Finally, a distinguishing trait of EUROPRISE is its PhD School of students from across Europe, a unique example in the world of science aimed at spreading excellence through training.EUROPRISE is a Network of Excellence sponsored from 2007 to 2011 by the European Commission within the 6th Framework Program. The Network encompasses a wide portfolio of activities ranging from an integrated research program in the field of HIV vaccines and microbicides to training, dissemination and advocacy. The research program covers the whole pipeline of vaccine and microbicide development from discovery to early clinical trials. The Network is composed of 58 partners representing more than 65 institutions from 13 European countries; it also includes three major pharmaceutical companies  involved in HIV microbicide and vaccine research. The Network displays a dedicated and informative web page: EUROPRISE held its second annual conference in Budapest in November, 2009. The conference had 143 participants and their presentations covered aspects of vaccine and microbicide research, development and discovery. Since training is a major task of the Network, the students of the EUROPRISE PhD program summarized certain presentations and their view of the conference in this paper. This is one of the first e-newsletters about HIV and the first to simultaneously cover the broad fields of prevention, science and technology, as well as policy aspects. It covers most scientific publications on HIV research and the most relevant news from the media.Budapest, Hungary, hosted the second annual conference of the EUROPRISE Network of Excellence (NoE) from the 15th to the 18th of November 2009. The Network has organized several conferences, workshops and PhD courses on specific topics related to HIV vaccines and microbicides. To facilitate access to information, it provides a weekly newsletter edited by Anne-Marie Prieels from GlaxoSmithKline BIO that is freely accessible on the web homepage The PhD School has 20 students directly receiving stipends from EUROPRISE and about 30 additional students, who, through their supervisors or collaborations, attend courses and meetings given by the network. The EUROPRISE training program has enhanced the students' possibilities to get involved in new collaborations with other scientific groups in Europe. This provides invaluable opportunities for students to prepare and deliver their scientific work in the form of abstracts, posters and oral presentations at meetings, including this annual conference.http://www.europrise.org. Overview lectures concentrated on microbicide use, HIV vaccine design and trials in developed and developing countries. The lectures addressed the biological and medical aspects of vaccine and microbicide research, which are fundamental for basic research.The complete conference program is available at the EUROPRISE website This article presents the students' own selection of presentations and is not meant to be a comprehensive coverage of the EUROPRISE second annual conference.An important issue for this large network is the standardization and reproducibility of assays to facilitate cross comparison and validation of data produced by the partners.One workpackage of the network is devoted to harmonize assays for the measurement of cytokine secretion as a marker of cellular immune responses . Richardhttp://www.europrise.org/neutnet.html, utilizing uncloned virus supernatant (virus infectivity assays-VIA) or Env pseudotyped viruses (PSV assays) [in vivo has not been defined. It is therefore recommended that more than one assay is used to obtain optimal information on the virus neutralisation potential of a serum or agent.Another workpackage of the network aims to develop, standardize and compare relevant assays for the detection of antibody responses. A previously initiated consortium, the NeutNet coordinated by Gabriella Scarlatti from San Raffaele Scientific Institute in Milan involves 18 independent laboratories from 12 EU countries. These participants showed in a first stage of activity that the sensitivity of different neutralisation assays differ, depending on both the antibodies and the virus used [ assays) . Target in vitro in interleukin-2 enriched medium after initial mitogenic stimulation.Guido Poli from the San Raffaele Scientific Institute in Milan presented post-entry events of viral infection. The discovery of CCR5 and CXCR4 as obligatory entry co-receptors in CD4 + cells  has resulted in creation of novel antiretroviral agents targeting these host determinants. Some of these are also in development as potential microbicide candidates. This knowledge has also contributed to an understanding of the distribution of HIV-1 variants, both world-wide and inter-individually. In acute infection there is a disproportionate distribution of HIV-1 strains using exclusively CCR5 as entry coreceptors (R5 viruses), while CXCR4 utilization  mostly occurs in subtype B infection in late stages of disease [Using a modified version of this protocol, Poli and collaborators have analysed a transcriptome of the host genome of about 22,000 genes, 11% of which were found to be activated by either R5 or X4 infection (productive and non-productive). The group is also optimizing a similar model system using peripheral CD4 + T-cells obtained from both healthy children and from children affected by primary immunodeficiency collected before and after gene therapy. The results confirm the original finding that a post-entry permissive signal for HIV replication is delivered by R5, but not by X4 viruses.The role of dendritic cells (DCs) in the detection, spread and control of HIV-1 infection has been under investigation for quite some time. At the EUROPRISE conference, Dominique Kaiserlian from INSERM in Paris and Maryse Peressin (PhD student) from the University of Strasbourg emphasized the role of DCs in HIV-1 infection. Since DCs play a major role in the induction of an adaptive immune response within the mucosa , it is cin vivo ability of mucosal and skin DCs to induce CD8 + CTL response and tolerance within monostratified  and pluristratified  epithelia. Indeed, the nasal, buccal and intestinal mucosal tissues provide ideal surfaces for vaccine delivery to induce mucosal immune responses. However, the efficacy by which a vaccine can be delivered to such surfaces is hampered by mucosal tolerance, particularly in the gastrointestinal tract [in vivo cross-priming of CD8 + CTL responses. In addition, testing of adjuvants shown to induce local secretion of CCL20 was followed by strong antigen specific CD8 cross-priming. This suggests that targeting of specific DC populations in conjunction with local secretion of specific substances can lead to the induction of CD8 T-cells. One such pathway was shown to be the up-regulation of CCL20 in the epithelium followed by DC recruitment via CCR6. By a series of experiments Kaiserlian and collaborators concluded that newly recruited DCs, rather than tissue resident LCs or dDCs, prime CD8 + CTLs. As the balance between immune response and tolerance is important in the design of an HIV vaccine, Kaiserlian's group investigated the mechanism of oral tolerance against antigen delivered intragastrically. They showed that antigen leakage from the gastrointestinal tract to the liver allowed antigen uptake and presentation by tolerogenic pDCs in the liver and mesenteric lymph nodes. pDCs are essential for oral tolerance in that they induce T-cell hypo-responsiveness. Bypassing antigen uptake by pDC might thus be a way to circumvent oral tolerance and generate an anti-infectious oral vaccine.Kaiserlian drew attention to the al tract . Hence, In vivo, DCs such as LCs or interstitial DCs (iDCs), located in mucosal epithelium or sub-mucosal tissue respectively, are considered to be the first HIV targets [in vitro. Using an in vitro model of LCs and iDCs Peressin was able to show that immunoglobulin G (IgG) can prevent HIV infection of LCs and iDCs by two mechanisms: first, neutralisation of the virus via the Fab fragment of the antibody; second, inhibition of virus and/or infected cells infection mediated by the action of the antibody Fc receptor. Peressin demonstrated that non-neutralising IgGs also inhibited HIV infection of the LCs/iDCs via an Fcγ receptor dependant mechanism. This mechanism may also involve effector cells binding to the antibody Fc receptor. HIV transfer from LCs/iDCs to CD4 + T lymphocytes can be efficiently inhibited by non-neutralising IgGs, highlighting the need for vaccines to induce mucosal neutralising as well as non-neutralising Igs in order to prevent the initial establishment of infection [Since DCs are implicated in the sexual transmission of HIV-1 , Maryse  targets . Moreovenfection .Annette Sköld from the Karolinska Institute in Stockholm presented her work on the effect of different Toll-Like Receptor (TLR) ligands on monocyte-derived DC (moDC) evaluated by upregulation of CD80 surface expression. moDC are known to express TLR1 to TLR6 and TLR8, possibly also TLR9. Sköld showed that the TLR3 agonist Poly(I:C) and the TLR4 agonist LPS, but not CpG DNA (a TLR9 agonist), can induce maturation of moDCs. TLR9 activation worked together with TLR4 but not with TLR3 activation. The TLR3 activation induced the production of cyto/chemokines IL-12, IL-4, TNF-α, MCP-1, MIP-1α and MIP-1β. Phosphorylation of IRF3 occurred after TLR3 activation, but not after TLR9 activation by CpG DNA. The fast timing of these events suggests that the inhibitory effect of TLR9 ligands must occur early in the transduction pathway. Finally, Sköld presented a CpG DNA molecule with a modified backbone which did not induce moDC maturation, and was only partially able to inhibit the TLR3 Poly(I:C)-induced maturation. This implies that the structure of the fine structure of the oligonucleotide CpG DNA is essential for its inhibitory function .Streptococcus gordonii and Adenovirus), to prime local CD4 + and CD8 T-cells. Vaccine formulations were administered either nasally or vaginally and T helper and CTL proliferation, expression of activation and migration markers were analysed [S. gordonii vaccine vector allowed efficient internalization of the vaccine by DCs, followed by DC maturation and activation. The intranasal immunisation induced primed CD4 + and CD8 + T-cells in the lymph nodes draining the respiratory, genital and intestinal tract. This response was maintained post immunisation. Using this type of vaccination strategy, Medaglini and her group were able to observe activation of proliferating T-cells as measured by the up-regulation of CD69. At the same time, a modulation of migration markers such as CCR7 and CD62L could be detected on proliferating CD4 + and CD8 + T-cells. A comparison of routes for vaccine administration showed differences between intranasal and intravaginal administration. Although good proliferative responses were found in both scenarios in lymph nodes draining the immunisation site at day 5 post vaccination, proliferation in distal sites was observed primarily following intranasal vaccination. Overall, it was concluded that the adoptive transfer model is a powerful tool for studying priming by mucosal adjuvants and delivery systems in vivo.Human mucosa displays a surface area of more than 400 m2 and contains roughly 80% of all immune cells. Hence, research has been focused on the development and improvement of mucosal adjuvants and routes of vaccine delivery to elicit mucosal immune responses. In the context of HIV-1 infection, mucosal priming events in particular are essential to elicit memory cells at sites of pathogen entry. As underscored by Donata Medaglini from the University of Siena, T-cell priming results may be important as early markers of vaccine immunogenicity and immunological memory. However, the study of T-cell priming is hampered by the low precursor frequency of naïve T-cells. Using an adoptive transfer model of naïve antigen-specific transgenic T-cells in singenic recipient mice, Medaglini's group tested the ability of various mucosal TLR-dependent as well as -independent adjuvants , or of different vaccine delivery systems , which are TLR9 ligands, and α-GalCeramide as potential mucosal adjuvants in the murine female genital tract. He reported that mice given an immunogen  together with CpG ODN were protected against HSV challenge . SimilarSafety is critical in the improvement of adjuvants and vaccines. Hence, the degree of inflammation and IL-1 production in the presence of such adjuvants in mucosal tissue was analysed α-GalCer was described to induce low levels of inflammation, while CpG oligonucleotides up-regulated a larger number of genes in vaginal tissue and induced inflammation to a greater extent . Since iOne of the major challenges in the field of HIV-1 prevention is to elicit both systemic and mucosal protection. To address this question, a novel approach of mucosal immunisation was evaluated by Quentin Sattentau from the University of Oxford; he combined an Env-based experimental vaccine antigen (gp140CN54) with PRO2000, a candidate topical microbicide . This vaccine - microbicide combination, tested in mice and rabbits, significantly increased the titres of Env specific mucosal IgA (mice) and systemic and mucosal IgG (rabbits) compared to immunisation with Env alone. Furthermore, rabbit vaginal IgG was able to neutralise the virus. Moreover, PRO2000 was shown to be a robust TLR4 antagonist which may create a mucosal anti-inflammatory environment by skewing the mucosal immune response towards a Th2-type and suppressing the production of inflammatory mediators. This anti-inflammatory environment in combination with the induction of locally produced neutralising antibodies may provide a primary barrier to mucosal HIV-1 infection.Nicolas Ruffin's PhD project at the Karolinska Institute, Stockholm, concerns the adaptive immune response. Increase of CD28-CD8 + T-lymphocytes is characteristic for ageing and for chronic inflammatory infections such as HIV-1 . The incDonato Zipeto from the University of Verona talked about broad-spectrum neutralising antibodies against HIV-1 elicited by fusion complexes and CD4-independent gp120/41s. Due to the extraordinarily high variability of the HIV envelope glycoprotein, it is essential to focus on conserved epitopes. Such conserved epitopes are exposed transiently during fusion of the gp41 transmembrane region with the target cell. Immunisation of mice or rabbits with fusion complex intermediates or CD4-independent gp120/41s demonstrated that such complexes are immunogenic and induced antibodies . Indeed,One project focused on the possible role that broadly-neutralising antibodies might have in limiting HIV disease progression. However, Zelda Euler (PhD student) from the Academic Medical Center at the University of Amsterdam found no correlation between cross-reactive HIV-1 specific neutralising activity in serum and the clinical course of HIV-1 infection . This stPresentations by the PhD students Tessa Dieltjens, Lara Mainetti, Marie Borggren, Evelien Bunnik and Marit van Gils concerned the evolution of HIV-1 virus in response to the humoral immune pressure. A range of viruses from patients at various stages of disease progression were studied for susceptibility to neutralisation, using autologous sera or monoclonal antibodies. All viruses were shown to escape from the neutralising antibody response mounted by the host -23. InteAn HIV-1 vaccine solely based on inducing cellular immunity appears to be insufficient to protect against HIV-1 infection . A particular challenge for the induction of a neutralising antibody response is to improve immunogen capturing, processing and presentation by antigen presenting cells (APC). Hans Wolf, from the University of Regensburg, presented a new approach that uses a novel technique to reactivate virus-specific cytotoxic T-cells and T-helper cells by means of cross-presentation of soluble proteins mediated by urea adjuvants.de novo intracellular protein synthesis. Exogenous proteins, despite being internalised by macrophages, do not usually enter this pathway and vaccines based on inactivated viruses or purified proteins are generally poor inducers of the CTL response. However, certain types of antigen presenting cells are able to facilitate the induction of CTLs upon exposure to protein antigens via the process of cross-presentation. Novel modifications of proteins have been developed to take advantage of this phenomenon. It was shown that dissolution of proteins in high molar urea followed by pulsing of cells in low urea concentrations can overcome the barrier to endogenous processing. The urea treatment facilitated protein translocation into both the MHC-I and the MHC-II presentation pathways. Using the Epstein-Barr Virus (EBV) BZLF1 protein, Wolf's group showed that the urea-treated EBV protein (uBZLF1) undergoes temperature-dependent uptake by APC and also that different sub-populations and mononuclear cells from EBV-seropositive individuals pulsed with uBLZF1 were efficiently induced BLZF1-specific cytotoxicity and T-helper cells by means of cross-presentation [in vivo priming of cytotoxic T-cells was demonstrated in mice using urea-treated HIV-1 p24 combined with CpG oligonucleotides. Overall, Wolf stressed that urea treatment of proteins successfully induces antigen-specific CTL, and that this technology may be considered as a new strategy to increase protein-specific CTLs. This innovation may have prominent implications for in vivo priming of HIV-1-specific CTLs in therapeutic vaccine studies.Priming and re-stimulation of CD8 + CTL requires the endogenous processing of proteins and the expression of relevant fragments within the context of the MHC-I molecule, a process usually resulting from entation . Similarin vitro. This suggests a role for recombinant human GH as a possible immunomodulatory therapy, complimentary to the benefits of effective antiretroviral drug therapy, for HIV-1 infection [Margarita Bofill from Irsicaixa in Barcelona investigated the effects of the administration of growth hormone (GH) on immune reconstitution of HIV-infected adults. HIV-infection causes a severe down-regulation of virus-specific CD4 + and CD8 + T-cells that is not restored upon treatment with highly active antiretroviral therapy (HAART) . Bofill nfection . Patientnfection , this stA special guest of the meeting was Jerome Kim (U.S. Military HIV Research Program), who presented the recently concluded Phase III Prime Boost HIV Vaccine Trial performed in Thailand . The ALVin vivo, with peak viremia slightly lower and kinetics similar to SIVmac 239Δnef [Vaccination of Rhesus macaques with live attenuated SIV provides immediate protection against wild-type virus. While the use of attenuated HIV in humans is unlikely, the model may provide important insights on the mechanisms of protection. In order to investigate the role of adaptive immune responses and to understand whether the persistence of the vaccine virus is central to protection in macaques, a conditional live attenuated (Δnef) SIV has been developed that is dependent on the presence of doxycycline (SIVrtTA) . SIVrtTA 239Δnef . The virIt is difficult to cross-calibrate the human and macaque models for immunisation studies; it is therefore crucial to design challenge experiments that resemble the human system as closely as possible. Hence, to reflect human transmission routes such as sexual intercourse, repeated low-dose mucosal challenges in monkeys may be performed instead of intravenous challenges . HIV dynex vivo with influenza M1-expressing vectors demonstrated that these cells induced a strong expansion of autologous, antigen-specific CD8 + T-cells. This suggests that replication-defective vectors could be used for safe and efficient transduction of human antigen-presenting cells for vaccination purposes.Andrea Cara from the National AIDS Center in Rome summarised his work aimed at increasing the safety of lentiviral vectors. The use of such vectors for vaccination is currently cautious because of the potential dangers posed by integration of vector nucleic acids into the host genome. Integrase-defective lentiviral vectors (IDLV) based on HIV or SIV with mutational inactivation of the catalytic sites do not integrate. Transduction of both dividing and non-dividing cells results in transcription of episomal forms of the vector and a strong expression of the gene of interest. In non-dividing cells, episomal vector expression continues over a long period of time, whereas in dividing cells the expression decreases as the cells proliferate . A singlCaroline Weber (PhD student) presented her work on the use of nanoparticles as a vaccine vehicle for the delivery of adjuvants and antigens. For this purpose, biodegradable synthetic Poly D, L-lactic acid (PLA) or chitosan (CNP) nanoparticles, which are readily phagocytosed by DCs, were used . Phagocyin vitro with HIV VLPs did not show differences in the expression of cell surface markers such as CD83, HLA, CD80 or CD14. However, a difference in CD86 expression levels was observed between the two groups, suggesting that immune cells from seropositive individuals are capable of responding to the HIV antigens incorporated in the VLPs. The study of the pattern of cytokine production showed that IL-10 and IL-6 were more expressed in seropositive individuals than in seronegative controls. In the presence of VLPs, low doses of IL-10 were measured, suggesting that VLPs support a Th2 or a T regulatory pathway rather than a switch to Th1 response. Finally, the activation of both lymphokine clusters and IFN- stimulated gene clusters was confirmed in the PBMCs of infected individuals.Luigi Buonaguro from the National Cancer Institute in Naples emphasized the advantage of virus-like particles (VLPs) in HIV-1 vaccine development. The concept of using VLPs to induce cellular immune responses has already been used in many areas of virus vaccine research, the reasons being their ability to structurally mimic the actual pathogenic agent and their potential to express multiple epitopes in order to induce a broad immune response. Buonaguro's group engineered VLPs expressing HIV-1 gp120 and Pr55gag and used them for intraperitoneal and intranasal immunisation of mice . IntranaJulianna Lisziewicz and Esther Natz from Genetic Immunity in Budapest described the DermaVir Patch, a novel therapeutic vaccine against HIV/AIDS. The DermaVir Patch is their lead therapeutic vaccine candidate and originates from a development pipeline for plasmid- based vaccines . The aimThe emerging field of microbicide research is evolving rapidly, with several new innovative approaches, such as colorectal explants, freeze-dried tablets, new potential inhibitory molecules, and a vaccine-microbicide combination for mucosal immunisation. Carolina Herrera from St. George's Hospital, University of London, explored the use of drug combinations for colorectal application. To this end, the antiviral efficacy of two nucleoside reverse transcriptase inhibitors (NRTI) and two non- NRTIs (NNRTI), alone or in combination, were assessed in a colorectal explant model. The results clearly depicted the higher inhibitory activity of drug combinations compared to each drug alone. Furthermore, triple and quadruple combinations showed higher inhibitory activity than two drugs even against RTI escape mutants .In vitro testing of the gels and their freeze-dried variants demonstrated an advantage of the freeze-dried tablets. The latter displayed better stability, in addition to the higher viscosity and muco-adhesive properties compared to gels. Furthermore, there was no significant difference between the two formulations in the release of the antiviral compound dapivirine. In summary, due to their physicochemical properties, the freeze-dried tablets offer a prolonged vaginal residence time and a sustained release of antiviral compounds.Freeze-dried muco-adhesive tablets, designed to overcome problems of poor mucosal retention and maintained gel structure, were presented by Manish Umrethia from Queen's University in Belfast. Carbopol, dapivirine and other polymer components were mixed to form multiple polymeric gels, and freeze-dried. in vitro. In addition, Olipiphat™ showed synergistic effects with the nucleoside RT inhibitor AZT. Paxton reported two molecules that can block HIV-1 capture and transfer through binding to DC-SIGN expressed on DCs. One molecule, bile-salt stimulated lipase (BSSL), was isolated from human milk and the other, mucin6 (MUC6), from the seminal plasma. They have similarities in structure and in their specific glycosylation patterns, which likely facilitates their binding efficiencies [New inhibitory molecules based on natural substances in body fluids or sulfonamides were identified by Edward Karamov, Sylvaine Blois and William Paxton. Olipiphat™, a humic substance that is only moderately toxic, had a pronounced dose-dependent activity towards both AZT-sensitive and resistant HIV strains ciencies .Camelidae family, devoid of the light chains [Andrea Gorlani (PhD student) from Utrecht University emphasised the importance of microbicide development and introduced the use of llama heavy-chain antibody fragments (VHH) combined with topical microbicides. Immunoglobulin of the t chains , have bet chains . Gorlaniin vitro, requiring only a few amino acid changes while displaying cross-resistance with other CD4 bs inhibitors. However, this should not be a problem as these inhibitors are not used in therapy. Reverse transcriptase inhibitors are, however, essential in HIV-1 therapy and their use as microbicides could narrow systemic therapeutic options, as demonstrated by Philippe Selhorst (PhD student). Thus, RTI microbicides might promote the selective transmission of resistant virus [A series of posters addressed various hurdles in microbicide research. Since microbicides will inevitably be used also by undiagnosed HIV + women, there is growing concern about acquiring resistance to HIV if antiviral agents are incorporated in the microbicide. Katrijn Grupping (PhD student) showed that high level resistance to two microbicide candidate CD4 binding site (bs) inhibitors was easily induced nt virus ,45. Therin vitro of different pharmaceutical excipients in vaginal microbicide formulations. Their study revealed that excipients show a distinct hierarchy in their potential to exert toxic effects and that this should be addressed when considering their inclusion in developing new formulations. One novel approach to optimize mucosal protection would be to combine microbicides with a mucosal vaccine. To this end, Katja Klein (PhD student) screened different permeation enhancers as potential antigen carriers for mucosal delivery. She demonstrated that at least some of the compounds were able to increase the bioavailability of vaccine antigens through the vaginal route. Donatella Negri et al. , demonstrated that sublingual immunisation also showed promise as an alternative route for vaccine delivery as it induces a persistent immune response in mice. Umrethia et al. focused on optimizing the administration of mucosal vaccines or microbicides containing gp41 constructs. Freeze-dried formulations were developed which are suitable for administration via sublingual and vaginal routes. The lyophilized formats can release gp41 molecules at a high rate, have an increased antigen stability and are easy to apply, representing a useful tool for the development of microbicides.The next important issue is microbicide delivery. Youssef Gali et al.  have measured the toxicity profile ex vivo with replication defective SIVmac239ΔEnv/VSVEnv pseudovirus. Activated but not resting apoptotic cells proved to be adequate adjuvants for systemic IgG and mucosal IgA production. Three intradermal immunisations induced IFN-γ production, Th1 and CD8 + T-cell responses as well as neutralising antibodies and no detectable levels of virus replication. The second strategy involves biocompatible microspheres (H1D) as a delivery system for DNA and protein vaccines.Two approaches for HIV vaccination have been investigated. One strategy is based on the activation of DC by apoptotic cells . ApoptotMicrospheres have been hypothesised to favour the uptake of protein, induce maturation of APC, protect and permit a controlled release of antigen. Cynomolgus monkeys immunised with biologically active HIV-1 Tat protein adsorbed on H1D microspheres showed a significant control of viremia after challenge which correlated with the preservation of CD4 + T-cells. One hypothesis is that vaccine modalities that specifically improve T-helper cell responses might lead to better protection. Sieghart Sopper  showed in a macaque/SIV model for AIDS that expression of activation markers are related to higher viral load and disease progression as early as four weeks after infection. Vaccination using different prime boost regimens, which reduced acute and post-acute viral load, resulted in earlier activation of CD4 + T-cells . These rThe conference presentations focused on common goals of developing effective HIV prevention strategies. EUROPRISE brings together scientists from both microbicide and vaccine fields. The program focuses on the premise that vaccines and microbicides that target multiple stages of viral transmission through the mucosa will have the best chances of success. To demonstrate such approaches several partners are involved in clinical trials. The meeting in Budapest was focused on collaborative work between partners and was largely presented by the shared PhD students of the network. The showcasing of presentations by PhD students at the meeting promises a bright future for HIV research within Europe.The authors declare that they have no competing interests.All authors participated at the EUROPRISE conference as to be able to report on it. MB, AC, KDC, WDH, TD, SD, KG, DH, JH, KK, LM, PP, MR, JS, PS, AS, MJVG, and CW were in charge of the writing of dedicated chapters covering the different sessions of the conference. GS, BW and RS organized the sessions and the writing. Together with PB they wrote, corrected and revised the manuscript. All authors read and approved the final manuscript.


A soft-agar diffusion-chamber technique was used to grow colonies from human melanoma xenografts. Plating efficiencies ranged from 0-042% to 75% and increased with serial passage of some tumours. Cells in colonies were similar to human melanoma cells in morphology, histochemistry and ultrastructure, and were shown by immunofluorescence to contain human antigens. Xenograft tumours could be regrown from the colonies when re-implanted into immune-deprived mice. Cell-survival curves were constructed from 5 xenograft lines treated with 4 cytotoxic drugs. All lines were resistant to adriamycin, but each line appeared to have an individual spectrum of sensitivity to the more effective drugs. The responses were compatible with the clinical pattern of response in melanoma, and in 2 cases the objective response of lung metastases to treatment with melphalan was consistent with the xenograft cell-survival data. Dose-response curves were exponential for treatment with methyl-CCNU and melphalan, but distinct plateaux were seen for 2 xenografts treated with doses of DTIC over 100 mg/kg. These were thought to be due to resistant subpopulations of clonogenic cells within the tumours.


They typically feature an N-terminal effector domain, a central nucleotide-binding domain (NACHT) and a C-terminal ligand-binding region that is composed of several leucine-rich repeats (LRRs). NLRs are believed to initiate or regulate host defense pathways through formation of signaling platforms that subsequently trigger the activation of inflammatory caspases and NF-kB. Despite their fundamental role in orchestrating key pathways in innate immunity, their mode of action in molecular terms remains largely unknown. Here we present the first comprehensive sequence and structure modeling analysis of NLR proteins, revealing that NLRs posses a domain architecture similar to the apoptotic initiator protein Apaf-1. Apaf-1 performs its cellular function by the formation of a heptameric platform, dubbed apoptosome, ultimately triggering the controlled demise of the affected cell. The mechanism of apoptosome formation by Apaf-1 potentially offers insight into the activation mechanisms of NLR proteins. Multiple sequence alignment analysis and homology modeling revealed Apaf-1-like structural features in most members of the NLR family, suggesting a similar biochemical behaviour in catalytic activity and oligomerization. Evolutionary tree comparisons substantiate the conservation of characteristic functional regions within the NLR family and are in good agreement with domain distributions found in distinct NLRs. Importantly, the analysis of LRR domains reveals surprisingly low conservation levels among putative ligand-binding motifs. The same is true for the effector domains exhibiting distinct interfaces ensuring specific interactions with downstream target proteins. All together these factors suggest specific biological functions for individual NLRs.Innate immunity represents an important system with a variety of vital processes at the core of many diseases. In recent years, the central role of the Nod-like receptor (NLR) protein family became increasingly appreciated in innate immune responses. NLRs are classified as part of the  Eukaryotes have evolved complex systems to detect microbial infection and other potential threats to the host. Recognition of microbes relies on the sensing of microbe associated molecular patterns (MAMPs) by germline-encoded host pattern recognition molecules (PRMs), which include various families of leucine-rich repeat (LRR) bearing proteins in plants and animals. While Toll-like receptors (TLRs) constitute the main sensors for detection of extracellular microbes, recent findings suggest that two distinct protein families, the RIG-like helicases (RLHs) and the Nod-like receptors (NLRs), act as intracellular surveillance molecules Like TLRs, NLRs are thought to recognize microbial products, as well as other intracellular danger signals, thereby initiating host defense pathways through the activation of the NF-kB response and inflammatory caspases Structurally, NLRs are large multi-domain proteins with a tripartite architecture. NLR proteins typically contain a central nucleotide-binding domain termed NACHT domain , N-terminal effector domains  for binding downstream signaling molecules, while the C-terminal part consists of a receptor domain, which is characterized by a series of leucine-rich repeats (LRRs). It is hypothesized that the crucial step in NLR activation lies in the oligomerization of the NACHT-domain, thereby forming an active signaling platform  In respect to the C-terminal LRR region NALP10 is the only NLR member that has no or only a very short occurrence of LRR repeats. NALP1 on the other hand shows a typical LRR region, but contains two additional domains C-terminally of those LRRs; a FIIND domain of yet unknown function and a C-terminal CARD domain  that disDetailed sequence comparisons of 22 human NLR members reveal an overall sequence identity in the range of 10–30% amongst pairs. Since domain shuffling is a eukaryotic hallmark and has created a large complexity of functions in proteins, it hampers evolutionary analysis of full length proteins. In particular effector domains are subject to domain accretion, and/or domain shuffling or duplication for the acquisition of new domain architecture. Taking this into account, we have chosen the NACHT domain to conduct a phylogenetic analysis, addressing the possible evolutionary history of NLR proteins.By comparing the NACHT region of the NLR family members, we observed that the phylogenetic distribution clearly correlates with their respective effector domain composition . For insMembers of the AAA+ superfamily feature a so called ATPase, P-loop or Rossman-fold which adopts a three-layered α-β sandwich configuration. This fold contains recurring regulatory units with the β-strands forming a central, parallel β-sheet, which is embedded between α-helices on both sides (scop id C.37.1.20). The parallel β-sheet forming the core of the ATPase domain assumes a 51432 topology To decipher the structure and mechanism of the NLR protein family we performed a multiple sequence alignment of NLR proteins and Apaf-1  focusingThe Walker A motif is composed of the characteristic consensus pattern GxxxxGKT/S (x represents any amino acid), where the lysine residue directly interacts with a phosphate moiety of ATP The Walker B motif of ATPases, located in the nucleotide-binding site, is characterized by the conserved sequence pattern hhhhDD/E (h represents a hydrophobic amino acid). The proximal aspartate residue is crucial for coordinating binding of the Mg2+ cation, which has been shown to be required for nucleotide hydrolysis. The second acidic residue, usually glutamate, primes a water molecule for the hydrolysis of ATP et al reports that NALP12, which also contains a modified Walker B box, is capable of both, ATP hydrolysis and oligomerization Generally, the Walker A/P-loop and Walker B motifs are well conserved amongst ATPases (e.g. Apaf-1 and CED-4). However, our multiple sequence alignment revealed that all NLRs, with the exception of NAIP and NALP11, contain a modified Walker B motif, where the second acidic residue is missing. Thus, it remains elusive if NLRs harbouring these substituting amino acids  within the Walker B motif, hhhhD[GAS]hDE, are still capable of nucleotide hydrolysis . InteresThe Sensor 1 motif is typically found adjacent to the Walker A and B motifs and interacts with or “senses” the γ-phosphate of ATP  Additional NLR sequence motifs are the cysteine rich region in the NACHT domain, containing a VCWxVCT motif located adjacent to the nucleotide-binding site , which pSince our detailed sequence analyses revealed that most NLRs and Apaf-1 share the same domain architecture and many secondary structure features, the availability of structural and mechanistic data for Apaf-1 provides the opportunity to link conserved sequence features of NLRs to functional aspects of NLR signaling. Cytochrome c activated Apaf-1 has been shown to undergo an ATP-hydrolysis-dependent conformational rearrangement in order to form heptamers through an interaction of its NACHT domains. Interestingly, the heptamers were proposed to arrange in a ring-like structure, which is usually found in AAA+ ATPases such as RuvB or NtrC1 Based on the fact that all structural features required for oligomerization are present , we hypoStructural and mutational studies of the CARD domains of Apaf-1 and procaspase-9 have identified the essential motifs for procaspase-9 activation by Apaf-1 Based on these findings, we examined whether these residues, which are necessary for homophilic CARD-CARD interactions, are conserved among NLR CARDs and the CARDs of their effector proteins, respectively, by means of multiple sequence alignment . AlthougTo date, neither information from crystal structures nor mutational analysis of PYRIN domains or PYRIN-PYRIN interactions have been reported By utilizing multiple sequence alignments  of both,The proposed model of CARD-CARD and PYD-PYD interactions is that the acidic patch of one domain interacts with the basic patch of the other protein. Hydrophobic residues of adjacent regions are also suggested to be important in this interaction. Nevertheless, it is not clear so far, if there is a limited repertoire of structurally conserved motifs that may mediate interactions among death domain superfamily members. Therefore, more structural studies and mutational analysis of complexes built of those domains are necessary to define the motifs and interacting residues involved.Similarly to the WD-40 repeats in Apaf-1, leucine-rich repeats are the ligand sensing motif of NLR proteins, a property they share with members of the TLR and RLR (RIG-I-like receptors) families. LRRs in general consist of 2–45 motifs of 20–30 amino acids in length and exhibit a typical curved horseshoe-like structure with a parallel beta sheet on the concave side and helical elements on the convex side In NLRs the C-terminal LRR domain is thought to act as a sensor of bacterial products. Yet, little is known about how the PAMP is interacting with the LRR or even how the LRR region interacts with the remainder of the NLR, since no structural data is available on these questions. Recently, some insight into the possible mechanism of ligand-receptor binding was provided by the two LRR-ligand complex structures of TLR1:TLR2 In order to augment our understanding of the molecular mechanism of ligand recognition we generated a homology model of the NOD2 LRR domains based on the structure of the ribonuclease inhibitor  as a template. Additionally, we utilized the Consurf Server for the identification of functional regions in NLRs by surface mapping of phylogenetic information. Our findings are in accordance with recently published work by Tanabe and colleagues, which showed loss of function mutations in the LRR domain to be located on the convex surface with additional residues on the concave region Our homology model suggests a putative ligand-binding pocket situated in the concave surface and supports earlier observations, where the predicted loss-of function-mutations W907L, V935M, E959K, C961Y, K989E, S991F as well as the Crohn's disease related mutation G908 have been mapped to the same area Several diseases were found to arise from aberrant NLR function In summary, our study clearly shows that the overall architecture and secondary structure features of most NLRs resemble those of Apaf-1. From the structural point of view, most of the NLR family members are therefore Apaf-1-like, with deviations including NOD2 (2 CARD domains), NALP1 , NALP10 (missing LRR region), NOD5 and CIITA . Analyses of multiple sequence alignments revealed that all NLRs contain the crucial features for ATP-binding. In comparison to Apaf-1, most NLRs display a modified Walker B box. Since all NLRs, except for NAIP and NALP11, do not contain the crucial Walker B glutamate or aspartate required to activate the water molecule, they seem to have developed a new motif, the extended Walker B box to retain ATP hydrolysis activity. This is supported by the observation that NALP12 is able to bind and hydrolyze ATP Additionally, we identified one of the most intriguing features, which is the conserved histidine in the WH-domain, to be conserved among members of the NLR family. NLRs displaying this feature most likely assemble similar to Apaf-1 and activate their targets by oligomerization. Interestingly, NOD5, CIITA, NALP6, and NALP8 do not contain the conserved histidine in their WH domain. Whether their oligomerization mechanism and ATP hydrolysis capacity differ remains an open issue.Our analyses of the effector domains of NLRs as well as those of their adaptors and target caspases, or kinases reveal a common interface, which is composed of charged surface patches. The presence of acidic and basic surface patches theoretically renders all CARD and PYD domains compatible for interaction with each other. Yet their distinct profile and that of surrounding residues found in the described interfaces ensure the specificity for each interaction. This selectivity allows a well-balanced fine-tuning of the elicited immune response.Finally, sequence comparison of LRRs in human NLRs does not reveal one particular region that serves as the general ligand-binding site. This suggests that individual NLRs evolved highly specialized modes to recognize specific ligands. However, conserved residues found within this domain may contribute to the intramolecular interaction or backfolding of the LRR region in order to regulate NLR activation. Our results serve as a basis for further mutational and functional analyses required to more precisely define the role of LRRs in ligand recognition and NLR activation.http://ffas.ljcrf.edu) http://www.predictprotein.org) NLR protein sequences see  were subThe alignments were used to run phylogenetic probabilistic analyses using the parallel implementation of MrBayes http://www1.jcsg.org/scripts/prod/scwrl) https://prosa.services.came.sbg.ac.at/prosa.php) manually inspected, analyzed, and figures were prepared using Pymol (http://pymol.sourceforge.net). The degree of conservation was calculated using the ConSurf Server (http://consurf.tau.ac.il).The protein structure with the highest scoring alignment from the FFAS-search was used as a template for modeling the structures with the SCWRL-Server (


Fifteen post-menopausal patients with advanced breast cancer were treated with the LH-RH agonist leuprorelin (D-leu6-des-gly10-Gn-RH-ethylamide) given in a dosage of 7.5 mg as a monthly subcutaneous depot injection, to assess the clinical activity and endocrine response to treatment. None of the 15 patients showed an objective response to treatment, although four patients had stable disease for at least 6 months. No toxicity was demonstrated. Endocrine effects after 4 weeks' treatment were as follows: mean levels of serum gonadotrophins fell to 10% of their pretreatment values; there were no significant changes in the levels of prolactin on treatment; there was a significant decrease in the levels of serum testosterone in 12 out of 14 patients; there were no significant changes in the levels of oestradiol, androstenedione and oestrone. The lowering of serum testosterone suggests that androgens in post-menopausal women may be partly produced by the ovaries, stimulated by LH and FSH. This fall in testosterone may explain why some post-menopausal breast cancer patients in other studies have been reported to respond to treatment with LH-RH agonists, as it would decrease the substrate for the peripheral synthesis of oestrogens.


Using defined lipids and purified proteins, Billen et al. offer a new model to reconcile the two currently opposing models for how Bcl-2 family member interactions regulate cell death. Every day, a healthy human loses billions of cells by design . Cell deMuch of this attention has focused on the Bcl-2 protein family, which has been recognized as the principal regulatory component of the intracellular apoptotic machinery for over two decades . Bcl-2 fPart of the difficulty in resolving the functions of Bcl-2 family proteins is the inherent complexity of the cellular apoptotic network. An additional hurdle is that Bcl-2 protein action likely occurs at membranes, and membrane proteins can be made to reveal their secrets only with difficulty, as lipid-based systems are more challenging to study. Fortunately, basic aspects of the regulation of OMM permeability by Bcl-2 family proteins appear amenable to reconstitution in vitro, using simplified systems made up of purified proteins and lipids. As a prominent example, early studies from several laboratories using recombinant proteins and pure lipid membranes revealed that pro-apoptotic (activated) Bax has an intrinsic pore-forming activity . These aBax is often found as a monomeric protein in the cytosol of healthy cells. In cells committed to death, Bax appears to change conformation, translocate to the OMM, and assemble into intramembrane complexes . BH3-onlLargely based on these notions, two major competing models have been proposed . On the PLoS Biology, Billen et al. elegantly address this problem using a powerful combination of approaches applied to in vitro reconstitution systems appropriately consisting of full-length purified proteins and relevant membrane targets  [In this issue of posomes) . One impposomes) . MoreoveBillen et al.  also proAnother intriguing outcome of this study is the observation that interaction of tBid and/or activated Bax with Bcl-XL triggers translocation of Bcl-XL to the membrane. Structural studies with the Bcl-XL homologue Bcl-w  revealedNotwithstanding these important contributions, the model presented in this study also raises fascinating new questions. Chief among these: what is the structure adopted by Bcl-XL in the membrane, and how does this structure explain the inhibition of Bax permeabilizing function? One possibility is that membrane-associated Bcl-XL adopts an overall structure similar to that found in solution, except with its C-terminal helix inserted into the lipid bilayer in a transmembrane orientation  and 2B. Alternative possibilities exist. Indeed, biochemical labeling of Bcl-2 and Bax in cells previously led to the conclusion that both proteins insert a central helical hairpin into the lipid bilayer of the OMM ,18. ThisAnother exciting avenue for future research relates to the possible implication of OMM lipids in the function of Bcl-2 family proteins. Indeed, results obtained in model systems examining the lipid-dependence of Bax permeabilizing function lend support to a model whereby Bax permeabilizes the OMM by forming proteolipidic pores ,28,29, a


In previous clinical Positron Emission Tomography (PET) studies novel approaches for application of Principal Component Analysis (PCA) on dynamic PET images such as Masked Volume Wise PCA (MVW-PCA) have been introduced. MVW-PCA was shown to be a feasible multivariate analysis technique, which, without modeling assumptions, could extract and separate organs and tissues with different kinetic behaviors into different principal components (MVW-PCs) and improve the image quality.In this study, MVW-PCA was applied to 14 dynamic 11C-metomidate-PET (MTO-PET) examinations of 7 patients with small adrenocortical tumours. MTO-PET was performed before and 3 days after starting per oral cortisone treatment. The whole dataset, reconstructed by filtered back projection (FBP) 0–45 minutes after the tracer injection, was used to study the tracer pharmacokinetics.Early, intermediate and late pharmacokinetic phases could be isolated in this manner. The MVW-PC1 images correlated well to the conventionally summed image data (15–45 minutes) but the image noise in the former was considerably lower. PET measurements performed by defining "hot spot" regions of interest (ROIs) comprising 4 contiguous pixels with the highest radioactivity concentration showed a trend towards higher SUVs when the ROIs were outlined in the MVW-PC1 component than in the summed images. Time activity curves derived from "50% cut-off" ROIs based on an isocontour function whereby the pixels with SUVs between 50 to 100% of the highest radioactivity concentration were delineated, showed a significant decrease of the SUVs in normal adrenal glands and in adrenocortical adenomas after cortisone treatment.In addition to the clear decrease in image noise and the improved contrast between different structures with MVW-PCA, the results indicate that the definition of ROIs may be more accurate and precise in MVW-PC1 images than in conventional summed images. This might improve the precision of PET measurements, for instance in therapy monitoring as well as for delineation of the tumour in radiation therapy planning. Adrenal tumours are common incidental radiological findings in computed tomography (CT) examinations that are performed because of non-adrenal disease. For these adrenal tumours the term generally used is incidentaloma. As a result of the more frequent use and improved spatial resolution of modern multi-detector CT scanners, incidentalomas are being found more often. Because of their malignancy potential and because these tumours may cause hormonal hypersecretion, the discovery of an incidentaloma necessitates biochemical and radiological work-ups. Larger tumours (> 3–4 cm), tumours with high degree of suspicion of malignancy, and hormone-hypersecreting lesions are surgically removed -5.The initial work-up for adrenal incidentalomas is based on laboratory testing and CT or Magnetic Resonance Imaging (MRI) examinations or both ,3. Howev11C labeled metomidate, [11C]-MTO (MTO-PET), has been introduced as a sensitive method to determine the adrenocortical origin of adrenal tumours [11C]-MTO has a high affinity for the 11β-hydroxylase enzyme in the adrenal cortex and MTO-PET can therefore differentiate adrenocortical tumours from those of non-adrenocortical origin.In previous studies, positron emission tomography (PET) with  tumours -11. [11C11C]-MTO positive are generally readily depicted as a rounded, usually homogenously [11C]-MTO accumulating lesion. Detection is facilitated when the uptake in the lesion is higher or lower than the normal adrenal cortex; however, when the accumulation is similar in both, the tumour is generally recognized because it distorts the normal anatomy of the gland. Because of technical limitations related to camera resolution -MTO accumulation in the tumour and the normal adrenal can be determined over time. The time frames representing the highest tracer uptake in the tumour, relative to the normal adrenal cortex, may then be summed to a so-called "summed image" to optimize the image reading. Dynamic MTO-PET images from 15 to 45 minutes after the tracer injection are typically summed to create transaxial images of regional radioactivity concentration. However, the summation of image data is generally performed routinely and is not based on a pharmacokinetic analysis of the tumour in the individual patient since such a procedure is time consuming. In addition to visualizing the lesion, it is essential for its characterization to perform accurate PET measurements by outlining regions/volume of interest (ROIs/VOIs) with high precision, especially when the tumour is small. It has previously been demonstrated that the magnitude and variation of the noise, and partial volume effect in the PET images might affect the precision of defining ROIs and the resulting SUV measurements -MTO per kilogram of body weight, corresponding to 715 ± 199 MBq (mean ± SD) range 370–1050 MBq, was administered. After a rapid intravenous bolus injection of the tracer, a 45-minute dynamic examination sequence was started which comprised 14 time frames: 5 × 1 minute, 5 × 3 minutes, 3 × 5 minutes and 1 × 10 minutes.The study was approved by the local ethics committee (No 02-262) and was performedin compliance with the Helsinki Declaration. Inclusion into the study was by informed consent. Each patient had a 1- to 2-cm adrenocortical adenoma as characterized by CT attenuation measurements. In addition to the initial study, MTO-PET was performed a second time on each patient after 3 days of per oral cortisone medication (dexametasone 0.5 mg three times daily). The patients were examined after a minimum of four hours of fasting, allowing free intake of clear liquids. Approximately 10 MBq/kg of -MTO is the slow, gradual accumulation of the radioactivity into the gastric juice. This intermediate pharmacokinetic event was isolated and displayed in the MVW-PC3 images which, however, were devoid of diagnostic information related to visualization of the adrenocortical tumours. MVW-PC1 corresponds to the kinetic with the largest variation and most predominant behavior of the [11C]-MTO in this study. This means that MVW-PC1 images contain organs and tissues with the largest and mostly preserved kinetic behavior during the whole scanning time. This component isolates the pharmacokinetic phase in the dynamic PET sequence, which best resembled the conventional summed images based on data 15 to 45 minutes after [11C]-MTO injection but the image noise was lower and the organ and tissue separation was better. In these images the adrenocortical tumours were readily depicted with qualitatively better lesion delineation than in the summed images.MVW-PCA identified three separate pharmacokinetic events in the dynamic MTO-PET examinations. The kidneys, pancreas, and spleen were clearly delineated in the MVW-PC2 images corresponding to the early and fast pharmacokinetic events in these organs. No qualitative diagnostic information about the depiction of the adrenocortical tumours could be retrieved from the MVW-PC2 component. A characteristic feature of -MTO, leading to a dominance of metabolites in protein-free plasma, but with intact tracer bound to plasma proteins. Therefore, with a simplified approach of using a standardized summation of the image data, there is a risk that the pharmacokinetic pattern of the individual patient is ignored and potential diagnostic information may be missed.Previous analyses on the metabolite spectrum in plasma have indicated rather complex conditions with a rapid metabolism of -MTO with 18F-labeled analogs presently being developed. These analogs will provide more time for the tracer to accumulate in adrenocortical tumours as well as be eliminated from normal tissues. However, the radiation dose to the patient will be higher than for [11C]-MTO, which is approximately 3 mSv.A limitation with this study is the small number of patients in whom no dramatic impact was shown on the precision of the PET measurements by applying MVW-PCA. Moreover, in this first study applying MVW-PCA on PET of organs other than the brain, merely FBP was used. In future, work should also include automation of this method and MVW-PCA should be applied to PET examinations reconstructed by the OSEM method. Similarly, the output of the ROI analysis, including graphical illustrations, may be generated and illustrated automatically. The ongoing trend in PET tracer development to replace tracers with short half-lives, such asIn conclusion, MVW-PCA applied on FPB reconstructed dynamic PET examinations of patients with small adrenocortcal adenomas improved the image quality by decreasing the image noise and increased the precision by which the ROIs were defined for PET measurements. The decrease in tracer accumulation induced by cortisone treatment was more evident in the TAC analysis when MVW-PCA was used as compared to when ROIs were defined in the conventionally summed images.The authors declare that they have no competing interests.Authors PR, JH, AM AS participated in the design, and performed the image and data analysis, and drafted the manuscript. PH and BL contributed with some of the practical approaches and the writing of the paper. All authors read and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2342/9/6/prepub


The hypothalamic-pituitary-adrenal axis is known to be related to abdominal symptoms, and the relationship between abdominal pain and cortisol secretory patterns has been previously investigated using a cross-sectional approach. Here, we investigated the effect of day-to-day variations in salivary cortisol and dehydroepiandrosterone-sulfate levels on abdominal symptoms in healthy individuals.Eleven college students  participated in this study. The participants were asked to collect their saliva immediately after awakening and before bedtime for eight consecutive days. They also completed a questionnaire about abdominal symptoms before bedtime. The linear mixed model was applied to analyze the effects of the day-by-day variability or the 8-day average adrenal hormone level  on abdominal symptoms.The day-to-day variability of cortisol levels before bedtime was negatively related with loose stool, while the day-to-day variability of the cortisol slope was positively correlated with loose stool. A low 8-day average dehydroepiandrosterone-sulfate level at awakening was positively related with frequent bowel movements, loose stool, and long bouts of severe abdominal pain. Likewise, a low 8-day average dehydroepiandrosterone-sulfate slope was positively related with long bouts of abdominal pain.Low cortisol levels before bedtime and a steeper diurnal cortisol slope during the day may be related to bouts of diarrhea during the day. The effect of changes in the hypothalamic-pituitary-adrenal (HPA) axis on abdominal symptoms has been previously reported. On the basis of the results of previous studies, Mayer et al. 2002) suggested that complex interrelationships exist between gut-associated immune tissue, the central nervous system, and the enteric nervous system . Psychos002 suggePrevious studies have suggested a relationship between abdominal pain and the cortisol response levels in the morning and evening. Patients with functional gastrointestinal disorder have been shown to have lower cortisol levels 60 min after awakening as compared with healthy controls although there was no significant difference in the cortisol response after awakening between these groups. Furthermore, total secretion (area under the curve) of cortisol during the first hour after awakening was also found to be lower in patients with functional gastrointestinal disorder than healthy controls . MoreoveIt has been reported that variations in the HPA axis marker have an impact on abdominal symptoms, and the relationship between abdominal pain and cortisol secretory patterns has been investigated using the cross-sectional approach. However, the following problems must be addressed. First, in addition to investigating the effect of cortisol secretory patterns on abdominal symptoms, we must examine day-to-day intraindividual variations in cortisol secretion in relation to the severity of abdominal symptoms. Second, it is important to clearly elucidate the relationship between abdominal symptoms and dehydroepiandrosterone (DHEA). DHEA is the most important androgen secreted by the adrenal cortex and is a precursor for sex steroids; the main secretagogue for DHEA is thought to be adrenocorticotrophic hormone. DHEA is thought to affect immunological function in a manner opposite to that of cortisol . DHEA isWe aimed to examine the relationship between physiological factors and abdominal symptoms by assessing day-to-day intraindividual variations in cortisol and DHEA-S secretion patterns. For this, we investigated the effect of daily changes in adrenal hormone  levels on abdominal symptoms by monitoring the subjects for eight days.2. No one used any habitual medications or dietary supplements that affect HPA activity or had any history of gastrointestinal surgery for the past five years. Written informed consent was obtained, and the study was approved by the university's ethics committee.The participants were 11 college students . Mean body mass index (BMI) of the participants was 20.9 kg/mThe following items were assessed: "Bowel movement," "Stool properties," "Duration of abdominal pain (minutes)," and "Degree of abdominal pain.""Bowel movement" was assessed by asking the following question: "How many bowel movements did you have today?;" "Stool property" was based on a rating of 1 to 8 ; "Duration of abdominal pain" was assessed by asking the following question: "How long did you experience continuous abdominal pain today?;" and "Degree of abdominal pain" was rated from 0  to 10 (very severe).Sleep duration was calculated from the time subjects went to sleep to the time they awakened. Previous research has noted a correlation between cortisol secretion levels and sleep duration .The Perceived Stress Scale 4 (PSS4) ,13 was dParticipants were asked to collect their saliva samples at awakening and immediately before bedtime for eight consecutive days. The samples were used to determine cortisol and DHEA-S levels. Saliva samples used to determine cortisol levels were collected using Salivette swabs . For samples for determining DHEA-S levels patients were asked to expectorate saliva through a short plastic straw into a collection vial. Saliva samples were stored at room temperature (10-20°C) and centrifuged at 3000 rpm for 5 min within three days after sampling. The concentrations of cortisol and DHEA-S in the saliva were determined by an enzyme immunoassay using an EIA Kit .Participants were asked to collect their saliva on awakening and immediately before bedtime. They were also asked to complete the diaristic questionnaire before bedtime for eight consecutive days. The participants were asked to not drink alcohol during the experimental period. Further, we avoided selecting female subjects who were menstruating during the period so that menstrual pain would not be confused with bowel pains.t test for repeated measures was applied to analyze the cortisol and DHEA-S slopes between awakening and bedtime. The linear mixed model was applied to analyze the effect of adrenal hormones on abdominal symptoms. The abdominal symptoms were dependent variables and the 8-day average adrenal hormone level, the daily intraindividual variability of adrenal hormones , the PSS4 score, and the sleep duration were covariates. Data analysis was performed using the SPSS 15.0 software (SPSS Japan Inc.).For the analysis of adrenal hormone data, cortisol and DHEA-S concentration values were square root transformed. The relationship between adrenal hormone, abdominal symptoms, stress, and sleep duration was analyzed using Pearson's product-moment correlation coefficient. Student's t = 11.91, p < 0.001; DHEA-S: t = 9.21, p < 0.001).The mean sleep duration was 5.67 ± 1.56 hours. The mean PSS4 score was 5.97 ± 2.80. As shown in Table r = 0.34, p < 0.001) and bowel movement . Cortisol levels on awakening were positively correlated with sleep duration  and stool property . Cortisol levels before bedtime were negatively correlated with sleep duration  and stool property . The cortisol slope from awakening to bedtime was positively correlated with sleep duration  and stool property .As shown in Table r = -0.31, p < 0.01; before bedtime: r = -0.37, p < 0.001), sleep duration , bowel movement , stool property , and the degree of abdominal pain .DHEA-S levels at awakening and before bedtime were negatively correlated with the PSS4 score , stool property , the duration of abdominal pain , and the degree of abdominal pain .The results of the linear mixed model with abdominal symptoms as dependent variables and the day-to-day intraindividual variability, the 8-day average adrenal hormone levels at awakening, the PSS4 score, and sleep duration as covariates are shown in Table t = -2.13, p < 0.05), while the 8-day average DHEAS level was negatively related to bowel movement  and stool property .The results of the linear mixed model with abdominal symptoms as dependent variables and the day-to-day intraindividual variability, the 8-day average adrenal hormone levels before bedtime, the PSS4 score, and sleep duration as covariates are shown in Table t = 2.07, p < 0.05), while the 8-day average DHEA-S slope was negatively related to the duration of abdominal pain .The results of the linear mixed model with abdominal symptoms as dependent variables and the day-to-day intraindividual variability, the 8-day average adrenal hormone slopes, the PSS4 score, and sleep duration as covariates are shown in Table This is the first study to investigate the effect of the day-to-day variability of adrenal hormone levels on abdominal symptoms in healthy individuals. There is a possibility that a steeper diurnal cortisol slope during the day and lower cortisol levels in the evening is related to bouts of diarrhea. The results of this study are similar to those of two previous studies ,14. PrevWe found no significant effects of day-to-day variability of DHEA-S on bowel symptoms. However, the 8-day average DHEA-S level at awakening was negatively related to all variables of abdominal symptoms. These results suggest that chronically reduced levels of DHEA-S at awakening or baseline levels are related to the degree and duration of abdominal pain, diarrhea, and infrequent bowel movements. In addition, the 8-day average of the DHEA-S slope was related to the duration of abdominal pain. Therefore, a flat circadian rhythm of DHEA-S can increase the duration or frequency of abdominal pain. Moreover, the 8-day average DHEA-S level before bedtime was negatively related to the frequency of bowel movements and stool property. In fact, we assert that low DHEA-S levels may have an affect on the frequency of bowel movements and stool property to a higher degree than the slope between the morning and the evening.Previous research has suggested the possibility that higher cortisol and lower DHEA (or DHEA-S) levels may result in altered immune function, which in turn may cause abdominal pain ,5,10. HoThis study provides some new insights and provides valuable information for future research; nevertheless, there were three limitations to the present work. First, we were unable to consider the relation between the participants dietary habits and their abdominal symptoms. Although inclusion of items about dietary habits may yield more accurate results, we feel that future studies should employ a less demanding saliva sampling process over the 8-day period. Second, the diurnal cortisol slope was calculated by only determining cortisol levels immediately after awakening and before bedtime; samples were not obtained for any other times. Hence, our results may not be useful for determining the cause of abdominal pain taking place early in the day. In the future, the relationship between the circadian rhythm of adrenal hormones and abdominal symptoms must be determined by taking multiple saliva samples throughout the day. Therefore, future studies should aim to maintain a good balance between consideration for the participants and collecting multiple samples. Third, the number of participants in this study was rather small.This is the first study to investigate the effects of day-to-day variations in salivary cortisol and DHEA-S secretion on abdominal symptoms. The results of our study suggest that variations in adrenal hormone levels affect the duration and severity of abdominal symptoms. Day-to-day variations in cortisol levels before bedtime and a steeper cortisol slope may be positively related to a tendency toward bouts of diarrhea. On the other hand, a persistent lower baseline and a flatter slope with regard to DHEA-S may also be negatively related to abdominal symptoms. The results of our study suggested the possibility that in the future the accurate clinical condition of abdominal symptoms may be able to be revealed by a device that can take multiple saliva samples throughout the day and by better controlling various daily habits.The authors declare that they have no competing interests.NS conceptualized and designed the study, collected the data, conducted the saliva analysis, performed the statistical analysis, and drafted the manuscript. SI, NO, KS, HK, KY, and HT conceptualized and designed the study, collected the data, and conducted the saliva analysis. SN and HS conceptualized and designed the study. All authors read and approved the final manuscript.


Studies have shown that in the genome of human immunodeficiency virus (HIV-1) regions responsible for interactions with the host's immune system, namely, cytotoxic T-lymphocyte (CTL) epitopes tend to cluster together in relatively conserved regions. On the other hand, "epitope-less" regions or regions with relatively low density of epitopes tend to be more variable. However, very little is known about relationships among epitopes from different genes, in other words, whether particular epitopes from different genes would occur together in the same viral genome. To identify CTL epitopes in different genes that co-occur in HIV genomes, association rule mining was used.Pol epitope LFLDGIDKA is found to be significantly associated with epitopes GHQAAMQML and FLKEKGGL from Gag and Nef, respectively, and this association rule is observed even among circulating recombinant forms.Using a set of 189 best-defined HIV-1 CTL/CD8+ epitopes from 9 different protein-coding genes, as described by Frahm, Linde & Brander (2007), we examined the complete genomic sequences of 62 reference HIV sequences . The results showed that despite inclusion of recombinant sequences that would be expected to break-up associations of epitopes in different genes when two different genomes are recombined, there exist particular combinations of epitopes (epitope associations) that occur repeatedly across the world-wide population of HIV-1. For example, We have identified CTL epitope combinations co-occurring in HIV-1 genomes including different subtypes and recombinant forms. Such co-occurrence has important implications for design of complex vaccines (multi-epitope vaccines) and/or drugs that would target multiple HIV-1 regions at once and, thus, may be expected to overcome challenges associated with viral escape. In the course of viral infection, recognition of viral peptides by class I major histocompatibility complex (MHC) molecules and subsequent interactions of the peptide/MCH complex with the cytotoxic T lymphocytes  plays an important role in the control of the infection ,2. ViralThe epitope-vaccine approach has been suggested as a strategy to circumvent the rapid rate of mutations in HIV-1 and the subsequent viral escape from the host's immune system as well as the development of resistance to anti-viral drugs -14. The in silico, focusing on those capable of binding to multiple HLA alleles [in-silico epitope predictions is generating a rather large number of initially predicted epitopes, many of which are false positives; and hence, there exists a need for subsequent experimental validation of many potential candidates [Several strategies can be used to develop multi-epitope vaccines, including (a) the generation of tetramer epitope vaccines with epitopes being chosen based on the presence of principal neutralizing determinant , (b) the alleles , (c) the alleles  with epi alleles . Howeverndidates -24. Furtndidates ,25,26, wndidates . Other mndidates  and hiddndidates , also handidates . For exandidates , there wGag, Pol and Nef, respectively. We also identified several combinations of 3 to 5 CTL epitopes that are frequently found together in the same viral genome despite high mutation and recombination rates found in HIV-1 genomes, and thus, can be used as likely candidates for multi-epitope vaccine development.Briefly, an association rule mining technique, which is a method that can detect association between items (frequent item sets) and formulate conditional implication rules among them -33, is uGenomic nucleotide sequences of 9 protein-coding genes of HIV-1 were collected for 62 HIV-1 reference genomes from the 2005 subtype reference set of the HIV sequence database by Los Alamos National Laboratory (LANL) ,35 (TablThe summary of the average numbers of breakpoints in the CRF genomes was based on the breakpoint maps summarized at the HIV database at Los Alamos .K03455) [The set of 218 CTL epitopes, described as "the best-defined HIV CTL epitopes" by Frahm, Linde & Brander (2007)  that incK03455) , are desOf the 218 "best-defined" CTL epitopes, the subset of the most evolutionary conserved epitopes that are present across a majority of surveyed reference sequences was included according to the following criteria: (a) The epitope was present in at least one out of 62 reference sequences. (b) If two or more epitopes were completely overlapping with each other and there were no amino acid sequence differences, the longer epitope was selected. However, if overlapping epitopes harbored one or more amino acid difference from each other, all such epitopes were included , three data sets were created. The first sequence set  included all 62 HIV-1 reference sequences used in the study, the second set included only 44 non-recombinant sequences ("44-non-CRFs") and the third set included 18 CRFs (designated as "18-CRFs"). Because of the requirement that an epitope be present as a "perfect match" in at least one sequence as described above, 1 and 29 epitopes were removed from the epitope lists for the second and third data sets, respectively. This resulted in lists of 188 and 160 epitopes, respectively  was created by randomly selecting sequences from the original sequence set (random sampling with replacement). Similarly to the bootstrap test widely used in phylogenetics , these pAssociation rule mining is a data mining technique that discovers relationships  that exist within a data set -33,40. OAssociation rule mining was conducted using the Apriori algorithm  implemenThe relative degree of sequence divergence among reference sequences and different genomic regions was evaluated by comparing the number of synonymous and nonsynonymous substitutions. In particular, the number of synonymous nucleotide substitutions per synonymous site (dS) and the number of nonsynonymous nucleotide substitutions per nonsynonymous site (dN) were estimated by the Nei-Gojobori method  as impleIn order to identify CTL epitope regions that consistently co-occur together in the HIV-1 genomes, 189 CTL epitopes were mapped in 62 HIV-1 reference sequences , but only 52 association rules included an epitope from the Nef gene. Since Gag and Pol are located in adjacent genomic positions (and are somewhat overlapping), the physical proximity of the genes in the genome may be responsible for the existence of some association rules that involve epitopes from both of these genes . However, given the extremely high recombination rate in HIV-1, which was estimated to be as high as 2.8 crossovers per genome per replication cycle [Pol and Nef) would not be expected to be involved in many association rules, particularly those that occur with high support and confidence. Notably, our results identified at least 23 associations that involved epitopes located in 3 different genes, namely, Gag, Pol and Nef  and 23 association rules that involve epitopes from three different genes . The Venn diagram shown on Figure Gag and Pol, while among other categories of multi-gene association rules, majority involved combinations of epitopes from Pol and Nef. Similar results were obtained with the smaller 44-non-CRFs and 18-CRFs data sets, identifying 246 and 5 epitope associations from two and three genes, respectively, for the former data set, and 153 and 7 associations for the latter data set  [Interestingly, one of the frequently associated epitopes found in three genes associations Figure , Nef FLKepitope) ,8, also epitope) ,58. ThisNef epitope (B8-FL8 FLKEKGGL). The Pol gene had the highest number of associated epitopes (9) that differ from each other, while the Gag gene had 3 different epitopes involved in multiple association rules. Some of these associations included epitopes from the same adjacent/overlapping regions, e.g., Gag GLNKIVRMY is associated with the Pol IVTDSQYAL epitope and other adjacent/overlapping epitopes in at least 9 association rules , as well as associations that include epitopes from distantly located parts of the genome, although further studies are necessary. Overall, this approach allows us to identify co-evolving regions in viral genomes that are highly conserved at the amino acid level and are subjected to strong purifying selection eliminating the majority of amino acid changes that may occur in such regions.As Figure s Figure . Other eTo assess the extent of evolutionary sequence conservation of the CTL epitopes that participated in the association rules, we compared the levels of nonsynonymous  and synonymous substitutions in all pairwise comparisons of 62 HIV-1 genomes. The results are shown in Table Gag CRAPRKKGC and Pol LVGPTPVNI while occurring in over 75% of the analyzed sequences, were not part of any association rule). As Table By design, our study was focused on the identification of highly supported association rules (support >= 75%), i.e., those that involve epitopes present in at least 75% of the sequences analyzed. Notably, not all CTL epitopes that are present in over 75% of sequences can be found in association rules . Of these highly conserved epitopes, Pol epitope LFLDGIDKA (recognized by HLA-B81) is also found to be a part of several association rules identified in this study, including association rules spanning three genes and four CTL epitopes , and as such, represents a promising candidate for multi-epitope vaccine development.Overall, we were able to identify several highly conserved epitopes that are relatively widely spread across the worldwide HIV-1 population, and present not only in non-recombinant subtypes, but also in the circulating recombinant forms. Such highly conserved epitopes may be considered promising candidates for multi-epitope vaccine design, as they are likely to be targeted in a majority of HIV lineages, thereby increasing population coverage. However, in addition to being highly conserved, there are additional benefits in utilizing CTL epitopes identified as participants in association rules . Therefore, inclusion of these epitopes may potentially enhance the efficiency of a multi-epitope vaccine across a broader range of host HLA haplotypes . FurWhile our results demonstrated presence of several highly conserved – and identified to exist in association with each other – CTL epitopes in multiple HIV-1 reference genomes, including CRFs, the underlying functional significance of these regions for the virus remains poorly understood. Very few of the epitope regions found in association rules had such molecular features as glycosylstion, myristoylation, amidation, or phosphorylation sites. They also lacked any cell attachment motif or Leucine Zipper motif ,62. Yet,It is also noteworthy that some epitopes are not involved in any association despite being present in more than 75% of the reference sequences, hinting at some underlying mechanism that holds the "associated epitopes" together. It is possible that the associated epitopes from different genes co-evolve together because of functional and structural constraints due to protein-protein interactions that are necessary for many viral processes . Since sOur results revealed the presence of multiple associated co-evolving CTL epitope regions in HIV-1 genomes that are also significantly conserved across a broad range of HIV-1 subtypes and sub-subtypes. However, further studies are needed to ascertain the efficiency of these associated epitopes in multi-epitope vaccines as well as to uncover the underlying structural and/or functional constraints behind co-occurrences of the highly conserved epitopes.Application of association rule mining revealed that certain CTL epitope combinations (including epitopes from three different genes) consistently co-occur in HIV-1 genomic sequences present in major geographic regions around the world. Such epitopes that are both well supported by experimental evidence and highly conserved across different non-recombinant and recombinant forms of HIV-1 genomes can be considered as ideal candidates for multi-epitope vaccines against HIV-1.HLA: Human Leukocyte Antigen; HTL: Helper T-Lymphocyte.The authors declare that they have no competing interests.SP did the analyses and wrote the manuscript. HP conceived and coordinated the study nd wrote the manuscript. All authors read and approved the final manuscript.Table S1 – Epitopes included in the study. There were 218 epitopes in the "best defined CTL epitopes list" by Frahm et al. (2007). From this, 29 epitopes were removed from the 62-all data set because they did not satisfy the inclusion criteria. Additionally, one epitope from the 44-non-CRFs data set and 29 epitopes from the 18-CRFs data set were removed because of the inclusion criteria.Click here for fileTable S2 – Number of epitopes included in the study. The number of epitopes included in the study as well as epitopes found in association rules in each gene.Click here for fileTable S3 – Unique epitope associations in the 62-all data set. Each row represents one epitope association. Associated epitopes in each association are shown in adjacent columns. Presence of the association in other data sets is represented by 1 in respective columns. There are 484 unique associations in the 62-all data sets. Among them, 172 associations are present in the 44 – non-CRFs data set and 102 associations are present in the 18 – CRFs data set. The yellow color marks presence of the epitopes from all three genes in the 62-all data set. There are 23 associations of that kind.Click here for fileTable S4. Stage of HIV-1 infection where the epitopes have been shown to be immunogenic, if known.Click here for file


Enzymes that depend on vitamin B6  are of great relevance to biology and medicine, as they catalyze a wide variety of biochemical reactions mainly involving amino acid substrates. Although PLP-dependent enzymes belong to a small number of independent evolutionary lineages, they encompass more than 160 distinct catalytic functions, thus representing a striking example of divergent evolution. The importance and remarkable versatility of these enzymes, as well as the difficulties in their functional classification, create a need for an integrated source of information about them. contains documented B6-dependent activities and the relevant protein families, defined as monophyletic groups of sequences possessing the same enzymatic function. One or more families were associated to each of 121 PLP-dependent activities with known sequences. Hidden Markov models (HMMs) were built from family alignments and incorporated in the database. These HMMs can be used for the functional classification of PLP-dependent enzymes in genomic sets of predicted protein sequences. An example of such analyses (a census of human genes coding for PLP-dependent enzymes) is provided here, whereas many more are accessible through the database itself.The B6 database The B6 database is a curated repository of biochemical and molecular information about an important group of enzymes. This information is logically organized and available for computational analyses, providing a key resource for the identification, classification and comparative analysis of B6-dependent enzymes. The term 'vitamin B6' refers to a collective of six biologically interconvertible 3-hydroxy-2-methylpyridine compounds: pyridoxal, pyridoxine, pyridoxamine, and their respective 5'-phosphates. Among these, pyridoxal 5'-phosphate (PLP) is the main metabolically active form, serving as a cofactor for a variety of enzymes in all organisms -7.Nearly all PLP-dependent enzymes, with the exception of glycogen phosphorylases, are associated with biochemical pathways involving amino compounds - mostly amino acids. The reactions catalyzed by the PLP-dependent enzymes that act on amino acids include transamination, decarboxylation, racemization, and eliminations or replacements at the β- or γ-carbons. Such versatility arises from the fact that PLP can covalently bind the substrate and then act as an electrophilic catalyst, stabilizing different types of carbanionic reaction intermediates  Figure .) lists more than 140 PLP-dependent activities, corresponding to ~4% of all classified activities [The Enzyme Commission  includes 184 activities and over 2000 sequences of B6-dependent enzymes, subdivided into 149 families. For each family, the database provides a multiple sequence alignment and the derived hidden Markov model.The B6 database was constructed based on an inventory of documented B6-dependent activities, most but not all of which have been catalogued by the Enzyme Commission and are therefore associated to an official EC number. A systematic examination of the literature showed that 121 of these activities could be associated to enzymes of known sequences, and in these cases we proceeded to the creation of protein families, that we define as monophyletic groups of sequences all possessing the same enzymatic activity. Each given activity was associated to one or more families based on this criterion.The number of sequences in individual families was then increased by homology searches, i.e. by scanning the GenBank with BLAST  or with -10 were generally considered (this limit could be somewhat lowered for families composed of short sequences).(1) Only sequences yielding an E value < 10(2) Sequences showing a >90% identity to a protein of known function were usually not included, to diminish redundancy.(3) Sequences being substantially (>30%) shorter than the shortest functionally validated sequence in the family were discarded. Sequences lacking the PLP-binding lysine residue were also discarded (except for rare cases in which the protein is known not to bind PLP via a lysine).(4) Sequences showing a higher similarity to other characterized PLP-dependent enzymes  were discarded.(5) Finally, sequences from taxa in which the enzymatic activity of the family was not documented, were also generally discarded.Multiple alignments were constructed with ClustalW . Given tThe ProDom program  was usedA family HMM is a probabilistic model, constructed from a multiple alignment, which describes the sequence conservation within a protein family. In comparison to consensus sequences or similar regular expressions, HMMs provide a more articulated modeling of the features of a protein family. Such higher complexity is responsible for the greater discriminatory power of the HMM methodology in the identification of other putative family members . Dependiversus all comparison of the families in the database using an HMM-HMM alignment software [-5) between HMMs, the analysis identified seven separated clusters of PLP-dependent families  or sequence-HMM (e.g. HMMPFAM) methods. The HMM-HMM analysis, for example, indicates a significant similarity between Prosc (a family of proteins with unknown function) and diaminopimelate decarboxylase (EC: 4.1.1.20) - a relationship that is not apparent through Blast or HMMPFAM comparisons.Inter-families distances deriving from HMM-HMM comparisons served as a guide to build alignments representative of the seven distinct structural groups. Distance matrices among families were analyzed with an UPGMA algorithm and a rapid multiple sequence alignment method  was usedThe B6 database is a repository in which detailed  information about an important group of enzymes is concentrated, organized and made available for computational analyses. We expect that the B6 database will be a valuable tool for experimental researchers in the PLP field, but also a reference point for the design of theoretical studies by bioinformaticians.In particular, the sequence information accumulated in the database can be used to facilitate the identification and functional assignment of B6-dependent enzymes. To illustrate this point, we employed the family and fold-type HMMs (constructed as described above) to search and preliminarily classify PLP-dependent enzymes in genomic sets of predicted proteins. The results of such analyses have also been incorporated in the database. or from similar ftp repositories. The classification of protein sequences was achieved through a two-step procedure. First, each sequence was compared with our database of PLP-dependent sequences by performing a HMM search with the seven fold-type HMMs, using relaxed significance criteria . This step served as a quick filter to sift out genes that were likely to code for PLP-dependent enzymes. Candidates were subsequently compared with the library of family HMMs using HMMPFAM. This step was more time-consuming and served for a preliminary functional classification of the proteins.Complete sets of protein sequences deduced from genomic data were generally obtained from NCBI -3) and a score above a 'trusted' cut-off established by the family curator. Sequences with a score below this threshold were marked as 'low-score' to indicate their modest similarity to the family model. These sequences were not considered as possessing the enzymatic function of the family, but were regarded as possessing an uncharacterized, possibly related, activity. According to this analysis, very few sequences exhibited a significant similarity to a fold-type HMM (E ≤ 10-3) but no significant similarity to any family HMMs. In such cases, sequences were considered as potential PLP-dependent enzymes with an uncharacterized catalytic activity.A protein was considered to possess the same activity as its best-hit family if it exhibited a significant similarity to the family HMM  to obtain an inventory of the human genes coding for PLP-dependent enzymes. The initial output of the program (69 sequences recognized as probable PLP-dependent proteins) was further analyzed to identify pseudogenes, false positives and entries representing alternative protein isoforms.By employing the approach outlined above, we searched the latest draft of the human genome . Thirteen more proteins were recognized as isoforms deriving from some of the genes above. To appreciate the rate of false negatives in our analysis, we performed an extensive text search in the GenBank database of human genes, to identify all those genes annotated (directly or indirectly) to code for B6-dependent proteins. However, we found no hits other than the 56 genes listed in Table The search identified 56 expressed genes coding for PLP-dependent proteins  will be associated to specific sequences, while many new activities are likely to be discovered . Accordi. Users are asked to cite the present article.The B6 database, which is based on the web-oriented Perl package Woda, is publicly available over the Internet PLP: pyridoxal 5'-phosphate; HMM: hidden Markov model; EC: enzyme Commission.RP designed and implemented the B6 database and website, carried out the genomic analysis and revised the manuscript. AP collected the literature included in the database, selected the functionally validated sequences, helped to build the families, drafted and revised the manuscript. Both authors read and approved the final manuscript.