Chemokines cooperate with TNF to provide protective anti-viral immunity and to enhance inflammation.

The role of cytokines and chemokines in anti-viral defense has been demonstrated, but their relative contribution to protective anti-viral responses in vivo is not fully understood. Cytokine response modifier D (CrmD) is a secreted receptor for TNF and lymphotoxin containing the smallpox virus-encoded chemokine receptor (SECRET) domain and is expressed by ectromelia virus, the causative agent of the smallpox-like disease mousepox. Here we show that CrmD is an essential virulence factor that controls natural killer cell activation and allows progression of fatal mousepox, and demonstrate that both SECRET and TNF binding domains are required for full CrmD activity. Vaccination with recombinant CrmD protects animals from lethal mousepox. These results indicate that a specific set of chemokines enhance the inflammatory and protective anti-viral responses mediated by TNF and lymphotoxin, and illustrate how viruses optimize anti-TNF strategies with the addition of a chemokine binding domain as soluble decoy receptors.

Full Interchangeability in Regard to Immunogenicity Between the Infliximab Reference Biologic and Biosimilars CT-P13 and SB2 in Inflammatory Bowel Disease.

Background: Infliximab (IFX) biosimilars CT-P13 and SB2 have comparable efficacy, safety, and immunogenicity to the originator Remicade (RMC). However, concerns about cross-switching patients between the 3 brands were raised in the absence of cross reactivity data between them. We aimed to determine whether antibodies to infliximab (ATI) in inflammatory bowel disease (IBD) patients cross-react with RMC, CT-P13, and SB2. Methods: Based on previous ATI status, samples from 34 patients participating in the BIOSIM01 study (13 RMC, 9 CT-P13, and 12 switchers) were selected. Patients were treated with either RMC only, or CT-P13 only, or with RMC switched to CT-P13. Additionally, 28 IFX-naïve patients were assayed as controls. In total, 180 samples were analyzed. ATI trough levels were measured in parallel with 3 different bridging Enzyme Linked Immunosorbent Assays constructed using the 3 drugs. Spearman's coefficient and percentages of agreement were used to study the correlation between each assay. Results: In total, 76 samples out of 152 IFX-treated patient samples were ATI-positive (30 RMC, 14 CT-P13, and 32 switchers). All resulted ATI-positive when either CT-P13 or SB2 bridging assays were used. The overall percentage of agreement was 100% when compared either with CT-P13 or SB2 assays. No significant differences were found among ATI levels and coefficients (Spearman's 0.98 to 1.0, P < 0.0001). Conclusions: ATI of RMC-treated, CT-P13-treated or RMC to CT-P13 switched patients show full cross-reactivity with CT-P13 and SB2. Findings suggest that immunodominant epitopes in the reference and CT-P13 drugs are equally present in SB2. Data support full interchangeability between biosimilars in regard to immunogenicity.

Antibodies to infliximab in Remicade-treated rheumatic patients show identical reactivity towards biosimilars.

OBJECTIVES: The aim of this study was to determine whether antibodies to infliximab (IFX) in Remicade-treated patients cross-react with the biosimilar CT-P13. METHODS: 250 consecutive patients with rheumatic diseases under Remicade and 77 controls were retrospectively selected for the study. Anti-IFX antibodies at drug through levels were measured in parallel with three different bridging ELISA assays: Promonitor-ANTI-IFX kit, which uses Remicade to detect antibodies, and two more assays that use either Inflectra or Remsima with the same format. Correlation and association between each assay was studied. RESULTS: 50.4% of patients were tested positive with Promonitor-ANTI-IFX. All were antibodies to IFX (ATI)-positive when either Inflectra or Remsima assays were used. In all comparisons positive and negative percentage agreements were 100%, and correlation coefficients were ≥0.995. No differences between rheumatoid arthritis and spondyloarthritis, or between concomitant immunosuppressives, were observed. CONCLUSIONS: Anti-IFX antibodies of Remicade-treated patients cross-react with either Inflectra or Remsima. Although additional epitopes may be present in the biosimilar, results suggest that epitopes influencing the immune response to IFX are also present in the biosimilar. Antibody-positive patients treated with Remicade should not be switched to the biosimilar, since antibodies will interact with the new drug and potentially lead to loss of response. This finding supports the utility for therapeutic drug monitoring before a switching strategy is considered.

A new biomarker panel in bronchoalveolar lavage for an improved lung cancer diagnosis.

INTRODUCTION: The enormous biological complexity and high mortality rate of lung cancer highlights the need for new global approaches for the discovery of reliable early diagnostic biomarkers. The study of bronchoalveolar lavage samples by proteomic techniques could identify new lung cancer biomarkers and may provide promising noninvasive diagnostic tools able to enhance the sensitivity of current methods. METHODS: First, an observational prospective study was designed to assess protein expression differences in bronchoalveolar lavages from patients with (n = 139) and without (n = 49) lung cancer, using two-dimensional gel electrophoresis and subsequent protein identification by mass spectrometry. Second, validation of candidate biomarkers was performed by bead-based immunoassays with a different patient cohort (204 patients, 48 controls). RESULTS: Thirty-two differentially expressed proteins were identified in bronchoalveolar lavages, 10 of which were confirmed by immunoassays. The expression levels of APOA1, CO4A, CRP, GSTP1, and SAMP led to a lung cancer diagnostic panel that reached 95% sensitivity and 81% specificity, and the quantification of STMN1 and GSTP1 proteins allowed the two main lung cancer subtypes to be discriminated with 90% sensitivity and 57% specificity. CONCLUSIONS: Bronchoalveolar lavage represents a promising noninvasive source of lung cancer specific protein biomarkers with high diagnostic accuracy. Measurement of APOA1, CO4A, CRP, GSTP1, SAMP, and STMN1 in this fluid may be a useful tool for lung cancer diagnosis, although a further validation in a larger clinical set is required for early stages.

An ectromelia virus protein that interacts with chemokines through their glycosaminoglycan binding domain.

Poxviruses encode a number of secreted virulence factors that modulate the host immune response. The vaccinia virus A41 protein is an immunomodulatory protein with amino acid sequence similarity to the 35-kDa chemokine binding protein, but the host immune molecules targeted by A41 have not been identified. We report here that the vaccinia virus A41 ortholog encoded by ectromelia virus, a poxvirus pathogen of mice, named E163 in the ectromelia virus Naval strain, is a secreted 31-kDa glycoprotein that selectively binds a limited number of CC and CXC chemokines with high affinity. A detailed characterization of the interaction of ectromelia virus E163 with mutant forms of the chemokines CXCL10 and CXCL12alpha indicated that E163 binds to the glycosaminoglycan binding site of the chemokines. This suggests that E163 inhibits the interaction of chemokines with glycosaminoglycans and provides a mechanism by which E163 prevents chemokine-induced leukocyte migration to the sites of infection. In addition to interacting with chemokines, E163 can interact with high affinity with glycosaminoglycan molecules, enabling E163 to attach to cell surfaces and to remain in the vicinity of the sites of viral infection. These findings identify E163 as a new chemokine binding protein in poxviruses and provide a molecular mechanism for the immunomodulatory activity previously reported for the vaccinia virus A41 ortholog. The results reported here also suggest that the cell surface and extracellular matrix are important targeting sites for secreted poxvirus immune modulators.

A chemokine-binding domain in the tumor necrosis factor receptor from variola (smallpox) virus.

Variola virus (VaV) is the causative agent of smallpox, one of the most devastating diseases encountered by man, that was eradicated in 1980. The deliberate release of VaV would have catastrophic consequences on global public health. However, the mechanisms that contribute to smallpox pathogenesis are poorly understood at the molecular level. The ability of viruses to evade the host defense mechanisms is an important determinant of viral pathogenesis. Here we show that the tumor necrosis factor receptor (TNFR) homologue CrmB encoded by VaV functions not only as a soluble decoy TNFR but also as a highly specific binding protein for several chemokines that mediate recruitment of immune cells to mucosal surfaces and the skin, sites of virus entry and viral replication at late stages of smallpox. CrmB binds chemokines through its C-terminal domain, which is unrelated to TNFRs, was named smallpox virus-encoded chemokine receptor (SECRET) domain and uncovers a family of poxvirus chemokine inhibitors. An active SECRET domain was found in another viral TNFR (CrmD) and three secreted proteins encoded by orthopoxviruses. These findings identify a previously undescribed chemokine-binding and inhibitory domain unrelated to host chemokine receptors and a mechanism of immune modulation in VaV that may influence smallpox pathogenesis.

Thermostability of the human respiratory syncytial virus fusion protein before and after activation: implications for the membrane-fusion mechanism.

Anchorless fusion (F) proteins () of human respiratory syncytial virus (RSV) are seen by electron microscopy as unaggregated cones when the proteolytic cleavage at two furin sites required for membrane-fusion activity is incomplete, but aggregate into rosettes of lollipop-shaped spikes following cleavage. To show that this aggregation occurred by interactions of the fusion peptide, a deletion mutant of lacking the first half of the fusion peptide was generated. This mutant remained unaggregated even after completion of cleavage, supporting the notion that aggregation of involved the fusion peptide. As exposure of the fusion peptide is a key event that occurs after activation of F proteins, the uncleaved and cleaved forms of may represent the pre- and post-active forms of RSV F protein. In an analysis of the structural differences between the two forms, their thermostability before and after proteolytic cleavage was examined. In contrast to other viral proteins involved in membrane fusion (e.g. influenza haemagglutinin), the pre-active (uncleaved) and post-active (cleaved) forms of were equally resistant to heat denaturation, assessed by spectrofluorimetry, circular dichroism or antibody binding. These results are interpreted in terms of the proposed structural changes associated with the process of membrane fusion mediated by RSV F protein.

Sphingomyelinase cleavage of sphingomyelin in pure and mixed lipid membranes. Influence of the physical state of the sphingolipid.

Sphingomyelin hydrolysis by sphingomyelinase is essential in regulating membrane levels of ceramide, a well-known metabolic signal. Since natural sphingomyelins have a gel-to-fluid transition temperature in the range of the physiological temperatures of mammals and birds, it is important to understand the influence of the physical state of the lipid on the enzyme activity. With that aim, large unilamellar vesicles consisting of pure egg sphingomyelin (gel-to-fluid crystalline transition temperature ca. 39 degrees C) were treated with sphingomyelinase in the temperature range 10-70 degrees C. The vesicles were also examined by differential scanning calorimetry (DSC). Shingomyelinase was active on pure sphingomyelin bilayers, leading to concomitant lipid hydrolysis, vesicle aggregation, and leakage of aqueous liposomal contents. Enzyme activity was found to be much higher when the substrate was in the fluid than when it was in the gel state. Sphingomyelinase activity was found to exhibit lag times, followed by bursts of activity. Lag times decreased markedly when the substrate went from the gel to the fluid state. When egg phosphatidylcholine, or egg phosphatidylethanolamine were included in the bilayer composition together with sphingomyelin, sphingomyelinase activity at 37 degrees C, that was negligible for the pure sphingolipid bilayers, was seen to increase with the proportion of glycerophospholipid, while the latency times became progressively shorter. A DSC study of the mixed-lipid vesicles revealed that both phosphatidylcholine and phosphatidyletanolamine decreased in a dose-dependent way the transition temperature of sphingomyelin. Thus, as those glycerophospholipids were added to the membrane composition, the proportion of sphingomyelin in the fluid state at 37 degrees C increased accordingly, in this way becoming amenable to rapid hydrolysis by the enzyme. Thus sphingomyelinase requires the substrate in bilayer form to be in the fluid state, irrespective of whether this is achieved through a thermotropic transition or by modulating bilayer composition.

Membrane restructuring via ceramide results in enhanced solute efflux.

The capacity of ceramides to modify the permeability barrier of cell membranes has been explored. Membrane efflux induced either by in situ generated ceramides (through enzymatic cleavage of sphingomyelin) or by addition of ceramides to preformed membranes has been studied. Large unilamellar vesicles composed of different phospholipids and cholesterol, and containing entrapped fluorescent molecules, have been used as a system to assay ceramide-dependent efflux. Small proportions of ceramide (10 mol % of total lipid) that may exist under physiological conditions of ceramide-dependent signaling have been used in most experiments. When long chain (egg-derived) ceramides are used, both externally added or enzymatically produced ceramides induce release of vesicle contents. However, the same proportion of ceramides generated by sphingomyelinase induce faster and more extensive efflux than when added in organic solution to the preformed vesicles. Under our conditions 10 mol % of N-acetylsphingosine (C(2)-ceramide) did not induce any efflux. On the other hand, sphingomyelinase treatment of bilayers containing 50 mol % sphingomyelin gave rise to release of fluorescein-derivatised dextrans of molecular mass approximately 20 kDa, i.e. larger than cytochrome c. These results have been discussed in the light of our own previous data (Ruiz-Argüello, M. B., Basañez, G., Goñi, F. M., and Alonso, A. (1996) J. Biol. Chem. 271, 26616-26621) and of the observations by Siskind and Colombini (Siskind, L. J., and Colombini, M. (2000) J. Biol. Chem. 275, 38640-38644). Our spectroscopic observations appear to be in good agreement with the electrophysiological studies of the latter authors. Furthermore, some experiments in this paper have been designed to explore the mechanism of ceramide-induced efflux. Two properties of ceramide, namely its capacity to induce negative monolayer curvature and its tendency to segregate into ceramide-rich domains, appear to be important in the membrane restructuring process.