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In this opinion paper, we suggest that the scheme of the complement system should be redrawn in order to better illustrate its potencies. This can be achieved by putting the amplification loop of the alternative complement pathway at the center of the complement system. This arrangement emphasizes that C3b molecules, generated by any pathway, can stimulate complement amplification. Furthermore, it allows one to differentiate between this type of stimulation of amplification and that driven by those immune complexes that capture dimeric C3b molecules, which are more potent C3 convertase precursors than C3b. Schemes similar to the one drawn may help to better illustrate the interplay of the pathways and convey a clearer comprehension of the mechanics of the complement system.

Asparagine-linked sugar chains were quantitatively released from chimpanzee, Rhesus monkey and rat IgA proteins as oligosaccharides by hydrazinolysis, converted to radioactive oligosaccharides by reduction with NaB3H4, and separated into neutral and two acidic fractions by paper electrophoresis. The acidic oligosaccharides were converted to neutral ones by sialidase digestion, indicating that they are sialyl derivatives. However, the content of N-acetyl and N-glycolyl neuraminic acids was different among three species. The neutral and sialidase-treated acidic oligosaccharides were fractionated by Bio-Gel P-4 column chromatography in combination with linkage-specific sequential exoglycosidase digestion. Although IgA molecules from these species have mainly biantennary complex-type sugar chains, the contents of fucose and bisecting N-acetylglucosamine residues displayed marked species differences. In addition to these sugar chains, a small amount of the high mannose-type sugar chains was detected in chimpanzee and rat, but not in Rhesus monkey IgA. These results indicated that the processing of asparagine-linked sugar chains of IgA is different in each species.

CNBr-cleavage of a human IgM protein results in the formation of two types of L-chain-containing fragments (designated as CI and CII, respectively). These are different in size as well as charge properties. The L-chain material, which comprises approximately 16 per cent of the CI fragment and 41 per cent of the CII fragment, can be readily isolated from the reduced-alkylated fragments by the combination of gel filtration and isoelectric focusing techniques. In order to specifically localize the L-chains in the CI and CII fragments, tryptic peptides of μ-chain that are linked to the cycteinyl residue in the C-terminal tripeptide of κ-chain are purified from the enzyme digests of both fragments by the combination of gel filtration, ion exchanger chromatography, paper chromatography and high voltage paper electrophoresis. The data of amino acid analyses and partial N-terminal sequence analyses of the purified tryptic peptides indicate that the L-chain present in the CI fragment is linked by a disulfide bond to a cysteinyl residue which has been assigned by Miekka and Deutsch (1970) to the intersubunit linkage and reported by Putnam et al. (1973) to occupy the position 414 of the μ-chain, while the L-chain present in the CII fragment is linked to a cysteinyl residue at the position 140.

Thin layer and paper fingerprints of tryptic digests of Fc and pFc' fragments from an IgGl Gm (1,2,17) myeloma protein (Mit) and an IgGl Gm (1,17) myeloma protein were compared to locate differences that might be due to Gm(2). None were found among the 22 peptides that were recovered. Similarly, there were no differences in amino acid content of the 20 peptides whose amino acid content could be determined. However, the octadecapeptide (obtained by cyanogen bromide cleavage) from the Gm (1,2,17) protein had two Gly residues and no Ala residues while previous analyses of Gm(−2), including our own, had shown one of each. The Ala residue is at 431 (Eu numbering). Two heavy chain disease proteins analysed by others showed Gly at 431. We have tested both of these proteins and both are Gm(2). We suggest that Gly at residue 431 is associated with Gm(2) activity. Evidence is presented indicating that some anti-Gm(1) and some anti-Gm(2) antibodies detect two antigenic determinants. One is the allotype, the second is associated with residues between 216 and residues 229 to 236, i.e. essentially the hinge region.

We recently published a paper in Molecular Immunology (Leo and Goldman, 2009) showing that the Eib proteins from certain E. coli strains bind IgG Fc, in contrast to a previous report (Ghumra and Pleass, 2007). Richard J. Pleass has commented on our paper (Pleass, 2009) and expressed a series of unjustified misgivings about our conclusions. Here, we address the points raised by Pleass, and reassert our claim that the Eibs are indeed genuine receptors for IgG Fc.

The author regrets that in the above published the following errors occurred: In Fig. 2, the expression of TLR2, 3, 4, 7, NOD1 and NOD2molecules in human epithelial cells has been revised to show negative control stained with control IgG; the results and conclusions of the paper remain unchanged. The correct figure is shown below. Due to a technical error, in Fig. 4, the expression of -defensin 2 in human epithelial cells upon stimulation with synthetic PAMPs has been revised; the results and conclusions of the paper remain unchanged. The correct figure is shown below.

At present, the occurrence and development of inflammatory diseases are closely related to the abnormal changes of the content and function of many cytokines. At the same time, targeting related cytokines to prevent and treat diseases has also achieved good results. Therefore, it is very important to explore the role of various cytokines in inflammatory diseases. As an inflammation related protein, Tumor necrosis factor alpha stimulating gene-6 (TSG-6) has attracted more and more attention. In the process of disease, it's like a double-edged sword, showing different responses. It is constitutively expressed in some tissues with high metabolic activity and barrier protection. The diversity of its functions depends on the binding of TSG-6 with a variety of ligands, including matrix molecules, autoimmune regulatory factors and growth factors, participating in extracellular matrix remodeling and regulating protease network. This paper reviews the structure, biological function and research progress of TSG-6 in inflammatory diseases, in order to provide reference for drug development in the future.

With worsening air pollution brought by global social development, the prevalence of allergic diseases has increased dramatically in the past few decades. The novel Lck/yes-related protein tyrosine kinase (Lyn) belongs to the Src kinase family (SFK) and plays a pivotal role in the pathogenesis of inflammation, tumor, and allergy. This signaling molecule is vital in the IgE/FcεRI signaling pathway that regulates allergy. The Lyn-FcεRIβ interaction is essential for mast cell activation. The signaling pathway of Lyn has become the focus of immune, inflammatory, tumor, and allergy research. This molecule has positive and negative regulatory effects, which have attracted researchers' attention. This paper reviews the basic characteristics of Lyn and its regulatory mechanism and role in tumor and other diseases, specifically in allergies.

We previously reported that an area encompassing amino acids 147–162 of the envelope region of the simian (type D) retrovirus serotype 1 (SRV-1) constitutes an antigenic site for the binding of murine and rhesus neutralizing antibodies. Neutralizing antibodies to SRV-2 are directed to a different area, encompassing residues 96–102 of SRV-2. This paper presents data on the activity of an SRV-1 recombinant envelope protein (rEP) and of monoclonal hybridoma cell line, C11B8, produced from murine spleen cells immunized with SRV-1 rEP. Purified monoclonal antibodies from C11B8 bind to the SRV-1 rEP and to both SRV-1 and SRV-2. However, the monoclonal antibody exhibits strain specificity in the capacity to neutralize SRV-1 infection in vitro . Thus, C11B8 neutralizes SRV-1 infection but fails to neutralize four other known serotypes of the virus. C11B8 also binds to an SRV-1 synthetic peptide representing residues 142–167, which encompasses the previously defined antigenic site of recognition for neutralizing antibodies to SRV-1. This paper also contains evidence that the SRV-1 rEP construct binds the site for SRV-1 attachment to the cell receptor. This is indicated by the ability of SRV-1 rEP to compete with SRV-1 (but not with SRV-2) and inhibit its infectivity in vitro . In addition, SRV-1 rEP inhibits the neutralizing activity of C11B8 against SRV-1 infection in vitro . SRV-1 rEP has no inhibitory effect on rhesus neutralizing antibodies to SRV-2. Taken together, the above findings indicate that immunity conferred at the level of neutralizing antibodies during SRV infection is strain-specific and involves the recognition of envelope sequences unique to each strain.

MR1 is an MHC class I-like molecule with unique structural and biological features that make it an important member among the molecules involved in antigen presentation to T cells. Distinctive features include ubiquitous expression of the MR1 gene and its monomorphism. Another relevant property is that the MR1 protein appears at very low levels on the plasma membrane and its surface expression is regulated by antigen binding. Finally, the nature of presented antigens differs from those that bind other presenting molecules and includes small metabolites of microbial and self-origin, small drugs and tumor-associated antigens. This opinion paper describes in detail some of those features and discusses recent literature in the field.

Background Smalotrophomonas maltophilia(S. maltophilia) is common in nosocomial infections. However, few studies have revealed the effect of S. maltophilia on cellular immunity in the host's immune system up to now. In clinical work, we accidentally discovered that S. maltophilia directly stimulated T cells to secrete IFN-γ. Materials and methods S. maltophilia was co-cultured with PBMCs to detect secretion of cytokines (IFN-γ, TNF-α and IL-2) and expression of cell surface molecules (CD3, CD4, CD8, CD69, CD147 and CD152) of T cells. We used light microscopy and electron microscopy to observe the cell morphology and subcellular structure of S. maltophilia co-cultured with lymphocytes. Flow cytometry and Western Blot were used to detect the expression of PD-1/PD-L1 and annexin V in cells. Results T cells stimulated by S. maltophilia secreted a large amount of IL-2, IFN-γ, and TNF-α. The expression of CD4 and CD8 on the cell surface were declined, accompanied by the activation of the PD-1/PD-L1 pathway, which eventually led to the massive apoptosis of T cells. Electron microscopy showed that cells showed significant apoptotic morphology. Blocking the PD-1/PD-L1 pathway can inhibit the apoptosis-inducing effect of S. maltophilia on T cells. Conclusions These indicates that T cells are inhibited after being stimulated by S. maltophilia, and then accelerated to induce death without the initiation of an immunologic cascade. This paper demonstrates for the first time the inhibitory effect of S. maltophilia on cellular immunity, and the immunosuppressive effect induced by infection of S. maltophilia should be considered.

It has long been known that the complement cascade is activated in various forms of glomerulonephritis. In many of these diseases, immune-complexes deposit in the glomeruli and activate the classical pathway. Researchers have also identified additional mechanisms by which complement is activated in the kidney, including diseases in which the alternative and lectin pathways are activated. The kidney appears to be particularly susceptible to activation of the alternative pathway, and this pathway has been implicated as a primary driver of atypical hemolytic uremic syndrome, C3 glomerulopathy, anti-neutrophil cytoplasmic antibody-associated vasculitis, as well as some forms of immune-complex glomerulonephritis. In this paper we review the shared and distinct mechanisms by which complement is activated in these different diseases. We also review the opportunities for using therapeutic complement inhibitors to treat kidney diseases.

Humanization of monoclonal antibodies by complementary determinant region (CDR)-grafting has become a standard procedure to improve the clinical usage of animal antibodies. However, antibody humanization may result in loss of activity that has been attributed to structural constraints in the framework structure. In this paper, we report the complete humanization of the 6.7 anti-human CD18 monoclonal antibody in a scFv form. We used a germline-based approach to design a humanized VL gene fragment and expressed it together with a previously described humanized VH. The designed humanized VL has only 14 mutations compared to the closest human germline sequence. The resulting humanized scFv maintained the binding capacity and specificity to human CD18 expressed on the cell surface of peripheral blood mononuclear cells (PBMC), and showed the same pattern of staining T-lymphocytes sub-populations, in comparison to the original monoclonal antibody. We observed an unexpected effect of a conserved mouse–human framework position (L37) that hinders the binding of the humanized scFv to antigen. This paper reveals a new framework residue that interferes with paratope and antigen binding and also reinforces the germline approach as a successful strategy to humanize antibodies.

Objective Spinal cord injury (SCI) is a neurological disease with high incidence and disability. In this paper, we made a foundational study in long non-coding RNA paternally expressed gene 10 (lncRNA PEG10) at PC-12 cells. Methods We used CCK8, flow cytometry, migration and invasion assay to detect changes at the cellular level. PEG10, microRNA-34a-5p (miR-34a-5p) and TLX transfected by transfection assay and amplified by real-time quantitative PCR (qRT-PCR). TLX expression and proteins about apoptosis and pathways were investigated by Western blot. Additionally, the relationship between PEG10 and miR-34a-5p, miR-34a-5p and TLX were examined through luciferase assay. Results H2O2-injury model was established. Knockdown PEG10 deteriorated H2O2-injury. miR-34a-5p was confirmed as a target of PEG10 and miR-34a-5p inhibitor remitted PEG10-induced H2O2-injury. Moreover, TLX was confirmed as a target miR-34a-5p and TLX remitted H2O2-injury. At last, TLX worked in H2O2-injury via Smad and Wnt/β-catenin pathways. Conclusion Knockdown PEG10 remitted H2O2-injury of PC-12 cells by targeting miR-34a-5p/TLX, and the behavior may be involved in Smad and Wnt/β-catenin pathways.

Research on the complement system, like most areas of immunology, has seen tremendous progress over the last decades. Further advances in the complement field will rely on the next generation of scientific leaders, which are today's early-career researchers (ECRs). ECRs are emerging scientists who obtained their PhD degree within the past five years. They represent a distinct population within the scientific community, and accordingly have unique needs. Unfortunately, ECRs are faced with significant challenges that require customized solutions. The current paper provides a snapshot of the major obstacles ECRs face, such as an unhealthy work-life balance, lack of mentor and peer support, and uncertain career prospects. Efforts must consequently be taken to ensure stability and success of ECRs, not only to benefit these researchers in the early stages of their career, but the entire field of complement research. The Early-Career Complementologists (ECCO) was, therefore, launched as a new Task Force to support ECRs in the complement field. This new initiative aims to support and connect ECRs in the complement field worldwide. Furthermore, ECCO is supported by both the International Complement Society (ICS) and the European Complement Network (ECN); two professional societies led by scientists investigating the complement system.

The author regrets that in the above published paper the following error occurred:In Fig. 3 of the paper cited above, trace amounts of protein A in the affinity chromatography-purified anti-syndecan-2 mAb 186C(protein Awhichleachedfromtheaffinityresin),whosepresencewentunnoticed,interferedwiththeanti-CD3 mAb(mouseIgG2a)usedto stimulate the T cells. Based on the original results we suggested that syndecan-2 could have an inhibitory effect on T cells. However, re-evaluation ofsimilarexperimentswithproteinA-freeanti-syndecan-2mAb186CindicatesthatthemAbdoesnotinhibitT-cellproliferationor IL-2 mRNA synthesis.

Insects lack an acquired immune system and rely solely on the innate immune system to combat microbial infection. The innate immunity of insects mainly depends on the interaction between the host's pattern recognition receptor (PRR) and pathogen-associated molecular pattern (PAMP). The peptidoglycan recognition proteins (PGRPs) family is the most important pattern recognition receptor (PRR) for insects. It can recognize the main component of the cell wall of the pathogenic microorganism, peptidoglycan (PGN), and plays an important role in the innate immunity of insects. In this paper, the structure, classification, and function of PGRPs is summarized, and the role of PGRPs in the innate immunity of insects is also discussed.

CD40 is a crucial signal mediating factor in T-dependent B cell responses and involved in many aspects of cellular and humoral immunity. In this paper, recombinant protein of CD40 in flounder (Paralichthys olivaceus) and its antibodies (Abs) were produced, native CD40 molecules in flounder tissues were identified, then the CD40+ leukocytes in T/B lymphocytes were characterized, and the variations of CD40+ leukocytes in flounder after Hirame novirhabdovirus (HIRRV) infection and immunization were investigated, respectively. The results showed that the Abs could specifically recognize native flounder CD40 molecule at 32 kDa. The proportions of CD40+ leukocytes were varied by flounder tissues. CD40+/IgM+ B lymphocytes, CD40+/CD4-1+ T lymphocytes, CD40+/CD4-2+ T lymphocytes and CD40+/CD8+ T lymphocytes in peripheral blood leukocytes (PBLs) were 1.18 ± 0.27%, 0.69 ± 0.17%, 0.75 ± 0.14% and 0.25 ± 0.14%; were 2.80 ± 0.32%, 0.71 ± 0.19%, 0.88 ± 0.23% and 0.33 ± 0.17% in spleen; 4.11 ± 0.47%, 0.92 ± 0.18%, 1.09 ± 0.17% and 0.9 ± 0.17% in head kidney; 1.92 ± 0.39%, 1.02 ± 0.23%, 1.33 ± 0.38% and 0.67 ± 0.24% in intestine; 1.24 ± 0.36%, 1.21 ± 0.24%, 1.70 ± 0.3% and 0.97 ± 0.21% in gill, respectively. The percentages of CD40+ leukocytes in PBLs were significantly increased in both HIRRV infection and immunization groups, and reached their peak levels at 3rd day with 5.70 ± 0.16% and 6.40 ± 0.13%, respectively. Concluded with our previous study, these data first reported that CD40 molecules were expressed on both B and T lymphocytes in teleost, and had a coordination with T and B lymphocytes in immune responses.

Interferon regulatory factor 8 (IRF8), also known as interferon consensus sequence-binding protein (ICSBP), is a negative regulatory factor of interferon (IFN) and plays an important role in cell differentiation and innate immunity in mammals. In recent years, some irf8 homologous genes have been cloned and confirmed to take part in innate immune response in fish, but the mechanism still remains unclear. In this paper, a grass carp (Ctenopharyngodon idella) irf8 gene (Ciirf8) was cloned and characterized. The deduced protein (CiIRF8) possesses a highly conserved N-terminal DNA binding domain but a less well-conserved C-terminal IRF association domain (IAD). Ciirf8 was widely expressed in all tested tissues of grass carp and up-regulated following poly(I:C) stimulation. Ciirf8 expression was also up-regulated in CIK cells upon treatment with poly(I:C). To explore the molecular mechanism of how fish IRF8 regulates ifn1 expression, the similarities and differences of grass carp IRF8 and IRF2 were compared and contrasted. Subcellular localization analysis showed that CiIRF8 is located both in the cytoplasm and nucleus; however, CiIRF2 is only located in the nucleus. The nuclear-cytoplasmic translocation of CiIRF8 was observed in CIK cells under stimulation with poly(I:C). The interaction of CiIRF8 and CiIRF2 was further confirmed by a co-immunoprecipitation assay in the nucleus. Dual-luciferase reporter assays showed that the promoter activity of Ciifn1 was significantly inhibited by co-transfection with CiIRF2 and CiIRF8. The transcription inhibition of Ciifn1 was alleviated by competitive binding of CiIRF2 and CiIRF8 to CiIRF1. In conclusion, CiIRF8 down-regulates Ciifn1 expression via interaction with CiIRF2 in cells.

The RIP family plays a key role in mediating cell inflammation, oxidative stress and death. Among them, RIPK1, as an important regulatory factor in the upstream of the NF-κB pathway, is involved in multiple pathways of cell inflammation and death. Epidermal cells constitute the outermost barrier of the human body. Radiation can induce epidermal cell death, inflammation and oxidative stress to cause damage. Therefore, this paper selected HaCaT cell and used CRISPR/Cas technology to construct a cell model of stable knockout of RIPK1 gene, to analyze the effect and regulation of RIPK1 knockout on the function and death of HaCaT cells induced by UVB or TNF-α. The results showed that knockout of RIPK1 had no significant effect on the morphology of HaCaT cells at rest, but it led to slowing cell proliferation and blocking the G2M phase of cell cycle. Compared with HaCaTWT, HaCaTRIP1KO was abnormally sensitive to TNF-α-induced cell death and apoptosis, and may be associated with inhibition of NF-κB pathway. Knocking out RIPK1 led to a more significant inhibition of cell growth by UVB, and up-regulation of the expression of the inflammatory factor IL-1α. P38 MAPK and NF-κB pathways may be involved this process. This study further found that RIPK1 in epidermal cell has a regulatory function on pro-survival signals.

Systemic lupus erythematosus (SLE) is a chronic inflammatory disease. Endothelial cell injury plays an important role in the inflammatory processes associated with SLE. CD4+Foxp3+regulatory T cells (Tregs) reduce the injury to endothelial cells induced by inflammatory factors. As a newly identified regulatory T cell, we previously reported that CD8+CD103+iTregs had similar effects to those of CD4+iTregs in the process of immunoregulation. In this paper, we further explored the effect and mechanism of CD8+iTregs on endothelial cell injury. The expressions of vascular cellular adhesion molecule-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM-1), interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) in MRL/lpr mouse glomerular endothelial cells (lupus-MGECs) were estimated by quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assay and Western blotting. The lupus-MGEC apoptosis rate was detected by flow cytometry and the adhesion of monocyte-like cells to lupus-MGECs exposed to lipopolysaccharide (LPS) was determined by the adhesion assay. Additionally, the expressions of P-p38, P-NF-κB and P-IκBα were detected by Western blotting. The results showed that LPS increased the expressions of VCAM-1, ICAM-1, IFN-γ, TNF-α, IL-6 and MCP-1 in lupus-MGECs, while CD8+iTregs significantly decreased the levels of these adhesion molecules and inflammatory mediators. Furthermore, CD8+iTregs alleviated lupus-MGEC apoptosis and inhibited the adhesion of monocyte-like cells to lupus-MGECs. Both nuclear factor-κB (NF-κB) and p38 mitogen-activated protein kinase (MAPK), activated by LPS, were suppressed by CD8+iTregs. These findings suggest that CD8+iTregs attenuate LPS-induced glomerular endothelial cell injury through blocking the activation of p38 MAPK and NF-κB in lupus-MGECs. The protective effect of CD8+iTregs indicates their possible therapeutic application in Lupus nephritis.

Heartwater is a tick-borne non-infectious fatal disease of wild and domestic ruminants caused by the bacterium Ehrlichia ruminantium, transmitted by Amblyomma ticks. Although there is evidence that interferon-gamma (IFN-γ) controls E. ruminantium growth and that cellular immune responses could be protective, an effective recombinant vaccine for this disease is lacking. An overall analysis of which immune pathways are up- or down-regulated in sheep peripheral blood mononuclear cells is expected to lead to a better understanding of the global immune response of sheep to E. ruminantium infection. Therefore, a systems biology oriented approach following the infection with E. ruminantium was investigated from peripheral blood mononuclear cells to aid recombinant vaccine development. In this study, heartwater naïve sheep were infected and challenged by allowing E. ruminantium infected ticks to feed on them. After primary infection, all the animals were treated with antibiotic during the resulting febrile response. Blood was collected daily for E. ruminantium detection by qPCR (pCS20 assay). The pCS20 assay only detected the pathogen in the blood one day prior to and during the febrile stage of infection confirming infection of the sheep. IFN-γ real-time PCR indicated that this cytokine was expressed at specific time points: post infection, during the febrile stage of the disease and after challenge. These were used as a guide to select samples for transcriptome sequencing. This paper focuses on transcripts that are associated with innate activating pathways that were identified to be up- and down-regulated after primary infection and the subsequent challenge. These included the CD14 monocyte marker, toll-like receptor (TLR), nod-like receptor, chemokine, cytosolic and cytokine-cytokine interaction receptor pathways. In particular, TLR4, TLR9 and CD14 were activated together with DNA detection pathways, suggesting that vaccine formulations may be improved if CpG motifs and lipopolysaccharides are included. This data indicates that innate immune activation, perhaps by using adjuvants, should be an important component for consideration during future heartwater recombinant vaccine development.

Infiltration of macrophages plays a critical role in the connection between inflammation and cancer invasion; however, the molecular mechanism that enables this crosstalk remains unclear. This paper investigates a molecular link between infiltration of macrophages and metastasis of lung cancer cells. In this study, the macrophage density and cyclooxygenase-2 (COX-2) protein were examined in surgical specimens by immunohistochemistry (IHC), and the prostaglandin E2 (PGE2) levels were determined in the blood of 30 non-small cell lung cancer (NSCLC) patients using enzyme-linked immunosorbent assay (ELISA). We demonstrated that macrophage infiltration was significantly associated with elevated tumour COX-2 expression and serum PGE2 levels in NSCLC patients. Interestingly, the COX-2 and PGE2 levels as well as macrophages were poor predictors of NSCLC patient survival. THP-1-derived macrophages were co-cultured in vitro with A549 and H1299 lung cancer cells. In the co-culture process, interleukin-6 (IL-6) induced the COX-2/PGE2 pathway in lung cancer cells, which subsequently promoted β-catenin translocation from the cytoplasm to the nucleus, resulting in epithelial-mesenchymal transition (EMT) and lung cancer cell invasion. Our findings show that the IL-6-dependent COX-2/PGE2 pathway induces EMT to promote invasion of tumour cells through β-catenin activation during the interaction between macrophages and lung cancer cells, which suggests that inhibition of COX-2/PGE2 or macrophages has the potential to suppress metastasis of lung cancer cells.

This paper aims to investigate the effect of Toll-like receptors 3 (TLR3)/TIR-domain-containing adapter-inducing interferon-β (TRIF) signal pathway on the airway inflammation and remodeling in asthmatic mice. C57BL/6 and TLR3-/- mice were randomly divided into three groups (10 mice per group), including Control group (mice inhaled phosphate buffer saline (PBS)), Asthma group (mice inhaled ovalbumin (OVA)) and polyriboinosinic-ribocytidylic acid (poly (I: C)) group (asthmatic mice were injected intraperitoneally with TLR3 agonist poly (I: C)). Hematoxylin-eosin (HE) staining, Wright-Giemsa staining, Enzyme-linked immunosorbent assay (ELISA), Immunohistochemistry, Hydroxyproline assay, quantitative real time polymerase chain reaction (qRT-PCR) and Western blot were used to assess for the indices of airway inflammation and remodeling. In terms of WT mice, all asthma groups with or without the addition of poly (I: C) showed exaggerated inflammation and remodeling in the airways as compared to Control group, which were more seriously in poly (I: C) group than Asthma group. Furthermore, we observed the significant inhibition of airway inflammation and remodeling in the TLR3-/- mice in both Asthma no matter with or without addition of poly (I: C) than the WT mice. TLR3 knockout could obviously relieve the airway inflammation and remodeling in asthma through inhibiting TLR3/TRIF signaling pathway.

Proinflammatory cytokines and type I IFNs were produced by TLR signaling and these responses are crucial for host defensive responses against pathogens. In order to avoid harmful and inappropriate inflammatory responses, there are multiple mechanisms to negatively regulate TLR signaling. In this paper, we have firstly studied IRF9 functions as a negative regulator involved in TRIF-mediated NF-κB pathway. Moreover, we found inhibitory effect of IRF9 primary depends on DBD domain. Interestingly, we also demonstrated that else mutants of IRF9, except for IRF9-ΔDBD, have different inhibitory effects upon TRIF-mediated NF-κB pathway. This study provides a novel evidence about the negatively regulation of innate immune signaling pathway in teleost fish. In addition, this finding provides new insights into the regulatory mechanism in mammals.

Most of the complement proteins in circulation are, by and large, synthesized in the liver. However data accumulated over the past several decades provide incontrovertible evidence that some if not most of the individual complement proteins are also synthesized extrahepatically by activated as well as non-activated cells. The question that is finally being addressed by various investigators is: are the locally synthesized proteins solely responsible for the myriad of biological functions in situ without the contribution of systemic complement? The answer is probably "yes". Among the proteins that are synthesized locally, C1q takes center stage for several reasons. First, it is synthesized predominantly by potent antigen presenting cells such as monocytes, macrophages and dendritic cells (DCs), which by itself is a clue that it plays an important role in antigen presentation and/or DC maturation. Second, it is transiently anchored on the cell surface via a transmembrane domain located in its A chain before it is cleaved off and released into the pericellular milieu. The membrane-associated C1q in turn, is able to sense danger patterns via its versatile antigen-capturing globular head domains. More importantly, locally synthesized C1q has been shown to induce a plethora of biological functions through the induction of immunomodulatory molecules by an autocrine- or paracrine- mediated signaling in a manner that mimics those of TNFα. These include recognition of pathogen- and danger- associated molecular patterns, phagocytosis, angiogenesis, apoptosis and induction of cytokines or chemokines that are important in modulating the inflammatory response. The functional convergence between C1q and TNFα in turn is attributed to their shared genetic ancestry. In this paper, we will infer to the aforementioned "local-synthesis-for-local function" paradigm using as an example, the role played by locally synthesized C1q in autoimmunity in general and in systemic lupus erythematosus in particular.

Rhodanese homology domains (RHODs) are the structural modules of ubiquitous tertiary that occur in three major evolutionary phyla. Despite the versatile and important physiological functions of RHODs containing proteins, little is known about their invertebrate counterparts. A novel HSP67B2-like single-domain rhodanese homologue, MdRDH1 from Musca domestica, whose expression can be induced by bacterial infection or oxidative stress. Silencing MdRDH1 through RNAi causes important accumulations of reactive oxygen species (ROS) and malondialdehyde (MDA), and increases mortality in the larvae treated with bacterial invasion. The E. coli with MdRDH1 and the mutant MdRDH1C135A are transformed, with significant rhodanese activity of the recombinant protein of MdRDH1 in vitro found, without no detection of enzyme activity of the mutant MdRDH1C135A, revealing that catalytic Cys135 in the active-site loop is essential in the sulfurtransferase activity of MdRDH1. When oxidative stress is insulted by phenazine methosulfate (PMS), the MdRDH1 transformed E. coli shows enhanced survival rates compared with those bacteria transformed with MdRDH1C135A. Our research indicates that MdRDH1 confers oxidative stress tolerance, thus rendering evidence for the idea that rhodanese family genes play a critical role in antioxidant defenses. This paper yields novel insights into the potential antioxidative and immune functions of HSP67B2-like rhodanese homologues in invertebrate.

T cell response is crucial to the pathogenesis and progression of rheumatoid arthritis (RA). IL-7/IL-7R axis has significant effect on CD4+ T cell response, including proliferation, differentiation, survival and migration. However, whether blockade of IL-7/IL-7R axis signaling can relieve RA and what is the potential treatment mechanisms are still remaining unclear. In this paper, we established collagen-induced arthritis (CIA) model and observed the effect of IL-7Rα antibody in the treatment of CIA mice. It is demonstrated that IL-7Rα antibody significantly alleviated clinical symptoms of CIA mice, accompanied with reduced CD4+ T cell number in both spleen and joints. Decreased CII-specific CD4+ T cell proliferation and reduced mRNA expression of inflammatory cytokines in IL-7Rα antibody-treated mice were observed. Subsequently, IL-7Rα antibody treatment in vivo downregulated the percentages of Th1 and Th17 cells and the mRNA expression of T-bet and RORγt gene. Moreover, it was found that IL-7 promoted Th1 cell differentiation in vitro, while having no effect on Th17 cell differentiation. In addition, administration of IL-7Rα antibody reduced the mRNA expression of chemokine receptors (CCR7, CXCR3, CXCR6 and XCR1) on CD4+ T cells and chemokine CXCL2 in joints. The results suggested that IL-7Rα antibody treated CIA mice via the inhibition of CII-specific CD4+ T cell proliferation, the reduction of Th1 cell differentiation and the restrain of CD4+ T cell migration to joint lesion site. This investigation indicates that IL-7Rα is a potential therapeutic target for RA.

The advent of high throughput sequencing has permitted to investigate the genome and the transcriptome of novel non-model species with unprecedented depth. This technological advance provided a better understanding of the evolution of adaptive immune genes in gnathostomes, revealing several unexpected features in different fish species which are of particular interest. In the present paper, we review the current understanding of the adaptive immune system of the coelacanth, the elephant shark and the Atlantic cod. The study of coelacanth, the only living extant of the long thought to be extinct Sarcopterygian lineage, is fundamental to bring new insights on the evolution of the immune system in higher vertebrates. Surprisingly, coelacanths are the only known jawed vertebrates to lack IgM, whereas two IgD/W loci are present. Cartilaginous fish are of great interest due to their basal position in the vertebrate tree of life; the genome of the elephant shark revealed the lack of several important immune genes related to T cell functions, which suggest the existence of a primordial set of TH1-like cells. Finally, the Atlantic cod lacks a functional major histocompatibility II complex, but balances this evolutionary loss with the expansion of specific gene families, including MHC I, Toll-like receptors and antimicrobial peptides. Overall, these data point out that several fish species present an unconventional adaptive immune system, but the loss of important immune genes is balanced by adaptive evolutionary strategies which still guarantee the establishment of an efficient immune response against the pathogens they have to fight during their life.

Acute liver failure is a devastating clinical syndrome with extremely terrible inflammation reaction, which is still lack of effective treatment in clinic. Suppressor of Cytokine Signaling 1 protein is inducible intracellular negative regulator of Janus kinases (JAK)/signal transducers and activators of transcription (STAT) pathway that plays essential role in inhibiting excessive intracellular signaling cascade and preventing autoimmune reaction. In this paper, we want to explore whether dendritic cells (DCs) with overexpression of SOCS1 have a therapeutic effect on experimental acute liver failure. Bone marrow derived dendritic cells were transfected with lentivirus encoding SOCS1 and negative control lentivirus, thereafter collected for costimulatory molecules analysis, allogeneic Mixed Lymphocyte Reaction and Western blot test of JAK/STAT pathway. C57BL/6 mice were randomly separated into normal control and treatment groups which respectively received tail vein injection of modified DCs, negative control DCs and normal saline 12 h earlier than acute liver failure induction. Our results indicated that DCs with overexpression of SOCS1 exhibited like regulatory DCs (DCregs) with low level of costimulatory molecules and poor allostimulatory ability in vitro, which was supposed to correlate with block of JAK2/STAT1 signaling. In vivo tests, we found that infusion of modified DCs increased survival rate of acute liver failure mice and alleviate liver injury via inhibition of TLR4/HMGB1 pathway. We concluded that DCs transduced with SOCS1 gene exhibit as DCregs through negative regulation of JAK2/STAT1 pathway and ameliorated lipopolysaccharide/ d -galactosamine induced acute liver failure via inhibition of TLR4 pathway.

Nanosecond fluorescence depolarization studies reported in the accompanying companion paper showed that the long rotational correlation time, φL, increased somewhat when rabbit IgG anti-dansyl antibodies were anchored in staphylococcal protein A (SpA) soluble complexes. The increases in φL upon anchoring IgG probably resulted from “global coupling” effects caused by: (1) increased steric hindrance of the antibody segments in the SpA complexes and (2) intrinsic structural constraints already present in the monomeric IgG. Global coupling results from a restriction in the angular range of a flexible segment and is manifest when flexible motions alone cannot depolarize all of the fluorescence, so that the slower global tumbling of the entire particle is also required. Such effects cannot be resolved directly from experimental anisotropy data, however, because only a single long correlation time, φL, is well defined over the limited time range of most fluorophores. In this paper, estimates of the anisotropy contributions from flexible and global motions of the IgG-SpA complexes are determined by contrasting theoretical and measured decays. For this analysis it was assumed that each of the experimental φL-values is a weighted composite of the rotational correlation time associated with the less restricted flexible motions of the Fab arms, φF, and the correlation time associated with global tumbling of the entire particle, φG. A general two-exponential expression was used to relate φF and φG to φL. This approach was meaningful because φG-values of the various SpA complexes had been calculated from hydrodynamic measurements. The theoretical decays clearly show that, even if φG is much longer than φF, these two rotational motions still cannot be resolved over the experimentally accessible time range. Families of emission anisotropy decay curves for IgG antibodies with different amounts of intrinsic global coupling and for anchored antibodies with different amounts of steric hindrance were simulated by varying the preexponential weighting factors of the flexible and global terms. By comparing the calculated curves with the measured decays, it is evident that the rabbit IgG anti-dansyl antibodies do not have much intrinsic global coupling, but rather they are highly flexible. The curves also indicate that even for the exceptionally compact IgG4-SpA2 17-S complex, which showed the most steric hindrance in electron micrographs, the appropriate φG weighting factor is only 0.28. Thus, as supposed earlier, the anchored antibodies exhibit considerable segmental flexibility. In closing, the above concepts are used to examine the results of other reported depolarization studies with mouse monoclonal IgG1 antibodies.

This paper presents the initial characterization of a mouse monoclonal antibody (RH1-38) which blocks, in the absence of complement, three different systems of cell-mediated cytotoxicity. This monoclonal antibody markedly inhibits cytotoxicity mediated by human natural killer cells, a monocyte-like cell [phorbol myristate acetate (PMA) stimulated HL-60], and cytotoxic T-lymphocytes generated in a mixed leukocyte reaction. RH1-38 is not nonspecifically toxic to cells since antibody-dependent cellular cytotoxicity was not inhibited and viability as assessed by trypan blue exclusion was not affected. Inhibition is specific since control hybridoma culture supernatants, parent (NS-1) ascites supernatant, monoclonal anti-HLA and normal mouse IgG were not significantly inhibitory. In the NK system, the inhibitory effect appears to be due to binding of monoclonal antibody to effector cell surface since exposure of targets to antibody followed by washing yielded no inhibition of killing. Inhibition requires the antigen-binding portion of the antibody molecule and thus appears to be related to steric hindrance of an effector cell surface molecule which is important in the expression of cell-mediated cytotoxicity. Immunoprecipitation of surface-radioiodinated membranes from PMA-stimulated HL-60 cells and analysis on sodium dodecyl sulfate-polyacrylamide gels revealed a bimolecular complex (195,000 and 125,000 daltons) without significant change under reducing conditions. Control immunoprecipitates yielded no peaks of activity. This monoclonal antibody should serve as a useful probe of the function and biochemistry of a killer cell surface antigen important in the expression of cell-mediated cytotoxicity. Since RH1-38 inhibits cytotoxicity mediated by at least three apparently unrelated effector cells, the relevant antigen may be part of a common mechanistic step. As the companion paper demonstrates, this monoclonal antibody does not affect the conjugation step, but appears to block a late step in the NK cytolytic mechanism. Thus, RH1-38 recognizes either an epitope district from previously-described anti-LFA-1 antibodies or alternatively recognizes a distinct functional killer cell surface molecule.

A 4-hydroxy-3-iodo-5-nitrophenacetyl (NIP) binding mouse plasmacytoma (ABPC 24) protein was shown to have a monovalent component of the molecular weight of about 66 000. This component was allowed to bind on NIP coupled paper in order to measure the amount of binding as a function of NIP density on paper. This plasmacytoma protein bound to a significantly higher degree on solid phase NIP than to NIP in solution. Further, the calculated standard free energy change for the binding reaction on solid phase NIP exhibited a linear relationship to the average distance between NIP molecules on paper. Thus it was possible to quantitate the effect of steric hindrance on binding on solid phase hapten.

Allergic reactions caused by 9kDa Lipid Transfer Proteins (9k-LTP), such as Pru p 3, have been widely investigated, whereas a possible contribution of components of 7kDa LTP (7k-LTP) sub-family in triggering allergic symptoms has been overlooked so far. With the aim to investigate the contribution of 7k-LTPs to the food allergies, we have identified, isolated and characterised a tomato seed 7k-LTP (Sola l 7k-LTP). The protein was purified by chromatographic separations, identified by direct protein sequencing and mass spectrometry and a molecular model was built. Functional evaluation of the allergen has been performed by skin testing. Sola l 7k-LTP consists of 68 amino acids producing a molecular mass of 7045Da and displays 41% sequence identity with Pru p 3, the allergenic 9k-LTP from peach. IgE antibodies specifically recognising Sola l 7k-LTP were found within the population claiming tomato ingestion-related symptoms, but also in subjects tolerant on tomato exposure. A few subjects were mono-sensitised to Sola l 7k-LTP, which is biologically active as shown by the positive skin test. In line with the immunological results, the molecular model shows structural similarities between the IgE binding regions of the two sub-families. Therefore, Sola l 7k-LTP shares some structural and immunological features with Pru p 3, but it also displays individual features that could be responsible for mono-specific IgE binding. In conclusion, Sola l 7k-LTP is a new identified allergenic LTP, the description of which may contribute to the improvement of allergy diagnosis and to the formulation of a safe and personalised diet. In addition, to avoid current confusing classifications, a new nomenclature policy for LTP sub-families is proposed in this paper. We now suggest that 7-kDa LTP (so far named LTP2) be renamed 7k-LTP and 9-kDa LTP (so far named LTP1) be renamed 9k-LTP.

T and B lymphocytes are closely related to immunization and pathogen infection. Our previous study confirmed the CD3+, CD4-1+, CD4-2+, CD8β+ T lymphocytes and IgM+ B lymphocytes presented in the peripheral blood leukocytes (PBLs) of flounder (Paralichthys olivaceus), in this paper, the variations of T and B lymphocytes of flounder after Hirame novirhabdovirus (HIRRV) infection or immunization were investigated. The flounders were injected with live or inactivated HIRRV, then the percentages of T and B lymphocytes in PBLs were analyzed by Flow cytometry (FCM), total antibodies and HIRRV-specific antibodies in serum were detected by Enzyme-linked immunosorbent assay (ELISA), and expression of twelve immune-related genes in the head kidneys were determined using q-PCR. The results showed that the percentages of CD3+, CD4-1+, CD4-2+, CD8β+ T lymphocytes and IgM+ B lymphocytes significantly increased in both infection and immunization groups, in infection group they decreased rapidly after the peak and significantly lower than control levels at the end of infection, in immunization group they went down steadily to the control levels at the end of immunization. The total antibodies and HIRRV-specific antibodies increased first and peaked on the 7th day post infection and on the 14th day post immunization, respectively, then gradually decreased to the control levels. Additionally, twelve immune-related genes were up-regulated in both groups. These results demonstrated that the HIRRV induced both humoral and cellular immunity of flounder, the lymphocytes varied more sharply in infection group than those in immunization group and CD8+ T lymphocytes responded much more than CD4+ T lymphocytes to HIRRV antigen.

Suppressor of cytokine signalling (SOCS) family members are crucial in the control and attenuation of cytokine induced responses via activation of the JAK/STAT, TLR and NF-kB signalling pathways. SOCS proteins orchestrate the termination of many types of immune responses and are often the targets of microbial pathogens exploiting SOCS mechanisms to evade the host's immune response. Through whole and lineage specific genome duplication events, the teleost cytokine/SOCS network is complex. Not only are the orthologues of all mammalian SOCS members present, namely cytokine inducible Src homology 2 (SH2)-containing protein (CISH) and SOCS-1 to -7, but multiple gene copies exist that may potentially become functionally divergent. In this paper we focus on the CISH genes in rainbow trout (Oncorhynchus mykiss), and have cloned two further paralogues, CISHa2 and CISHb2, additional to the known CISHa1 and CISHb1 genes. We present for the first time a comparative expression analysis of these four paralogues, to establish whether subfunctionalisation is apparent. In vivo examination of gene expression revealed a higher constitutive expression level of CISHa paralogues compared to CISHb expression in adult trout tissues. All CISHs were relatively highly abundant in immune tissues but CISHa2 and CISHb2 had highest expression in the heart and muscle. An inverse picture of CISH abundance during trout ontogeny was seen, and further hints at differential roles of the four genes in immune regulation and development. Stimulation of head kidney (HK) leukocytes with trout recombinant interleukin (rIL)-15 and rIL-21 had a major effect on CISHa2 and to a lesser extent CISHa1 expression. In HK macrophages rIL-1β, phytohemagglutinin, and phorbol 12-myristate 13-acetate also had a strong impact on CISHa2 expression. Yersinia ruckeri infection caused a temporally and spatially differential onset of CISH expression that may be viewed in the context of pathogen evasion strategies. These data, against the backdrop of fish specific whole genome duplication events and functional divergence, provide the first evidence for differential roles of the four trout CISH genes in immune control and development.

Highlights • There is evidence that the common mucosal immune system exists in pigs. • Vaccination at an easily accessible mucosal site may assist in providing protection at other mucosal sites. • Local and distal mucosal sites should be sampled after vaccinations to define the optimal dose and formulation which promotes the common mucosal immune system in pigs., The majority of lymphocytes activated at mucosal sites receive instructions to home back to the local mucosa, but a portion also seed distal mucosa sites. By seeding distal sites with antigen-specific effector or memory lymphocytes, the foundation is laid for the animal's mucosal immune system to respond with a secondary response should to this antigen be encountered at this site in the future. The common mucosal immune system has been studied quite extensively in rodent models but less so in large animal models such as the pig. Reasons for this paucity of reported induction of the common mucosal immune system in this species may be that distal mucosal sites were examined but no induction was observed and therefore it was not reported. However, we suspect that the majority of investigators simply did not sample distal mucosal sites and therefore there is little evidence of immune response induction in the literature. It is our hope that more pig immunologists and infectious disease experts who perform mucosal immunizations or inoculations on pigs will sample distal mucosal sites and report their findings, whether results are positive or negative. In this review, we highlight papers that show that immunization/inoculation using one route triggers mucosal immune system induction locally, systemically, and within at least one distal mucosal site. Only by understanding whether immunizations at one site triggers immunity throughout the common mucosal immune system can we rationally develop vaccines for the pig, and through these works we can gather evidence about the mucosal immune system that may be extrapolated to other livestock species or humans.

SAP is a small cytosolic adaptor protein expressed in hematopoietic lineages whose main function is to regulate intracellular signaling pathways induced by the triggering of members of the SLAM receptor family. In this paper, we have identified the adhesion molecule PECAM-1 as a new partner for SAP in a conditional yeast two-hybrid screen. PECAM-1 is an immunoglobulin-like molecule expressed by endothelial cells and leukocytes, which possesses both pro- and anti-inflammatory properties. However, little is known about PECAM-1 functions in T cells. We show that SAP directly and specifically interacts with the cytosolic tyrosine 686 of PECAM-1. We generated different T-like cell lines in which SAP or PECAM-1 are expressed or down modulated and we demonstrate that a diminished SAP expression correlates with a diminished PECAM-1-mediated adhesion. Although SAP has mainly been shown to associate with SLAM receptors, we evidence here that SAP is a new actor downstream of PECAM-1.

Dysregulation of cell death signaling pathways in many cell types such as B lymphocytes (B-cells) can lead to cancer, for example to B-cell lymphomas. Rituximab (RTX) and glucocorticoids such as dexamethasone (Dex) are widely used to treat hematological malignancies including B-cell lymphomas. Although the combination of Dex and RTX improves the treatment outcome of lymphoma patients, most lymphomas remain incurable diseases. Therefore, a detailed investigation of Dex- and RTX-induced signaling might provide new insights into the therapeutic benefits of these drugs. In this paper, we describe Dex- and RTX-induced signaling pathways and their downstream target proteins/cells. In addition, we also overview how the signaling initiated by Dex and RTX modulate the outcome of Dex- and RTX-mediated cell death in lymphoma cells. The combination of Dex and RTX results in massive cell death in lymphoma cells. However, pretreatment of lymphoma cells or mononuclear cytotoxic cells with Dex followed by RTX leads to a decrease in apoptosis or it impairs antibody-dependent cellular cytotoxicity (ADCC). RTX-mediated ADCC is impaired by Dex-induced depletion of cytotoxic cells, whereas RTX-mediated short-term ERK1/2 activation decreases Dex-induced apoptosis. Therefore, the timing of the combination of Dex and RTX is a determining factor for the synergistic effect of these cell death inducing agents.

Highlights ► All the structural B-cell epitopes we examined are discontinuous. ► Only 18% of structural epitopes are spanned by a peptide fragment of 40 residues. ► Centralized and random distributions were considered for key functional residues. ► Fragments with only 7 residues will successfully span most key functional residues., Although it is widely acknowledged that most B-cell epitopes are discontinuous, the degree of discontinuity is poorly understood. For example, given that an antigen having a single epitope that has been chopped into peptides of a specific length, what is the likelihood that one of the peptides will span all the residues belonging to that epitope? Or, alternatively, what is the largest proportion of the epitope's residues that any peptide is likely to contain? These and similar questions are of direct relevance both to computational methods that aim to predict the location of epitopes from sequence (linear B-cell epitope prediction methods) and window-based experimental methods that aim to locate epitopes by assessing the strength of antibody binding to synthetic peptides on a chip. In this paper we present an analysis of the degree of B-cell epitope discontinuity, both in terms of the structural epitopes defined by a set of antigen–antibody complexes in the Protein Data Bank, and with respect to the distribution of key residues that form functional epitopes. We show that, taking a strict definition of discontinuity, all the epitopes in our data set are discontinuous. More significantly, we provide explicit guidance about the choice of peptide length when using window-based B-cell epitope prediction and mapping techniques based on a detailed analysis of the likely effectiveness of different lengths.

Aspergillus versicolor is common on moldy building materials. Asp v 13, the principal allergen is produced by strains collected from across Canada. In this paper, we report a 1833 bp Asp v 13 open reading frame predicted to encode a protein of 403 amino acids in length with three introns. A BLAST search of Asp v 13, a phylogenic tree calculation and alignment with its homologous proteins from other species indicated that Asp v 13 is a secretory, subtilisin-like serine protease widely distributed in Aspergillus species. His-tagged Asp v 13 was over-expressed in Escherichia coli and purified using Ni-NTA columns with a yield of 1 mg/L. Based on immuno binding assay of recombinant protein both antibodies developed against the natural protein, and human sera IgE, the recombinant protein was similar to the natural form. Six IgE- and seven IgG-binding epitopes were also identified with selected human sera along the entire amino acid sequence of Asp v 13. Most residues binding these epitopes are exposed on the surface and correspond to charged regions of the molecule.

An approach for enhancing antibody affinity is to engineer Chelating Recombinant Antibodies (CRAbs) which consist of two tandemly linked single-chain Fvs (scFvs) that bind to distinct non-overlapping epitopes on the antigen molecule leading to a synergistic decrease in K(D). In order to develop this technology, the aim of this present study was to identify scFvs which can simultaneously bind to the tetanus toxin heavy chain C-terminal sub-domain (H(c)), characterise their bio-physical properties and determine their functional efficacy. Over 50 antibodies specific for Hc were isolated from a human scFv phagemid library and found to bind specifically to the C-terminal sub-domain of H(c) (H(c)C clones), the N-terminal sub-domain (HcN clones) or junctional epitopes on the whole Hc fragment only (HcJ clones). Fifteen clones were assayed in a pairwise competition binding study. The revealed, with few exceptions, that H(c)C clones were able to simultaneously bind to the toxin with H(c)N or H(c)J clones. All other combinations competed for binding. Interestingly, we also observed cooperative binding with many non-competing scFv pairings which may impact upon the binding mechanism of CRAbs. We found that 14/15 clones neutralised toxin activity in a ganglioside binding assay and this effect was strongly related to affinity. This included clones that did not bind to the H(c)C sub-domain which is responsible for direct interaction with gangliosides on nerve cells. For 7 scFvs that underwent further characterisation we found broad variations in propensity for multimerisation, affinity and potency. The diverse array of clones characterised in this paper can be used to construct CRAbs and will prove useful in further characterisation of toxin biology and in measuring the effects of polyclonal antibody therapy.

This paper reports the cloning and sequencing of interleukin (IL)-22 in two gadoid fish, cod (Gadus morhua) and haddock (Melanogrammus aeglefinus). The complete transcript of this gene was 1002 and 1154 bp respectively, of which 492 bp was the open reading frame (ORF) in both genes. High amino acid identity (88.3%) was found between these genes but was less than 50% aa identity to other known genes. The gene organisation of haddock IL-22 consisted of five exons and four introns, as with other IL-10 family members. Expression studies showed that IL-22 is constitutively expressed in gill, with low level expression also observed in gut, gonad and head kidney. In a vaccination experiment, haddock were injected intraperitoneally with formalin-killed Vibrio anguillarum or with PBS, and 2 months later challenged by immersion in 107 cfu/ml V. anguillarum for 30 min. Head kidney and gill samples were collected prior to challenge and 24, 48 and 72 h post-challenge (hpc) for Real-time PCR analysis of IL-22 expression. No significant changes in IL-22 expression were observed in head kidney tissue but vaccinated fish showed a significantly increased expression of IL-22 24hpc in gill and no mortalities were seen in these fish. In contrast, control fish, which started to succumb to the disease from 72hpc, showed no significant increase in gill expression after challenge.

A 2-phase method is described for the determination of the intrinsic affinity constants (K-values) for the interaction between monoclonal antibodies (MAbs) against carcinoembryonic antigen (CEA) and CEA. The Mabs were coupled covalently to CNBr-activated paper discs. MAb coupled discs and a 2-fold dilution series of 125I-CEA were incubated at 20 degrees C until equilibrium was reached. Nonlinear curve-fitting was used for estimation of K-values and different calculation models were thereby tested. The K-values for 12 different anti-CEA MAbs were determined to be 0.3-52 X 10(6) M-1, which is 1-2 orders of magnitude lower than the values obtained in a previous study with some of these MAbs using the ammonium sulphate method to separate free from bound antigen. The K-values obtained by the paper disc method are probably better estimates of the intrinsic association constant than those obtained previously. There are two main reasons for this: (1) free and bound antigen are separated from each other under physiological conditions and (2) the opportunity for multipoint interaction between MAb and CEA is minimized when the MAb is coupled to a rigid carrier substance. Thus, even when the MAb reacts with greater than or equal to 2 epitopes in CEA, as seems to be the case with several of our anti-CEA MAbs, the intrinsic K-value should be obtained. The fundamental validity of 2-phase assays, sometimes questioned in the literature, is demonstrated.

This paper describes a relatively simple procedure, the identical pair method, for using existing data to estimate the total number of different antibody V region sequences or isoelectric focusing spectrotypes. Since the calculations required can be performed without a programable computer the method should be useful to all interested biologists, regardless of previous mathematical training. Using this procedure we have determined that the probable repertoire of isoelectric focusing spectrotypes of antistreptococcal group A antibodies from mouse strains A/J and SWR/J are 200 and 300, respectively. The paper also describes how the identical pair method can be used to estimate the number of different V region amino acid sequences.