Use Of Synthetic Peptides Research Articles

Fluorimetric sandwich affinity assay for Staphylococcus aureus based on dual-peptide recognition on magnetic nanoparticles

A novel dual-peptide recognition strategy was designed for sandwich fluorimetric detection of Staphylococcus aureus (S. aureus) utilizing the affinity of bacterial cells for certain peptides. A phage-display peptide derived from a random peptide library was used to functionalize magnetic particles and to specifically capture S. aureus. Magainin I is a broad-spectrum antimicrobial peptide that binds to cell membranes of most bacteria. It was used as a second recognition peptide to form a sandwich complex with S. aureus. By using fluorescein isothiocyanate as the fluorescent label and working at excitation/emission wavelengths of 488/525 nm, S. aureus can be directly detected in the 10 to 10,000 cfu·mL−1 concentration range, with a detection limit as low as 9 cfu·mL−1. The whole detection process can be completed within 90 min. This strategy was successfully applied to the detection of S. aureus in spiked lake water, human urine and apple juice. Respective recovery values ranged from 81% to 110%. The assay is highly sensitive and specific, and fast. The use of synthetic peptides shows many advantages such as lower cost, higher stability and ease of chemical modification compared to other molecular recognition agents such as antibodies, bacteriophages, or aptamers. In our perception, this detection scheme may be extended to many other pathogens if phage-displayed peptides specific for other bacteria are available.

Neurotoxicity of Prion Peptides on Cultured Cerebellar Neurons.

Prion neurotoxicity has been modeled in vitro using synthetic peptides derived from the PrPC sequence. The major region of neurotoxicity has been localized to the hydrophobic domain located in the middle of the PrP protein. Neurotoxicity assays are typically performed on cultured mouse cerebellar neurons derived from neonatal pups, and cell viability can be monitored by assays including MTT or MTS, cell death by LDH release, or apoptosis by caspase cleavage assays. These neurotoxicity studies have been useful in identifying cofactors, such as PrPC and metals, as modulators of PrP peptide-mediated neurotoxicity. Given the biosafety issues associated with handling and purifying infectious prions, the use of synthetic peptides, which display a dependence upon PrPC expression for toxicity, as per the PrPSc agent for infectivity, supports the relevance of using these synthetic peptides for understanding PrP-mediated neurotoxicity.

Chemical synthesis of transmembrane peptide and its application for research on the transmembrane-juxtamembrane region of membrane protein.

Membrane proteins possess one or more hydrophobic regions that span the membrane and interact with the lipids that constitute the membrane. The interactions between the transmembrane (TM) region and lipids affect the structure and function of these membrane proteins. Molecular characterization of synthetic TM peptides in lipid bilayers helps to understand how the TM region participates in the formation of the structure and in the function of membrane proteins. The use of synthetic peptides enables site-specific labeling and modification and allows for designing of an artificial TM sequence. Research involving such samples has resulted in significant increase in the knowledge of the mechanisms that govern membrane biology. In this review, the chemical synthesis of TM peptides has been discussed. The preparation of synthetic TM peptides is still not trivial; however, the accumulated knowledge summarized here should provide a basis for preparing samples for spectroscopic analyses. The application of synthetic TM peptides for gaining insights into the mechanism of signal transduction by receptor tyrosine kinase (RTK) has also been discussed. RTK is a single TM protein and is one of the difficult targets in structural biology as crystallization of the full-length receptor has not been successful. This review describes the structural characterization of the synthetic TM-juxtamembrane sequence and proposes a possible scheme for the structural changes in this region for the activation of ErbBs, the epidermal growth factor receptor family. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 613-621, 2016.

Laminin-111 Peptides Conjugated to Fibrin Hydrogels Promote Formation of Lumen Containing Parotid Gland Cell Clusters.

Previous studies showed that mouse submandibular gland cells form three-dimensional structures when grown on Laminin-111 gels. The use of Laminin-111 for tissue bioengineering is complicated due to its lack of purity. By contrast, the use of synthetic peptides derived from Laminin-111 is beneficial due to their high purity and easy manipulation. Two Laminin-111 peptides have been identified for salivary cells: the A99 peptide corresponding to the α1 chain from Laminin-111 and the YIGSR peptide corresponding to the β1 chain from Laminin-111, which are important for cell adhesion and migration. We created three-dimensional salivary cell clusters using a modified fibrin hydrogel matrix containing immobilized Laminin-111 peptides. Results indicate that the YIGSR peptide improved morphology and lumen formation in rat parotid Par-C10 cells as compared to cells grown on unmodified fibrin hydrogel. Moreover, a combination of both peptides not only allowed the formation of functional three-dimensional salivary cell clusters but also increased attachment and number of cell clusters. In summary, we demonstrated that fibrin hydrogel decorated with Laminin-111 peptides supports attachment and differentiation of salivary gland cell clusters with mature lumens.

Identification of protective B-cell epitopes of Atroxlysin-I: A metalloproteinase from Bothrops atrox snake venom

Atroxlysin-I (Atr-I) is a hemorrhagic snake venom metalloproteinase (SVMP) from Bothrops atrox venom, the snake responsible for the majority of bites in the north region of South America. SVMPs like Atr-I produce toxic effects in victims including hemorrhage, inflammation, necrosis and blood coagulation deficiency. Mapping of B-cell epitopes in SVMPs might result in the identification of non-toxic molecules capable of inducing neutralizing antibodies and improving the anti-venom therapy. Here, using the SPOT-synthesis technique we identified two epitopes located in the N-ter region of Atr-I (AtrEp1—22YNGNSDKIRRRIHQM36; and AtrEp2—55GVEIWSNKDLINVQ68). Based on the sequence of AtrEp1 and AtrEp2 a third peptide named Atr-I biepitope (AtrBiEp) was designed and synthesized (23NGNSDKIRRRIH34GG55GVEIWSNKDLINVQ68). AtrBiEp was used to immunize BALB/c mice. Anti-AtrBiEp serum cross-reacted against Atr-I in western blot and was able to fully neutralize the hemorrhagic activity of Atr-I. Our results provide a rational basis for the identification of neutralizing epitopes on Atr-I snake venom toxin and show that the use of synthetic peptides could improve the generation of immuno-therapeutics.

Immunogenicity of twenty peptides representing epitopes of the hepatitis B core and surface antigens by IFN-γ response in chronic and resolved HBV.

BackgroundPatients with chronic hepatitis B virus infection (CHB) usually mount a modest T cell response against HBV epitopes. In order to determine immunogenic epitopes of HBV recognized by HBV-specific T cells, previous studies focused on previously confirmed HBV epitopes and assessed the T cell response by the number of HBV-specific T cells by IFN-γ ELISPOT.MethodsWe studied T cell functionality by combined in silico methods predicting HBV-specific epitopes and experimental investigations on the recognition of these epitopes. 30 chronic CHB patients and 10 patients with resolved HBV (RHB) were included in the study. We identified epitopes from the literature and by in silico analysis. These were evaluated for immunogenicity by use of synthetic peptides representing the epitopes through exposure to PBMCs from patients with CHB or RHB by IFN-γ ELISPOT. The number of IFN-γ producing cells (SFC), mean spot size (MSS) and stimulation index (SI) were recorded.ResultsThe frequency of HBV-specific T cells producing IFN-γ after stimulation with HBV epitopes was similar in CHB and RHB patients. CHB patients had a higher MSS SI than RHB patients. Patients not carrying the HLA-A2 genotype had higher SFC SI and MSS SI. Patients with HLA-A11 had higher MSS SI compared to non- HLA-A11 allele patients. HBeAg-positive patients had a lower MSS SI, and none of the HBeAg positive patients had the HLA-A11 genotype. We found 3 immunogenic epitopes not described previously.ConclusionIFN-γ ELISPOT-determined MSS is an efficient marker for T cell recognition of epitopes. This experimental measure showed the in silico analysis for epitope prediction to be a valuable tool in future studies on HLA genotypes and HBV epitopes. This way our study now points to previously unappreciated consequences of carrying the HLA-A11 allele in terms of stronger immunity to HBV.

Trifluoroacetic acid as excipient destabilizes melittin causing the selective aggregation of melittin within the centrin-melittin-trifluoroacetic acid complex.

Trifluoroacetic acid (TFA) may be the cause of the bottleneck in high resolution structure determination for protein-peptide complexes. Fragment based drug design often involves the use of synthetic peptides which contain TFA (excipient). Our goal was to explore the effects of this excipient on a model complex: centrin-melittin-TFA. We performed Fourier transform infrared, two-dimensional infrared correlation spectroscopies and spectral simulations to analyze the amide I'/I'* band for the components and the ternary complex. Melittin (MLT) was observed to have increased helicity upon its interaction with centrin, followed by the thermally induced aggregation of MLT within the ternary complex in the TFA presence.

Peptide-based allergen specific immunotherapy for the treatment of allergic disorders.

Allergen specific immunotherapy (ASIT) and environmental control are the only etiologic treatments of allergic rhino-conjunctivitis, asthma and atopic dermatitis. The clinical benefit of ASIT relies on the selection of the patients and the identification and administration of the allergen, or allergens. Different routes of administration have been investigated, including subcutaneous, intradermal, epicutaneous, sublingual, inhaled, or intra-lymphatic. While subcutaneous and sublingual allergen specific immunotherapy may require from 3 to 5 years of treatment, clinical efficacy with intra-lymphatic treatment can be achieved after 3 injections. The most severe side effect of ASIT is anaphylaxis. Novel approaches are being investigated to reduce the allergenicity of immunotherapy vaccines, maintaining immunogenicity. Peptide immunotherapy has been directed mostly against autoimmune diseases, but the use of synthetic peptides for ASIT is a promising field in basic science, applied immunology and in clinical development. Short synthetic peptides bear allergen-specific CD4 T-cell epitopes which induce tolerance by stimulating regulatory (Treg) and Th1 cells. In the present patent review, we describe new trends in allergen immunotherapy using peptides, which, from a clinical point of view, are promising.

The use of citrullinated peptides for the diagnosis and prognosis of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune disease that causes joint inflammation and extra-articular manifestations. To prevent progressive and irreversible structural damage, early diagnosis of RA is of paramount importance. Antibodies directed against citrullinated proteins and peptides (ACPAs) are the most specific serological markers available for diagnosing RA. ACPAs may be detected several years before symptoms appear, and their presence at disease onset is a good predictor of the development of erosive joint lesions. Synthetic peptides can replace cognate proteins in solid-phase assays for specific autoantibody recognition in RA patients. The use of synthetic peptides instead of proteins represents an advantage in terms of the reproducibility of such immunoassays. They give absolute control over the exact epitopes presented. Furthermore, it is difficult to prepare sufficient amounts of high-quality antigenic proteins with a well-defined degree of citrullination. Synthetic citrullinated peptides, in contrast, are easily obtained in a pure form with a well-defined chemical structure and the epitopes can be precisely oriented in the plate by covalent binding of the peptides. We have recently obtained and highlighted the application of chimeric peptides bearing different citrullinated protein domains for the design of RA diagnosis systems. Our results indicate that more than one serological test is required to classify RA patients based on the presence or absence of ACPAs. Each of the target molecules reported (fibrin, vimentin and filaggrin) helps to identify a particular subset of RA patients.

Immunodiagnosis of canine visceral leishmaniasis using mimotope peptides selected from phage displayed combinatorial libraries.

ELISA and RIFI are currently used for serodiagnosis of canine visceral leishmaniasis (CVL). The accuracy of these tests is controversial in endemic areas where canine infections by Trypanosoma cruzi may occur. We evaluated the usefulness of synthetic peptides that were selected through phage display technique in the serodiagnosis of CVL. Peptides were chosen based on their ability to bind to IgGs purified from infected dogs pooled sera. We selected three phage clones that reacted only with those IgGs. Peptides were synthesized, polymerized with glutaraldehyde, and used as antigens in ELISA assays. Each individual peptide or a mix of them was reactive with infected dogs serum. The assay was highly sensitive and specific when compared to soluble Leishmania antigen that showed cross-reactivity with anti-T. cruzi IgGs. Our results demonstrate that phage display technique is useful for selection of peptides that may represent valuable synthetic antigens for an improved serodiagnosis of CVL.

Identification and characterization of B-cell epitopes of 3FTx and PLA2 toxins from Micrurus corallinus snake venom

The main goal of this work was to develop a strategy to identify B-cell epitopes on four different three finger toxins (3FTX) and one phospholipase A2 (PLA2) from Micrurus corallinus snake venom. 3FTx and PLA2 are highly abundant components in Elapidic venoms and are the major responsibles for the toxicity observed in envenomation by coral snakes. Overlapping peptides from the sequence of each toxin were prepared by SPOT method and three different anti-elapidic sera were used to map the epitopes. After immunogenicity analysis of the spot-reactive peptides by EPITOPIA, a computational method, nine sequences from the five toxins were chemically synthesized and antigenically and immunogenically characterized. All the peptides were used together as immunogens in rabbits, delivered with Freund's adjuvant for a first cycle of immunization and Montanide in the second. A good antibody response against individual synthetic peptides and M. corallinus venom was achieved. Anti-peptide IgGs were also cross-reactive against Micrurus frontalis and Micrurus lemniscatus crude venoms. In addition, anti-peptide IgGs inhibits the lethal and phospholipasic activities of M. corallinus crude venom. Our results provide a rational basis to the identification of neutralizing epitopes on coral snake toxins and show that their corresponding synthetic peptides could improve the generation of immuno-therapeutics. The use of synthetic peptide for immunization is a reasonable approach, since it enables poly-specificity, low risk of toxic effects and large scale production.

CD8+ T-cell recognition of a synthetic epitope formed by t-butyl modification.

We set out to clone Bax-specific CD8+ T cells from peripheral blood samples of patients with primary chronic lymphocytic leukaemia. A number of clones were generated using a Bax peptide pool and their T-cell epitope was mapped to two peptides sharing a common 9-amino-acid sequence (LLSYFGTPT), restricted by HLA-A*0201. However, when these T-cell clones were tested against highly purified syntheses (> 95%) of the same peptide sequence, there was no functional response. Subsequent mass spectrometric analysis and HPLC fractionation suggested that the active component in the original crude peptide preparations (77% pure) was a peptide with a tert-butyl (tBu) modification of the tyrosine residue. This was confirmed by modification of the inactive wild-type sequence to generate functionally active peptides. Computer modelling of peptide:HLA-A*0201 structures predicted that the tBu modification would not affect interactions between peptide residues and the HLA binding site. However, these models did predict that the tBu modification of tyrosine would result in an extension of the side chain out of the peptide-binding groove up towards the T-cell receptor. This modified product formed < 1% of the original P603 crude peptide preparation and < 0·05% of the original 23-peptide mixture used for T-cell stimulation. The data presented here, illustrate the potential for chemical modifications to change the immunogenicity of synthetic peptides, and highlight the exquisite capacity of T-cell receptors to discriminate between structurally similar peptide sequences. Furthermore, this study highlights potential pitfalls associated with the use of synthetic peptides for the monitoring and modulating of human immune responses.

Abstract 2896: Identification of novel tumor associated antigens in colorectal cancer

Abstract The prognosis of colorectal carcinoma (CRC) patients is dependent on the establishment of distant metastasis, which have been shown to arise from a small population of long term tumor initiating cells (LT-TIC). Another major prognostic parameter is the T cell infiltrate in the primary tumor of CRC patients, suggesting that tumor specific T cell responses occur and that the T cell mediated immune surveillance might play an important role in controlling tumor relapse, metastasis and response to treatment. Based on these observations we hypothesize that T cell responses against antigens expressed on TIC might trigger more efficient immune surveillance. So far little is known about the target antigens of the spontaneous T cell responses in CRC patients. In order to identify those antigens in a systematic and unbiased manner, we established a two-dimensional protein separation technique (PF2D). This technique allowed to fractionate lysates from primary and metastatic tumor tissue and to select the fractions which were recognized by the patient's T cell repertoire, as assessed by IFN-γ ELISpot assays. The reactive fractions were further characterized with mass spectrometry (MS) and candidate proteins were verified by use of synthetic peptides in ELISpot. As a result, we have identified 21 novel CRC-associated target antigens of spontaneous T cell responses. We have shown in a cohort of 20 patients that the newly identified target antigens not only triggered responses more frequently than “canonical” tumor antigens that are commonly used for immunotherapy (up to 50% of the tested patients), but the frequency of the T cells specific for the novel tumor associated antigens (TAA) was significantly higher. Furthermore, as shown by gene expression analysis some of the newly identified TAAs were selectively overexpressed on LT-TIC of CRC, and by means of PF2D of LT-TIC enriched spheroid cultures and subsequent MS of the immunogenic fractions, we have demonstrated that they represent target antigens of patients' endogenous T cell responses in all of the tested LT-TIC cultures. In addition, we have identified 30 amino acids long sequences of the these TAA, which elicit CD8+ or CD4+ T cell responses and we are currently working on identification of T cell epitopes and generation of T cell clones specific for these antigens. These would be further used to test the hypothesis that LT-TIC specific T cell clones have the capability to reduce the metastatic potential and tumorigenicity of TIC enriched spheroids in vitro and in vivo. In conclusion we have shown that novel set of antigens expressed in a therapeutically relevant subset of CRC cells, can be highly immunogenic and might serve as better targets for immune monitoring and could be utilized for more efficient T cell based immunotherapy. Citation Format: Slava Stamova, Christoph Schlude, Saskia Rösch, Christel Herold-Mende, Taronish D. Dubash, Claudia R. Ball, Hanno Glimm, Martin A. Schneider, Philipp Beckhove. Identification of novel tumor associated antigens in colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2896. doi:10.1158/1538-7445.AM2014-2896

EndoProteoFASP: A novel FASP approach to profile salivary peptidome and disclose salivary proteases

The salivary peptidome, which can represent up to 20% of total secreted proteins in human saliva, is highly influenced by proteolytic events. However, the development of strategies to understand the dynamics underlying the generation of salivary peptides has been a challenging task. In order to disclose in more detail the proteolytic events taking place in saliva, we aimed to characterize salivary peptidome and predict salivary proteases by applying, for the first time, a filter-aided sample preparation (FASP) approach to saliva. Thus, as a proof-of-concept of this application, harvested saliva samples from healthy individuals were incubated in 30kDa cut-off spin filters for 18 or 115h, at 37°C, to promote saliva autolysis and the attained peptidome was characterized and compared with the naturally occurring one. In ex vivo conditions, proline-rich proteins, P-B peptide, histatin 1 and statherin were found to be the most susceptible salivary proteins to proteolysis. Peptide fragments were mainly attributed to the activity of cathepsin L1 and K at 18h, whereas at 115h, the attained peptide fragments were attributed to the activity of cathepsins K and L1, and MEP1A. Overall, the described endoProteoFASP approach makes the most of saliva׳s own protease pool and avoids the use of synthetic peptides and exogenous proteases to understand the proteolytic events occurring in the oral fluid. Hence, it could be very helpful in future studies targeting the characterization of salivary proteases and peptidome from different pathophysiological conditions.

Updating the Use of Synthetic Peptides as Inhibitors of HIV-1 Entry

The use of synthetic peptides as HIV-1 inhibitors has been the object of research over recent years. A large number of peptides that affect different stages of the HIV-1 life cycle have been and continue to be studied due to their possible clinical application in the fight against HIV-1 infection. The main advantages of synthetic peptides as therapeutic agents are their low systemic toxicity, the fact that structural modifications can be made to them and their resulting capacity to mimic certain substrates or epitopes. HIV-1-inhibiting peptides have been identified and/or developed using different methods. Some therapeutic peptides such as enfuvirtide-already approved for clinical use-are derived from HIV-1 itself. Others are natural peptides such as chemokines, defensins or the "virus inhibitory peptide"; while still others have been designed and synthesized based on crystallographic data on HIV-1 proteins or from peptide libraries. Initial attempts at therapeutic applications focused on HIV-coded enzymes (reverse transcriptase, protease and, more recently, integrase). However, structural HIV proteins and, more specifically, the mechanisms that involve the virus in cell infection and replication are now also considered therapeutic targets. Several chemical strategies to improve both the stability of peptides and their pharmacokinetics, including prolonging their half-life, have recently been described in the literature. There is growing an interest in inhibitors that prevent HIV entry into the host cell (fusion inhibitors) which could lead to the development of new antiviral agents. Knowledge of the mechanism of action of fusion inhibitors is essential not only for the development of future generations of entry inhibitors, but also to gain an understanding of the form and kinetics of membrane fusion induced by the virus. The physico-chemical processes involved at the interface between the lipid surface of cells and enveloped viruses (such as HIV-1) are essential to the action of peptides that prevent HIV-1 entry into the host cell. The interaction of these peptides with biological membranes may be related to their inhibition efficiency and to their mechanism of action, as the HIV-1 gp41 glycoprotein is bound and confined between the cellular membrane and the viral envelope.

Utility of B-cell epitopes based peptides of RD1 and RD2 antigens for immunodiagnosis of pulmonary tuberculosis

Tuberculosis (TB) continues to be a major health problem due to lack of accurate, rapid, and cost-effective diagnostic tests. Serodiagnostic tests incorporating highly specific region of difference (RD) antigens (early secretory antigenic target 6 [ESAT-6], culture filtrate protein 10 [CFP-10], culture filtrate protein 21 [CFP-21], and mycobacterial protein from species tuberculosis 64 [MPT-64]) have recently been shown to be promising for specific diagnosis of TB in our lab. However, only few studies have reported the use of synthetic peptides of RD antigens, and none has used them to differentiate TB from sarcoidosis, a close mimic of smear-negative pulmonary TB (PTB) with entirely different management. The present study was conducted with an aim to study the utility of B-cell epitopes based peptides of RD1 (ESAT-6, CFP-10) and RD2 (CFP-21, MPT-64) antigens for immunodiagnosis of PTB for which sputum smear-positive PTB patients, sputum smear-negative PTB patients, sarcoidosis patients, and healthy controls (n = 24/group) were recruited. Bioinformatic software Bcepred was used to predict linear B-cell epitopes, using physico-chemical properties on a non-redundant dataset. Seven peptides as representative B-cell epitopes of ESAT-6, CFP-10, CFP-21, and MPT-64 were evaluated as targets of the antibody responses in TB patients and controls by enzyme-linked immunosorbent assay (ELISA). The current study showed sensitivity with individual peptides ranging from 37.5% to 83.3% for smear positive, 25% to 58.3% for smear negative as compared to 4.16% to 20.8% for sarcoidosis. Four out of 7 peptides that showed higher reactivity with TB patients and better discrimination from sarcoidosis patients representing ESAT-6, CFP-10, CFP-21, and MPT-64 were selected for multiepitope ELISA. The combination of peptides yielded 83.3% sensitivity for smear positive, 62.5% for smear negative, and only 4.16% for sarcoidosis. The specificity, however, for all the peptides/combination was 100%. Combination of peptides has proven to be better than individual peptides as per the latest criteria of the World Health Organization according to which a test that can replace smear microscopy with sensitivity of >90% for smear-positive patients and >65% for smear-negative TB patients with a specificity >95%, and thus, the present study suggests that a test based on combination of peptides selected from mycobacterial RD1 and RD2 antigens could be important for promoting an early diagnosis and management of otherwise difficult to diagnose smear-negative PTB patients. Moreover, it can also be used to discriminate sarcoidosis from PTB, thus preventing the misdiagnosis and mismanagement.

Synthetic peptides from heat-shock protein 65 inhibit proinflammatory cytokine secretion by peripheral blood mononuclear cells from rheumatoid arthritis patients.

1. Rheumatoid arthritis (RA) is a systemic autoimmune disease mediated by T cells. Proinflammatory cytokines plays a critical role in the pathogenesis of RA. The aim of the present study was to investigate the effects of synthetic peptides (HP-R1, HP-R2 and HP-R3), derived from the sequence of 65 kDa mycobacterial heat shock protein (HSP), on the proliferation of and cytokine secretion by peripheral blood mononuclear cells (PBMC) from RA patients. 2. The PBMC were obtained from RA patients and collected by Ficoll-Hypaque density centrifugation. Peripheral blood mononuclear cells were treated with one of the three synthetic peptides for 4 h, after which time proliferation and cytokine production were determined. The effects of the three peptides on the proliferation of PBMC were analysed by the colorimetric cell proliferation (CCK-8) assay. Cytokine production was measured in culture supernatants using specific ELISAs. 3. None of the three peptides had any significant effect on the proliferation of PBMC from healthy controls. However, the proliferation of PBMC from RA patients was inhibited by all three peptides. The production of tumour necrosis factor-α from RA patients was significantly inhibited by all three peptides. The secretion of interferon-γ was significantly suppressed by HP-R1 and HP-R2. Unlike the other two peptides, HP-R2 increased the secretion of interleukin (IL)-4. None of the peptides had any significant effect on the production of IL-10. 4. The results of the present study suggest that the synthetic peptides derived from HSP65 exhibit antiproliferative and anti-inflammatory activity, and support the potential use of synthetic peptides as therapeutic drugs in RA patients.

Citrullinated Peptides in the Diagnosis of Rheumatoid Arthritis

Antibodies directed against citrullinated proteins and peptides (ACPAs) are the most specific serological markers available for diagnosing rheumatoid arthritis (RA). ACPAs may be detected several years before symptoms of RA appear, and their presence at disease onset is a good predictor of the development of erosive joint lesions. RA patients can be classified into two major groups: those who have ACPAs and those who do not. The presence of ACPAs at early stages of RA predicts the development of earlier and more widespread joint erosions, and low remission rates.Synthetic peptides can replace cognate proteins in solid-phase assays for specific autoantibody recognition in RA patients. The use of synthetic peptides instead of proteins represents an advantage in terms of the reproducibility of such immunoassays. Proteins also contain non-citrullinated epitopes that are recognized by non-RA sera and this could reduce the specificity of the test. The use of synthetic citrullinated peptides gives absolute control over the exact epitopes presented. Furthermore, it is difficult to prepare sufficient amounts of high-quality antigenic proteins with a well-defined degree of citrullination. Synthetic citrullinated peptides, in contrast, are easily obtained in a pure form with a well-defined chemical structure and the epitopes can be precisely oriented in the plate by covalent binding of the peptides.Chimeric peptides bearing different citrullinated protein domains have recently been used in the design of RA diagnosis systems. The results of the application of those systems indicate that more than one serological test is required to classify RA patients based on the presence or absence of ACPAs. Each of the target molecules reported (fibrin, vimentin and filaggrin) helps to identify a particular subset of RA patients.

Clinical relevance of antibody development in renal transplantation

The detection and characterization of anti-HLA antibodies and the clinical impact of their appearance following renal transplantation are areas of immense interest. In particular, de novo development of donor-specific antibodies (DSA) has been associated with acute and chronic antibody-mediated graft rejection (AMR). Recently, methods for antibody detection have evolved remarkably from conventional cell-based assays to advanced solid phase systems. These systems have revolutionized the art of defining clinically relevant antibodies that are directed toward a renal graft. While anti-HLA DSAs have been widely associated with poor graft survival, the role of non-HLA antibodies, particularly those directed against endothelial cells, is beginning to be realized. Appreciation of the mechanisms underlying T cell recognition of alloantigens has generated great interest in the use of synthetic peptides to prevent graft rejection. Hopefully, continued progress in unraveling the molecular mechanisms of graft rejection and posttransplant monitoring of antibodies using highly sensitive testing systems will prove beneficial to immunological risk assessment and early prediction of renal allograft failure.

Characterization of a human toll-like receptor 3 antibody designed to block ligand binding and ligand mediated signaling (180.16)

Abstract Toll-like receptors (TLRs) play key roles during pathogen invasion to initiate innate immune responses. Toll-like receptor 3 (TLR3) recognizes double-stranded RNA (microbial and endogenous) and transmits signals to activate NF-kappa B. TLR3 is restricted to acidic pH+ intracellular compartments of innate immune cells (viz immature DC). Ligand binding and formation of multimeric signal complexes, to activate down-stream signaling, are facilitated by acidic pH in these compartments. Mutational and co-crystallization studies determined the interaction of TLR3 &amp; ligand binding at the molecular level and also individual residues important in ligand recognition and signal transduction. Besides, neutralizing antibodies were utilized as tools for better understanding of ligand binding and TLR3 signaling. In the present study, we report results of a TLR3 antibody which blocks ligand (poly IC) binding and its' effect on functional activity. We tested multiple TLR3 antibodies, generated through use of synthetic peptides designed to include various regions of protein and also the residues playing important role in ligand binding. The binding and blocking (ligand) activities were analyzed by flow cytometry using TLR3 transfectant cells in presence/absence of poly IC. We also tested blocking activity in a functional assay by measuring secreted alkaline phosphatase (SEAP) in cultures of TLR3-NF-kB-SEAP co-transfectants stimulated wih poly IC in the presence of antibody.