Non-contact induction heating and SAAP-148 eliminate persisters within MRSA biofilms mimicking a metal implant infection.

Implant-associated infections are the primary cause of complications following orthopaedic surgery. Due to biofilm and persister formation, current treatments, i.e. surgical debridement followed by antibiotics, often fail. There is an urgent need for alternative strategies to combat such infections. Therefore, the present study investigated the effects of non-contact induction heating (NCIH), the antimicrobial peptide SAAP-148 and combinations thereof on bacterial counts in 7 d mature biofilms and in persister-enriched biofilms of methicillin-resistant Staphylococcus aureus (MRSA) on titanium-aluminium-niobium (TAN) discs. Enrichment of persisters was achieved by daily exposure of mature biofilms to high doses of rifampicin and ciprofloxacin for 3 consecutive days. To heat up the TAN discs, a miniaturised induction heater was built and successfully validated. Using this apparatus, NCIH resulting in surface temperatures up to 85 °C eradicated all the bacteria in immature biofilms but not in mature biofilms, whereas persisters were already eliminated at surface temperatures ≥ 70 °C. SAAP-148 at concentrations > 25.6 µmol/L reduced the persister counts in antibiotics-exposed, mature biofilms. As surface temperatures > 60 °C can have detrimental effects on the surrounding tissues, the maximum temperature of NCIH used in combination with SAAP-148 on persisters was set to 60 °C. Results revealed that this combination was slightly more effective than the peptide or NCIH alone in eliminating biofilm-embedded persisters. NCIH and SAAP-148 can be applied both invasively and non-invasively in various treatment scenarios. Together, combinations of NCIH and SAAP-148 might be a promising treatment strategy to combat metal-implant-associated infections.

Antibodies and B cells recognising citrullinated proteins display a broad cross-reactivity towards other post-translational modifications.

OBJECTIVE: Autoantibodies against antigens carrying distinct post-translational modifications (PTMs), such as citrulline, homocitrulline or acetyllysine, are hallmarks of rheumatoid arthritis (RA). The relation between these anti-modified protein antibody (AMPA)-classes is poorly understood as is the ability of different PTM-antigens to activate B-cell receptors (BCRs) directed against citrullinated proteins (CP). Insights into the nature of PTMs able to activate such B cells are pivotal to understand the 'evolution' of the autoimmune response conceivable underlying the disease. Here, we investigated the cross-reactivity of monoclonal AMPA and the ability of different types of PTM-antigens to activate CP-reactive BCRs. METHODS: BCR sequences from B cells isolated using citrullinated or acetylated antigens were used to produce monoclonal antibodies (mAb) followed by a detailed analysis of their cross-reactivity towards PTM-antigens. Ramos B-cell transfectants expressing CP-reactive IgG BCRs were generated and their activation on stimulation with PTM-antigens investigated. RESULTS: Most mAbs were highly cross-reactive towards multiple PTMs, while no reactivity was observed to the unmodified controls. B cells carrying CP-reactive BCRs showed activation on stimulation with various types of PTM-antigens. CONCLUSIONS: Our study illustrates that AMPA exhibit a high cross-reactivity towards at least two PTMs indicating that their recognition pattern is not confined to one type of modification. Furthermore, our data show that CP-reactive B cells are not only activated by citrullinated, but also by carbamylated and/or acetylated antigens. These data are vital for the understanding of the breach of B-cell tolerance against PTM-antigens and the possible contribution of these antigens to RA-pathogenesis.

CD8 T cell autoreactivity to preproinsulin epitopes with very low human leucocyte antigen class I binding affinity.

Beta cells presenting islet epitopes are recognized and destroyed by autoreactive CD8 T cells in type 1 diabetes. These islet-specific T cells are believed to react with epitopes binding with high affinity to human leucocyte antigen (HLA) expressed on beta cells. However, this assumption might be flawed in case of islet autoimmunity. We evaluated T cell recognition of the complete array of preproinsulin (PPI) peptides with regard to HLA binding affinity and T cell recognition. In a comprehensive approach, 203 overlapping 9-10mer PPI peptides were tested for HLA-A2 binding and subjected to binding algorithms. Subsequently, a high-throughput assay was employed to detect PPI-specific T cells in patient blood, in which conditional HLA ligands were destabilized by ultraviolet irradiation and HLA molecules refolded with arrays of PPI peptides, followed by quantum-dot labelling and T cell staining. Analysis of patient blood revealed high frequencies of CD8 T cells recognizing very low HLA binding peptides. Of 28 peptides binding to HLA-A2, a majority was predicted not to bind. Unpredicted peptides bound mainly with low affinities. HLA binding affinity and immunogenicity may not correlate in autoimmunity. Algorithms used to predict high-affinity HLA peptide binders discount the majority of low-affinity HLA binding epitopes. Appreciation that peptides binding HLA with very low affinity can act as targets of autoreactive T cells may help to understand loss of tolerance and disease pathogenesis and possibly point to tissue-specific immune intervention targets.

Anti-citrullinated protein antibodies have a low avidity compared with antibodies against recall antigens.

OBJECTIVES: Anti-citrullinated protein antibodies (ACPA) are highly specific for rheumatoid arthritis (RA) and have been implicated in disease pathogenesis. Recent ongoing evidence indicates that the ACPA response broadens before precipitation of full-blown RA, as indicated by a more extensive isotype usage and an increased citrullinated epitope recognition profile. Nonetheless, the evolution of the ACPA response is still poorly understood and might intrinsically differ from the protective responses against pathogens. METHODS: The avidity and the avidity maturation of ACPA in relation to the avidity of antibodies against recall antigens were analysed. RESULTS: The avidity of ACPA was significantly lower than the avidity of antibodies to the recall antigens tetanus toxoid and diptheria toxoid. Moreover, ACPA did not show avidity maturation during longitudinal follow-up and ACPA avidity was also relatively low in patients who displayed extensive isotype switching. CONCLUSIONS: These observations indicate that the natural evolution of ACPA differs from the development of antibodies against recall antigens. These data also indicate that ACPA avidity maturation and isotype switching are disconnected, whereby extensive isotype switching occurs in the setting of restricted avidity maturation. Intrinsic differences between the RA-specific autoantibody system and protective antibody responses against pathogens could be of relevance for designing novel B cell-targeted therapies for RA.

Simultaneous humoral and cellular immune response against cancer-testis antigen NY-ESO-1: definition of human histocompatibility leukocyte antigen (HLA)-A2-binding peptide epitopes.

A growing number of human tumor antigens have been described that can be recognized by cytotoxic T lymphocytes (CTLs) in a major histocompatibility complex (MHC) class I-restricted fashion. Serological screening of cDNA expression libraries, SEREX, has recently been shown to provide another route for defining immunogenic human tumor antigens. The detection of antibody responses against known CTL-defined tumor antigens, e.g., MAGE-1 and tyrosinase, raised the question whether antibody and CTL responses against a defined tumor antigen can occur simultaneously in a single patient. In this paper, we report on a melanoma patient with a high-titer antibody response against the "cancer-testis" antigen NY-ESO-1. Concurrently, a strong MHC class I-restricted CTL reactivity against the autologous NY-ESO-1-positive tumor cell line was found. A stable CTL line (NW38-IVS-1) was established from this patient that reacted with autologous melanoma cells and with allogeneic human histocompatibility leukocyte antigen (HLA)-A2(-), NY-ESO-1-positive, but not NY-ESO-1-negative, melanoma cells. Screening of NY-ESO-1 transfectants with NW38-IVS-1 revealed NY-ESO-1 as the relevant CTL target presented by HLA-A2. Computer calculation identified 26 peptides with HLA-A2-binding motifs encoded by NY-ESO-1. Of these, three peptides were efficiently recognized by NW38-IVS-1. Thus, we show that antigen-specific humoral and cellular immune responses against human tumor antigens may occur simultaneously. In addition, our analysis provides a general strategy for identifying the CTL-recognizing peptides of tumor antigens initially defined by autologous antibody.

Identification of a graft versus host disease-associated human minor histocompatibility antigen.

Minor histocompatibility antigen disparities between human leukocyte antigen (HLA)-matched bone marrow donors and recipients are a major risk factor for graft versus host disease (GVHD). An HLA-A2.1-restricted cytotoxic T cell clone that recognized the minor histocompatibility antigen HA-2 was previously isolated from a patient with severe GVHD after HLA-identical bone marrow transplantation. The HLA-A2.1-bound peptide representing HA-2 has now been identified. This peptide appears to originate from a member of the non-filament-forming class I myosin family. Because HA-2 has a phenotype frequency of 95 percent in the HLA-A2.1-positive population, it is a candidate for immunotherapeutic intervention in bone marrow transplantation.

H1N1 hemagglutinin-specific HLA-DQ6-restricted CD4+ T cells can be readily detected in narcolepsy type 1 patients and healthy controls.

Following the 2009 H1N1 influenza pandemic, an increased risk of narcolepsy type 1 was observed. Homology between an H1N1 hemagglutinin and two hypocretin sequences has been reported. T cell reactivity to these peptides was assessed in 81 narcolepsy type 1 patients and 19 HLA-DQ6-matched healthy controls. HLA-DQ6-restricted H1N1 hemagglutinin-specific T cell responses were detected in 28.4% of patients and 15.8% of controls. Despite structural homology between HLA-DQ6-hypocretin and -H1N1 peptide complexes, T cell cross-reactivity was not detected. These results indicate that it is unlikely that cross-reactivity between H1N1 hemagglutinin and hypocretin peptides presented by HLA-DQ6 is involved in the development of narcolepsy.

Antigen arrays in T cell immunology.

The screening of compound arrays in in vitro bioassays has developed into a powerful tool for the identification of biologically active substances. In the past decade, this technology has increasingly been applied to immunology. As the specificity of the immune system is determined by antigen detection via receptors on B and T cells, targeting the specificity of these immune receptors with random arrays is unique in its ability to generate general and quantitative information on cellular (cross-)reactivity. Synthetic array studies have been useful for identification of epitopes and antigens from databases by defining recognition patterns, isolation of synthetic peptides capable of modulating T cell responsiveness, characterisation of TCR promiscuity, and identification of functionally cross-reacting peptides that are potentially involved in molecular mimicry.

Human clonal CD8 autoreactivity to an IGRP islet epitope shared between mice and men.

Type 1 diabetes (T1D) is a multifactorial disease characterized by the infiltration and subsequent destruction of the pancreatic insulin-producing beta cells by autoreactive T cells. CD8(+) T cells play an essential role in this beta cell destruction. However, little is known about the target antigens of CD8(+) T cells in human T1D patients. The aim of this study was to assess whether an epitope derived from the islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), IGRP(265-273,) which has recently been identified as a target in non-obese diabetic (NOD) mice and is fully homologous to the human epitope, is a target of human diabetogenic CD8(+) T cells. We isolated a human CD8 T cell clone against this epitope, which confirms that this IGRP epitope is shared across species.

BCR-ABL fusion regions as a source of multiple leukemia-specific CD8+ T-cell epitopes.

For immunotherapy of residual disease in patients with Philadelphia-positive leukemias, the BCR-ABL fusion regions are attractive disease-specific T-cell targets. We analyzed these regions for the prevalence of cytotoxic T lymphocyte (CTL) epitopes by an advanced reverse immunology procedure. Seventeen novel BCR-ABL fusion peptides were identified to bind efficiently to the human lymphocyte antigen (HLA)-A68, HLA-B51, HLA-B61 or HLA-Cw4 HLA class I molecules. Comprehensive enzymatic digestion analysis showed that 10 out of the 28 HLA class I binding fusion peptides were efficiently excised after their C-terminus by the proteasome, which is an essential requirement for efficient cell surface expression. Therefore, these peptides are prime vaccine candidates. The other peptides either completely lacked C-terminal liberation or were only inefficiently excised by the proteasome, rendering them inappropriate or less suitable for inclusion in a vaccine. CTL raised against the properly processed HLA-B61 epitope AEALQRPVA from the BCR-ABL e1a2 fusion region, expressed in acute lymphoblastic leukemia (ALL), specifically recognized ALL tumor cells, proving cell surface presentation of this epitope, its applicability for immunotherapy and underlining the accuracy of our epitope identification strategy. Our study provides a reliable basis for the selection of optimal peptides to be included in immunotherapeutic BCR-ABL vaccines against leukemia.

Immunization with human papillomavirus type 16 (HPV16) oncoprotein-loaded dendritic cells as well as protein in adjuvant induces MHC class I-restricted protection to HPV16-induced tumor cells.

Human papillomavirus (HPV) E6 and E7 oncoproteins are attractive targets for T-cell-based immunotherapy of cervical cancer. In this study, we demonstrate that dendritic cells (DCs) pulsed with HPV16 E7 protein are not only recognized in vitro by E7-specific CTLs but also elicit E7-specific CTL responses in vivo, associated with protection against a challenge with syngeneic HPV16-induced tumor cells. Vaccination with soluble E7 protein in incomplete Freund's adjuvant likewise induces E7-specific CTL responses associated with tumor protection. The presence of HPV16 E7-specific CTLs in vivo and the observation that depletion of CD8+ cells completely abolishes tumor protection demonstrate that CTLs are the major effector cells in mediating antitumor activity. The in vivo involvement of DCs in the activation of protective CTLs is suggested by the surface display of E7 peptide-loaded MHC class I molecules on these cells after E7 protein immunization. These data show that HPV16 E7 protein-pulsed DCs, as well as the administration of E7 protein antigen in adjuvant, can effectively stimulate tumor-specific MHC class I-restricted CD8+ T-cell-mediated protective immunity to HPV16-induced cancers.

Prevention of Staphylococcus aureus biomaterial-associated infections using a polymer-lipid coating containing the antimicrobial peptide OP-145.

The scarcity of current antibiotic-based strategies to prevent biomaterial-associated infections (BAI) and their risk of resistance development prompted us to develop a novel antimicrobial implant-coating to prevent Staphylococcus aureus-induced BAI. We incorporated the antimicrobial peptide OP-145 into a Polymer-Lipid Encapsulation MatriX (PLEX)-coating to obtain high peptide levels for prolonged periods at the implant-tissue interphase. We first confirmed that OP-145 was highly effective in killing S. aureus and inhibiting biofilm formation in vitro. OP-145 injected along S. aureus-inoculated implants in mice significantly reduced the number of culture-positive implants. OP-145 was released from the PLEX coating in a controlled zero-order kinetic rate after an initial 55%-burst release and displayed bactericidal activity in vitro. In a rabbit intramedullary nail-related infection model, 67% of rabbits with PLEX-OP-145-coated nails had culture-negative nails after 28days compared to 29% of rabbits with uncoated nails. In rabbits with PLEX-OP-145-coated nails, bone and soft tissue samples were culture-negative in 67% and 80%, respectively, whereas all bone samples and 71% of the soft tissue samples of rabbits with uncoated nails were infected. Together, PLEX-OP-145 coatings, of which both compounds have already been found safe in man, can prevent implant colonization and S. aureus-induced BAIs.

HLA-DR binding analysis of peptides from islet antigens in IDDM.

HLA molecules are essential for thymic education and HLA restriction of T-cell responses. We therefore analyzed the HLA-DR binding affinities of synthetic peptides covering the entire sequences of GAD65, islet cell antigen 69 (ICA69), and (pro)insulin, which are candidate antigens in the autoimmune process of T-cell-mediated destruction of the pancreatic beta-cells. Subsequently, peptide HLA-DR binding was correlated to peptide antigenicity by comparing known T-cell epitopes with their HLA-binding affinities defined in this study. The results demonstrate the following. 1) (Pro)insulin peptides display a strong binding affinity for HLA-DR2, which is associated with negative genetic predisposition to IDDM, whereas poor binding was observed for HLA-DR molecules neutrally or positively associated with IDDM. This suggests that the absence of insulin-reactive T-cells in DR2+ individuals may be explained by negative selection on high-affinity DR2 binding insulin peptides. 2) Most autoantigenic peptides display promiscuous HLA-DR binding patterns. This promiscuity in itself is not sufficient to explain the genetic association of HLA-DR with development of IDDM. 3) HLA-DR3 binding of autoantigenic GAD65 peptides is relatively weak compared with that of other known T-cell epitopes. 4) All peptide epitopes recognized by HLA-DR-restricted T-cells from either IDDM patients or GAD65-immunized HLA-DR transgenic mice bind with high affinity to their HLA-DR restriction molecule (P < 0.0006). In contrast, T-cell epitopes recognized by nondiabetic controls bind DR molecules with weak or undetectable affinity. These results thus indicate a strong correlation between antigenicity and HLA-DR binding affinity of GAD65 peptides in IDDM. Furthermore, negative thymic selection of insulin peptides in low-risk (HLA-DR2 expressing) subjects may explain the lack of autoreactivity to insulin in such individuals.

T-cell lines reactive to an immunodominant epitope of the tyrosine phosphatase-like autoantigen IA-2 in type 1 diabetes.

Type 1 diabetes is the result of destruction of the insulin-secreting beta-cells of the pancreas by a process in which T-cells play a central role. A tyrosine phosphatase-like protein, IA-2, is a major target for autoantibodies and T-cells in the disease. In this study, we have further characterized the T-cell response to IA-2 by the generation and characterization of T-cell lines. T-cell lines responsive to IA-2 antigen were generated from 17 of 32 patients and 3 of 10 control subjects. Antigen specificity was confirmed in lines from six diabetic patients and one control individual by demonstration of responses to synthetic IA-2 peptides and epitope mapping. Five lines from diabetic patients responded to one of two peptides representing amino acids 831-850 and 841-860 of IA-2. The overlapping portion may therefore represent an immunodominant region of the molecule. The sixth patient-derived line responded to a peptide representing amino acids 751-770 of IA-2 presented by the DR 4 (DRB1*0401) allele that confers susceptibility to type 1 diabetes. Primary T-cell responses to peptides of the immunodominant region were detected in 9 of 19 (47%) type 1 diabetic patients and 16 of 22 (73%) nondiabetic siblings, consistent with this region having immunostimulatory properties. The study reports for the first time T-cell lines reactive to IA-2 from diabetic patients and defines an immunodominant region of the molecule.

Synaptojanin 2 is recognized by HLA class II-restricted hairy cell leukemia-specific T cells.

Hairy cell leukemia (HCL) is a chronic mature B-cell leukemia characterized by malignant B cells that have typical hairy protrusions. To characterize possible HCL-associated tumor antigens, we generated an HCL-specific and HLA class II (DPw4)-restricted proliferative CD4+ T-cell clone. To identify the target antigen of these T cells, we constructed a synthetic peptide library dedicated to bind HLA DPw4, and identified a mimicry epitope recognized by the T-cell clone. With this epitope, the recognition motif of the T-cell clone was deduced and a peptide of human synaptojanin 2 (Syn 2) was identified that stimulated the HCL-reactive T-cell clone. Both Northern and Western blot analyses showed that Syn 2 expression was increased in HCL samples compared to other B cells. Besides, the Syn 2-expressing cell line AML193, with the introduced restrictive HLA-DPw4 molecules, was recognized by the HCL-specific T-cell clone. These results indicate that Syn 2 is a target of autoreactive HCL-specific T cells. Since Syn 2 is a phosphatidylinositol 4,5-biphosphatase involved in cell growth and rearrangement of actin filaments, the increased Syn 2 expression may correlate with the disease etiology or the characteristic morphologic alterations caused by the disease.

Clip binds to HLA class II using methionine-based, allele-dependent motifs as well as allele-independent supermotifs.

The invariant chain (Ii) region that interacts with class II and inhibits premature peptide binding has been mapped to amino acids 82-107, known as CLIP. It is unclear whether CLIP binds directly to the class II peptide binding groove and thus competitively blocks binding of other peptides, or whether it binds to conserved class II sites and indirectly inhibits peptide binding by inducing conformational changes in class II. Here we show evidence that strongly suggests that CLIP binds within the peptide binding groove, as CLIP binds to various HLA-DR alleles using allele-dependent as well as allele-independent, methionine-based binding motifs. First, a core sequence of 12 amino acids was identified within CLIP which is required for optimal binding to DR1, DR2, DR3(17) and DR7. This sequence is composed of CLIP p88-99 (SKMRMATPLLMQ). By substitution analysis, all three methionine residues appeared to control CLIP binding to HLA-DR. However, whereas M90 controlled binding to all four alleles, M92 and M98 were of different importance for the various alleles: M92 is involved in CLIP binding to DR1 and DR3(17) but not to DR2 or DR7, and M98 controls CLIP binding to DR2, DR3(17) and DR7 but not DR1. Also, CLIP competes with known immunogenic peptides for class II binding in a manner indistinguishable from regular, class II binding competitor peptides. Finally, the dissociation rates of CLIP-class II complexed are similar to those of antigenic peptide-class II complexes. Thus, CLIP most likely binds to the class II peptide binding groove, since most allelic class II differences are clustered here. CLIP uses unconventional methionine anchor residues representing an allele-independent supermotif (M90) as well as allele-dependent motifs (M92 and M98).

Highly increased levels of active stromelysin in rheumatoid synovial fluid determined by a selective fluorogenic assay.

Stromelysin-1 (MMP-3) is an important member of the matrix metalloproteinase family. In joint-degrading diseases like arthritis, elevated levels of MMP-3 protein are detected in synovial fluid using immunological methods. However, these methods do not discriminate between active and inactive enzyme. In the present study, a specific stromelysin activity assay was developed using the selective fluorogenic substrate TNO003 (Dabcyl-Gaba-Arg-Pro-Lys-Pro-Val-Glu / Nva-Trp-Arg-Glu-(EDANS)-Ala-Lys-NH2, / =cleavage site). For its use in biological media, cleavage of TNO003 by enzymes other than stromelysin was effectively blocked by a proteinase inhibitor cocktail. Spiking of MMP-3 to synovial fluid resulted in an MMP-3 concentration-dependent linear increase in activity. The measured MMP-3 activity was not affected by the addition of MMP-13, even in a 5-fold excess over MMP-3. Synovial fluid from rheumatoid arthritis patients demonstrated 100-fold higher levels of active stromelysin than control synovial fluids.

Liposome-mediated peptide loading of MHC-DR molecules in vivo.

Amino acid residues 3-15 of mycobacterial HSP60 define a dominant T-cell epitope for HLA-DR3+ve humans and Mamu-DR3+ve rhesus monkeys. Our results show that Mamu-DR3 molecules on PBMC can be efficiently loaded in vivo with the above-mentioned peptides when they are intravenously injected encapsulated in liposomes, but not in the free form. Mamu-DR3 loading is abolished by encapsulation of a nonstimulatory peptide. These results have implications for the delivery of therapeutic peptides in vivo.

Convenient fluorometric assay for matrix metalloproteinase activity and its application in biological media.

Matrix metalloproteinases (MMPs) are involved in physiological tissue remodeling and pathological conditions like tumour metastasis and joint destruction. Until now, no convenient and sensitive MMP-activity assay in crude media like synovial fluid has been available. Therefore, the highly soluble fluorogenic substrate TNO211 (Dabcyl-Gaba-Pro-Gln-Gly-Leu-Glu(EDANS)-Ala-Lys-NH2), containing the MMP cleavable Gly-Leu bond and EDANS/Dabcyl as fluorophore/quencer combination, was synthesized and characterized as an MMP specific substrate. We show that the fluorogenic assay using TNO211 is sensitive and can detect MMP activity in culture medium from endothelial cells and untreated synovial fluid (SF) from RA and OA patients, and control subjects. MMP activity in SF significantly increased in the order C < OA < RA, thus the frequent use of OA samples as control in studies on RA is debatable.

A novel, highly efficient peptide-HLA class I binding assay using unfolded heavy chain molecules: identification of HIV-1 derived peptides that bind to HLA-A*0201 and HLA-A*0301.

A novel cell-free, highly automated peptide-HLA binding assay has been designed during which a mixture of unfolded recombinant HLA heavy chain molecules, beta 2-microglobulin and a fluorescent labeled standard peptide is allowed to form peptide-HLA complexes. The binding of a peptide of interest is monitored as the ability to inhibit the formation of fluorescent peptide-HLA complexes. The assay was validated using published, known HLA-A* 0201 and HLA-A* 0301 binding peptides. In addition a selected set of HIV-1LAI reverse transcriptase derived 10-mer peptides, that had been selected on the basis of HLA-A* 0201 or HLA-A* 0301 binding motifs, were tested for HLA-A* 0201/A* 0301 binding. In that set we identified 8 peptides which bound with high affinity to HLA-A* 0201 and 5 peptides which bound with high affinity to HLA-A* 0301. The major advantage of the use of denatured heavy chain is the improved economy and efficiency, as unfolded protein material is in principle easily accessible by recombinant technology.