslin (2012), this distinction between cue-sensitivity and cue-relevance is what was earlier referred to as Problem 3—the presence of contextual ambiguity. That is, learners must be open to the possibility that a cue serves as a contextual signal for a change of structure, but TCL not overly willing to assume that every cue that is discriminable signals such a contextual cue. Problem 3 has a further implication for

what a learner should do after they have partitioned (or not) the environmental input into separate structural representations. If a learner has a stationarity bias and treats multiple structures as being generated by a single representation, then they will incorrectly generalize across those multiple structures. This overgeneralization is a common property of early language productions for certain grammatical morphemes (e.g., the –ed ending on verbs). In contrast, if a learner has a nonstationarity bias and falsely infers multiple structures when they are not present in the input, then they will incorrectly restrict generalization. This undergeneralization is seen in 5-month-old infants who, after exposure to multiple views of a single person’s face, fail to generalize to a novel view of that same person’s face (Fagan, 1976).

Thus this special issue tries to cover some of the major areas of neural repair and regeneration and by so Small molecule library clinical trial doing highlight the potential for such treatments to be used to great effect in the clinic. However each article also underlines the limitations of the different approaches as well as the challenges they present for the future. Nevertheless understanding what is being investigated and how it may work, means that in the future, the treatment of many disorders of the CNS may not simply rely on symptomatic agents but the use of synergistically combined regenerative

therapies. “
“We first reported ubiquitin-positive tau-negative intraneuronal inclusions in the hippocampal granular cell layer and entorhinal cortices in patients with amyotrophic lateral sclerosis (ALS). We then found that those inclusions

occur Selleckchem INCB024360 frequently in patients with presenile dementia and motor neuron disease. The ultrastructure of the inclusions consists mainly of granules with a few filaments. In 2006, TDP-43 was identified as a major component of the inclusions specific for frontotemporal lobar degeneration and ALS. Here, we review the current knowledge regarding ubiquitin-positive tau-negative intraneuronal inclusions. In 1964, Yuasa1 described a patient with both neurological features typical of amyotrophic lateral sclerosis (ALS) and behavioral and psychiatric symptoms of frontotemporal dementia. However, autopsy findings were not reported. In 1985, Mitsuyama2 reviewed the clinicopathological findings of 26 patients with presenile dementia and motor neuron next disease (MND) in Japan. Pathologically, there were nonspecific mild degenerative changes throughout the CNS, and he suggested the possibility of a new disease. Thereafter, we used (mainly in Japan) the term “Yuasa–Mitsuyama-type” dementia with MND to describe these patients.3 MND and ALS were used almost synonymously. At that time, we studied the pathological findings of senile changes in the autopsied brains from 21 patients with sporadic ALS, aged 42–81 years. Paraffin-embedded sections were examined with the Bielschowsky

method and by imunohistochemical staining with antibodies directed against β-protein, tau and ubiquitin. We suggested that aged ALS patients accelerate senile plaque formation.4 During these studies, we chanced to find ubiquitin-positive tau-negative intracytoplasmic inclusions in the hippocampal granular cells of some patients with sporadic ALS. These inclusions had not been previously reported, and similar inclusions are not found in routinely autopsied brains. Therefore, we studied their morphology and their specificity to ALS. We studied the brains of 27 patients with clinically and pathologically confirmed sporadic ALS (aged 42–84 years), including one patient with dementia and ALS. Fifty non-ALS patients were also studied.

In the presence of either TGF-β alone or in combination of TGF-β and IL-12, the changes in the expression levels were more modest. These results are in agreement with previous data showing that TGF-β is a critical factor for the maintenance of the Th17 phenotype 35. The expression levels of Ifng and Tbx21 mRNAs were

increased significantly only in the presence of IL-12 (Fig. 3B), yet were significantly lower than in 1-wk differentiated Th1 cells (Fig. 3C). In accordance with the mRNA measurements, the presence of the polarizing cytokines during restimulation influences the number of IL-17A+ cells. Six-day differentiated Th17 cells were either left unstimulated or were restimulated with anti-CD3 and anti-CD28 antibodies in the presence or absence of the Th17-polarizing cytokines for 18 h (Fig. DAPT concentration 3D). Then all cells were restimulated again with PMA and ionomycin for intracellular flow cytometric analysis of IL-17A and IFN-γ expression. Approximately 19% of the cells

that were not restimulated for 18 h were IL-17A+. Following Erlotinib clinical trial 18 h of restimulation without cytokines only ∼11% were IL-17+ cells, and following 18 h of restimulation in the presence of cytokines ∼25% of the cells were IL-17A+. These results show that the fraction of the IL-17A+ cells increased in the presence of polarizing cytokines during restimulation, but also that in their absence less cells express IL-17A. All together, these results show that shortly after restimulation (2 h) TGF-β is unnecessary for the inducible expression of the Th17 cytokines Il17a and Il17f and the lineage specifying transcription factors Rorc and Rora. However, enough a longer restimulation of 18 h requires a continuous presence of TGF-β to maintain the transcriptional program of Th17 cells. At these stages, IL-12 is mostly required for the upregulation of the Th1-specific genes Tbx21 and Ifng. Next we wanted to assess whether the polarizing cytokines modulate the expression of PcG proteins or their binding activity at the Il17a promoter. The expression levels of Mel-18 mRNA (Fig. 4A) or protein (Fig. 4B) following restimulation were comparable in either the

presence or absence of Th17 polarizing conditions, or even in the presence of IL-12. However, the binding of Mel-18 at the Il17a promoter was significantly diminished if the restimulation occurred in the absence of the polarizing cytokines, regardless of the presence or absence of IL-12 (Fig. 4C). We did not observe significant decrease in the binding activity of Mel-18 at the Rorc promoter in the absence of cytokines, which in general was lower than at the Il17a promoter (Fig. 4D). Although we previously showed that Mel-18 is associated with Ifng promoter in correlation with gene expression 66, we neither observed significant changes in the binding activity at the Ifng promoter nor at Tbx21 promoter in the presence of IL-12 (Fig. 4E and F).

This allows the use of ACT technology for antigen delivery and tumor immunotherapy. Diagnosis and treatment of autoimmune

diseases and allergies Autoimmune diseases are common and debilitating, but their severe manifestations could be reduced if biomarkers were available to allow individual tailoring of the potentially toxic immunosuppressive therapy required for their control. Clinically Deforolimus useful biomarkers have been identified using DNA microarrays in cancer but not autoimmunity. Ken Smith (Cambridge, UK) showed that transcriptional profiling of purified CD8 T cells, but not unseparated T cells, identifies two distinct patient subgroups predicting

long-term prognosis in four different autoimmune diseases: 17-AAG cost anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, systemic lupus erythematosus, ulcerative colitis, and Crohn’s disease. Ongoing work is also examining renal transplantation, and the underlying mechanism driving these transcriptional signatures. Ken Smith showed that genes defining the poor prognostic group are enriched for those of the IL-7R pathway, TCR signaling, and, in some diseases, those expressed by memory T cells 7. These subgroups can be identified by measuring expression of only three genes, raising the prospect of individualized therapy and

suggesting novel potential therapeutic targets for autoimmunity. Mattias Flucloronide Collin (Lund, Sweden) suggested antibody glycan hydrolysis as a novel therapy against autoimmunity. The enzyme EndoS from Streptococcus pyogenes is an immunomodulatory molecule hydrolyzing the conserved glycans in the effector part of immunoglobulin G (IgG) 8. EndoS is remarkably specific for IgG, and hydrolysis has profound effects on IgG effector functions. EndoS pre-treatment of IgG, or direct administration to animals with experimental antibody-mediated autoimmune diseases, inhibits development of disease or cures animals from established disease. The properties of EndoS make it a unique experimental tool and an attractive alternative to current therapies of conditions involving pathogenic antibodies, including antibody-mediated autoimmune diseases and acute transplant rejections. Mattias Collin described ongoing studies of the biotechnological potential of EndoS, as well as the outcomes of EndoS treatment in several, both passive and active, animal models of autoimmunity. Jörg Köhl (Lübeck, Germany) presented data on novel roles of complement in the regulation of adaptive immunity.

Antibodies against the following molecules coupled to the indicated fluorochromes were purchased from BD Pharmingen (San Diego, CA, USA): CD4-FITC, CD8-PE, CD3-biotin, CD25-biotin, CD44-FITC, CD62L-biotin, CD69 PECy7. Biotin-conjugated-anti-CD24, APC-Cy7-conjugated-anti-CD8, anti-CD3ε and anti-CD28 were purchased from Biolegend (San Diego, CA, USA). A700-conjugated-anti-CD4 and PercP-conjugated-anti-CD8 buy Veliparib were purchased from eBioscience (San Diego, CA, USA). The determination of

cell survival in fresh or cultured thymocytes was conducted by staining with Annexin V (BD Biosciences) and propidium iodide (Sigma-Aldrich, St Louis, MO, USA) after surface staining for CD4 and CD8. The anti-cylindromatosis 1 (E-4), (E-10), anti-p65/RelA (A), anti-p50/NF-kB1(C-19), anti-IKK2 (T-20) and anti-JNK (D-2) antibodies were obtained from Santa Cruz Biotechnology. The anti-pJNK

(9251) antibody was obtained from Cell Signaling. The anti-actin mouse monoclonal antibody was purchased from MP Biomedical (Solon, OH, USA). Single-cell suspensions were obtained from thymus, spleen and lymph nodes by the dissociation of isolated tissues through a 60-μm mesh. Red blood cells were excluded by Gey’s lysis solution and debris was removed by cell strainer. Cells were stained for a panel of cell markers by incubation in PBS, 0.1% NaN2, 2% FBS for 20 min on ice by titrated concentrations of reagents. Cell-associated fluorescence was analyzed by an FACSCantoII flow cytometer and the DIVA V6 software (Becton Dickinson). Flow cytometry figures were see more prepared using the FlowJo

Software (Tree Star, Ashland, OR, USA). Differences in lymphocyte populations were analyzed statistically with unpaired Student’s t-test using the Sigmaplot 9 statistical software. Immunoblotting assays were performed as previously described 28. Nuclear extracts were prepared Lonafarnib in vivo by thymocytes and EMSA was performed as previously described 26. The sequences of the oligonucleotides used to detect Oct-1 DNA-binding activity were the following: Oct-1 F: 5′-TGT CGA ATG CAA ATC ACT AG-3 Oct-1 R: 5′-TTC TAG TGA TTT GCA TTC G-3′. The sequences of the oligonucleotides with two tandemly repeated NF-κB-binding sites (underlined) that were used to detect NF-κB DNA-binding activity were the following: NF-κBf: 5′-ATC AGG GAC TTT CCG CTG GGG ACT TT-3 NF-κBr: 5′-CGG AAA GTC CCC AGC GGA AAG TCC CT-3′. Total RNA was isolated from total thymocytes or DP cells with Trizol (Invitrogen, Carlsbad, CA, USA), and oligo-dT-primed cDNA was prepared using Improm Reverse Transcriptase (Promega, Madison, WI, USA) according to the manufacturer’s instructions. A. T. performed the experiments and analyzed the results. S. G. performed the FACS sorting and prepared the extracts that were used in the experiments presented in Supporting Information Fig. 3. A. T. and G. M. designed the experiments and wrote the manuscript. G. M. coordinated the research.

rubrum-specific primers. Of the scale samples, 16% were positive for T. rubrum in the culture and PCR as well, 9% were positive in the PCR only and 3% in the culture only, whereas 5% were only KOH-positive. The corresponding results for nail samples were 17%, 20%, 3% and 7%. PCR results were available after 2–5 days, culture results after 2–3 weeks. Our results show that a specific PCR assay can successfully be used to detect T. rubrum directly in samples collected from superficial skin lesions and nails under routine

conditions. Compared with conventional methods, it is faster and more sensitive. We recommend its complementary use. Superficial tinea including onychomycosis is the most frequent cutaneous fungal infection in Germany with Trichophyton rubrum as the causative agent in about 80–90% of all cases.1,2 However, the

clinical picture of tinea caused by T. rubrum is not diagnostic because a multitude of other diseases can cause phenotypic changes Quizartinib datasheet identical to those induced by various dermatophytes, including T. rubrum. Therefore, a definite diagnosis of T. rubrum-tinea needs a positive proof of T. rubrum within the tissue. The most common I-BET-762 price and approved methods to detect dermatophytes in skin samples are KOH-mounts that allow a rapid demonstration of fungal elements, but no species identification and mycological cultures for species recognition. However, for various reasons, cultures can remain false negative, a positive culture can easily take 3 weeks and occasionally even a positive culture may not allow a definite identification. On the other hand, T. rubrum can nowadays unambiguously be identified by molecular analysis3,4 and modern PCR-based genetic methods to detect dermatophytes reliably and rapidly in infected skin and nails are currently proposed.5–11 In our study, we systematically analysed unselected skin samples collected under routine conditions from suspected tinea lesions by KOH-mounts, dermatophyte cultures and a

T. rubrum-specific PCR to check the Ureohydrolase applicability and benefit of the latter method in the daily routine. Unselected samples of skin scales and nail scrapings obtained from dermatological patients that were submitted to our laboratory for mycological testing were employed. No particular instructions had been given for the collection of these samples and all samples had been taken under routine conditions from skin lesions or nails to prove or exclude a fungal infection. The samples included scrapings from lesional stratum corneum and from nails (almost exclusively toe nails) and were submitted in glass tubes without any additives. Samples from nails were taken by scraping off material from the destructed nail plate and/or subungual debris at a site as closely as possible to the proximal margin of the lesional area by use of a curette. The time period of collection was from April 2007 to November 2008 and all submitted samples with a sufficient amount of material were included.

However, it is Adriamycin cell line now recognized that DC are also important for the induction and maintenance of peripheral T cell tolerance [15]. For

instance, mice in which both conventional and plasmacytoid DC subsets have been ablated develop severe, fatal autoimmunity [16]. Notably, patients with the recently identified combined mononuclear cell deficiency DCML [DC, monocyte, B and natural killer (NK) lymphoid-deficient], virtually lacking DC in the blood and interstitial tissues, have a reduced number of Tregs, and a quarter of these patients develop autoimmune disorders [17]. The dual function of DC in initiating immunity, on one hand, and maintaining T cell tolerance on the other hand, can be explained, in part, by the different maturation stages selleck products of DC [18, 19]. In the absence of danger signals provided by infection or inflammation (also referred to as ‘steady state’), DC are largely in an immature differentiation state. They can capture and present antigens to T cells, but in so doing will induce tolerance rather than immunity [20-22]. Maturation of DC can be induced by pathogen-associated molecular patterns (PAMP), e.g.

bacterial lipopolysaccharide (LPS) or viral double-stranded RNA [23]. The process of DC maturation enhances their immunogenicity by up-regulation of major histocompatibility complex (MHC)–peptide complexes and T cell co-stimulatory molecules (e.g. CD80, CD86) on the plasma membrane, and by inducing the production of proinflammatory cytokines (e.g. IL-12) that help and polarize T cell differentiation [24, 25]. However, the notion that immature DC induce tolerance and mature

DC induce immunity has been revised in recent years, as it has become clear that mature DC can also exert pro-tolerogenic effects. For example, DC matured in response to certain PAMP display Rucaparib a typical mature DC surface phenotype but produce anti-inflammatory IL-10 and promote the development of IL-10-producing Tregs [26, 27]. It is now generally accepted that the tolerogenic function of DC is determined by the signals that they receive during maturation; these signals can be derived either from the microenvironment in which DC maturation takes place or from invading pathogens. For instance, anti-inflammatory cytokines [IL-10, transforming growth factor (TGF)-β], immunosuppressive substances (e.g. corticosteroids) or certain PAMP (e.g. schistosomal lysophosphatidylserine) have all been shown to promote the tolerogenic function of DC [27-31]. Several mechanisms by which tolDC induce immune peripheral tolerance have been described, including blocking of T cell clonal expansion and induction of T cell anergy, deletion of T cells and the induction of Tregs. Two major groups of Tregs have been defined: naturally occurring Tregs (nTregs) that arise in the thymus, and adaptive Tregs, that are induced in the periphery (iTregs) [32, 33].

Moreover, Dlg1 loss has been linked to increased rates of cell proliferation [7]. Given the involvement of Dlg1 in signaling molecule assembly in neural synapses [2, 3], we and others proposed it could also play a role in regulating Ag receptor-mediated signaling in T cells [8-12]. Indeed, several published reports implicate cell polarity proteins in regulation of T-cell development and function. For example, Scribble has been shown to be involved in T-cell migration and immunological synapse formation [9] as well as T-cell development [13], while Par6 and aPKC

may contribute to the ability of T cells to efficiently scan dendritic cells [14], and PALS1 has been implicated in regulation of TCR-driven T-cell proliferation [15]. Recently, several reports suggested a function for Dlg1 as an important scaffolding adaptor involved in modulation of signaling

networks at the immunological synapse [8, 11, Selleck KU 57788 16-18]. Dlg1 was shown to be recruited to the immunological synapse and to colocalize with ZAP70, Lck, Vav1, TCR-ξ, and Kv.1.3 potassium channel, which collectively coordinate signaling cascades from TCR receptor to the nucleus [8, 19]. Nonetheless, R428 nmr the requirement and function of Dlg1 in T-cell activation and TCR signal transduction remains to be clarified. Because deletion of Dlg1 from the murine germline is lethal [20], we employed a conditional KO mouse in which Dlg1 loss is restricted to the T-cell lineage only, or all hematopoietic cells. Using this system, we showed that Dlg1 is not required for Ag activation of T cells harboring transgenic TCR in vitro and in vivo. Surprisingly, however, we found that Dlg1 is required for normal regulation of memory T-cell generation in response to immunization with conventional Ag. Our previous studies using RAG-deficient complementation approaches indicated that Dlg1 is dispensable for development of all major αβ-lineage thymocyte subsets [17].

To verify this finding we generated Lck-Cre+ Dlg1flox/flox and Vav1-Cre+ Dlg1flox/flox mice, in which AZD9291 transgenic Cre expression is driven by the Lck [21], or the Vav promoter [22], respectively. We observe efficient deletion of Dlg1 in both models, as ascertained by Western blotting with Dlg1-specific antibodies using lysates from either thymocytes or T-cell blasts obtained from purified and activated peripheral T cells, which show a complete deletion of Dlg1 protein (Supporting Information Fig. 1, and top panel in Fig. 2). We analyzed T-cell development in Dlg1-deficient (Lck-Cre+ Dlg1flox/flox or Vav1-Cre+ Dlg1flox/flox, further referred to as KO) and control (Lck-Cre+ Dlg1flox/+ or Vav1-Cre+ Dlg1flox/+, further referred to as WT) mice and find no obvious abnormalities (Supporting Information Fig. 2). We note, however, that the requirement for Dlg1 in T-cell development has not yet been assessed in thymocytes harboring functionally rearranged TCR transgenes.

They were finally prepared by critical-point drying, mounted on an aluminum stub and covered with a thin layer of gold

(20–30 nm). Examinations were carried out using a scanning electron microscope (XL-20; Philips, Eindhoven, the Netherlands) at the Unité Interfacultaire de Microscopie Electronique (University of Namur, Belgium). Recently, a bioinformatic screen of Brucella genomes was carried out to find AHL-acylase homolog(s). One gene encoding a protein with Fostamatinib clinical trial 24.8% identity to AiiD, the AHL-acylase from Ralstonia sp. strain XJ12B, was identified and called aiiD (Lin et al., 2003). It has been shown that AiiD from Brucella is a functionally secreted Quorum-Quenching enzyme displaying a broad-range AHL-acylase activity (J. Lemaire, unpublished data). We observed that a B. melitensis 16M strain (MG210) overexpressing aiiD exhibits a strong clumping phenotype in liquid culture. As the MG210 strain reached a high density in broth culture, bacteria aggregated and

formed a pellicle-like structure that settled to the bottom of the culture tube. A similar phenotype was already described in B. melitensis vjbR-defective strains unresponsive to AHL (Uzureau et al., 2007). Because in these QS mutants, the clumps contain exopolysaccharide labeled by the Concanavalin A (ConA) www.selleckchem.com/products/BKM-120.html lectin (Uzureau et al., 2007), which is specific for α-mannopyranosyl and α-glucopyranosyl residues (Naismith & Field, 1996), we wondered whether the strain MG210 could produce a similar exopolysaccharide. To this end, we attempted to label exopolysaccharide using ConA-FITC. Propidium iodide was used to counterstain bacteria in red. As shown in Fig. 1, strain MG210 produced a ConA-FITC-labeled matrix not observed in the wild-type strain. This result shows that the MG210 aiiD-overexpressing strain is also able to produce exopolysaccharide containing α-mannopyranosyl and/or α-glucopyranosyl residues, like B. melitensis vjbR-defective alleles did (Uzureau et al., 2007). Although

all Baricitinib these QS mutants display a similar phenotype, the MG210 strain formed larger and more stable clumps than the previously described strains. Thus, we focused our further characterization on the clumping phenotype of this MG210 strain. We were interested in solving the nature of B. melitensis exopolysaccharide(s). Exopolysaccharide was extracted from MG210 cultures as described in Materials and methods. We first tested the purity of the exopolysaccharide preparation. To this end, we carried out a dot-blot analysis using specific MAbs (Cloeckaert et al., 1990) to compare the abundance of the lipopolysaccharide O-chain and two outer membrane proteins (OMPs) described on the OMVs formed by Brucella (Omp25 and Omp31) (Gamazo & Moriyon, 1987; Boigegrain et al., 2004) in exopolysaccharide samples taken before the first dialysis step in the phenol phase of the lipopolysaccharide removal step and in the final exopolysaccharide sample.