Comprehensive exploratory autoantibody profiling in patients with early rheumatoid arthritis treated with methotrexate or tocilizumab.

BACKGROUND: We sought to identify immunoglobin G autoantibodies predictive of early treatment response to methotrexate, the recommended first-line therapy for patients with newly diagnosed rheumatoid arthritis, and to the interleukin-6 receptor inhibitor biologic tocilizumab, initiated as the first disease-modifying anti-rheumatic drug. MATERIALS AND METHODS: In baseline sera of a subset of patients with newly diagnosed rheumatoid arthritis in the U-Act-Early study, selected based on specific responder/non-responder criteria using the Disease Activity Score assessing 28 joints (DAS28) within the first 20 weeks, we measured immunoglobin G antibody reactivity against 463 protein antigens and performed supervised cluster analysis to identify predictive autoantibodies for treatment response. The analysis subset comprised 56 patients in the methotrexate arm (22 responders, 34 non-responders) and 50 patients in the tocilizumab arm (34 responders, 16 non-responders). For comparison, these analyses were also performed in 50 age- and gender-matched healthy controls. RESULTS: Increased reactivity in responders versus non-responders was found in the methotrexate arm against two antigens-DOT1-like histone lysine methyltransferase (p = 0.009) and tropomyosin (p = 0.003)-and in the tocilizumab arm against one antigen-neuro-oncological ventral antigen 2 (p = 0.039). Decreased reactivity was detected against two antigens in the methotrexate arm-G1 to S phase transition 2 (p = 0.023) and the zinc finger protein ZPR1 (p = 0.021). Reactivity against the identified antigens was not statistically significant in either treatment arm for patients with rheumatoid factor-positive versus-negative or anti-cyclic citrullinated test-positive versus test-negative rheumatoid arthritis (p ≥ 0.06). CONCLUSIONS: Comprehensive profiling of baseline sera revealed several novel immunoglobin G autoantibodies associated with early treatment response to methotrexate and to tocilizumab in disease-modifying anti-rheumatic drug-naive patients with rheumatoid arthritis. These findings could eventually yield clinically relevant predictive markers, if corroborated in different patient cohorts, and may facilitate future benefit in personalised healthcare.

Immunization of Vγ2Vδ2 T cells programs sustained effector memory responses that control tuberculosis in nonhuman primates.

Tuberculosis (TB) remains a leading killer among infectious diseases, and a better TB vaccine is urgently needed. The critical components and mechanisms of vaccine-induced protection against Mycobacterium tuberculosis (Mtb) remain incompletely defined. Our previous studies demonstrate that Vγ2Vδ2 T cells specific for (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) phosphoantigen are unique in primates as multifunctional effectors of immune protection against TB infection. Here, we selectively immunized Vγ2Vδ2 T cells and assessed the effect on infection in a rhesus TB model. A single respiratory vaccination of macaques with an HMBPP-producing attenuated Listeria monocytogenes (Lm ΔactA prfA*) caused prolonged expansion of HMBPP-specific Vγ2Vδ2 T cells in circulating and pulmonary compartments. This did not occur in animals similarly immunized with an Lm ΔgcpE strain, which did not produce HMBPP. Lm ΔactA prfA* vaccination elicited increases in Th1-like Vγ2Vδ2 T cells in the airway, and induced containment of TB infection after pulmonary challenge. The selective immunization of Vγ2Vδ2 T cells reduced lung pathology and mycobacterial dissemination to extrapulmonary organs. Vaccine effects coincided with the fast-acting memory-like response of Th1-like Vγ2Vδ2 T cells and tissue-resident Vγ2Vδ2 effector T cells that produced both IFN-γ and perforin and inhibited intracellular Mtb growth. Furthermore, selective immunization of Vγ2Vδ2 T cells enabled CD4+ and CD8+ T cells to mount earlier pulmonary Th1 responses to TB challenge. Our findings show that selective immunization of Vγ2Vδ2 T cells can elicit fast-acting and durable memory-like responses that amplify responses of other T cell subsets, and provide an approach to creating more effective TB vaccines.

Identification of a peptide-pheromone that enhances Listeria monocytogenes escape from host cell vacuoles.

Listeria monocytogenes is a Gram-positive facultative intracellular bacterial pathogen that invades mammalian cells and escapes from membrane-bound vacuoles to replicate within the host cell cytosol. Gene products required for intracellular bacterial growth and bacterial spread to adjacent cells are regulated by a transcriptional activator known as PrfA. PrfA becomes activated following L. monocytogenes entry into host cells, however the signal that stimulates PrfA activation has not yet been defined. Here we provide evidence for L. monocytogenes secretion of a small peptide pheromone, pPplA, which enhances the escape of L. monocytogenes from host cell vacuoles and may facilitate PrfA activation. The pPplA pheromone is generated via the proteolytic processing of the PplA lipoprotein secretion signal peptide. While the PplA lipoprotein is dispensable for pathogenesis, bacteria lacking the pPplA pheromone are significantly attenuated for virulence in mice and have a reduced efficiency of bacterial escape from the vacuoles of nonprofessional phagocytic cells. Mutational activation of PrfA restores virulence and eliminates the need for pPplA-dependent signaling. Experimental evidence suggests that the pPplA peptide may help signal to L. monocytogenes its presence within the confines of the host cell vacuole, stimulating the expression of gene products that contribute to vacuole escape and facilitating PrfA activation to promote bacterial growth within the cytosol.

A protein nanofiber hydrogel for sensitive immunoassays.

Amyloid-like protein nanofibers were assembled in vitro using a recombinant protein fusion, with yeast Sup35 and human SSB/La proteins as assembly units, where the length of the nanofibers was mostly between 100 and 400 nm. The protein nanofibers, hereafter referred to as Sup35-based protein nanofiber probes (SuPNPs), were used to sensitively detect anti-SSB/La antibodies [Sjögren's syndrome (SS)-specific marker]. After, the SuPNPs were vinylated and subsequently linked to acrylamide. The polymerization reaction with acrylamide formed a SuPNP-hydrogel with uniform porosity, where the SuPNPs were directly cross-linked to polyacrylamide. Alternatively, biotinylated SuPNPs (bt-SuPNP) were attached to a streptavidin-hydrogel, resulting in the formation of a bt-SuPNP-hydrogel. When both the SuPNP-hydrogel and bt-SuPNP-hydrogel were used as 3D assay platforms for the detection of anti-SSB/La antibodies in a buffer solution, the LODs (limit of detection) were found to be 10 pM for both, showing 100-fold enhancement in sensitivity compared to conventional 2D polystyrene (PS) plate-based assays. It seems that the exposed surface and uniform distribution of the SuPNPs within the 3D space of the porous hydrogel matrix interacted more effectively with the anti-SSB/La antibodies, leading to more sensitive detection. The equal sensitivity demonstrated by the SuPNP- and bt-SuPNP-hydrogels above indicates that the target binding activity of the SuPNPs remains unchanged when either directly cross-linked to the hydrogel or indirectly immobilized to the hydrogel via streptavidin. When used to detect anti-SSB/La antibodies in human serum, the SuPNP-hydrogel is 1000 times more sensitive than a 2D PS plate. It seems that non-specific adsorption of the serum proteins occurs heavily on the 2D PS plate. While diagnostic assays for Sjögren's syndrome were demonstrated as proof-of-concept in this study, the SuPNP-hydrogel can be generally applied for the sensitive and specific detection of many other disease markers.

Multieffector-functional immune responses of HMBPP-specific Vγ2Vδ2 T cells in nonhuman primates inoculated with Listeria monocytogenes ΔactA prfA*.

Although Listeria monocytogenes can induce systemic infection causing spontaneous abortion, septicemia, and meningitis, studies have not been performed to investigate human anti-L. monocytogenes immune responses, including those of Ag-specific Vγ2Vδ2 T cells, a dominant human γδ T cell subset. L. monocytogenes is the only pathogen known to possess both the mevalonate and non-mevalonate isoprenoid biosynthesis pathways that produce metabolic phosphates or phosphoantigens activating human Vγ2Vδ2 T cells, making it interesting to explore in vivo anti-L. monocytogenes immune responses of Vγ2Vδ2 T cells. In this study, we demonstrated that subclinical systemic L. monocytogenes infection of rhesus macaques via parenteral inoculation or vaccination with an attenuated Listeria strain induced multieffector-functional immune responses of phosphoantigen-specific Vγ2Vδ2 T cells. Subclinical systemic infection and reinfection with attenuated L. monocytogenes uncovered the ability of Vγ2Vδ2 T cells to mount expansion and adaptive or recall-like expansion. Expanded Vγ2Vδ2 T cells could traffic to and accumulate in the pulmonary compartment and intestinal mucosa. Expanded Vγ2Vδ2 T cells could evolve into effector cells producing IFN-γ, TNF-α, IL-4, IL-17, or perforin after L. monocytogenes infection, and some effector Vγ2Vδ2 T cells could coproduce IL-17 and IFN-γ, IL-4 and IFN-γ, or TNF-α and perforin. Surprisingly, in vivo-expanded Vγ2Vδ2 T effector cells in subclinical L. monocytogenes infection could directly lyse L. monocytogenes-infected target cells and inhibit intracellular L. monocytogenes bacteria. Thus, we present the first demonstration, to our knowledge, of multieffector-functional Vγ2Vδ2 T cell responses against L. monocytogenes.

Monoclonal antibodies against human translation termination factor eRF3 and their utilization for sub-cellular localization of eRF3.

Eukaryotic translation termination is triggered by peptide release factors eRF1 and eRF3. eRF1 recognizes the stop codon and promotes nascent peptide chain release, while eRF3 facilitates this peptide release in a GTP-dependent manner. In addition to its role in termination, eRF3 is involved in normal and nonsense-mediated mRNA decay. Despite extensive investigation, the complete understanding of eRF3 function have been hampered by the lack of specific anti-eRF3 monoclonal antibodies (Mabs). The purpose of the study was production of recombinant eRF3a/GSPT1, development of anti-eRF3a/GSPT1 Mabs and their utilization for eRF3a/GSPT1 sub-cellular localization. Plasmid encoding C-terminal part of human GSPT1/eRF3a was constructed. Purified protein, which was predominantly present in the inclusion bodies, was used for the development of Mabs. Characterization of the regions recognized by Mabs using GSPT1/eRF3a mutants and its visualization in the 3D space suggested that Mabs recognize different epitopes. Consistent with its function in translational termination, immunostaining of the cells with developed Mabs revealed that the endogenous GSPT1/eRF3a localized in endoplasmic reticulum. Taking into account the important role of eRF3 for the fundamental research one can suggests that developed Mabs have great prospective to be used as a research reagent in a wide range of applications.

Characterization of Ers, a PrfA-like regulator of Enterococcus faecalis.

We have identified a transcriptional regulator, named Ers (for enterococcal regulator of survival), of Enterococcus faecalis, an important opportunistic bacterium commonly recovered from hospitalized patients. Ers is a member of the Crp/Fnr family and is 69% similar to Srv, a PrfA-like regulator of Streptococcus pyogenes implicated in virulence, and is the E. faecalis protein most closely related to PrfA, a positive regulator of virulence genes in Listeria monocytogenes. In an in vivo-in vitro macrophage infection model, the survival of an ers mutant was highly significantly decreased compared with that of the parental strain JH2-2. This mutant was more than 10-fold more sensitive to oxidative challenge by hydrogen peroxide. In order to identify genes whose expression was under Ers control, the RNA levels of 31 likely candidates were measured by real-time quantitative PCR. The results indicate that ers may be autoregulated and that the locus ef0082 appears to be positively regulated by Ers. Nevertheless, mutation of ef0082 did not result in any detectable changes in the survival of the bacterium within murine macrophages.

Overexpression and purification of recombinant eRF1 proteins of rabbit and Tetrahymena thermophila.

The polypeptide release factor (eRF1) gene was cloned from rabbit and its overexpression and purification system was established in parallel with that of the eRF1 gene of Tetrahymena thermophila that has been cloned recently in this laboratory. The rabbit eRF1 (Ra-eRF1) is composed of 437 amino acids and is completely identical to human eRF1 though 3% distinct in the nucleotide sequence. This is in sharp contrast to Tetrahymena eRF1 (Tt-eRF1) that is only 57% identical to human eRF1. The recombinant Ra-eRF1 was marked with a histidine tag, overexpressed, and purified to homogeneity by two-step chromatography using Ni-NTA-agarose and Mono Q columns. In contrast to Ra-eRF1, Tt-eRF1 formed aggregates upon overexpression in Escherichia coli, hence it was purified under denaturing conditions, and used to raise rabbit antibody. The resulting anti-Tt-eRF1 antibody proved useful for examining conditions for soluble Tt-eRF1 in test cells. Finally, a soluble Tt-eRF1 fraction was purified from Saccharomyces cerevisiae transformed with the Tt-eRF1 expression plasmid by three steps of affinity and anion exchange chromatography. The cloned Ra-eRF1 gene complemented a temperature-sensitive allele in the eRF1 gene, sup45 (ts), of S. cerevisiae, though the complementation activity was significantly impaired by the histidine tag, whereas Tt-eRF1 failed to complement the sup45 (ts) allele.

Immunochemical determination of the cellular content of polypeptide chain release factor RF3 in Escherichia coli.

Polypeptide chain termination in Escherichia coli is known to require two codon specific release factors, RF1 and RF2. A third factor, RF3, has been described to stimulate the termination. Earlier investigations have estimated the cellular content of factors RF1 and RF2. Two different immunological techniques for measuring the amount of RF3 per cell in crude E coli cell extracts are reported here, using a sensitive immunoblotting method and a sandwich assay by ELISA. Monoclonal murine antibodies and polyclonal rabbit antibodies were raised against extensively purified recombinant E coli RF3. The immunoblotting involves a specific monoclonal antibody (mAb), biotinylated second antibody and finally radioactive iodinated streptavidin. In the sandwich assay polyclonal antibodies are immobilised on a polystyrene surface before addition of crude cell extract; a specific mAb serves as primary antibody and an HRP-labelled anti-mouse Ig as secondary antibody. Both methods are accurate and rapid to perform. The number of RF3 molecules per cell in exponentially growing E coli cells was found to vary considerably according to the K12 strain examined and depended on the culture medium (from 20 to 500 molecules per cell), faster growth being positively correlated with the number of RF3 molecules per cell.

[Glycoprotein D (5-23) specific Th2-T-cell line induces HSV-1 keratitis]. / Glykoprotein-D (5-23)-spezifische Th2-T-Zell-Linie induziert HSV-1-Keratitis.

BALB/c inbred Igh-1-disparate mice exhibit different susceptibility to the development of HSV-1 stromal keratitis (HSK), which may be due to the differential immune regulation. CD4+ T lymphocytes may be critical for the disease induction. A T-cell line (CD4+, T-cell receptor V beta 8+, interleukin-4+) specific for the N-terminal amino acids 5-23 of glycoprotein D from HSV-1 [gD(5-23)] was established from HSK susceptible C.AL-20 mice. HSK-resistant C.B-17 mice, and HSK-susceptible BALB/c mice were injected intraperitoneally with cells (5 x 10(5)/mouse) alone or combined with HSV-1 corneal inoculation (10(5) PFU, KOS strain). Control groups were injected with HSV-antigen-unrelated cells (PPD specific), or were only HSV-1 infected. Migration of the adoptively transferred gD(5-23) Th2 cells was analyzed by histology, by immunohistochemistry and by cell membrane labelling (PKH26). The transfer of gD(5-23) cells accelerated the disease onset (day 2, compared to day 7 without cells). The transfer of gD(5-23) cells increased the incidence of HSK (BALB/c 100%, C.B-17 20%) compared to mice that were only infected with HSV-1 (BALB/c 75%, C.B-17 0%). Keratitis was more severe in mice injected with gD(5-23) cells. In contrast, the transfer of PPD-specific cells did not influence the disease patterns. Mice injected with gD(5-23) cells and not inoculated with HSV-1 did not develop keratitis. The results suggest that CD4+ MHC class II, V beta 8+, IL-4 expressing T-cells (T helper 2) may be important for the induction of HSK.(ABSTRACT TRUNCATED AT 250 WORDS)

Are the tryptophanyl-tRNA synthetase and the peptide-chain-release factor from higher eukaryotes one and the same protein?

Recently, cDNA clones encoding the bovine (b) [M. Garret, B. Pajot, V. Trézéguet, J. Labouesse, M. Merle, J.-C. Gandar, J.-P. Benedetto, M.-L. Sallafranque, J. Alterio, M. Gueguen, C. Sarger, B. Labouesse and J. Bonnet (1991) Biochemistry 30, 7809-7817] and human (h) [L. Yu. Frolova, M. A. Sudomoina, A. Yu. Grigorieva, O. L. Zinovieva and L. L. Kisselev (1991) Gene 109, 291-296] tryptophanyl-tRNA synthetases (TrpRS) were sequenced; the deduced amino acid sequences exhibit typical structural features of class I aminoacyl-tRNA synthetases [G. Eriani, M. Delarue, O. Poch, J. Gangloff and D. Moras (1990) Nature 237, 203-206] and limited, although significant, similarity with bacterial TrpRS. Independently, it was shown that a major protein whose synthesis is stimulated in human cell cultures by interferon gamma [J. Fleckner, H. H. Rasmussen and J. Justesen (1991) Proc. Natl Acad. Sci. USA 88, 11,520-11,524], and interferons gamma or alpha [B. Y. Rubins, S. L. Anderson, L. Xing, R. J. Powell and W. P. Tate (1991) J. Biol. Chem. 226, 24,245-24,248], exhibits TrpRS activity and an amino acid sequence identical to that of hTrpRS. The amino acid sequences of bTrpRS and hTrpRS are highly similar and are surprisingly very similar to the amino acid sequence deduced from a cloned and sequenced cDNA reported to encode rabbit (r) peptide-chain-release factor (RF) [C. C. Lee, W. J. Craigen, D. M. Muzny, E. Harlow and C. T. Caskey (1990) Proc. Natl Acad. Sci. USA 87, 3508-3512]. This close similarity between mammalian TrpRS and cloned RF is unexpected given the distinct functional properties of these proteins. Consequently, the question arises as to whether the mammalian TrpRS and RF activities reside on identical or very similar polypeptides. Alternatively, one may assume that the cloned rabbit cDNA encodes a protein other than rRF. Several properties (immunochemical, biochemical and physico-chemical) of mammalian TrpRS and RF have been compared. rTrpRS and rRF have distinct thermostability behaviours, and dissimilar chromatographic profiles on phosphocellulose. Both the anti-bTrpRS polyclonal antibodies and the monoclonal antibody Am2 strongly inhibit the bTrpRS and hTrpRS aminoacylation activities, but not the rRF activity. In addition, neither bTrpRS nor hTrpRS exhibit RF activity.(ABSTRACT TRUNCATED AT 400 WORDS)

[The N-terminal peptide of the hexon of human adenovirus type 2: its chemical synthesis and antigenic properties]. / N-kontsevoi peptid geksona adenovirusa cheloveka 2-go tipa: khimicheskii sintez i antigennye svoistva.

Peptides corresponding to the N-terminal section of protein of a hexone of the 2nd type human adenovirus have been synthesized. Being conjugated with a carrier of BSA they induced formation of antibodies which reacted both with peptides and with viral proteins. Sera to native proteins were also bound to synthetic peptides. Immunogenicity strengthening in peptides conjugated with synthetic polyelectrolytes is shown. The N-terminal section of hexon is supposed to participate in formation of an antigenic determinant possessing group specificity.

Interaction of the release factors with the Escherichia coli ribosome: structurally and functionally-important domains.

There are two major domains of interaction between the Escherichia coli release factors (RF-1 and RF-2) and each subunit of the ribosome. RF-2 has a binding domain on the shoulder and lower head region of the small subunit at the small lobe distant from the decoding site. This is in close proximity to one of the domains on the large subunit which includes the body dimer of L7/L12 and L11. The other domains of interaction, at the decoding site on the small subunit, and at the peptidyltransferase centre of the large subunit of the ribosome, are some distance from the first two, although the evidence for direct contact with the ribosome is less comprehensive. The release factors may therefore have two distinct structural domains, and in support of this concept RF-1 and RF-2 can both be cleaved into two fragments by papain. Region-specific antibodies, and antibodies against defined peptide within the RF sequences have given an indication that a significant part of an interacting RF molecule is in close proximity to the ribosome surface, confirming an observation by immunoelectron microscopy which suggested that the RF penetrates deeply into the cleft between the two subunits. A region of highly conserved primary sequence between the two release factors from E coli is also conserved in those from B subtilis suggesting it forms an important structural or functional domain. Antibodies against peptides from the N-terminal end of this region strongly inhibit binding of the RF to the ribosome.

Immunological characterization of the role of adenovirus terminal protein in viral DNA replication.

The function of the adenovirus-coded terminal protein and its precursor in viral DNA replication was studied by raising an antiserum against the adenovirus type 5 (Ad5) terminal protein isolated from virions. This antiserum reacted with both the terminal protein and its precursor as measured by a radioimmunoassay. In an in vitro DNA replication system employing nuclear extracts the addition of antiserum inhibits replication when a DNA-terminal protein complex from adenovirions is used as template. The replication of a 3.8% terminal fragment of the Ad2 genome with a protein-free origin (derived from the plasmid XD-7) is also inhibited by the antiserum. This observation confirms a role of the terminal protein precursor in DNA replication. The antiserum completely inhibited the formation of a covalent complex between the precursor terminal protein and dCMP, which is essential for initiation. A function of the terminal protein in the elongation reaction was shown by the inhibitory effect of antiserum on DNA chain elongation in isolated nuclei from Ad5-infected cells. Also in the in vitro DNA replication system employing nuclear extracts the elongation reaction is strongly reduced by addition of the antiserum. These results indicate that the terminal protein and/or its precursor are not only involved in initiation of DNA replication but also in DNA chain elongation.

Production of anti-glucagon sera with a C-terminal fragment of pancreatic glucagon.

The C-terminal region-sepcific anti-glucagon sera were raised in rabbits using as immunogen, and conjugate of BSA and a C-terminal fragment of pancreatic glucagon. The hapten was prepared by trypsin digestion of the glucagon, which was proved to be a 1:3 mixture of glucagon (18--29) and (19--29). Six rabbits were immunized by subcutaneous injection of an emulsion of the conjugate with complete Freund's adjuvant and five of the rabbits produced antibodies to the glucagon (GC-1, GC-2, GC-3, GC-5 and GC-6). For comparison, rabbit antisera were also produced against glucagon polymer (GA-10) and syrupy glucagon fibrils (PGA-2). All these antisera as well as the pancreatic glucagon-specific antiserum 30 K were characterized with dog gut-extract (gut-GLI) and glucagon-related peptide fragments in the radioimmunoassay systems. The assay systems utilized 125 I-monosubstituted pancreatic glucagon as tracer and human mono-component glucagon as standard. All sera of the GC-series crossreacted with the dog gut-extract very weakly and antisera GC-5 and GC-6 exhibited the lowest crossreactivities with the extract, which were shown to be as low as that of 30k. Characterization of the antiserum GC-5 with purified glucagon related fragments indicated that the major antigenic determinant located exactly in the C-terminal region of glucagon. The present results clearly showed high efficiency of the use of the glucagon C-terminal fragment as hepatenic immunogen in obtaining the C-terminal region-specific, i.e., pancreatic glucagon-specific antisera.