TNF 308 G/A polymorphism and risk for type 2 diabetes mellitus (T2DM). However, the sample sizes of most of the studies were small. The aim of this study is to evaluate the precise association between this variant and risk for T2DM in a large-scale meta-analysis.Many investigations have focused the association between TNF 308 G/A polymorphism and T2DM. The key words were as follows: diabetes, tumor necrosis factor and polymorphism/variant/genotype. This meta-analysis was assessed by Review manager 5.0.All publications were searched on the association between TNF 308 G/A polymorphism were 1.03 (0.95–1.12), 1.03 (0.94–1.13) and 1.03 (0.78–1.36) in overall, Caucasian and Asian populations, respectively. The sensitivity analysis further strengthened the validity of this association. No publication bias or heterogeneity was observed in this study.There were 18 studies identified. The odd ritos (ORs) and 95% confidence intervals (CI) for GA+AA versus GG genotype of TNF 308 G/A polymorphism and risk for T2DM.In summary, there was no significant association detected between the  The prevalence of diabetes is high, with high rates of morbidity and mortality. World Health Organization (WHO) estimations predicted that by the year 2030, 350 million individuals worldwide would suffer diabetes TNF gene, such as −238, −308, −857, −1031 in human TNF 308 G/A polymorphism found guanine (G) replaced by adenine (A) in TNF 308 position TNF gene transcription than that of wild-type in vitro expression studies TNF 308 G/A polymorphism as a risk factor for type 1 diabetes mellitus (T1DM) et al.As genetic variations in the promoter region may regulate TNF-α production, single nucleotide polymorphisms in the promoter region of the TNF 308 G/A polymorphism would be associated with the risk for T2DM.In this study, we have therefore conducted a meta-analysis from all eligible studies to confirm whether We searched several databases  through November, 2010 for all publications on the association between TNF 308 G/A polymorphism and T2DM. The key words were as follows: diabetes, tumor necrosis factor and polymorphism or variant or genotype. In addition, we also searched references of retrieved articles. Studies should meet the following criteria: (1) case-control study; (2) only diabetes as outcome, and (3) at least two comparison groups (diabetes vs. control groups) involved in a single study. Exclusive criteria: no report about the genotype frequency, or insufficient information for data extraction. The MOOSE Checklist and the flow chart for the studies were shown as TNF 308 G/A polymorphism and the risk for T2DM Finally, we identified 18 studies on the association between Two authors extracted data independently and in duplicate, and reached on all items, including: author's last name, journal and year of publication, country of origin, selection and characteristics of diabetes cases and controls, ethnicity of the study population, genotypes (rs1800629) and numbers of cases and controls. The results were compared and disagreements were discussed and resolved with consensus.2I statistics that was interpreted as the proportion of total variation contributed by between-study variation. If there was no statistical heterogeneity among studies (2I<50% and P>0.05), the ORs and 95% CI would be estimated for each study in a fixed-effects model. Otherwise, a random-effect model should be employed. In sensitivity analysis, relative influence of each study on the pooled estimate was assessed by omitting one study at a time. Funnel plots were used to evaluate publication bias. All P-values were two-tailed.The meta-analysis was performed by using Review manager 5.0. We pooled the odds Ratios (ORs) for TNF 308 GA+AA versus GG genotype and further conducted subgroup analyses by ethnicity. Heterogeneity among studies was examined with The detailed characteristics of the studies included were shown in TNF 308 G/A polymorphism in the overall 18 studies and subgroups did not reach the significant difference. The ORs of GA+AA versus GG for T2DM were 1.03 (0.95–1.12) for the overall, 1.03 (0.94–1.13) for Caucasian and 1.03 (0.78–1.36) for Asian, respectively. There was no evidence of heterogeneity among the overall 18 studies or subgroups .It has been shown in TNF 308 G/A polymorphism and T2DM  in the promoter region of TNF was found to increase the expression of this pro-inflammatory cytokine in culture cells and positive associated with risk for T1DM TNF 308 G/A polymorphism and T2DM, but the results were still unclear.Inflammation has been widely known as an important feature of T2DM, with high levels of pro-inflammation cytokines, including IL-1, IL-6 and TNF-α. As TNF-α can impair insulin signal pathways and lead to B-cell destruction, elevated TNF-α is considered playing a central role in the development of T2DM. The TNF 308 G/A polymorphism did not contribute to the development of T2DM. However, T2DM is a complex disease, and both environmental and genetic factors are involved in the development of T2DM. There are some points should be concerned for the inconsistent results in early reports. Firstly, ethnic differences may attribute to these different results, since the distributions of the TNF 308 G/A polymorphism were different between various ethnic populations. For instance, the frequencies of TNF 308 G/A polymorphism allele differs from 9% in Chinese population TNF 308 G/A polymorphism. Thus, even if the variation has a causal effect on T2DM, it may take a long time to be observed. Supporting this deduction, Ishii et al. reported that FPG in older men was higher and a trend for triglycerides to be higher and HDL cholesterol to be lower in the group with TNF 308 G/A polymorphism, but not in the young TNF 308 G/A polymorphism. The effects of the TNF 308 G/A polymorphism are so small that in the short period the difference can not be observed. In addition, interaction of the TNF gene with other pro- and anti- inflammatory cytokine genes plays an integrated role in destruction of pancreatic beta cells TNF 308 G/A polymorphism was found in susceptibility to T2DM.In this large-scale meta-analysis, the combined evidence suggested that et al. found that TNF-α levels were not significantly different between Japanese T2DM patients with and without this variation et al.TNF 308 G/A polymorphism and metabolic syndrome had indicated that individuals carrying TNFA allele had significantly higher fasting insulin level, systolic arterial blood pressure, higher risk of developing obesity and maybe HOMA-IR, but no significant association with BMI. WHR, fasting glucose and plasma leptin levels, which suggested TNFA allele would increase the risk of metabolic syndrome TNF 308 G/A polymorphism was more common in T2DM patients with than without macrovascular disease TNF 308 A allele conferred a significant risk for T1DM TNF 308 G/A polymorphism might not increase the development of T2DM, but it can enhance the risk for human metabolic disorder and some autoimmunity diseases.However, this variant was reported to be related to some metabolic disorders and T1DM. Furta conclusion, we did not find any evidence of association between TNF 308 G/A polymorphism and T2DM in this large-scale meta-analysis. Further prospective research, with larger numbers of participants and fully confounding risk factors considered, such as age, sex, ethnicity and life style, is warrant to examine the possible effects of this variation on T2DM to confirm our conclusion.In Checklist S1(DOC)Click here for additional data file.Figure S1The flow chart of the included studies.(DOC)Click here for additional data file.


We describe a 67-year-old woman who developed c-ANCA positive vasculitis with involvement in eyes, skin, kidney, peripheral nerves, and upper and lower airway during treatment with etanercept therapy for rheumatoid arthritis. A diagnosis of Granulomatosis with Polyangiitis was done. Thus, anti-TNF therapy may be associated with the development of ANCA positive vasculitis. Several anti-TNF-α biological agents have been approved for the management of RA [α therapies are infectious diseases, malignancies, demyelinating diseases, and drug-induced lupus [The tumor necrosis factor alpha  and methotrexate (15 mgr/weekly). After 8 months of this treatment and because of high activity disease score, a treatment with subcutaneous etanercept (50 mg weekly) was initiated three months before admission. The physical examination at admission revealed bilateral conjunctive injection, and the ophthalmologic exam showed bilateral scleritis. Deformity in hands with synovitis and nodules in the right elbow extensor aspect was also observed. Ulcerated lesions with necrotic center in lower limbs were evident . Laborat S. aureus superantigens or Gram-negative bacteria [α is implicated in the GPA development. This cytokine plays a critical role in the immune system development and response regulation. Indeed, TNF-α stimulation primed the PR3 expression in mononuclear cells. This activation is needed to start the physiopathological axis in vasculitis.GPA (formerly named as Wegener's granulomatosis) is a systemic granulomatous disease associated with small and medium vessel vasculitis of unknown origin that primarily affects the upper respiratory tract, lungs, glomeruli, skin, kidneys, and peripheral nerves. The diagnosis is established based on clinical, radiological findings, presence of c-ANCA (by immunofluoresce), and anti-Proteinase 3 (PR3) antibodies by ELISA, supported by histological evidence of granulomas and vasculitis . There ibacteria . In addiα in vasculitis, with the extended use of anti-TNF-α agents in diverse clinical scenarios, rare adverse events, including granulomatous diseases such as sarcoidosis and GPA, have been described. Thus, granulomatous diseases may be triggered or worsened by anti-TNF therapies [α may additionally trigger systemic autoimmune diseases as systemic lupus erythematosus [Although different to the expected action of TNF-herapies , 9. Antiematosus .α action between these TNF-α antagonist may be implicated in the genesis of granulomatous reaction. Etanercept, a biologic agent composed of two recombinant forms of the human TNF receptor P75 fused to an Fc portion of human immunoglobulin G1, blocks primordially soluble TNF-α and has poor effect on tissue TNF-α. The effect of Etanercept over dilution of granulomas seems to be less important that those induce by antibodies against TNF-α such as infliximab that block this cytokine both tissue and soluble forms [Differences in the mode of neutralizing TNF-le forms .α therapies such as etanercept [α exposure is not fully explained. Cytokine misbalance after TNF-α inhibition may induce granuloma formation in a compensatory way [Although less expected as an adverse event, GPA development has been described with anti-TNF-anercept  and golianercept . Howevertory way , 14.In conclusion, we reported a new case of GPA developing during etanercept therapy. This clinical observation must be considered in all patients treated with anti-TNF.


Current human papillomavirus (HPV) vaccines that are based on virus-like particles (VLPs) of the major capsid protein L1 largely elicit HPV type-specific antibody responses. In contrast, immunization with the HPV minor capsid protein L2 elicits antibodies that are broadly cross-neutralizing, suggesting that a vaccine targeting L2 could provide more comprehensive protection against infection by diverse HPV types. However, L2-based immunogens typically elicit much lower neutralizing antibody titers than L1 VLPs. We previously showed that a conserved broadly neutralizing epitope near the N-terminus of L2 is highly immunogenic when displayed on the surface of VLPs derived from the bacteriophage PP7. Here, we report the development of a panel of PP7 VLP-based vaccines targeting L2 that protect mice from infection with carcinogenic and non-carcinogenic HPV types that infect the genital tract and skin.L2 peptides from eight different HPV types were displayed on the surface of PP7 bacteriophage VLPs. These recombinant L2 VLPs, both individually and in combination, elicited high-titer anti-L2 IgG serum antibodies. Immunized mice were protected from high dose infection with HPV pseudovirus (PsV) encapsidating a luciferase reporter. Mice immunized with 16L2 PP7 VLPs or 18L2 PP7 VLPs were nearly completely protected from both PsV16 and PsV18 challenge. Mice immunized with the mixture of eight L2 VLPs were strongly protected from genital challenge with PsVs representing eight diverse HPV types and cutaneous challenge with HPV5 PsV.VLP-display of a cross-neutralizing HPV L2 epitope is an effective approach for inducing high-titer protective neutralizing antibodies and is capable of offering protection from a spectrum of HPVs associated with cervical cancer as well as genital and cutaneous warts. Human papillomaviruses (HPVs) are non-enveloped viruses with an icosahedral capsid of 55–60 nm in diameter. The HPV capsid is composed of 360 copies of the major capsid protein (L1) and up to 72 copies of the minor capsid protein (L2) which encapsidate a double-stranded circular DNA genome of about 8 kb Currently, two prophylactic vaccines have been licensed to prevent HPV infection. Cervarix® (GlaxoSmithKline) and Gardasil® (Merck) contain virus-like particles (VLPs) derived from HPV16 and HPV18 L1; in addition to these VLPs Gardasil® also contains HPV6 and HPV11 L1 VLPs. Both vaccines are highly immunogenic and elicit high titer, long-lasting neutralizing antibody responses. One limitation of these vaccines is that L1 VLPs by and large induce type-specific antibody responses, meaning that the current L1 VLP-based vaccines provide protection against a subset of HPV types associated with cancer and only offer limited cross-protection against other clinically important carcinogenic HPVs As an alternative to multitype L1-VLP vaccines, we and others have been interested in developing pan-HPV vaccines targeting the HPV minor capsid protein L2. Although L2 is not required to make VLPs, it is an essential component of infectious virions, playing roles in virus entry and trafficking of the viral genome to the nucleus Because L2 neutralizing epitopes are exposed transiently, natural HPV infection fails to elicit anti-L2 neutralizing antibody responses. Thus, it is likely that there has been little evolutionary pressure for L2 to undergo antigenic variation. Correspondingly, amino acid sequence analysis of the amino terminus of HPV L2 reveals a great degree of homology amongst HPV and even animal papillomavirus types. Immunization with L2 not only elicits antibodies that are capable of homologous neutralization, but these antibodies can also neutralize heterologous HPV types Numerous studies have attempted to increase the immunogenicity of L2. For example, synthetic peptides of cross-neutralizing epitopes from HPV16 L2 have been conjugated to keyhole limpet hemocyanin (KLH) or linked to a ubiquitous T helper epitope in vivo against HPV PsVs that represent diverse HPV-types associated with three different clinical manifestations; cervical cancer, genital warts, and cutaneous warts.One of the reasons that the HPV L1-VLP vaccine is so effective is that it presents the target antigen (L1) in a highly multivalent format that is characteristic of the structure of virus particles. VLPs induce strong antibody responses because the regularity of their capsid structure presents viral epitopes as dense, highly repetitive arrays, which are strongly stimulatory to B cells We have previously shown that a peptide derived from amino acids 17–31 of HPV16 L2 can be genetically inserted into a surface-exposed loop of the PP7 bacteriophage coat protein and that this recombinant coat protein self-assembles into VLPs that display the L2 peptide on their surface 4 to 105 . No reactivity was observed with PP7 VLP-derived serum with all the synthetic L2 peptides tested. Thus, L2 peptides displayed on the surface of PP7 VLPs display the high immunogenicity that is characteristic of other VLP-displayed antigens.To test the immunogenicity of the recombinant L2 PP7 VLPs, mice were immunized twice with 5 µg of L2 PP7 VLPs or wild-type PP7 VLPs. In this experiment, immunizations were performed using incomplete Freund's adjuvant (IFA). Sera were analyzed for anti-L2 IgG by end-point dilution ELISA using five synthetic disulfide-constrained L2-peptides representing amino acids 14–40 from HPV1, 5, 6, 16, and 18 as target antigens. Based on amino acid sequence similarities in this region of L2, the 6L2 peptide was used as target antigen for both 11L2 PP7 VLP- and 58L2 PP7 VLP-derived sera and the 18L2 peptide was used as a target antigen for the 45L2 PP7 VLP-derived sera. As shown in As one of our goals was to generate an immunogen with the broadest cross-reactivity against other HPV types, we tested the ability of sera from mice immunized with L2 PP7 VLPs to react with all five synthetic HPV L2 peptides described above . At the Since the reactivity level of 16L2 PP7 VLP sera with 16L2 peptide was similar to that with 18L2 peptide, we were interested to know if mice immunized with 16L2 PP7 VLP will be cross-protected against genital challenge with PsV18; we were also interested to know how efficient this protection would be compared to the protection provided by immunization with 18L2 PP7 VLPs. We assessed whether L2 PP7 VLPs could protect mice from HPV challenge using the HPV pseudovirus (PsV) mouse genital challenge model developed by Roberts and colleagues 5 (for the 6L2 peptide). The trend of reactivity was similar to that observed with single L2 PP7 VLPs. The highest reactivity was observed with both 5L2 and 6L2 HPV L2 peptides followed by 18L2 and 16L2 peptides, likely reflecting the cross-reactivity between induced antibodies.In an attempt to broaden the protection conferred by vaccination, we also tested the immunogenicity of a combination vaccine consisting of all eight L2-PP7 VLPs that were constructed. Mice were immunized with a total of 10 µg of a mixture of 1L2, 5L2, 6L2, 11L2, 16L2, 18L2, 45L2, and 58L2 PP7 VLPs (mixed L2 PP7 VLPs). As shown in in vivo. Mice were immunized three times with mixed L2 PP7 VLPs and then were vaginally challenged with eight different HPV PsVs carrying encapsidated luciferase reporter genes, including one type (PsV31) that was not a component of our vaccine. As shown in p<0.05, by one-tailed t-test). There was some variability in the degree of protection. For example, mice immunized with mixed L2 PP7 VLPs were completely protected from infection with PsV31 and PsV45. We also observed almost complete protection from infection with PsV5, PsV18, and PsV52 (except that one mouse in the control group was not infected with PsV52). Mice immunized with mixed L2 PP7 VLPs and challenged with PsV6, PsV16, and PsV58 were substantially  protected from infection. The lack of complete protection from these PsV types may partially reflect the fact that we used somewhat higher challenge doses of these viruses. Thus, immunization with mixed L2 PP7 VLPs provides substantial and significant protection from genital challenge by diverse HPV PsVs.Given the fact that the broadest L2 peptide reactivity was observed upon immunization with mixed L2 PP7 VLPs, we asked whether binding of mixed anti-L2 IgG to HPV L2 peptides correlated with neutralization Due to the fact that HPV5 is associated with cutaneous warts and the primary site of infection is the skin, we also tested whether our mixed L2 PP7 VLPs vaccine would also protect against cutaneous challenge with PsV5. As shown in The current licensed HPV vaccines have great potential for providing protection against the HPV types that are associated with the majority of cases of cervical cancer and genital warts. Nevertheless, the fact that these vaccines largely provide protection against only two of the 15–18 carcinogenic HPVs means that vaccinated women are still at risk for cancer and will continue to require HPV DNA and/or Pap testing. Moreover, the cost of the current vaccines may be a barrier to global implementation. Thus, it remains a priority to develop an HPV vaccine that provides broad protection against diverse HPV types and at a cost that is affordable worldwide.The largely type-specific protection provided by L1 VLP vaccines is a significant hurdle to the development of similar L1-based vaccines that will provide protection against diverse HPVs associated with all of the clinical manifestations of infection. In contrast, immunization with L2 elicits broad (but often low titer) cross-neutralizing antibodies against multiple HPV types in vitro cross-neutralization with different HPV types Previous studies have documented low titer in vivo neutralization studies. Although a few studies have documented neutralization and cross-neutralization of HPV PsVs both in vitro and in vivoin vivo protection of PsVs from 8 diverse HPV types. We observed protection from vaginal infection by all eight HPV PsVs tested as well as protection from cutaneous infection with PsV5. Protection from PsV58 infection was somewhat lower compared to other PsV types tested. This was a little surprising given the fact that efficient protection from infection was observed with PsV52, which has the same amino acid sequence as HPV58 at this epitope. Moreover, serum anti-L2 IgG titers from the two groups of mice were similar (data not shown). There are three possibilities for this discrepancy; one could be the doses of the PsV used . Another possibility is that this epitope on HPV58 may not be exposed as readily as that of PsV52. The third formal possibility is that mixing of the eight L2 PP7 VLPs together may lead to the induction of both neutralizing and non-neutralizing antibodies that may compete for PsV binding. However, we believe that this is unlikely given the protection levels we observed against PsV18; mixing of 18L2 PP7 VLPs with other L2 PP7 VLPs did not compromise the ability of the vaccine to protect from PsV18 challenges given the fact that mice immunized with single 18L2 PP7 VLPs or mixed L2 PP7 VLPs were equally protected from PsV18 infection.The immunogenicity of the mixed L2 PP7 VLPs and their broad reactivity correlated with our Notably, we also observed complete protection from infection by PsV31, which was not included in our vaccine. This data provides tantalizing evidence of broad cross-neutralization, and we plan on continuing to assess the potential for broad cross-neutralization as pseudoviruses from additional HPV types become available to the scientific community. In summary, our data suggest that the mixed L2 PP7 VLPs vaccine generated in this study can serve as a pan-HPV vaccine targeting diverse HPV types. The phage-VLP approach is effective at low doses, which will be advantageous for use in resource-poor settings, where the vast majority of cases of cervical cancer arise.E. coli5′-GTTGTAAAACGACGGCCAGT-3′) was used as a reverse primer for all PCR amplifications. PCR fragments were cloned into either p2P7K32 or pET2P7K32 using KpnI and BamHI restriction sites. All constructs were confirmed by sequence analysis.Two previously described plasmids were used for the synthesis of L2-recombinant PP7 coat protein in 600 of 0.6. L2 PP7 protein expression was induced with 0.5 mM IPTG, grown for an additional 3 hours, and the culture was centrifuged to pellet the cells. Cell pellets were lysed and L2 PP7 VLPs were purified from the soluble fraction as previously described L2 PP7 VLPs were made by transforming CSH41F or C41 cells (Lucigen) with plasmids p2P7K32 and pET2P7K32, respectively, containing L2 peptides from different HPV types. Constitutive expression of p2P7K32 transformed CSH41F cells was done overnight at 37°C. C41 cells transformed with pET2P7K32 were grown at 37°C until they reached an ODTo assess the antigenicity of the L2 PP7 VLPs, 500 ng of recombinant L2 PP7 VLPs or wild-type PP7 VLPs were used to coat Immulon 2 ELISA plates (Thermo Scientific) at 4°C overnight. ELISA procedure was completed as previously described All mice work was done in accordance with the National Institutes of Health and the University of New Mexico Institutional Animal Care and Use Committee guidelines. In mice immunized with a single recombinant L2 PP7 VLPs, groups of 3 to 13 Balb/c mice were immunized twice at a two-week interval. Immunizations were performed intramuscularly (i.m.) using 5 µg of VLPs plus incomplete Freund's adjuvant (IFA). In addition, groups of 5 Balb/c mice were immunized i.m. three times with a total of 10 µg of mixed L2 PP7 VLPs  without IFA. The mixed L2 PP7 VLPs consisted of 2 µg each of 1L2 and 5L2 VLPs, 2.5 µg of 16L2 VLPs, and 0.7 µg each of the remaining 5 VLP types. Somewhat higher amounts of 1L2, 5L2, and 16L2 VLPs were added in this mixture because 1L2 VLPs elicited antibodies that were the least cross-type reactive, and 5L2 and 16L2 VLPs elicited antibody responses that were the most cross-type reactive . Sera fr405 greater than 2-fold higher than control sera at the same dilution.A peptide-based ELISA was used to assess the titer of anti-L2 IgG in sera. ELISA plates were coated with 500 ng of the appropriate target peptide  conjugated to streptavidin using a bifunctional cross-linker . The ELISA procedure was completed as described above, except that dilutions of mouse sera were used instead of mAb RG-1. Antibody titer was determined as the reciprocal of the highest sera dilution with an ODhttp://home.ccr.cancer.gov/lco/packaging.htm) were generously provided by Chris Buck, Susana Pang, John Schiller, Martin Muller, and Tadahito Kanda.HPV5, HPV6, HPV16, HPV18, HPV31, HPV45, HPV52, and HPV58 PsVs with encapsidated reporter plasmid (pClucf) encoding both luciferase and green fluorescence protein (GFP) genes were produced in 293TT cells as previously described 5– 6.5×106 infectious units (IU) of the above PsVs as previously described 2/sr) was determined from the images by drawing equally sized regions of interests surrounding the site of PsV instillation. Statistical significance was determined by one-tailed paired t-test.Prior to challenge, female Balb/c mice were given two i.m. immunizations with either 5 µg of 16L2 PP7 VLP, 18L2 PP7 VLP, or PP7 VLP in IFA, or three i.m. immunizations of 10 µg  of mixed L2 PP7 VLPs  or 10 µg of PP7 VLP. Three weeks after the last boost, mice were treated with 3 mg of Depo-Provera (Pharmacia Corp). Five days post-Depo-Provera treatment, mice were vaginally challenged with 1.3×105 IU PsV5  in 25 µl of 0.3% carboxymethylcellulose (Sigma-Aldrich). Three days post-challenged, mice were anesthetized again and 0.7 mg of luciferin was injected subcutaneously to the challenged region of the belly. Three minutes later, their images were taken at 10 minutes exposure with Caliper IVIS Lumina II . Average radiance (p/s/cm2/sr) was determined by drawing equally sized regions of interests surrounding the site of PsV instillation.Balb/c mice were immunized three times as described above with either PP7 VLPs or mixed L2 PP7 VLPs. Three weeks after the last immunization, they were challenged as previously described


Neutralization sensitivity of HIV-1 virus to antibodies and anti-sera varies greatly between the isolates. Significant role of V1/V2 domain as a global neutralization sensitivity regulator has been suggested. Recent X-ray structures revealed presence of well-defined tertiary structure within this domain but also demonstrated partial disorder and conformational heterogeneity.Correlations of neutralization sensitivity with the conformational propensities for beta-strand and alpha-helix formation over the entire folded V1/V2 domain as well as within sliding 5-residue window were investigated. Analysis was based on a set of neutralization data for 106 HIV isolates for which consistent neutralization sensitivity measurements against multiple pools of human immune sera have been previously reported.Significant correlation between beta-sheet formation propensity of the folded segments of V1/V2 domain and neutralization sensitivity was observed. Strongest correlation peaks localized to the beta-strands B and C. Correlation persisted when subsets of HIV isolates belonging to clades B, C and circulating recombinant form BC where analyzed individually or in combinations.Observed correlations suggest that stability of the beta-sheet structure and/or degree of structural disorder in the V1/V2 domain is an important determinant of the global neutralization sensitivity of HIV-1 virus. While specific mechanism is to yet to be investigated, plausible hypothesis is that less ordered V1/V2s may have stronger masking effect on various neutralizing epitopes, perhaps effectively occupying larger volume and thereby occluding antibody access. Neutralization by antibodies, along with cellular immunity, is a key defense mechanism against viral infection. Most clinical isolates of HIV-1 virus are notoriously difficult to neutralize by antibodies. This resistance is contributing to both, the inability of human immune system to control HIV infection in the vast majority of individuals and the fact that despite decades of concerted efforts to create an effective prophylactic HIV vaccine, only a rather limited success has been reported so far  Yet this resistance varies greatly between different virus isolates, and a ‘Tier’ system has been proposed to classify HIV strains and to provide a virus panel for objective evaluation of immune sera and monoclonal antibodies in terms of their neutralization potency. Importantly, strains that resist neutralization often do so across multiple antibody types targeting different epitopes.env trimer, i.e. its propensity to undergo conformational transition to lower-energy states from the initial native state, provides an important contribution to global inhibition sensitivity In principle, neutralization resistance variations should be determined by env sequence and ultimately by the structure and dynamics of the spike. It has been proposed that ‘intrinsic reactivity’ of the Intriguingly, it was demonstrated that V1/V2 region of gp120 is an important determinant of the overall neutralization sensitivity of the HIV-1: modifications and deletions often increase neutralization sensitivity Until recently, little has been known about the structure of V1/V2 domain and the two segments in it delineated by disulfide bridges were viewed as ‘loops.’ V1/V2 received limited attention in vaccine development efforts because of its high variability and apparent limited functional importance – V1/V2 deleted virus often remains replication competent Antibody responses to V1/V2 in the sera correlated with protection in the RV144 vaccine trial in Thailand Remarkably, linear epitopes of the mAbs CH58 and CH59, which localize to the stretch of the V1/V2 sequence corresponding to the C-strand in mAb PG9 complexes, are structurally distinct: CH58 binds a largely alpha-helical conformation of the antigen, while in CH59 complex it is a coil with elements of 3–10 helical and extended conformations .Can structural variability of V1/V2 domain underlie its influence on the neutralization sensitivity (NS) of the HIV strains? In the absence of the structural data for a range of V1/V2 domains from different strains and in the native context, it may still be possible to tease out certain structural correlates from simple sequence-based biophysical descriptors. Since the possibility of secondary structure switch between beta-strand, coil and/or alpha helix is indicated by the X-ray structures, propensities to form alpha-helix and beta-strand are of particular interest. While all 20 naturally occurring amino-acids are found within all types of secondary structures, preferences towards one or another structure that vary significantly between the amino-acids are well-established 10 of neutralization ID50 (50% inhibitory dose) by HIVIG (HIV immune globulins). Pierson correlation coefficient R was 0.34. While modest, correlation was highly statistically significant with the p-value estimate of 0.0003. For comparison, when the same calculation was performed for alpha-helical propensity, R = −0.14 with p-value estimate of 0.15 was obtained.Correlation of the neutralization sensitivity for 106 HIV isolates on the tiered neutralization assessment panel 10 ID50 evaluated. Short 5-residue segments rather than individual residues were analyzed because secondary structure formation is a cooperative process and therefore total propensities within a window are expected to be more structurally meaningful and less noisy. For 15 positions p-values of the correlations reached statistical significance (p<0.05). Importantly all of them had the same (positive) sign of the correlation, i.e. lower ID50s correlated with more negative propensity (i.e. more stable beta-strand structures). When plotted along the amino-acid sequence, two peaks of logID50/beta propensity correlation centered at positions T163 and Y177 were observed , as well as a weaker peak at S197 (p = 0.02). Notablyp = 0.02).10 ID50 of 0.35, similar to that of the total BSP of the entire conserved V1/V2 domain above, and p-value of 0.00002 – actually better than that for the entire domain. Third weaker peak was in the middle of the strand D, extending into V1/V2 stem.Remarkably, the two pronounced peaks of beta-strand propensity correlation corresponded well to the C-terminal parts of adjacent beta-strands (B and C) observed in the available X-ray structures (Protein Data Bank IDs 3U4E and 3U2S). It is therefore plausible that propensities from both segments combine to determine the overall stability of the beta-hairpin structure formed by the two strands, perhaps regulating the length of the stably folded part versus less ordered connecting loops. Indeed, total BSP from the central 32-residue V1/V2 segment (E153-I184) showed Pierson correlation coefficient with neutralization loga posteriori). The correlation plot log10 ID50/ΔGBSP is shown on Stronger correlation could be achieved if contributions from the top three independent 5 residue segments were combined , with R = 0.47  and p<0.000001  BC and 11 in CRF AG. Other clades and forms had only a few representatives each and were combined in a fourth group of 20 viruses, mostly related to clade A. Significant correlation was still present in the first two groups, with strongest correlation in clade B, and somewhat weaker in clade C . It shouConformational heterogeneity of V1/V2 region was recently demonstrated by the X-ray investigations of PG9/PG16, CH58 and CH59 mAb complexes with their epitopes: the C-strand of PG9 complex that encompasses the key residues involved in binding to all three mAbs exhibits beta-strand secondary structure in the first case, mostly alpha-helical secondary structure in the second case, and coiled conformation in the third. While this ability to adopt different conformations was so far directly demonstrated only in antibody binding, it may also have a functional role, i.e. the conformation may change during the transitions associated with attachment and fusion, and prevalence of different conformations may also vary from virus strain to virus strain or from clade to clade. Herein it is demonstrated that a simple sequence-based measure of the propensity to form beta-structure within B/C hairpin region of the V1/V2 domain significantly correlates with neutralization sensitivity of the virus.Modulation of this propensity likely either triggers switch from one conformation to another or affects equilibrium between multiple conformations that the domain can adopt. Another possibility is that beta-sheet propensity controls order/disorder transition or equilibrium within V1/V2 domain. Functional importance of intrinsic disorder in proteins Clearly, the described effect of V1/V2 beta-strand propensity is not the only factor that determines the neutralization resistance of HIV virus, as the wide spread of the correlation plot  indicateenv sequence of HIV1 strain HXB2) are extremely variable both in composition and length. They were excluded from the analysis. Also excluded were two short inserts present only in one sequence each (4 AAs between positions 169/170 in H078.14 and 1 AA between positions 165/166 in 9021.14.B2.4571). Because X-ray structures of V1/V2 domain show that C- and N- terminal secondary structure elements (beta strands A and D) extend through the C126-C196 disulfide bridge that formally separates V1/V2 domain itself and the so-called ‘stem’ (independently observed forming beta-strand in other gp120 X-ray structures), sequence segments belonging to the stem were also included in the analysis (up to the next disulfide C119-C205). As seen in the results, only minor signal was observed outside the V1/V2 domain proper.Gp120 sequences of 106 HIV isolates on the tiered neutralization assessment panel As a measure of neutralization sensitivity (NS) of the virus, neutralization data from ref Smith, Withka & Reagan env sequence:j(i)S is the amino acid at i-th position in j-th sequence according to the alignment, p is the appropriate propensity according to the scale and summation is over all well-aligned positions. Highly variable segments with insertions/deletions in many sequences were excluded from the analysis, leaving 60 well-aligned positions within N-terminal (in HXB2 numbering AA C119-C131), central (E153-I184), and C-terminal (Y191-C205) conserved elements.Initially, total beta-sheet propensity over the conserved elements of V1/V2 domain were evaluated for each Total beta-sheet or alpha-helix propensities for 5-residue segments within a window sliding along V1/V2 domain sequence were calculated:10 ID50 with total propensity and 5-residue segment propensities for each position was calculated. Randomization test Thus, vectors of propensities across 106 sequences were generated for each position within the alignment of V1/V2 domains. Pierson correlation coefficient of logFigure S1Plots of Pierson correlation coefficients R and p-values for the AHP/NS correlation. Plots are made the same way as for BSP on (TIFF)Click here for additional data file.


An immune correlates study of the RV144 vaccine trial demonstrated that elicitation of V1/V2-directed antibodies was inversely associated with infection risk. In addition, a sieve analysis of breakthrough infections identified residue 169 in the V2 loop as a site of immune pressure. Antibodies CH58 and CH59 were isolated from RV144 vaccinees and were shown to neutralize some Tier 1 isolates.The epitopes for CH58 and CH59 were identified by peptide-mapping and alanine-scanning. Antigen-binding fragments (Fabs) were generated by proteolysis, and crystal structures of the CH58 and CH59 Fabs in complex with a linear V2 peptide were determined to 1.7 and 1.5 Å, respectively. Surface plasmon resonance was used to determine the kinetics of antibody binding to recombinant env-derived proteins.10 helix. Both antibodies form hydrogen bond or salt-bridge interactions with the side chain of Lys169, the imputed site of immune pressure. The conformations of V2 recognized by CH58 and CH59 differ markedly from the β-strand conformation recognized by the broadly neutralizing antibody PG9. All three antibodies bound with high affinity to the same gp120 protein, suggesting that V2 residues 167-176 can adopt multiple conformations on a shed gp120.The crystal structures reveal that CH58 recognizes V2 residues 167-176 as an α-helix and residues 177-181 as an extended coil. In contrast, CH59 recognizes residues 168-173 as coil, with residues 174-176 as a short 3Since PG9 is broadly neutralizing and recognizes V2 as a β-strand, and because CH58 and CH59 neutralize only some Tier 1 isolates and recognize alternative conformations of V2, these data suggest that the β-strand conformation of V2 may be favored in the viral spike, whereas alternative V2 conformations may be favored on shed gp120s. If true, then vaccine immunogens may need to have the V1/V2 region stabilized in the β-strand conformation in order to elicit broadly neutralizing antibodies.


As the core of the G protein signal transduction machinery, the Gα subunits are required to interact with multiple partners. The GTP-bound active state of many Gα subunits can bind a multitude of effectors and regulatory proteins. Yet it remains unclear if the different proteins utilize distinct or common structural motifs on the Gα subunit for binding. Using Gα16/TPR1 association by testing a series of chimeras between Gα16 and Gαz. TPR1 co-immunoprecipitated with Gα16 and more tightly with its constitutively active Gα16QL, but not Gαz. Progressive replacement of Gα16 sequence with the corresponding residues of Gαz eventually identified a stretch of six amino acids in the β3 region of Gα16 which are responsible for TPR1 interaction and the subsequent Ras activation. Insertion of these six residues into Gαz allowed productive TPR1-interaction. Since the β3 region only minimally contributes to interact with PLCβ, several chimeras exhibited differential abilities to stimulate PLCβ and Ras. The ability of the chimeras to activate downstream transcription factors such as signal transducer and activator of transcription 3 and nuclear factor κB appeared to be associated with PLCβ signaling.We have examined the specificity of Gα16 can signal through TPR1/Ras and PLCβ simultaneously and independently. The β3 region of Gα16 is essential for interaction with TPR1 and the subsequent activation of Ras, but has relatively minor influence on the PLCβ interaction. Gα16 may utilize different structural domains to bind TPR1 and PLCβ.Our results suggest that Gα Heterotrimeric guanine nucleotide-binding proteins (G proteins) are multifaceted signaling modules that relay extracellular signals detected by G protein-coupled receptors (GPCRs) to intracellular effector -3. At thThe resolution of the crystal structures of several Gα subunits in their GDP- or GTP-bound states ,8 and asq subfamily have the capacity to activate phospholipase Cβ (PLCβ)  were from Pierce Biotechnology . ECL kit was from GE Healthcare Bio-Sciences . Ras activation kit was a product of Millipore .The human cDNAs of Gαcterized . Cell cu16 and Gαz by using PCR techniques. The N-terminal 102, 155, 188, 200, 210, 246, 266 and 295 residues of Gα16 were substituted by the corresponding regions of Gαz to generate N102, N155, N188, N200, N210, N246, N266 and N295 chimeras, respectively. Primer pairs were designed to cover the overlapping regions in forward and reversed directions. For each construct, the 5' fragment was generated with the reversed and T7 primers, whereas the 3' fragment was made with the forward and SP6 primer. The two half-products were then annealed together to generate a full-length fragment by another round of PCR using T7 and SP6 primers. Mirror-images of these constructs were generated analogously and were named C164, C174, C186, C219 and C272 chimeras. Primers for chimera construction are listed in Table Pfx SuperMix . The N200-C164 and N188-C164 chimeras were constructed using C164 as the initial template for the 3' half-products and those primers designed for N200 and N188, respectively. Likewise, zβ3 and zβ2β3 were constructed using the C174 and C164 primers with N210 as the initial templates. All chimeras were checked by restriction mapping and then subcloned into pcDNA3 at Hind III and Xho I sites. The constructs were fully sequenced by dideoxynucleotide sequencing to confirm the identities.Gα chimeras were constructed from the cDNAs encoding the human Gα2, 37°C with 10% fetal bovine serum (FBS), 50 units/ml penicillin and 50 μg/ml streptomycin. HEK 293 cells were grown to 80% confluency in 100-mm tissue culture plates and then co-transfected with 800 ng Gα and 800 ng FLAG-TPR1 cDNAs using 15 μl Plus™ and Lipofectamine™ reagents in Opti-MEM™. FBS was replenished 3 h after transfection. Cross-linking was performed one day after transfection. Transfected cells were washed with PBS twice and then treated with 0.5 mM dithiobis[succinimidylpropionate] in PBS for 15 min at room temperature. Cells were then washed again with PBS and maintained in quenching solution  for 5 min. Subsequently, cells were lysed in ice-cold RIPA buffer . Cell lysates were gently rocked with an anti-Gα16, anti-Gαz or anti-PLCβ antiserum at 4°C overnight, and then incubated in 30 μl protein G-agarose (50% slurry) at 4°C for 2 h. Alternatively, the cell lysates were incubated in 30 μl anti-FLAG affinity agarose gel (50% slurry) at 4°C overnight. Immunoprecipitates were washed with ice-cold RIPA buffer (400 μl) for four times, resuspended in 50 μl RIPA buffer and 10 μl 6 × sample buffer and then boiled for 5 min. Gα16, Gαz, PLCβ and FLAG-TPR1 proteins in the immunoprecipitates were analyzed by Western blots.HEK 293 cells were obtained from the American Type Culture Collection . They were maintained in Eagle's minimum essential medium at 5% CO2+ lysis buffer . Cell lysates were pre-cleaned with Protein G-agarose and activated Ras was immunoprecipitated with 20 μl Raf-1 Ras-binding domain-conjugated agarose (Millipore) for 45 min and followed by three washes of 400 μl ice-cold MLB. Immunoprecipitates was finally reconstituted in 50 μl MLB and 10 μl 6 × sample buffer and resolved in SDS-PAGE for detecting Ras using specific antibody.HEK 293 cells were co-transfected with 800 ng Gα, 800 ng FLAG-TPR1 and 400 ng Ras cDNAs. After 1 day, transfectants were serum-deprived for 4 h. Cells were then washed twice with ice-cold PBS and lysed with the 1 × Mg5 cells/well 1 day prior to transfection. Transfection was performed with 200 ng Gα and 200 ng FLAG-TPR1 cDNAs using 4 μl Plus™ and Lipofectamine™ reagents in Opti-MEM™. The transfectants were serum-deprived overnight one day after transfection. Cells were treated with pertussis toxin  and N6-cyclohexyladenosine  where appropriate, lysed, and then assayed for phosphorylation statuses of ERK, STAT3, and IKK as described previously inositol and subsequently assayed for CHA-induced [3H]IP3 formation as described previously [HEK 293 cells were seeded on a 12-well plate at 2 × 10eviously .1R-induced signals were analyzed, 10 ng receptor cDNA was included in the transfection. For NFκB-driven luciferase assay, HEK 293 cells stably transfected with pNFκB-luc luciferase reporter were seeded on a 96-well microplate at 15,000 cells/well. The setup and transfection were as described previously [For STAT3-driven luciferase assay , HEK 293eviously . One dayeviously .Protein samples were resolved on 12% SDS-PAGE and transferred to nitrocellulose membrane (GE Osmonics). Resolved proteins were detected by their specific primary antibodies and horseradish peroxidase-conjugated secondary antisera. The immunoblots were visualized by chemiluminescence with the ECL kit from GE Healthcare Bio-Sciences, and the images detected in X-ray films were quantified by densitometric scanning using the Eagle Eye II gel documentation system .q in the complex with p63RhoGEF and RhoA (PDB ID: 2rgnA) [q, and for creating a molecular model of Gα16 by homologous modeling using SWISS-MODEL [16 with Gαq in order to accommodate the extraordinarily long α4/β6 loop of Gα16. Molecular models of Gαz with or without the mutations at β2/β3 loop (194ELTFKM → KTNLRI) were generated using 3D-JIGSAW [i1 bound to AlF4- and GDP (PDB ID: 2hlbA) [Gα: 2rgnA)  was emplSS-MODEL , which aD-JIGSAW  based on: 2hlbA)  as selec: 2hlbA) .16 regions responsible for effector interaction have not been mapped, but there is evidence to suggest that the Switch III and the α3 helix may participate in the binding of p63RhoGEF [q mainly encompasses the α2-β4-α3-β5 regions [q/PLCβ complex formation has been revealed clearly in the very recently resolved crystal structure [q [16 that interact with TPR1, Gα16 sequences were progressively replaced by those of Gαz because the latter does not recognize TPR1  of N30 is composed of Gαz sequence  or C-terminus (Gα16C), were required in the co-immunoprecipitation assay. Although both mutants were expressed to comparable levels  exhibited elevated Ras activity whereas C174QL (β3 region from Gαz) did not activate Ras  was similarly tested .To confirm that the PLCβ-interacting regions of Gα2 Figure . Chimerad Figure . Due to s Figure . These r1R were stimulated with CHA, and the phosphorylation of STAT3 and inhibitor of κB kinase (IKK) monitored by western blot analysis using phosphoprotein-specific antibodies. As shown in Table 705 and Ser727) and IKK phosphorylations, with magnitudes similar to those of Gα16. These chimeras included N30, N188, C25, C44, zβ2β3, and zβ3. N200 failed to mediate the CHA-induced STAT3 phosphorylation but supported the IKK phosphorylation  of Gα16. The β2-β3 region lies between the Switch I (mainly β2 strand) and II (mainly α2 helix) regions [16WT or Gα16QL are comparably smaller, and TPR1 can obviously bind to Gα16WT. Such observation implies that TPR1 may bind to Gα16 at regions with relatively less drastic conformational changes. Our results suggested that the β3 strand of Gα16 alone appears to be sufficient for the interaction with TPR1. The mere incorporation of the β3 region of Gα16 confers upon Gαz the ability to bind TPR1 (N200-C164 chimera). N200-C164 has the amino acid sequence 201KTNLRIVDVG from Gα16 inserted into a Gαz backbone, but only the first six residues are different from the corresponding Gαz sequence (194ELTFKMVDVG). Based on the crystal structure of Gαi1 [z reveals that Glu194 is located at the "hook-shaped" β2-β3 turn  contain a contiguous helical core (αA-αF helices) in addition to the putative PLCβ-interacting domains. An early study of Gαs suggested that its helical domain could function as an internal GAP for the GTPase domain [16/z chimeras. The sudden regain of PLCβ-activating property of N188 which contains a helical domain completely derived from Gαz also implies that structural integrity of the helical domain is critical for PLCβ activation. As revealed in the very recent crystal structure of Gαq-PLCβ3 [q interact extensively with the C2 domain of PLCβ3, and these two strands also heavily contribute to the overall scaffold of the GTPase domain. Replacement of one or both of them, as in the N200 and N210 chimeras, might disrupt PLCβ interaction severely. Nonetheless, N200 can successfully form a heterotrimer with FLAG-tagged Gβγ dimer in co-immunoprecipitation experiment (unpublished data), excluding the possibility of improper folding of the chimera.Although the chimeras used in the present study were not tailored for the mapping of the PLCβ-interacting domain of Gαe of Gαq ,50, the 6 Figure . A more 6 Figure  which mae domain , and thee domain ,59. Furte domain . It is t16 is constructed by homologous modeling based on the crystal structure of Gαq to visualize the potential interacting surfaces for PLCβ and TPR1. Except for the unusually long α4-β6 loop and differences in the N-terminal helix (not shown in the model), Gα16 basically fit very well to the structure of Gαq. Assuming that Gα16 utilizes domains similar to those of Gαq for the binding of PLCβ [16 to simultaneously regulate PLCβ and TPR1. As shown in the right panel of Figure 16 (yellow-colored), while the putative TPR1-interacting β3 region is located in the lower quadrant of the right hand side without severe overlap with the PLCβ-interacting region. Preliminary co-immunoprecipitation studies indeed suggest the existence of Gα16/PLCβ and PLCβ/TPR1 complexes (unpublished data). Further studies will be required to confirm if a Gα16/PLCβ/TPR1 complex truly exists. TPR motif-containing adaptors such as Rapsyn are known to cluster signaling molecules for the efficient propagation of signals [13, Gαt1 [z  can bind TPR1  can interact with TPR1. Since many ligands that act on Gq-coupled receptors are mitogenic [q/TPR1 provides a means for the efficient stimulation of the Ras/Raf/MEK/ERK cascade for cell proliferation. Given that both PLCβ and TPR1 are ubiquitously expressed, the ability of Gα16 as well as other Gαq subfamily members to selectively activate one of the two pathways may have to rely on alternative means of signal segregation, such as spatial orientation [q subunit to the lipid bilayer [q signaling. It should also be noted that the presence of p63RhoGEF can affect the binding of PLCβ and TPR1 to Gα16 and differentially inhibit their signaling [16 has yet to be elucidated, but is expected to encompass the α2-β4-α3-β5 regions and the C-terminal α5 helix based on the crystal structure of Gαq-p63RhoGEF-RhoA complex complex (Figure 16/z chimeras represent useful tools to confirm such a prediction.The possibility of Gαubfamily ,63 or thubfamily ,65, withitogenic ,66, linkentation , formatientation , and comentation . Attachm bilayer , as well bilayer , have beignaling . The p6316 for the interaction with TPR1 and subsequent activation of Ras, but the β3 strand appears to be dispensable for PLCβ interaction. The integrities of both helical and GTPase domains are essential for PLCβ activation. Gα16 can signal through TPR1/Ras and PLCβ simultaneously and independently to regulate transcriptional events involving STAT3 and NFκB by utilizing different structural domains to bind TPR1 and PLCβ. Overall, Gα16 is able to interact with multiple molecular partners to convey different streams of signal transduction.This study provided evidence for the importance of the β3 strand of Gα1R: type 1 adenosine receptor; AGS: activator of G protein signaling; CHA: N6-cyclohexyladenosine; FBS: fetal bovine serum; G protein: heterotrimeric guanine nucleotide-binding proteins; GPCR: G protein-coupled receptor; GTPase: GTP hydrolase; IKK: inhibitor of κB kinase; IP3: inositol trisphosphates; NFκB: nuclear factor κB; PLCβ: phospholipase Cβ; PTX: pertussis toxin; RGS: regulator of G protein signaling; STAT3: signal transducer and activator of transcription 3; TPR1: tetratricopeptide repeat 1AAMFL and RKHL performed most biological experiments. EXG assisted in experiments. MKCH performed homology modeling. AMFL, MKCH and YHW conceived and designed the experiments and wrote the manuscript. RDY participated in the design of the study. All authors read and approved the final manuscript.


AMA1 is efficiently shed from the invading parasite surface, predominantly through juxtamembrane cleavage by a membrane-bound protease called SUB2, but also by limited intramembrane cleavage. We have investigated the structural requirements for shedding of Plasmodium falciparum AMA1 (PfAMA1), and the consequences of its inhibition. Mutagenesis of the intramembrane cleavage site by targeted homologous recombination abolished intramembrane cleavage with no effect on parasite viability in vitro. Examination of PfSUB2-mediated shedding of episomally-expressed PfAMA1 revealed that the position of cleavage is determined primarily by its distance from the parasite membrane. Certain mutations at the PfSUB2 cleavage site block shedding, and parasites expressing these non-cleavable forms of PfAMA1 on a background of expression of the wild type gene invade and replicate normally in vitro. The non-cleavable PfAMA1 is also functional in invasion. However – in contrast to the intramembrane cleavage site - mutations that block PfSUB2-mediated shedding could not be stably introduced into the genomic pfama1 locus, indicating that some shedding of PfAMA1 by PfSUB2 is essential. Remarkably, parasites expressing shedding-resistant forms of PfAMA1 exhibit enhanced sensitivity to antibody-mediated inhibition of invasion. Drugs that inhibit PfSUB2 activity should block parasite replication and may also enhance the efficacy of vaccines based on AMA1 and other merozoite surface proteins.The malarial life cycle involves repeated rounds of intraerythrocytic replication interspersed by host cell rupture which releases merozoites that rapidly invade fresh erythrocytes. Apical membrane antigen-1 (AMA1) is a merozoite protein that plays a critical role in invasion. Antibodies against AMA1 prevent invasion and can protect against malaria  The malaria parasite invades red blood cells. During invasion several parasite proteins, including a vaccine candidate called PfAMA1, are clipped from the parasite surface. Most of this clipping is performed by an enzyme called PfSUB2, but some also occurs through intramembrane cleavage. The function of this shedding is unknown. We have examined the requirements for shedding of PfAMA1, and the effects of mutations that block shedding. Mutations that block intramembrane cleavage have no effect on the parasite. We then show that PfSUB2 does not recognise a specific amino acid sequence in PfAMA1, but rather cleaves it at a position determined primarily by its distance from the parasite membrane. Certain mutations at the PfSUB2 cleavage site prevent shedding, and parasites expressing non-cleavable PfAMA1 along with unmodified PfAMA1 grow normally. However, these mutations cannot be introduced into the parasite's genome, showing that some shedding by PfSUB2 is essential for parasite survival. Parasites expressing shedding-resistant mutants of PfAMA1 show enhanced sensitivity to invasion-inhibitory antibodies, suggesting that shedding of surface proteins during invasion helps the parasite to evade potentially protective antibodies. Drugs that inhibit PfSUB2 may prevent disease and enhance the efficacy of vaccines based on PfAMA1. Apicomplexa contains several pathogens of major clinical and veterinary importance. These include the aetiological agents of coccidiosis in poultry, theileriosis and babesiosis in cattle, and toxoplasmosis, cryptosporidiosis and malaria in humans. In all cases, the causative agents are parasitic protozoa that share the feature of possessing several life-cycle stages that switch sequentially between replicative intracellular forms and transiently extracellular zoite stages equipped to seek out and invade suitable host cells. Invasion by apicomplexan zoites has been a subject of great attention for two major reasons: first, because it represents a step at which the parasite is exposed to host antibodies and other effector molecules capable of preventing invasion The phylum Plasmodium spp. in circulating erythrocytes. In the case of the most dangerous malarial species, Plasmodium falciparum, the parasite develops intracellularly over the course of∼48 hours to produce a mature schizont containing around 16 daughter merozoites. The schizont eventually ruptures, releasing the merozoites which rapidly invade fresh erythrocytes, thus perpetuating the cycle. Erythrocyte invasion comprises several discrete steps P. falciparum AMA1 (PfAMA1) and the MSP1/6/7 complex, as well as another micronemal protein called PTRAMP, is all mediated by the same membrane-associated, calcium-dependent parasite subtilisin-like serine protease called PfSUB2, which is itself released from micronemes onto the merozoite surface during invasion P. falciparum MSP1 and PfAMA1 has shown that the sequences that flank the scissile bond lack obvious similarity The clinical manifestations of malaria stem from replication of asexual blood stages of in vitro culture a small fraction of PfAMA1 is also shed by intramembrane cleavage P. falciparum. Intriguingly, however, shedding of the Toxoplasma gondii AMA1 orthologue, TgAMA1, takes place exclusively through intramembrane cleavage Toxoplasma orthologue of ROM4 Toxoplasma, so the relative importance of PfSUB2-mediated and intramembrane shedding of PfAMA1 remains unclear.In addition to PfSUB2-mediated shedding, under normal conditions of P. falciparum PfSUB2 gene (pfsub2) or its orthologue in the rodent malaria species P. berghei have been unsuccessful, suggesting that SUB2 plays an indispensable role in parasite asexual blood-stages Attempts to disrupt the single-copy Plasmodium species, AMA1 is conserved in most other apicomplexan genomes examined in vitro and protect against blood-stage malaria in vivo  are required to obtain a significant effect on invasion . Despite this, certain radical mutations at the PfSUB2 cleavage site can block shedding. Parasites expressing these non-cleavable forms of PfAMA1 on a background of expression of the wild type protein invade and replicate normally P. falciparum microneme protein EBA-175 Toxoplasma microneme proteins MIC2 pfama1 gene by targeted homologous recombination, replacing the P1 Ala550 with a Tyr residue  are sufficient for normal parasite growth.Precise mapping by us and others of the intramembrane cleavage sites in PfAMA1 and TgAMA1  residue . Parasit residue  showed tpfama1 transgene in P. falciparum. Expressed under the control of the pfama1 promoter, the transgene product , or the three residues downstream of the processing site , or all six residues   . As showy PfSUB2 . Under c48 product. If, on the other hand, the site of cleavage was dependent on its distance from the membrane, cleavage would now take place at a new site, probably at or around the position indicated in 48. As shown in P. falciparum resulted in the usual pattern of microneme localisation, and the expression as expected of larger forms of intracellular anti-HA-reactive PfAMA1, likely due to the presence of the 21 residue (∼2.6 kDa) insertion in AMA_ins. Examination of culture supernatants from this line , has never been observed in digests of wild-type PfAMA148To seek a more precise estimate of the position of PfSUB2-mediated cleavage in the AMA_ins transgene product, we decided to isolate the shed product from culture supernatants for more detailed structural examination. Attempts to affinity-purify it using anti-HA antibodies failed, possibly due to poor solvent accessibility of the domain III-located single HA epitope tag in the folded protein. However, using a previously described method P. falciparum is complicated by the fact that, even under continuous selective drug pressure, episomes generally segregate poorly in the parasite Expression of transgenes from episomal constructs in P. falciparum resulted in expression of all 7 transgene products at levels approximately equivalent to those of the parental construct AMA_C, with the same pattern of sub-cellular localisation (not shown) and intracellular proteolytic prosequence cleavage , integration of constructs intAMA_P1 and intAMA_P1-P1′ was predicted to introduce Pro substitutions of residues at either the P1 position or both the P1 and P1′ positions respectively relative to the PfSUB2 cleavage site , unambiguously demonstrating that the effect of R1 on invasion is solely a result of its capacity to bind PfAMA1.Having failed to obtain viable parasites in which the endogenous −1) that effectively blocks invasion by the parental 3F7 clone  flanking the PfAMA1 cleavage site, or mutation of the P1 Thr to Arg or Tyr, or the P2 Val to a Phe residue, or even substitution of both P1 and P1' residues with Tyr, had no detectable effect on the efficiency of shedding. The shed cleavage products from the AMA_M1, AMA_M2, AMA_M3, AMA_R1, AMA_Y1, AMA_Y1-Y1′ and AMA_F2 mutants co-migrated precisely on SDS PAGE with those of the parental AMA_C transgene product, leading us to conclude that cleavage of the mutants likely takes place at the same position relative to the TMD. The significance of the enhanced levels of the intramembrane cleavage product, PfAMA1The third part of our study addressed the effects of mutations at the processing site that interfere with cleavage. As previously observed for several other proteases, we found that the insertion of a single Pro residue at the P1 position, or two Pro residues flanking the scissile bond, very efficiently blocked shedding of the PfAMA1/DIII-HA transgene product. We had anticipated that, given the normally highly efficient shedding of PfAMA1 at invasion, expression of such episomal constructs in the parasite might be deleterious to parasite growth. In fact, we found that the cleavage-resistant AMA_del, AMA_P1, or AMA_P1-P1′ transgenes were expressed just as efficiently as the other transgene mutants, and that the lines carrying them showed no detectable growth defect. Focusing on the AMA_P1, or AMA_P1-P1′ mutants, we used antibodies to the ectodomain-located HA epitope tag to show that, consistent with the virtually complete absence of shed ectodomain released into culture supernatants, the mutant gene products were efficiently carried into host erythrocytes on the surface of invading merozoites. This proves unambiguously that complete shedding is not a prerequisite for invasion. Furthermore, using the R1 peptide, which blocks the invasion-related function of 3D7 PfAMA1 in an allele-specific manner, we showed that the FVO-based cleavage-resistant mutants complemented the function of R1-inactivated endogenous PfAMA1 as efficiently as the AMA_C-derived transgene product, convincingly demonstrating that PfSUB2-mediated shedding is not required for the function of PfAMA1 at invasion.pfama1 gene? One interpretation of this result is that although PfSUB2-mediated cleavage of PfAMA1 is not essential for the function of PfAMA1 during invasion, some shedding is essential at some point in the asexual blood-stage life cycle. This notion is supported by a recent study in Toxoplasma indicating that intramembrane cleavage of TgAMA1 is required not for invasion, but to trigger parasite replication following invasion P. falciparum performs an analogous role to that of TgROM4 in Toxoplasma? Work is underway to address this question.Given these findings, what is the explanation for our inability to introduce the P1 and P1-P1' mutations into the endogenous Toxoplasma using a tetracycline-regulated conditional system have indicated that TgAMA1 is normally expressed at levels well in excess of those necessary to sustain its role at invasion, since reduction of endogenous protein levels by ∼90% (upon replacement of the endogenous gene with a regulated copy) had no effect on invasion efficiency Plasmodium, and if only those PfAMA1 molecules involved in interactions with the RON complex play a productive role in invasion, it may be important for the parasite to rapidly discard excess surface PfAMA1 molecules in the presence of anti-PfAMA1 antibodies in order to prevent accumulation of antibody complexes on the parasite surface that interfere with the parasite's normally rapid passage into the nascent parasitophorous vacuole. Thus, even though our results clearly demonstrate that complete shedding from the merozoite surface is not mechanistically essential for invasion to go to completion, it may be evolutionarily advantageous for shedding of PfAMA1 to occur in an efficient manner in order to cope with the threat of host-derived anti-PfAMA1 antibodies.Unlike many other microneme proteins  into plasmid pST2A-sgPfa1 HpaI and KspI, followed by blunting and religation, producing pHH-TKΔMCS2. The P. berghei dhfr 3′ UTR (PbDT3′) was then PCR-amplified from pHH1 using primers PbDT3′_ClaI_MCS_F and PbDT3′_R and subcloned into pHH-TKΔMCS2, giving rise to plasmid pHH-TKmod. Sequence including the mutation at the rhomboid cleavage site in PfAMA1 was subcloned from AMA_A550Y into intermediate plasmid PSL1180_AMA_C (see below) with restriction enzymes SnaBI and StuI. The entire chimeric gene from this plasmid, as well as the control chimera from PSL1180_AMA_C was excised by restriction with XhoI and blunting the cut ends followed by digestion with BglII. This was cloned into pHH-TKmod pre-digested with BglII and HindII, producing intAMA_R-TKmod and intAMA_C-TKmod.To produce integration constructs intAMA_R-TKmod and intAMA_C-TKmod, one of the multiple cloning sites was first removed from pHH-TK with restriction enzymes pfama1 gene, excluding sequence encoding the first 4 Met residues, was amplified from 3D7 genomic DNA using primers AMAint_F and AMAint_R. This was cloned into plasmids AMA_C, AMA_P1 and AMA_P1-P1′ using restriction enzymes HpaI and AvrII, replacing the pfama1 promoter sequence and 3′ end of the synthetic FVO ama1 gene and resulting in a partial in-frame chimera between the targeting and synthetic coding sequences. To obtain the derivatives intAMA_C_TK, intAMA_P1_TK and intAMA_P1-P1′_TK the entire chimeric coding sequence of each construct plus its downstream P. berghei dhfr 3′ UTR (PbDT3′) was excised with restriction enzymes HpaI and NotI, cloned into plasmid PSL1180  pre-digested with EcoRV and NotI  then the inserts excised with restriction enzymes BglII and KspI and cloned into plasmid pHHT-TK digested with the same enzymes. All final constructs were confirmed by sequencing on both strands.Integration constructs intAMA_C, intAMA_P1 and intAMA_P1-P1′ were generated using AMA_C, AMA_P1 and AMA_P1-P1′ respectively. A 1071 bp 5′ targeting region of the P. falciparum clone 3D7 was maintained and synchronized as described previously pfama1 locus.-1 R1 peptide, with or without the addition of 0.2 mg ml-1 mAb 4G2 or Protein G-purified IgG from the rabbit anti-PfAMA1 antiserum Rb1, described previously For invasion assays, schizonts were enriched by centrifugation over cushions of 70% Percoll , added to fresh erythrocytes to obtained a parasitaemia of ∼4% and 1% haematocrit, and cultured overnight in the presence or absence of 100 µg mlXbaI  or NdeI (for lines transfected with intAMA_R-TKmod and intAMA_C-TKmod), electrophoresed on a 0.7% agarose gel, transferred to Hybond N+ nylon membrane  using standard procedures, and probed with [32P]-labelled DNA fragments, produced using the Prime-It II random primer labelling kit (Stratagene). The 3′ region of the endogenous pfama1 gene , digested to completion with −1 DAPI in PBS, then washed in PBS. Samples were mounted in Citifluor , and images collected using AxioVision 3.1 software on an Axioplan 2 Imaging system (Zeiss) using a Plan-APOCHROMAT 1006/1.4 oil immersion objective.Rabbit antibodies raised to correctly-folded recombinant FVO PfAMA1, and the polyclonal mouse anti-PfAMA1 antibody R5 have been described previously PfAMA1 fragments shed into culture supernatants were affinity-purified on a column of immobilised mAb 4G2 as previously described Text S1Supplemental Figures S1 to S8, and Table S1.(PDF)Click here for additional data file.


Proinflammatory cytokine tumor necrosis factor-alpha (TNF-α) is a crucial effector of immune responses in the brain that participates in the pathogenesis of several acute and chronic neurodegenerative disorders. Accumulating evidence has suggested that TNF-α negatively regulates embryonic and adult neurogenesis. However, the effect of TNF-α on cell fate decision in human neural progenitor cells (NPCs) has rarely been studied. Our previous studies have shown that recombinant TNF-α enhances astrogliogenesis and inhibits neurogenesis of human NPCs through the STAT3  pathway. In the current study, we further elucidated the specific mechanism involved in TNF-α-induced astrogliogenesis. We found that TNF-α activated STAT3 at delayed time points (6 h and 24 h), whereas conditioned medium collected from TNF-α-treated NPCs induced an immediate STAT3 activation. These data suggest TNF-α plays an indirect role on STAT3 activation and the subsequent NPC differentiation. Further, we showed that TNF-α induced abundant amounts of the IL-6 family cytokines, including Leukemia inhibitory factor (LIF) and Interleukin 6 (IL-6), in human NPCs. TNF-α-induced STAT3 phosphorylation and astrogliogenesis were abrogated by the addition of neutralizing antibody for LIF, but not for IL-6, revealing a critical role of autocrine secretion of LIF in TNF-α-induced STAT3 activation and astrogliogenesis. This study generates important data elucidating the role of TNF-α in neurogenesis and may provide insight into new therapeutic strategies for brain inflammation. Neural stem/progenitor cells (NSPCs) are self-renewing, multipotent cells that are capable of differentiating into neurons, astrocytes, and oligodendrocytes et al. have demonstrated that TNF-α reduced the number of NPCs adopting a neuronal phenotype under differentiating conditions Among these proinflammatory mediators, tumor necrosis factor (TNF)-α is an important effector of immune response in the brain and is involved in the pathogenesis of several acute and chronic neurodegenerative disorders, such as Parkinson's disease, Multiple Sclerosis, and stroke In the current study, we used human cortical NPC cultures to investigate the mechanism involved in TNF-α-mediated NPC differentiation. We found that TNF-α-induced STAT3 activation and subsequent astrogliogenesis is dependent upon the autocrine secretion of LIF.Human fetal brain tissue (12–16 weeks post-conception) was obtained from elective abortions carried out by the University of Washington in full compliance with the University of Washington, the University of Nebraska Medical Center, and the National Institutes of Health (NIH) ethical guidelines, with human subjects Institutional Review Board (IRB) approval no. 96-1826-A07 (University of Washington) and no. 123-02-FB . A written informed consent is obtained by the University of Washington using an IRB approved consent form. Human cortical NPCs were isolated as previously described To collect conditioned medium, dissociated NPCs were plated on poly-D-lysine-coated cell culture dishes in NPIM for 24 h. Cells were rinsed with fresh X-Vivo 15 and then treated with TNF-α (20 ng/ml) in X-Vivo 15 for 24 h. The NPC conditioned medium (NCM) was then harvested, cleared of free-floating cells by centrifugation for 5 min at 1200 rpm, and stored at −80°C.To block the soluble factors in NCM, it was pre-incubated with neutralizing antibodies for LIF  or IL-6  for 1 h at 37°C. Cells were then treated with NCM with or without neutralizing antibodies for 30 min. Whole-cell protein lysates were collected for Western blot or cells were fixed for immunocytochemical analysis.Neuronal differentiation of NPCs was performed as previously described Cells were fixed in 4% PFA and washed in PBS as previously described http://rsb.info.nih.gov/nih-image/).Cells were rinsed twice with PBS and lysed by M-PER Protein Extraction Buffer  containing 1× protease inhibitor cocktail . Protein concentration was determined using a BCA Protein Assay Kit (Pierce). Proteins (20–30 µg) were separated on a 10% SDS-polyacrylamide gel electrophoresis (PAGE) and then transferred to an Immuno-Blot polyvinylidene fluoride (PVDF) membrane . After blocking in PBS/Tween (0.1%) with 5% nonfat milk, the membrane was incubated with primary antibodies  overnight at 4°C followed by horseradish peroxidase-conjugated secondary antibodies  and then developed using Enhanced Chemiluminescent (ECL) solution (Pierce). For data quantification the films were scanned with a CanonScan 9950F scanner and the acquired images were then analyzed on a Macintosh computer using the public domain NIH image program  and RNeasy Kit  according to the manufacture's protocol. Primers used for real-time reverse-transcription polymerase chain reaction  include IL-6, LIF, Ciliary neurotrophic factor (CNTF) and Glyceraldehyde 3-phosphate dehydrogenase . Real-time RT-PCR was carried out using the one-step quantitative TaqMan assay in a StepOne™ Real-Time PCR system (Applied Biosystems Inc.). Relative IL-6, LIF, and CNTF mRNA levels were determined and standardized with a GAPDH internal control using comparative ΔΔCT method. All primers used in the study were tested for amplification efficiencies and the results were similar.Supernatants were collected for IL-6 and LIF determination by an in house ELISA. Briefly, 96-well micro titer plates (Costar) were coated overnight at room temperature with capture antibodies (R&D Systems) in PBS. Non-specific binding was blocked for 2 h with 1% BSA in PBS. Triplicate samples of cell supernatants or a serial dilution of standards of human recombinant IL-6 or LIF were applied to the wells and incubated overnight at 4°C. Samples were removed and wells were incubated with the biotinylated detection antibodies, followed by 1 h incubation with HRP-conjugated streptavidin (R&D Systems). TMB Substrate Solution (Sigma) was added and the absorbance was determined using a microplate reader  set at 450 nm.p values≤0.05 are regarded as statistically significant.Data were presented as means ± standard deviation (SD) unless otherwise noted. All experiments were repeated at least three times with different donors with triplicate or quadruplicate samples in each assay. All data were evaluated statistically by the analysis of variance (ANOVA), followed by Newman-Keuls multiple comparison tests using software . In the case of single mean comparison, data were analyzed by t test. Previous work in our laboratory has demonstrated that TNF-α increases astrocytic differentiation and inhibits neuronal differentiation of human NPCs. Furthermore, TNF-α induces astrogliogenesis through STAT3 signaling, since siRNA specifically targeting STAT3 (siSTAT3) inhibited TNF-α-induced astrogliogenesis The delayed STAT3 activation by TNF-α indicates that TNF-α may play an indirect role on STAT3 activation: secreted factors produced by TNF-α-treated NPCs activated the STAT3 pathway at later time points (6 h and 24 h). To test this hypothesis, human NPCs were treated with TNF-α for 30 min, 6 h and 24 h, and supernatants were collected as conditioned medium (CM). Parallel cultured NPCs were then treated with these different time point conditioned media (TNF-α-treated (TNF-α-CM) or control NPC-CM (Con-CM)) for 30 min and cell lysates were collected for Western blot. TNF-α-CM collected at 30 min did not induce a significant increase of STAT3 phosphorylation. In contrast, TNF-α-CM collected at 6 h moderately increased STAT3 phosphorylation; and TNF-α-CM collected at 24 h showed a significant increase of STAT3 phosphorylation as compared with Con-CM treatment . This reWe next studied the kinetics of CM-mediated STAT3 phosphorylation in NPCs. To exclude the effect of residual TNF-α in CM, human NPCs were treated with TNF-α for 6 h, rinsed twice with X-Vivo 15 and then maintained in fresh X-Vivo 15 medium. Twenty-four hours later, the TNF-α-free cell supernatants were collected as TNF-α-free-CM. TNF-α-free-CM treatment induced an immediate STAT3 phosphorylation at 30 min, but not at 6 h or 24 h . This reTo further characterize TNF-α-induced STAT3 activation in NPCs, we performed immunocytochemical studies with NPC culture using antibodies against phospho-STAT3 and nestin, a neural progenitor cell marker. Consistent with the Western blot result, TNF-α did not increase STAT3 phosphorylation or nucleus translocation at the early time point (30 min). However, at 24 h following TNF-α treatment, we observed apparent STAT3 phosphorylation and nucleus translocation . In addiMembers of the IL-6 cytokine family such as LIF, IL-6 and ciliary neurotrophic factor (CNTF) have been reported to activate the Jak-STAT signaling pathway and promote astroglial differentiation through the gp130-mediated signaling pathway To confirm that LIF is produced by human NPCs, we further assess the protein levels of LIF expression by immunocytochemistry. Human NPCs were treated with TNF-α (20 ng/ml) for 14 h. As shown in Because IL-6 and LIF were identified as the cytokines up-regulated by TNF-α stimulation in NPCs, we next studied their possible involvement in TNF-α-induced STAT3 activation and NPC differentiation. NPCs were pre-treated with neutralizing antibodies for LIF or IL-6 and then treated with TNF-α for 24 h. LIF neutralizing antibody, but not IL-6 neutralizing antibody, significantly inhibited TNF-α-induced STAT3 phosphorylation . NotablyTo test whether the presence of LIF and/or IL-6 was responsible for TNF-α-CM-induced STAT3 activation, we pre-incubated CM with neutralizing antibodies for LIF or IL-6 for 1 h and then treated NPCs with CM for 30 minutes. LIF neutralizing antibody, not IL-6 neutralizing antibody, significantly attenuated TNF-α-CM-induced STAT3 phosphorylation . Taken tTo test if LIF also contributes to TNF-α-induced astrogliogenesis, we differentiated NPCs with TNF-α in NB27 differentiation medium with or without LIF neutralizing antibody for 6 days. TNF-α significantly increased GFAP expression, a marker for astrocytes  and inhiTo further evaluate the effect of LIF neutralizing antibody on TNF-α-induced astrogliogenesis, we used immunocytochemistry to visualize the change of GFAP-positive cells . TNF-α tin situ during brain injury. The lack of significant neurogenesis in damaged brain areas may be due to the absence of molecules necessary for neuronal differentiation and/or the presence of molecules that favor the differentiation of NPCs toward other phenotypes. In pathological conditions of the CNS that are associated with neuroinflammation, activated resident immune cells (microglia and perivascular macrophages) produce a large number of proinflammatory cytokines and chemokines that affect the capacity of brain stem cells and alter neurogenesis Neural precursor cells, despite being multipotent, differentiate into astrocytes rather than neurons et al. also reported that TNF-α inhibited neuronal differentiation and increased astrocytic differentiation of hippocampal NPCs through increased expression of Hes1 Cell-fate determination during the differentiation of neural stem cells into specific neuronal and glial cell lineages is a highly orchestrated process. TNF-α has been shown to exert critical functions in survival, proliferation, and neuronal differentiation of NPCs, though the specific mechanisms through which TNF-α mediates these processes are not fully resolved due to conflicting results in the published literature The Jak-STAT3 signaling is a critical component of the astrogliogenic machinery during brain development Members of the IL-6 cytokine family such as, LIF, IL-6 and CNTF, are able to activate the Jak-STAT signaling pathway and promote astroglial differentiation LIF signals through the heterodimeric complex of common glycoprotein 130 (gp130) and LIF receptor (LIFR) subunits. A number of studies have shown that gp130/LIFR-mediated signaling has pleiotropic action on different cell types. LIF is well known for promoting mouse embryonic stem (ES) cell self-renewal In this study, we also observed the up-regulated expression of IL-6 in NPC. However, neutralizing antibody for IL-6 failed to block TNF-α-induced activation of STAT3, suggesting IL-6 may not be the main contributor for TNF-α-induced STAT3 activation and astrogliogenesis. This may be due to the lower expression of IL-6 receptor on NPCs .In conclusion, we have demonstrated that TNF-α activates STAT3 and promotes astrogliogenesis through the autocrine secretion of LIF. This study provides a novel mechanism by which pro-inflammatory cytokines affect neurogenesis and regulate the fate of NPCs. This information is important for developing transplantation therapies or promoting activation of endogenous NPCs for repair in neurodegenerative diseases with brain inflammation.


The aim of this study was to investigate the effect of three years of tumor necrosis factor-alpha (TNF-α) blocking therapy on bone turnover as well as to analyze the predictive value of early changes in bone turnover markers (BTM) for treatment discontinuation in patients with ankylosing spondylitis (AS).This is a prospective cohort study of 111 consecutive AS outpatients who started TNF-α blocking therapy. Clinical assessments and BTM were assessed at baseline, three and six months, as well as at one, two, and three years. Z-scores of BTM were calculated to correct for age and gender. Bone mineral density (BMD) was assessed yearly.After three years, 72 patients (65%) were still using their first TNF-α blocking agent. In these patients, TNF-α blocking therapy resulted in significantly increased bone-specific alkaline phosphatase, a marker of bone formation; decreased serum collagen-telopeptide (sCTX), a marker of bone resorption; and increased lumbar spine and hip BMD compared to baseline. Baseline to three months decrease in sCTX Z-score , AS disease activity score , and physician's global disease activity  were independent inversely related predictors of time to treatment discontinuation because of inefficacy or intolerance. Early decrease in sCTX Z-score correlated significantly with good long-term response regarding disease activity, physical function and quality of life.Three years of TNF-α blocking therapy results in a bone turnover balance that favors bone formation, especially mineralization, in combination with continuous improvement of lumbar spine BMD. Early change in sCTX can serve as an objective measure in the evaluation of TNF-α blocking therapy in AS, in addition to the currently used more subjective measures. Ankylosing spondylitis (AS) is a chronic inflammatory disease that mainly affects the axial skeleton. Bone formation and bone loss are both present in AS. New bone formation can lead to the formation of syndesmophytes, ankylosis of the spine and sacroiliac joints, and bone formations on enthesal sites ,2, whereRandomized controlled trials (RCTs) have shown that the tumor necrosis factor-alpha (TNF-α) blocking agents infliximab, etanercept and adalimumab are effective in controlling inflammation and improving clinical assessments in AS -8. PreviAlthough the majority of patients responds very well, a significant proportion of patients has to withdraw from TNF-α blocking therapy due to inefficacy or adverse events -14. Curret al. showed that an early increase in BALP was associated with significant increases in bone mineral density (BMD) of the spine and hip after two years of TNF-α blocking therapy [The early effect of TNF-α blocking therapy on bone turnover may be helpful in predicting treatment outcome. Bone turnover can be monitored using bone turnover markers (BTM) . The bon therapy .The first aim of the present study was to investigate the effect of three years of TNF-α blocking therapy on bone turnover. The second aim was to investigate whether the early effect of TNF-α blocking therapy on BTM can serve as an objective predictor of treatment discontinuation in patients with AS.n = 28) and the Medical Center Leeuwarden , were included in this longitudinal analysis. All patients participated in the Groningen Leeuwarden Ankylosing Spondylitis (GLAS) study, a prospective longitudinal observational cohort study with follow-up visits according to a fixed protocol. For the present analysis, patients with recent fractures and/or use of bisphosphonates were excluded because of major influence on bone metabolism. All patients were over 18 years of age, fulfilled the modified New York criteria for AS (n = 109) [n = 2) [n = 22), etanercept (n = 71), or adalimumab (n = 18) because of active disease (BASDAI ≥ 4 and/or expert opinion), according to the ASAS consensus statement [Between November 2004 and December 2007, 111 consecutive Dutch outpatients with AS, who started TNF-α blocking therapy at the University Medical Center Groningen   or the A [n = 2) . The pattatement . As desctatement , inflixiCRP  [Patients were evaluated at baseline and after three months , six months , one year , two years , and three years  of TNF-α blocking therapy. Disease activity was assessed using the BASDAI , physician's and patient's global assessment of disease activity , erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and ASDASand CRP) ,16. IncrBone turnover was studied by assessment of the bone formation markers bone-specific alkaline phosphatase (BALP) and procollagen type 1 N-terminal peptide (PINP), and the bone resorption marker serum type I collagen C-telopeptide (sCTX) . BALP waZ-scores of BTM were used to correct for the normal influence that age and gender have on bone turnover. Z-scores, the number of standard deviations (SD) from the normal mean corrected for age and gender, were calculated using matched 10-year-cohorts of a Dutch reference group (200 men or 350 women) checked for serum 25-hydroxyvitamin D levels > 50 nmol/liter as well as for the absence of osteoporosis (BMD T-score > -2.5) after 50 years of age. Z-scores were calculated as follows: /SD of matched reference cohort.BMD of the lumbar spine (anterior-posterior projection at L1 to L4) and hip  was assessed at baseline and after one year , two years , and three years  of TNF-α blocking therapy. BMD was measured using DXA (Hologic QDR Discovery (UMCG) or Hologic QDR Delphi (MCL), Waltham, MA, USA). T-scores, the number of SD from the normal mean obtained from young healthy adults, and Z-scores, the number of SD from the normal mean corrected for age and gender, were calculated using the NHANES reference database. According to the World Health Organization (WHO) classification, osteopenia was defined as a T-score between -1 and -2.5 and osteoporosis as a T-score ≤ -2.5 .P-value ≤ 0.3 in univariate Cox regression. The probability of F for entry was 0.05. Receiver Operating Characteristic (ROC) analysis was performed to determine the accuracy of early change in BTM to predict discontinuation of TNF-α blocking therapy during the first three years. Area under the curve (AUC) < 0.70 was interpreted as poor accuracy, 0.70 < AUC < 0.90 as moderate accuracy, and AUC > 0.90 as high accuracy [P-values < 0.05 were considered statistically significant.Statistical analysis was performed with PASW Statistics 18 . Results were expressed as mean ± SD or median (range) for normally distributed and non-normally distributed data, respectively. Predictor analysis of time to discontinuation of TNF-α blocking therapy (yes/no) was performed using forward conditional Cox regression of variables with a accuracy . PearsonThe mean age of the 111 AS patients was 42.2 years (SD ± 10.3), 70% were male, and median disease duration was 16 years (range 1 to 49). All baseline characteristics are shown in Table After three years, 72 patients (65%) continued to use their first TNF-α blocking agent. In these patients, all assessments of disease activity , adverse events ; infection (n = 3); allergic reaction (n = 2); cardio-vascular disease (n = 1); demyelization problems (n = 1)), both inefficacy and adverse events ; infection (n = 1); allergic reaction (n = 1); uveitis (n = 1)), or other reasons ; pregnancy wish (n = 2); lost to follow-up (n = 1)).The remaining 39 patients (35%) discontinued treatment after a median follow-up of 7.0 months (range 1.1 to 36.2). Reasons for discontinuation of TNF-α blocking therapy were inefficacy (P < 0.001) and BALP Z-score continued at a higher level up to three years. The bone formation marker PINP Z-score was found to be significantly increased only after three and 24 months of TNF-α blocking therapy (P < 0.05). The bone resorption marker sCTX Z-score decreased significantly after three months (P < 0.001) and remained decreased during three years of treatment . Subsequently, the lumbar spine BMD Z-score increased further after two and three years . The hip BMD Z-score tended to increase further after two years (P = 0.050), but remained stable after two to three years of treatment (P = 0.780)  were excluded from this analysis. Baseline to three months decrease in BASDAI, ASDASCRP, patient's GDA, physician's GDA and sCTX Z-score were inversely associated with time to discontinuation of TNF-α blocking therapy in univariate Cox regression. Multivariate analysis showed that baseline to three months decrease in sCTX Z-score , ASDASCRP , and physician's GDA  were independent inversely related predictors of time to discontinuation of TNF-α blocking therapy  in BTM for treatment discontinuation; 72 patients who continued versus 33 patients who discontinued their first TNF-α blocking agent because of inefficacy and/or adverse events. Patients who discontinued treatment due to other reasons , BASDAI , and physician's GDA  were selected during multivariate analysis.CRP and physician's GDA  were identified as independent inversely related predictors of time to treatment discontinuation.Since the number of female patients was relatively small, multivariate analysis using absolute BTM values was performed only in male patients. Baseline to three months decrease in sCTX , BASDAI  or alternatively, ASDASCRP  or in physician's GDA .The accuracy of baseline to three months change in sCTX Z-score to discriminate between patients who continued and discontinued TNF-α blocking therapy during the first three years was moderate, with an AUC of 0.741 (95% CI: 0.640 to 0.841), and comparable to the accuracy of early change in ASDASCRP, physician's and patient's GDA, ESR, and CRP), physical function (BASFI), spinal mobility (chest expansion), and quality of life (ASQoL) at last follow-up (defined as at three years of TNF-α blocking therapy or at the moment of treatment discontinuation)  Table .This is the first study that investigates the predictive value of early changes in bone turnover with regard to discontinuation of TNF-α blocking therapy in AS. Currently, in clinical practice, continuation of TNF-α blocking therapy is mainly based on subjective measures, such as the BASDAI and the global opinion of the patient and the physician. Recent studies showed the usefulness of the ASDAS as a more objective measure of disease activity -17,21. HUntil now, several studies investigated the influence of TNF-α blocking therapy on bone formation and bone resorption up to one year of treatment -23. The et al. showed no significant changes in BALP or sCTX during 24 weeks of placebo treatment [The changes in BTM over time found in this study cannot be specifically attributed to TNF-α blocking therapy because a placebo group is lacking. Visvanathan reatment , which iImportantly, the present results regarding BTM should not be extrapolated to any possible effect of TNF-α blocking therapy on radiographic progression in AS since no imaging method was used to measure new bone formation resulting in the formation of syndesmophytes and joint ankylosis. Furthermore, long-term observation is needed in order to see any effect of TNF-α blocking therapy on new bone formation in patients with AS.Interestingly, both lumbar spine and hip BMD improved significantly during three years of TNF-α blocking therapy, which can be explained by the increase in mineralization and decrease in bone resorption. Alternatively, the increase in lumbar spine BMD may in part be confounded by the progression of formation of ligament calcifications and fusion of facet joints ,29,30. HThis prospective longitudinal observational cohort study shows that three years of TNF-α blocking therapy results in a bone turnover balance that favors bone formation , in combination with continuous improvement of lumbar spine BMD. Furthermore, a baseline to three months' decrease in sCTX Z-score is identified as a significant inversely related predictor of time to treatment discontinuation, independent from ASDAS and physician's GDA. Based on these results, early change in the bone resorption marker sCTX seems useful as a purely objective biomarker in the evaluation of TNF-α blocking therapy in AS, in addition to the currently used more subjective measures.AS: ankylosing spondylitis; ASAS: assessments in ankylosing spondylitis; ASDAS: ankylosing spondylitis disease activity score; ASQoL: ankylosing spondylitis quality of life; AUC: area under the curve; BALP: bone-specific alkaline phosphatase; BASDAI: Bath ankylosing spondylitis disease activity index; BASFI: Bath ankylosing spondylitis functional index; BMD: bone mineral density; BTM: bone turnover markers; CI: confidence interval; CRP: C-reactive protein; ECLIA: electrochemiluminescence immunoassay; ELISA: enzyme-linked immunosorbent assay; ESR: erythrocyte sedimentation rate; GDA: global disease activity; GEE: generalized estimating equations; GLAS: Groningen Leeuwarden Ankylosing Spondylitis; HR: hazard ratio; IBD: inflammatory bowel disease; IE-CV: inter-assay coefficient of variation; MCL: Medical Center Leeuwarden; MRI: magnetic resonance imaging; OC: osteocalcin; PINP: procollagen type 1 N-terminal peptide; RCT: randomized controlled trial; RIA: radioimmunoassay; ROC: receiver operating characteristic; sCTX: serum type I collagen C-telopeptide; SD: standard deviation; sNTX: serum type I collagen N-telopeptide; TNF-α: tumor necrosis factor alpha; UMCG: University Medical Center Groningen; WHO: World Health OrganizationAS and PH have received unrestricted research grants from Wyeth. EB has received unrestricted research grants from Abbott, Schering-Plough, and Wyeth. The other authors declare that they have no competing interests.SA participated in the design of the study, performed the statistical analysis and interpretation of data, and drafted the manuscript. AS and EB participated in the design of the study, performed the acquisition of data, and critically revised the manuscript. PH, ML and RB contributed to the acquisition of clinical data and critically revised the manuscript. CK participated in the design of the study and critically revised the manuscript. HG contributed to the statistical analysis and interpretation of data and critically revised the manuscript. EV participated in the design of the study, contributed to the statistical analysis and interpretation of data and critically revised the manuscript. All authors approved the final manuscript.