T-cell growth factor, a mitogenic protein released from lectin- or antigen-activated mononuclear cells, maintains T-lymphocytes in continuous exponential proliferative culture. Studies which explored the parameters required for TCGF production revealed that both mature thymic-dependent T-cells and adherent cells participated in the release of TCGF. Further experimentation suggested that T-cells released TCGF after receiving two signals; (1) lectin or antigen cell membrane binding, and (2) a soluble product derived from adherent cells. A separate subset of T-cells, which did not require thymic influence, responded to TCGF. TCGF appeared to interact with the TCGF-responsive subset via specific membrane receptors: TCGF could be absorbed only by lectin- or antigen-activated living or glutaraldehyde-fixed T-cells. The T-cell response was initiated by lectin or antigen, but proliferation was mediated solely by purified TCGF. Thus, the T-cell immune response, which culminates in clonal expansion and differentiation of antigenreactive cells, appears to be controlled by an endogenously-derived proliferative hormone, which is obligatory for the realization of a competent effector T-cell.
The IFab2 molecule described is expressed by a single gene construct that encodes the VH-CH1 and the Vk-Ck modules of a humanized version of the anti-breast cancer antibody BrE-3, joined via a short peptide linker. It can be shown that this basic genetic construct can lead to the expression of monomers, dimers (IFab2s) and other multimer species, depending on the length of the linker. A 21-residue linker promotes the principal formation of the desired dimer IFab2 molecules. It was also shown that in binding competition assays, purified IFab2 completely retained the affinity and specificity of the original monoclonal antibody.
This study characterized the unique peptide-binding characteristics of HLA-DRB1*12:01 (DR1201), an allele studied in the context of various autoimmune diseases, using a peptide competition assay and structural modeling. After defining Influenza A/Puerto Rico/8/34 Matrix Protein M1 (H1MP) 40-54 as a DR1201 restricted epitope, the critical anchor residues within this sequence were confirmed by measuring the relative binding of peptides with non-conservative substitutions in competition with biotin labeled H1MP(40-54) peptide. Based on this information, a set of peptides was designed with single amino acid substitutions at these anchor positions. The overall peptide binding preferences for the DR1201 allele were deduced by incubating these peptides in competition with the reference H1MP(40-54) to determine the relative binding affinities of each to recombinant DR1201 protein. As expected, pocket 1 preferred methionine and aliphatic residues, and tolerated phenylalanine. Pocket 4 was mostly composed of hydrophobic residues, thereby preferentially accommodating aliphatic residues, but could also weakly accommodate lysine due to its slightly acidic environment. Pocket 6 accepted a wide range of amino acids because of the diverse residues that comprise this pocket. Pocket 9 accepted aliphatic and negatively charged amino acids, but showed a remarkable preference for aromatic residues due to the conformation of the pocket, which lacks the typical salt bridge between β57Asp and α76Arg. These binding characteristics contrast with the closely related DR1104 allele, distinguishing DR1201 among the alleles of the HLA-DR5 group. These empirical results were used to develop an algorithm to predict peptide binding to DR1201. This algorithm was used to verify T cell epitopes within novel antigenic peptides identified by tetramer staining and within peptides from published reports that contain putative DR1201 epitopes.
HLA molecules are cell-surface glycoproteins that present peptides, derived from intracellular protein antigens, for surveillance by T lymphocytes. Secreted HLA (sHLA) technology is a powerful approach for studying these peptides, since it facilitates large-scale production of HLA-bound peptides. We compared secreted and membrane-bound forms of HLA A2 in terms of intracellular trafficking and their bound peptide repertoire (termed the immunopeptidome). We demonstrate that sHLA and membrane bound HLA (mHLA) negotiate intracellular compartments with similar maturation kinetics. Moreover, mass spectrometry revealed a substantial overlap in the immunopeptidome was observed when HLA A2-bound peptides were purified from various sources of sHLA and mHLA. By combining machine based algorithms with manual validation, we identified 1266 non-redundant peptides. Analysis of these peptides revealed a number bearing post-translational modifications, although some of these may arise spontaneously others represent modifications performed within the cell that survive antigen processing. Peptides bearing some of these modifications have not previously been described for HLA ligands, therefore, this compendium of 1266 non-redundant peptide sequences adds greatly to the existing database of HLA A2 ligands. Peptides from all sources displayed comparable HLA A2 consensus binding motifs, peptide lengths, predicted HLA A2 binding affinities and putative source antigens. We conclude that sHLA is a valid and useful technique for studying the immunopeptidome.
Hybridomas producing monoclonal antibodies of different isotypes were isolated from BALB/c antibody responses to the capsid protein VP1 of the foot-and-mouth disease virus (FMDV) strain O1. According to antigen binding measured by ELISA a weak-binding (81D10, IgM) and a strong-binding antibody (113C12, IgG2a) were selected. As RNA sequencing of productive immunoglobulin VH and VK genes turned out, both chains of the weak-binding antibody (81D10) are encoded by germline (i.e. not mutated) genes whereas the gene encoding the strong-binding antibody (113C12) k chain is mutated at several sites. Therefore, rearranged VH and VK genes of 81D10 were cloned, expressed in immunoglobulin non-producing plasmacytoma cells, and mice transgenic for the 81D10 k gene were produced. These mice provide a first step in the development of a transgenic mouse model for genetical investigations in the affinity maturation of anti-viral immunoglobulin variable genes.
Monoclonal anti-DNA autoantibody BV 04-01 catalyzed hydrolysis of DNA in the presence of Mg2+ ions. DNA hydrolyzing activity was associated with BV 04-01 IgG, Fab, and SCA 04-01 proteins. Pronounced cleavage specificity for both ss and dsDNA was observed with efficient hydrolysis of the C-rich region of the oligonucleotide A7C7ATATAGCGCGT7 as well as preference for cleavage within CG-rich regions of double-stranded DNA. Data on specificity of ssDNA hydrolysis and kinetic data obtained from wild-type SCA 04-01 and two SCA 04-01 mutants (L32Phe and L27dHis) were used to model the catalytically active antibody site utilizing the previously resolved X-ray structure of (dT)3 liganded Fab 04-01. The resulting model suggested that BV 04-01 activates the target phosphodiester bond by induction of conformational strain. In addition, the antibody-DNA complex contained a potential Mg2+ ion coordination site composed of the L32Tyr and L27dHis amino acid side chains and a DNA 3'-phosphodiester group. Induction of strain and metal coordination could be constituents of a mechanism by which this antibody catalyzed DNA hydrolysis. Sequence data for BV 04-01 VH and VL genes suggested that the proposed catalytic antibody active site was germ-line encoded. This observation suggests the hypothesis that catalytic activity might represent an important but unspecified function of some antibody molecules.
Subgroup B adenovirus serotype 11 (Ad11) occasionally causes fatal infections in immunocompromised patients. The present study describes a novel Ad11 epitope presented by HLA-A*24:02 that could be used for adoptive immunotherapy. Ten synthetic Ad11 hexon protein-derived nonamer peptides that bound to HLA-A*24:02 were selected by a computer algorithm and MHC stabilization assay. Stimulation of peripheral blood mononuclear cells from HLA-A*24:02+ donors with each of these synthetic peptides induced peptide-specific CD8(+) T-cells for three peptides. Testing the reactivity of these peptide-specific CD8(+) T-cells against various target cells confirmed that peptide TYFNLGNKF is naturally processed in Ad11-infected cells and is presented by HLA-A*24:02. Emergence of TYFNLGNKF-specific CD8(+) T-cells coincided with the clearance of adenoviruses in a patient with Ad11 disease. Importantly, TYFNLGNKF-specific CD8(+) T-cells were suggested to be not serotype cross-reactive. The novel HLA-A*24:02-restricted Ad11 epitope could be used for anti-Ad11 adoptive immunotherapy and to monitor immunity to Ad11 using MHC tetramers.
HLA-E shares several peptide sequences with HLA-class Ia molecules. Therefore, anti-HLA-E antibodies that recognize the shared sequences may bind to HLA-class Ia alleles. This hypothesis was validated with a murine anti-HLA-E monoclonal antibody (mAb) MEM-E/02, which reacted with microbeads coated with several HLA-B and HLA-C antigens. In this report, the hypothesis was reexamined with another mAb 3D12, considered to be specific for HLA-E. The antibody binding is evaluated by measuring mean fluorescence index [MFI] with Luminex Multiplex Flow-Cytometric technology. The peptide-inhibition experiments are carried out with synthetic shared peptides, most prevalent to HLA-E and HLA-Ia alleles. The results showed that mAb 3D12 simulated MEM-E/02 in recognizing several HLA-B and HLA-C antigens. Both 3D12 and MEM-E/02 did not bind to HLA-A, HLA-F and HLA-G molecules. As observed with MEM-E/02, binding of 3D12 to HLA-E is inhibited by the peptides sequences (115)QFAYDGKDY(123) and (137)DTAAQI(142). Decrease in binding of mAb 3D12 to HLA class Ia, after heat treatment of antigen coated microbeads, supports the contention that the epitope may be located at the outside of the "thermodynamically stable" α-helix conformations of HLA-E. Several sequence and structure-based web-tools were employed to validate the discontinuous epitopes recognized by the mAbs. The scores obtained by these web-tools distinguished the shared peptide sequences that inhibited the mAb binding to HLA-E. Furthermore, ElliPro web tool points out that both mAbs recognize the conformational discontinuous epitopes (the shared inhibitory peptide sequences) in the secondary structure of the HLA-E molecule. The study favors the contention that the domain of the shared inhibitory peptide sequences may be the most immunogenic site of HLA-E molecule. It also postulates and clarifies that amino acid substitution on or near the binding domains may account for the lack of cross reactivity of 3D12 and MEM-E/02 with HLA-A, HLA-F and HLA-G molecules.
We investigated analogues of GP2 (IISAVVGIL), an HLA-A*0201-restricted T-cell epitope derived from residues 654-662 in the tumor-associated antigen (TAA) Her-2/neu. One limiting factor of GP2 is its poor affinity for HLA-A*0201. Conformational analysis revealed the P5-P7 region in GP2 appears to be linked to the stability of P9 side chain interaction with the MHC molecule. To identify variants of GP2 with enhanced presentation to HLA-A*0201, we tested V6S, V6T, V6Q, G7P, G7F, T6F7, and Q6F7 for their capacity to stabilize cell surface HLA-A*0201 molecules. Of the mono-substituted variants, V6Q and G7F exhibited superior stabilization as compared to GP2. Molecular dynamics simulations suggest the improved binding can be attributed to concerted motions in the central and C-terminal regions of the peptide. These data support the notion that amino acids in HLA-A*0201 epitopes may be inter-dependent. Priming HLA-A*0201 transgenic mice with G7F-loaded syngeneic dendritic cells stimulated mouse T cells to produce a higher level of INFgamma than mice immunized with GP2.
The synovial sarcoma X breakpoint (SSX) gene family contains nine members. The SSX proteins are CT (cancer/testis) antigens and can be expressed in many tumor types. T cell immune response against SSX protein can be detected in tumor patients and mice expressing any SSX. Screening predominant protective epitopes might improve the low immunogenicity against these "self" CT antigens. Herein, we predicted HLA-A*0201-restricted epitopes for all nine SSX family members, followed by validation with epitope molecular modeling, peptide/HLA-A*0201 affinity, and binding stability assays. We obtained four highly homologous candidate epitopes with the high immunogenicity scores designated P1, P4, P5 and P6, from the nine SSX members. Each of the four candidates could elicit strong epitope-specific CTL immune responses, but P4 could evoke more interferon gamma (IFN-gamma)-producing T cells and more potent CTLs that could lyse more target cells. Importantly, almost all of the four epitopes induced CTLs could cross-lyse the mutual targets both in vitro in human PBMCs and HLA-A2.1/K(b) transgenic mice, but P4 showed superiority to other epitopes in term of cross-cytolysis. All of these results demonstrate that P4 can induce anti-tumor immunity in a fashion superior to other candidates, and may be the "common" CTL epitope among all SSX-expressing tumors. Due to its documented responses herein, P4 has potential application in peptide-mediated immunotherapy.
The structure of the peptide-binding specificity of major histocompatibility complex (MHC) class I has been analyzed extensively in human and mouse. For fish, there are no crystallographic models of MHC molecules, neither are there data on the peptide-binding specificity. In this study, we describe for the first time the identification of a fish class I peptide-MHC ligand binding motif. Phage display technology using both 7 mer and 12 mer libraries enabled us to identify peptide ligands with unique specificity that interacts with the recombinant Salmon MHC class I molecule. The recombinant proteins, beta 2m/SasaUBA*0301, were produced in Escherichia coli, in which the carboxyl terminus of beta 2-microglobulin is joined together with a flexible (GGGGS)3 linker to the amino terminus of the heavy chain. One hundred and seven individual phages bound to beta 2m/SasaUBA*0301 were isolated after four rounds of panning from the 7 mer random-peptide library. The peptide encoding sequences were determined and peptide alignment led to the prediction of position-specific anchor residue. A prominent proline at position 2 was observed and we predict that it might be one of the anchors at the N-terminus. Meanwhile, phage display peptide library encoding random 12 mer peptides was also screened against beta 2m/SasaUBA*0301. Eighty-five percentages of the corresponding peptides have an enrichment of leucine, methionine, valine, or isoleucine at the C-terminus. We predict that this particular allele of Salmon class I molecule might have a very similar binding motif at the C-terminus compared with a known mouse class I molecule H2-Kb which has L, or I, V, M at p8. Previous work showed that Atlantic Salmon carrying the allele SasaUBA*0301 are resistant to infectious Salmon aneamia virus and there is a significant association between MHC polymorphism and the disease resistance. Therefore, our study might contribute to designing a peptide vaccine against this viral disease.
A novel TaqI restriction fragment length polymorphism (RFLP) of 4.15 kb is reported using a DR beta probe (pRTV1). This fragment corresponds to the DRB1 locus and allows the subdivision at the DNA level of the DRB1*0301 allele (DR3 antigen), which had not previously been reported. Both splits also distinguish each of the two DR3-bearing extended haplotypes (HLA-B8,SCO1,DR3,DQw2,Dw24 and B18,F1C30,DR3,DQw2,Dw25) found associated to several autoimmune diseases as insulin-dependent diabetes mellitus (IDDM), systemic lupus erythematosus (SLE) and myasthenia gravis. The fact that no polymorphism in the DRB1*0301 coding DNA sequence has been detected indicates that DRB1*0301 intronic, regulatory of neighbouring sequences might also contribute to differential disease associations (and pathogenic mechanisms) found linked to each of the two DR3-bearing haplotypes, i.e. IDDM and B8,DR3,Dw24 in North European/American Caucasoids vs IDDM and B18,DR3,Dw25 in Mediterraneans; SLE and B8,DR3,Dw24 in children vs SLE and B18,DR3,Dw25 in Spanish adults.
Human leukocyte antigens (HLA) are initially classified by serotyping but recently can be re-grouped by their peptide-presentation characteristics into supertypes. Both HLA-A*0301 and HLA-A*1101 are grouped into A3 supertype. Although a number of cross-presented T cell epitopes of HLA-A*0301 and HLA-A*1101 have been identified, the molecular mechanisms of cross-presentation remain elusive. Herein, the structures of HLA-A*0301 with two HIV-derived immunodominant T cell epitopes were solved and their characteristics in comparison with HLA-A*1101 presenting the same peptides were analyzed. The comparable structures of HLA-A*0301 and HLA-A*1101 with subtle differences illustrate the common modes of cross-presented peptides and the strict HLA-restriction of T cell receptor (TCR)-recognition.
To study variations in Rheumatoid Factor (RF) autoantibodies between and within healthy individuals, we have produced and analysed the variable heavy chain (VH) regions of 18 new monoclonal IgM RFs from the peripheral blood of two healthy subjects before and after immunization with tetanus toxoid (TT). The majority of these RFs used germline genes of the VH3 family, but RFs of the VH1, VH2 and VH7 families were also found. No RF of the VH4 RF is found. Fourteen different VH germline (GL) genes encoded the RFs, suggesting an extraordinary heterogeneity in structure. Consequently, changes in RF V region structures following immunization were difficult to identify. There were, however, structural differences between RFs from the two donors. RFs from one donor (IP) used more lambda light chains than RFs from the other donor and previously described RFs. In addition, 50% of the RFs from donor IP were encoded by GL genes frequently found to encode RFs from patients with Rheumatoid Arthritis (RA) but not described in RFs from other healthy subjects. The predominant use of VH3 RFs in the two healthy donors contrasts with the over-expression and expansion of VH1 RFs in one previously described healthy immunized donor. There are thus large individual differences in RF structures, which might be related to the immunological status, environment or genetic background of the donors. However, since these three donors are all HLA DRB1*0401, it is unlikely that this HLA type, associated with seropositive RA, accounts for the individual differences.
The aim of the present study was to assess the anti-inflammatory effects of selective ER beta (ER beta) agonist on lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) production in peritoneal macrophages (PMs) of endometriosis (EMS).
ER alpha (ER alpha) and ER beta expressions in PMs were analyzed by RT-PCR and immunoblot. The PMs of endometriosis were exposed to increasing concentrations of ER beta agonist ERB-041 over a period from 0.5 to 8h before stimulation with LPS and the levels of iNOS protein were evaluated by immunoblot. Subsequently, the PMs were pretreated with vehicle, ERB-041 or ER alpha agonist PPT before exposing to LPS. iNOS expression, p65 protein and active extracellular signal-regulated kinases (ERKs) level accumulated in the nuclear were detected by immunoblot. For experiment investigating the role of ERKs in LPS-induced iNOS expression, the PMs were pretreated with U0126, a specific ERK inhibitor, for 60 min before LPS treatment and iNOS expression was detected by immunoblot.
The PMs of EMS expressed ER beta to a greater extent compared with normal women. Pretreatment the PMs with ERB-041 resulted in a significant inhibition of LPS-induced iNOS expression and NF-kappaB activation by preventing its nuclear translocation. The ERKs pathway was involved in the LPS-induced iNOS production and was not repressed by the activation of ERs.
The inhibitory effect of ER beta agonist on LPS-induced iNOS production in PMs of EMS is likely mediated via repressing of nuclear factor-kappa B (NF-kappaB) but not ERKs signaling pathways.
We have re-examined the interaction of 1,10 phenanthroline with E, EAC1–8 and various other red cell intermediates. Two major findings emerged from this study: (1) 1,10-phenanthroline could mediate lysis of E, EAC4, EAC1–7 or EAC1–8. Under identical conditions of dose, pH and temperature, the rate (though not the extent) of lysis varied for different red cell intermediates, with EAC1–8 being lysed the most rapidly, followed by EAC1–7, EAC4 and E. (2) An intermediate could be isolated following the interaction of E and 1,10-phenanthroline that would lyse in the absence of fluid phase factors. This intermediate could be prevented from releasing its hemoglobin by addition of high molarity EDTA or EGTA. This cell was functionally similar to the E* state isolated during the lysis of E by anti-Forssman antibody and GPC.
1,25-Dihydroxyvitamin-D3 (1,25-D3) is known to inhibit DNA synthesis, immunoglobulin and lymphokine production [interleukin-2 (IL-2), gamma interferon (G-IFN), and granulocyte-monocyte colony-stimulating factor (GM-CSF)] by mitogen-stimulated human peripheral blood mononuclear cells (PBMCs). Recent data suggest these inhibitory effects are mediated at the gene level through inhibition of mRNA accumulation of specific lymphokines in the activated cells. In previous studies, we have demonstrated the CD8+ T cell population was less sensitive to the anti-proliferative actions of 1,25-D3 than CD4+ T cells. The purpose of this investigation was to further assess ability of 1,25-D3 to regulate CD4+ and CD8+ T cell functions. Initial experiments showed that 1,25-D3 inhibited both IL-2 production and mRNA accumulation in mitogen-stimulated PBMC. However, IL-2 receptor (IL-2R) expression and mRNA accumulation in stimulated PBMC was not affected by 1,25-D3. Both FACS sorted CD4+ and CD8+ T cells expressed IL-2R equally upon stimulation and neither showed an inhibitory effect on this expression by 1,25-D3. Human CD4+ and CD8+ T cells showed a stimulus-specific production of IL-2. CD4+ cells stimulated with mitogen and HLA-DR positive accessory cells produced measurable levels of IL-2 that were completely inhibited by 1,25-D3. CD8+ T cells did not generate measurable amounts of IL-2 in this system. However, CD4+ and CD8+ T cells produced large amounts of IL-2 when stimulated with mitogen and a protein kinase C activator, phorbol myristate acetate (PMA). Under these circumstances, both CD4+ and CD8+ T cell IL-2 production was inhibited completely by 1,25-D3. These data suggest that IL-2R expression in PBMCs and T cell subsets is equal and unaffected by 1,25-D3 while IL-2 production in T cell subsets is stimulus-specific and completely inhibited by 1,25-D3.
Human promyelocytic leukemia (HL-60) cells were induced by 1,25-dihydroxyvitamin D3 (calcitriol) to differentiate and examined using a panel of monoclonal antibodies (MoAbs) and functional assays. Although morphologically and histochemically these cells appeared to be of the monocyte-macrophage phenotype, there was a decline in Fc receptors for IgGl and no induction of class II HLA antigens. There was, however, dramatic induction of the antigen detected by the myeloid-specific MoAb AML-2-23. These data suggest that the phenotypic changes induced by calcitriol in HL-60 cells are consistent with myelomonocytic differentiation in that the resultant cells possess characteristics of both monocytes (morphology, non-specific esterase staining, high levels of AML-2-23 reactivity) and granulocytes (PMN 29 binding, decreased Fc receptors for IgGl, absence of class II HLA antigens). Perhaps more important, the ability of calcitriol-treated cells to perform antibody-dependent cellular cytotoxicity and phagocytosis was markedly augmented. Lysis of antibody-coated erythrocytes by HL-60 cells increased from 5% in controls to 30-35% with calcitriol treatment for 4 days. This enhanced effector cell function was seen despite a decline in Fc receptors measured by cytofluorography. These data suggest that calcitriol may be involved in both differential and functional activation of myeloid cells.
1,25-Dihydroxyvitamin D3 (1,25-D3) is known to have potent inhibitory effects on human peripheral blood mononuclear cell (PBMC) functions. Previous experiments suggest that addition of interleukin-2 (IL-2) to cell cultures can reverse the antiproliferative action of 1,25-D3. Previous studies have also shown that the CD4+ T-cell subset is more sensitive to the antiproliferative actions of 1,25-D3 than are the CD8+ T-cells. The objective of this study was to determine whether exogenous IL-2 could reverse the antiproliferative and immunoinhibitory action (inhibition of Ig production) in mitogen-activated PBMC cultures and in fluorescein-activated cell sorting (FACS) experiments where CD8+ T-cells were removed from PBMCs before mitogen stimulation with/without exogenous IL-2 added. In these studies, addition of IL-2 to mitogen-activated, 1,25-D3-treated PBMCs allowed the cells to overcome the 1,25-D3 suppressive effect on cell proliferation. However, exogenous IL-2 did not overcome the 1,25-D3-mediated inhibitory effect on PBMC Ig production. Using FACS lymphocyte populations (CD4+, CD8+ and B-cells), we showed that CD4+ T-cell-directed Ig synthesis in co-culture with autologous B-cells was inhibitable by incubation of cells with 1,25-D3, but Ig synthesis was restored to near-normal levels by addition of exogenous IL-2. This clearly contrasts with the inability of Il-2 to reverse the 1,25-D3 inhibitory effect on Ig synthesis in PBMCs. In other experiments, when CD8+ cells were removed from mitogen-stimulated, 1,25-D3-treated PBMCs, addition of exogenous IL-2 resulted in a full reversal of the 1,25-D3-mediated Ig inhibition. These data suggest that the inability of IL-2 to reverse the inhibitory effects of 1,25-D3 on PBMC Ig production is probably a result of a lack of sensitivity of CD8+ T-cells to the antiproliferative and immunoregulatory actions of 1,25-D3. This is possibly because of a differential expression of 1,25-D3 receptors on CD4+ and CD8+ T-cells.
The Vitamin D binding protein (DBP) is a multifunctional plasma protein that can significantly enhance the chemotactic response to complement fragment C5a. The chemotactic cofactor function of DBP requires cell surface binding in order to mediate this process. The goal of this study was to investigate the effect of ligating DBP with its two primary physiological ligands, Vitamin D and G-actin, on both binding to neutrophils and the ability to enhance chemotaxis to C5a. There was no difference in neutrophil binding between of the holo (bound) forms versus the apo (unbound) form of radioiodinated DBP, indicating that the cell binding region of DBP is likely distinct from the Vitamin D sterol and G-actin binding sites. Likewise, G-actin, 25(OH)D3, and G-actin plus 25(OH)D3 bound to DBP did not alter its capacity to enhance chemotaxis toward C5a. However, the active form of Vitamin D (1,25(OH)2D3) completely eliminated the chemotactic cofactor function of DBP. Dose-response curves demonstrated that as little as 1pM 1,25(OH)2D3 significantly inhibited chemotaxis enhancement. Moreover, at physiological concentrations 1,25(OH)2D3 needs to be bound to DBP to mediate the inhibitory effect. Neutrophil chemotaxis to optimal concentrations of C5a, formyl peptide, CXCL8 or leukotriene B4 was not altered by 1,25(OH)2D3, indicating that the active vitamin does not have a global inhibitory effect on neutrophil chemotaxis. Finally, inhibition of cell surface alkaline phosphatase (AP) with sodium orthovanadate completely reversed the inhibitory effect of 1,25(OH)2D3. These results indicate that the cell binding and co-chemotactic functions of DBP are not altered when the protein binds G-actin and/or Vitamin D. Furthermore, the co-chemotactic signal from DBP can be eliminated or counteracted by 1,25(OH)2D3.
Hymenoptera venom allergy is known to cause life-threatening and sometimes fatal IgE-mediated anaphylactic reactions in allergic individuals. About 30-50% of patients with insect venom allergy have IgE antibodies that react with both honeybee and yellow jacket venom. Apart from true double sensitisation, IgE against cross-reactive carbohydrate determinants (CCD) are the most frequent cause of multiple reactivities severely hampering the diagnosis and design of therapeutic strategies by clinically irrelevant test results. In this study we addressed allergenic cross-reactivity using a recombinant approach by employing cell lines with variant capacities of alpha-1,3-core fucosylation. The venom hyaluronidases, supposed major allergens implicated in cross-reactivity phenomena, from honeybee (Api m 2) and yellow jacket (Ves v 2a and its putative isoform Ves v 2b) as well as the human alpha-2HS-glycoprotein as control, were produced in different insect cell lines. In stark contrast to production in Trichoplusia ni (HighFive) cells, alpha-1,3-core fucosylation was absent or immunologically negligible after production in Spodoptera frugiperda (Sf9) cells. Consistently, co-expression of honeybee alpha-1,3-fucosyltransferase in Sf9 cells resulted in the reconstitution of CCD reactivity. Re-evaluation of differentially fucosylated hyaluronidases by screening of individual venom-sensitised sera emphasised the allergenic relevance of Api m 2 beyond its carbohydrate epitopes. In contrast, the vespid hyaluronidases, for which a predominance of Ves v 2b could be shown, exhibited pronounced and primary carbohydrate reactivity rendering their relevance in the context of allergy questionable. These findings show that the use of recombinant molecules devoid of CCDs represents a novel strategy with major implications for diagnostic and therapeutic approaches.
A series of substituted 2-aryl-1,3-indandiones were investigated for their ability to inhibit the complement system. Some of them were found to be considerably strong inhibitors. The inhibitory activity was mainly dependent on substitutions at positions 3 and 5 of the phenyl ring. 3,5-dichloro-(8), 3,5-bis(trifluoromethyl)- (7), 3,5-diisopropyl- (3) and 3,5-di-t-butyl- (5) phenylindandiones were the strongest inhibitors of the series. The generation of EAC1-5 cells from EAC1-3 cells and C5 was most strongly inhibited by these compounds although some inhibition of the interaction of EAC1-5 with C6-C9 and EAC1-6 with C7-C9 was also observed. Slight inhibition at other steps of complement activation was also seen but this was not considered to be appreciable. Dialysis of normal serum or purified C5 pre-incubated with compounds 3, 5, 7 and 8 did not cause recovery of the hemolytic activity of normal serum or purified C5. Thus, the main site of inhibition in the complement cascade appeared to be at C5. The total alternative pathway was also inhibited to some extent by these compounds, probably due to their interaction with C5.
Pattern recognition proteins (PRPs), such as lipopolysaccharide and beta-1,3-glucan binding protein (LGBP), have been identified in many animals and play a crucial role in invertebrate defense systems. In the current study, an LGBP gene was cloned from fleshy prawn (Fenneropenaeus chinensis, Fc-LGBP) utilizing homology cloning and RACE methods. The full cDNA of the Fc-LGBP gene in fleshy prawn was 1253bp in size with a deduced 366 amino acid protein that includes a glycosyl hydrolase domain. Northern blot and RT-PCR data suggested that Fc-LGBP mRNA was mostly synthesized in haemocytes and that the expression was down-regulated 24h post-injection of bacteria. In situ hybridization demonstrated that Fc-LGBP mRNA was only detected in haemocyte cytoplasm, with no detection in other tissues. The molecular weight of the purified recombinantly expressed Fc-LGBP was approximately 46kDa. Immunohistochemistry of haemocytes revealed that Fc-LGBP protein was localized on the membrane of most cells. Data from bacterial binding assays utilizing purified protein suggested that rFc-LGBP had strong binding activity to Gram-negative bacteria.
Nine distinct IgE-binding epitopes were identified along the entire amino acid sequence of the major latex allergen Hev b 2 (1,3beta-glucanase) using a set of synthetic 15-mer peptides frameshifted by 3 residues immobilized on cellulose membrane (Spot technique). Most of the amino acid residues building these IgE-binding epitopic regions are nicely exposed on the surface and the epitopes usually correspond to charged regions on the molecular surface of the protein. A smaller number of 5 IgE-binding epitopic areas was identified on the banana 1,3beta-glucanase, which exhibits a very similar overall conformation and charge distribution. The latter epitopes might be responsible for the IgE-binding cross-reactivity currently observed in the latex-fruit syndrome. Using rabbit polyclonal IgG anti-BanGluc as a probe instead of IgE from allergic patients the same epitopic regions were identified in both Hev b 2 and BanGluc. Additionally, surface-exposed regions with a very close conformation were predicted to occur on Ole e 9, the 1,3beta-glucanase allergen identified in olive pollen.
The VL amino acid sequence of an anti-lysozyme hybridoma protein, HyHEL-5, was determined. HyHEL-5 expresses a V region of the VK4 family and JK1. The VK4 family also includes light chains from galactan binding antibodies, although sequence comparisons suggest that a different member of this family is used to encode HyHEL-5. The HyHEL-5 light chain has a deletion of residue 96, such that L3 is one residue shorter than the majority of murine L3. Chain recombination experiments, employing H and L chains from different anti-galactan and anti-lysozyme binding antibodies, were performed to examine the contribution of the H and L chain in dictating specificity for either galactan or the lysozyme epitope recognized by HyHEL-5. The results indicate that, although the ability to bind galactan vs lysozyme is absolutely heavy-chain dependent, having the appropriate heavy chain is not sufficient for specific high affinity binding. Both the L chains from HyHEL-5 and J539 (a galactan-binding myeloma protein) were capable of supporting binding to galactan in combination with the J539 H chain, but affinity for galactan is less with the HyHEL-5 L chain. Only VK4 L chains supported binding of the HyHEL-5 heavy chain to the HyHEL-5 epitope, although binding with the J539 L chain was low affinity and relatively nonspecific.
A refined protocol for building a hypothetical model of the J539 Fv is described. Computer programs for positioning amino acid side chains and structure energy minimization [CHARMM program of Brooks et al., J. comp. Chem. 4, 187-217 (1983)] were employed. Computer modeling was accomplished on an Evans and Sutherland picture system which permitted structure visualization in three dimensions. Peptide backbone breaksites were rejoined by monitoring for correct distances and torsion angles. A physical model was then constructed and used as a basis for further refinements such as aligning conformations around remodeled sites, adjusting proline substitutions and optimizing hydrogen-bond-forming potentials. This structure (J539-ADO) was energy minimized and the final coordinates were obtained from the energy-refined model. The resulting hypothetical J539 structure can be compared to the structure of J539 now being determined by X-ray crystallography. The procedures described can be used for other Fv fragments.
The aim of this study was to produce the Bet v 1-related major hazelnut allergen Cor a 1.0401 and variants thereof as recombinant allergens, and to compare their immuno-reactivity with the major hazel pollen allergen using sera of patients whose hazelnut allergy recently was confirmed by double-blind placebo-controlled food challenges (DBPCFC) in a multicenter study. Total RNA was isolated from immature hazelnuts and transcribed into cDNA. Full length coding DNA obtained by PCR-strategy was subcloned into pTYB11 vector and expressed in E. coli ER2566 cells. Native non-fusion target proteins were purified by DTT-induced self-cleavage of the intein-tagged N-terminal fusion proteins. IgE reactivity of the recombinant allergens was tested by enzyme allergosorbent test (EAST), EAST-inhibition, immunoblot-inhibition and histamine release assays. Four recombinant allergens were produced showing deduced amino acid sequence identities among each other of 97-99%, and were considered as variants Cor a 1.0401 (GenBank Accession no.: AF136945), Cor a 1.0402 (AF323973), Cor a 1.0403 (AF323974) and Cor a 1.0404 (AF323975). Cor a 1.0402 and 03 only differed in a C4S exchange. Cor a 1.0404 had a unique proline residue in position 99. Surprisingly, only 63% identity was revealed with hazel pollen Cor a 1. EAST with 43 sera of patients with positive DBPCFC to hazelnut indicated IgE reactivity to Cor a 1.0401 in 95% of the sera, to Cor a 1.0402 in 93%, to Cor a 1.0403 in 91%, and in only 74% of the sera to the proline variant Cor a 1.0404. The allergenic activity of the four variants was confirmed by histamine release assays in 15 hazelnut-allergic patients stimulated with the four variants and controls. Eleven sera were positive with extract from native hazelnut, 13 with rCor a 1.0401, 12 with rCor a 1.0402, 11 with rCor a 1.0403, and only two with rCor a 1.0404 containing the proline exchange. The high IgE binding variant Cor a 1.0401 showed only partial IgE cross-reactivity with pollen Cor a 1. IgE-binding and histamine release capacity led to a concordant ranking of the allergenic activity of the recombinant variants: Cor a 1.0401>Cor a 1.0402 and 03>Cor a 1.0404 (the proline variant). Similar results for Cor a 1.0402 and 03 suggest a minor influence in IgE binding of cysteine in position 4, whereas proline in position 99 appears to be responsible for the decrease in IgE reactivity in Cor a 1.0404. It appears that the epitopes of hazelnut Cor a 1.04 are less related to pollen Cor a 1 than to Bet v 1 from birch pollen. Low IgE binding variants or mutants of Cor a 1.04 are candidate compounds for developing a novel and safe approach of specific immunotherapy of hazelnut allergy.
The binding properties and specificity of the SdJ5-1.17.15 (SdJ5) human IgM monoclonal antibody (mAb), prepared against S. minnesota R595 heat killed whole organisms, were assessed by dot blot assay in vitro. In that assay, lipopolysaccharide (LPS) related antigens, immobilized on nitrocellulose membrane, were reacted with the test and control human IgM antibodies. Results indicated that SdJ5 mAb reacted specifically with lipid A, a component of LPS, from a variety of bacterial species, but not with the whole LPS molecule. The inability of the mAb to react with whole LPS in dot blot assay was attributed to the possible effect of the solid phase on epitope exposure or structure. This hypothesis was tested by inhibition studies which indicated that liquid phase adsorption with LPS abolished or greatly reduced the specific recognition of solid phase lipid A by the mAb. These results indicated that LPS-associated antigens were recognized by the SdJ5 anti-lipid A mAb in liquid, but not solid, phase. In an attempt to identify the SdJ5-specific epitope on lipid A, the pattern of reactivities of different lipid A analogues with the mAb were examined by dot blot assay. Results indicated that a combination of the fatty acid side chains and the phosphate groups of lipid A (both groups being implicated as important for sepsis mediation) were either directly involved in the epitope structure or affected the exposure of epitope in solid phase. Because SdJ5 recognizes an LPS epitope on lipid A closely associated with endotoxin activity, this mAb could potentially be a useful therapeutic agent against septic shock.
A direct binding immunoradiometric assay (IRA) for Thy-1 antigen was developed to study the properties of membranous complexes shed from murine thymocytes and lymphoma cell lines. Monoclonal anti-Thy-1.2 antiserum was iodinated and used to study the shedding from AKR (Thy-1.1) and C3H(Thy-1.2) thymocytes, and S49.1(Thy-1.2), S49-Thy-1− and BW5147(Thy-1.1) continuous lymphoma cell lines. Culture supernatant fluids or purified shed complexes were allowed to bind to microtiter plates followed by measurement of the binding of iodinated anti-Thy-1.2. The assay was found to be completely specific for the Thy-1.2 allotype, and in conjunction with antibody coated wells could detect Thy-1 solubilized from cells with N-P40 detergent. Shed complexes containing Thy-1 from thymocytes and lymphoma cell lines were analysed by isopycnic centrifugation with continuous potassium tartrate gradients (5–40%). Shed complexes had a buoyant density of 1.06–1.10 g/cm3 as compared to 1.15–1.17 g/cm3 expected for murine leukemia virus or 1.20–1.24 g/cm3 expected for mycoplasma. We concluded that the shed membranous complexes had a buoyant density similar to plasma membrane and the complexes were similar from both thymocytes and lymphoma cell lines. Thy-1 was not associated with virus, mycoplasma or other particles found in the gradients and Thy-1 was not found in the unsedimented fraction. The release of Thy-1 from thymocytes and cultured cell lines results in only one defined density of particles which may participate in cellular communication or in the survival of malignant cells.
In non-excitable cells, store-operated Ca(2+) channels (SOCs) are the principal routes of Ca(2+) entry. Recently, store-independent Ca(2+) channels which are pharmacologically and/or immunologically similar to L-type Ca(2+) channels (LTCCs) have been shown to exist in various hematopoietic cells, including T cells, B cells and neutrophils. We previously reported that mast cells express LTCCs which regulate mast cell effector responses in a distinct manner from SOCs. In the present study, we examined the possible role for LTCCs in mast cell survival. Both RBL-2H3 mast cells and bone marrow-derived mast cells underwent considerable apoptosis after treatment with thapsigargin (Tg) but not stimulation through the high-affinity IgE receptor (Fc epsilon RI). The LTCC-selective antagonists such as nifedipine greatly augmented Fc epsilon RI-mediated apoptosis, while the LTCC-selective agonist (S)-BayK8644 blocked Tg-induced apoptosis. The modulation of apoptosis was accompanied by altered mitochondrial integrity, as measured with the mitochondrial membrane potential, cytochrome c release and caspase-3/7 activation. Fc epsilon RI stimulation induced mitochondrial Ca(2+) ([Ca(2+)](m)) entry through both SOCs and LTCCs, while Tg evoked [Ca(2+)](m) entry through LTCCs but not SOCs. The LTCC-selective antagonists blocked [Ca(2+)](m) entry, whereas (S)-BayK8644 augmented Tg-induced [Ca(2+)](m) entry. Moreover, blockade of the expression of the alpha(1C) subunit of Ca(v)1.2 LTCC using small-interfering RNA strongly augmented Fc epsilon RI-mediated apoptosis, mitochondrial integrity, and mitochondrial Ca(2+) collapse, and abolished the protective effects of (S)-BayK8644 against Tg-induced apoptosis. These findings suggest that Ca(v)1.2 LTCC protects mast cells against activation-induced cell death by preventing mitochondrial integrity disruption.
All cells continually survey their environment and make decisions based on cues encountered. This requires specific receptors that detect such cues, then transduce signals that initiate the appropriate responses. B lymphocytes provide an archetypal model for such 'adaptive' cellular responses, where signals transmitted by the B cell Ag-receptor (BCR) influence not only cellular selection, maturation, and survival, but are imperative in generating the ultimate effector function of B cells, i.e. antibody production. While other extracellular stimuli and their cognate receptor signals can also influence B cell development, BCR-mediated signals and the way in which they are integrated and regulated are paramount in defining the cell's physiological fate.
The circular dichroism (CD) spectra of five myeloma and six hybridoma proteins specific for phosphocholine were measured in the 250-310-nm range. The effect on the CD spectra of adding phosphocholine was also examined. The five myeloma proteins all had distinctive native spectra and, except for M603 and W3207, unique changes occurred on ligand binding. The hybridomas were chosen as pairs from each of the three known families of phosphocholine-specific immunoglobulins. Those from the T15 or M603 families resembled the appropriate prototype. However, the proteins from the M167 family were all distinctively different in their CD properties. In particular, the hybridoma protein 101.6G6 showed large CD changes on hapten binding and values for the association constant for phosphocholine of 1.1 X 10(5) M-1 and of 5.8 X 10(2) M-1 for acetylcholine were obtained by CD spectrophotometric titration. The CD properties of the proteins are interpreted in the light of the sequence data so far available, including the possible role of the D-segment.
We investigated the immunogenicity and the conformational properties of the non-repetitive sequences of the Plasmodium falciparum circumsporozoite (CS) protein. Two polypeptides of 104 and 102 amino acids long, covering, respectively, the N- and C-terminal regions of the CS protein, were synthesized using solid phase Fmoc chemistry. The crude polypeptides were purified by a combination of size exclusion chromatography and RP-HPLC. Sera of mice immunized with the free polypeptides emulsified in incomplete Freund's adjuvant strongly reacted with the synthetic polypeptides as well as with native CS protein as judged by ELISA and IFAT assays. Most importantly, these antisera inhibited the sporozoite invasion of hepatoma cells. In addition, sera derived from donors living in a malaria endemic area recognized the CS 104- and 102-mers. Conformational studies of the CS polypeptides were also performed by circular dichroism spectroscopy showing the presence of a weakly ordered structure that can be increased by addition of trifluoroethanol. The obtained results indicate that the synthetic CS polypeptides and the natural CS protein share some common antigenic determinants and probably have similar conformation. The approach used in this study might be useful for the development of a synthetic malaria vaccine.
The biological significance of TNF promoter polymorphism and infectious disease association prompted us to investigate whether TNF-α −308 G/A and −1031 T/C promoter polymorphisms are associated with Plasmodium vivax infection, cellular TNF-α level and possibly with clinical symptoms by employing PCR-RFLP methods. An overall significant elevation of serum TNF-α, IL-6 content (p = 0.0002, p = 0.002, respectively), whereas highly significant depletion of IL-10 content (p = 0.0001) was observed in vivax patients. In addition, TNF-α concentration in patients with and without fever were found to be significant (p = 0.0001, p = 0.0004, respectively). The genotypic distribution for −308 G/A and −1031 T/C positions were found non significant, but it was clinically potent to observe statistically significant distribution of genotypes (p = 0.032) in patients with and without fever. Furthermore, the TNF-α level in TNF1 and TNF2 genotype for −308 position was significantly higher (p = 0.010, p = 0.006 respectively). Incase of −1031 position TNF-α level was significant in ancestral (TT) genotype (p = 0.0007) in patients compared to healthy subjects and significantly higher in rare (CC) genotype (p = 0.021) as compared to ancestral genotype. In addition, the two polymorphisms 308G/A and −1031T/C were in highly significant LD (D′ = 0.7992, r² = 0.6005, p = 0.0001) in the patients as well as it is interesting to report that the distribution of novel 308A: 1031C alleles associated haplotypes are nearly the same in patients (0.2610) and in healthy subjects (0.2636).
Sera have been prepared in rabbits against the carboxy terminal cyanogen bromide fragment of horse cytochrome c (81-104) conjugated to bovine serum albumin (BSA). The specificity of these sera has been studied by radioimmunoassay and reveals that only peptide 81-104 binds the antibody. Neither whole cytochrome c nor peptides 1-65 and 66-80 displace 125I-81-104 from specific antibody, while cold peptides 81-104 is active in this regard, as expected. Fine specificity studies with isolated heterologous peptides 81-104 from the bovine, rabbit, canine, chicken, pigeon and tuna species of cytochrome c reveal that a major antigenic determinant is influenced by sequence positions 100 and 103 while a minor antigenic determinant is influenced by residue 92. These results are exactly mirrored and supported by inhibition studies with the isolated chymotryptic fragments 98-104 and 83-97. Studies with tryptic, chymotryptic and peptic digests of horse cytochrome c reveal that these natural proteases can generate peptides with the appropriate peptide sequences to react with antibodies produced against the peptide 81-104-BSA conjugate.
The BALB/c plasmacytoma 104E has been maintained in histocompatible mice for more than ten years. It secretes an IgM which binds α-1,3-dextran. The amino acid sequence of its λ light chain and μ heavy chain have been completely determined. Attempts to adapt 104E to continuous growth in culture led to the isolation of a ‘variant’ cell line (104-76), which produced IgM (μ,κ) and lacked dextran-binding specificity. We have purified the heavy chains from 104-76 and compared them with 104E μ. The 104-76 does secrete fully assembled IgM pentamers but the isolated μ chain has a different isoelectric focusing pattern from 104E μ. The cyanogen bromide fragments which orginate from the constant regions of 104E and 104-76 are the same size, but three of the four have a different charge, perhaps a result of the carbohydrate they bear. The cyanogen bromide fragments which originate all or in part from the variable region of 104-76 μ are completely different in both size and charge from the variable region fragments of 104E. These results indicate that 104E and 104-76 express completely different Vh genes. Therefore, 104-76 is unlikely to be a simple mutational derivative of 104E. Alternative explanations for the origin of 104-76 are considered.
Histones are frequent targets of self-reactive antibodies during autoimmune syndromes. We report the specificities and V region genes of three IgG anti-histone MAbs obtained from autoimmune mice. Each of the MAbs, named LG2-1, LG2-2 and BWA3, is directed against a different determinant located in the basic amino-terminal domain of core histones. LG2-1 reacts with a peptide from histone H3 (residues 30-45), LG2-2 recognizes the amino-terminus of H2B (residues 1-13) and BWA3 binds an epitope corresponding to a region of high sequence similarity between H2A and H4 (residues 1-20 and 1-29, respectively). The analysis of their V region sequences indicates that the H chain CDRs of these MAbs are remarkable for the presence of negatively charged amino acid residues that may play a role in the binding to cationic histones. The H chain importance in conferring reactivity to histones is corroborated by the observation that each of the VH gene segments of these MAbs is very similar to VH genes of previously described murine anti-histone antibodies.
In this study, a novel approach for the development of a peptide-based vaccine has been tested. We investigated the possibility of replacing an all-L amino acid peptide sequence corresponding to the protective B-cell epitope hemagglutinin (HA) 91-108 from influenza HA with a retro-inverso analogue encompassing this sequence. Retro-inverso peptides are composed of D-amino acids assembled in a reverse order from that of the parent L-sequence, thus maintaining the overall topology of the native sequence. This explains the observed antigenic cross-reactivity with anti-influenza virus antibodies. Mice immunized intranasally with the ovalbumin-conjugated retro-inverso analogue and cholera toxin as an adjuvant, produced strong systemic (serum IgG) and mucosal (lung IgA) antibody responses, and were protected against intranasal challenge with a lethal dose of influenza virus. The weight loss pattern in the protected group indicated that the vaccinated animals developed a disease of low severity resulting in a quick recovery. Furthermore, splenocytes of the immunized mice cultured in the presence of inactivated influenza virus, secreted high levels of IFN-gamma. The half-life of the retro-inverso analogue in the presence of lung homogenate proteases was at least 700 times greater than that of the parent L-peptide. These results demonstrate that peptidomimetic analogues with high resistance to proteolytic degradation are very effective immunogens when administered via the intranasal route, inducing protective immunity against a viral infection. This approach might be advantageous for vaccine development.
Hereditary angioedema (HAE) is an autosomal dominant disorder characterized by the deficiency of the inhibitor of the first component of complement system (C1-INH), which is due to mutations in its structural gene. There are two phenotypic variants: HAE type I, with reduced plasma antigen levels and HAE type II with normal antigen levels and reduced functional C1 inhibitor activity. The aim of this study was to determine the disease-causing mutations in 108 unrelated HAE families, followed at a single center in Italy, and in 50 normal controls by a genetic screening strategy of the C1-INH gene (SERPIN1G). To detect small mutations we either used fluorescence assisted mismatch analysis, followed by sequencing, or direct sequencing. Patients negative for mutations at this screening were further analyzed by long-range PCR to detect the presence of large deletions or insertions. Overall we identified 81 different mutations possibly responsible for the disease in 102 families, in the remaining 6 families no mutation was detected except for a synonymous substitution in a single probant. Sixty-seven of these mutations (23 missense, 22 frameshift, 8 splicing defects, 8 nonsense and 6 large insertion/deletions) had not been previously published. In addition, 4 rare variants, 2 synonymous alterations and 1 new polymorphism in the 3'UTR of the C1-INH gene were found. Mutations were distributed over all exons, at splice sites and in introns. Our study identified a large number of new mutations related to HAE providing additional evidence of the genetic heterogeneity of this disease. Our results also point toward particular amino acid residues important for protein function that may represent mutation hot spots.
The complement (C) inflammatory cascade is part of the phylogenetically ancient innate immune response and is crucial to our natural ability to ward off infection. It has three critical physiologic activities: (i) defending against microbial infections by triggering the generation of a membranolytic complex (C5b9 complex) at the surface of the pathogen and C fragments (named opsonins, i.e., C1q, C3b and iC3b) which interact with C cell surface receptors (CR1, CR3 and CR4) to promote phagocytosis. Soluble C anaphylatoxins (C4a, C3a and C5a) greatly control the local pro-inflammatory response through the chemotaxis and activation of leukocytes; (ii) bridging innate and adaptive immunity (essentially through C receptor type 2, CR2, expressed by B cells) and (iii) disposing of immune complexes and the products of the inflammatory injury (i.e., other danger signals, e.g., toxic cell debris and apoptotic corpses) to ensure the protection and healing of the host. The regulatory mechanisms of C are finely balanced so that, on the one hand, the deposition of C is focused on the surface of invading microorganisms and, on the other hand, the deposition of C on normal cells is limited by several key C inhibitors (e.g., CD46, CD55 and CD59). Knowledge of the unique molecular and cellular innate immunological interactions that occur in the development and resolution of pathology should facilitate the design of effective therapeutic strategies to fight selectively against intruders.
The immune response to a 37-amino acid synthetic peptide analogous to the carboxyl-terminal part (109-145) of the human chorionic gonadotropin beta subunit (beta hCG) was studied with monoclonal antibodies selected from 31 cell fusion experiments. Analysis of the immunogenic determinants borne on the synthetic peptide (CTP) showed a prevailing response to two immunodominant regions. The first was located on the 110-116 amino acid sequence of the CTP which is also the most hydrophilic region: 50% of anti-CTP antibodies selected for their high binding to 125I beta hCG were directed to this sequence. A second immunodominant portion was recognized by four antibodies, and comprised amino acids 134 to 139, representing a highly O-glycosylated region on the native protein. Moreover, a unique antibody designated FB13 bound to a region located on the last seven amino acids (139-145) of beta hCG. Finally, a hypothetical conformational determinant was recognized by antibody FB02 within the 121-145 region. Thus, the immune response to CTP was directed against two major and two minor regions. These antigenic determinants were demonstrated to be accessible for antibody binding on both the hCG molecule and its beta subunit. Localization of these epitopes suggests a relationship between the hydrophilicity and the immunological potency of different CTP regions.
Monoclonal antibodies (mAbs) are being increasingly used in cancer therapy owing to their ability to recognize specifically cancer cells and to activate complement- and cell-mediated cytotoxicity and/or to induce growth arrest or apoptosis. The therapeutic potential of anticancer antibodies is significantly limited due to the ability of cancer cells to block killing by complement. Of the multiple resistance strategies exploited by cancer cells, the expression of membrane complement regulatory proteins (mCRPs), such as CD46 (membrane cofactor protein (MCP)), CD55 (decay-accelerating factor (DAF)), CD35 (complement receptor type-1 (CR1)) and CD59, has received most attention. CD46, CD55 and CD35 block the complement cascade at the C3 activation stage and CD59 prevents assembly of the membrane attack complex of complement (MAC). These proteins protect normal tissues from accidental injury by activated complement, but also confer resistance on cancer cells, thereby limiting the effect of complement-fixing monoclonal antibodies. Expression of mCRPs on malignant cells is highly variable, yet there is clear indication that certain tumors express higher mCRP levels than the normal tissue from which they have evolved. mCRP level of expression and cellular location may also vary during malignant transformation and between differentiated and undifferentiated tumors. Neutralizing anti-mCRP mAbs have been used in vitro to elucidate the significance of mCRP expression to the tumor complement resistance phenotype. In general, CD59 appears to be the most effective mCRP protecting tumor cells from complement-mediated lysis. Nevertheless, it acts additively, and in certain tumors even synergistically, with CD55 and CD46. It is envisaged that treatment of cancer patients with mCRP blocking antibodies targeted specifically to cancer cells in combination with anticancer complement-fixing antibodies will improve the therapeutic efficacy.
The allergen Act d 11, also known as kirola, is a 17kDa protein expressed in large amounts in ripe green and yellow-fleshed kiwifruit. Ten percent of all kiwifruit-allergic individuals produce IgE specific for the protein. Using X-ray crystallography, we determined the first three-dimensional structures of Act d 11, produced from both recombinant expression in Escherichia coli and from the natural source (kiwifruit). While Act d 11 is immunologically correlated with the birch pollen allergen Bet v 1 and other members of the pathogenesis-related protein family 10 (PR-10), it has low sequence similarity to PR-10 proteins. By sequence Act d 11 appears instead to belong to the major latex/ripening-related (MLP/RRP) family, but analysis of the crystal structures shows that Act d 11 has a fold very similar to that of Bet v 1 and other PR-10 related allergens regardless of the low sequence identity. The structures of both the natural and recombinant protein include an unidentified ligand, which is relatively small (about 250Da by mass spectrometry experiments) and most likely contains an aromatic ring. The ligand-binding cavity in Act d 11 is also significantly smaller than those in PR-10 proteins. The binding of the ligand, which we were not able to unambiguously identify, results in conformational changes in the protein that may have physiological and immunological implications. Interestingly, the residue corresponding to Glu45 in Bet v 1 (Glu46), which is important for IgE binding to the birch pollen allergen, is conserved in Act d 11, even though it is not in other allergens with significantly higher sequence identity to Bet v 1. We suggest that the so-called Gly-rich loop (or P-loop), which is conserved in all PR-10 allergens, may be responsible for IgE cross-reactivity between Bet v 1 and Act d 11.
The effect of viral hemorrhagic septicemia virus (VHSV) was studied on the established rainbow trout (Oncorhynchus mykiss) monocyte/macrophage-like cell line RTS11. The virus was not able to complete its replication cycle as infectious viral particles were not released from the cells. However, in RTS11, the virus was capable of producing mRNA from at least its N and G genes. At the protein level, only N protein was detected 2 days post-infection, whereas a faint band corresponding to the G protein was also observed after 5 days post-infection. These results suggest an interruption of viral protein translation at some point. The expression of N mRNA was significantly inhibited in cells pre-treated with Poly I:C, but not affected by 2-aminopurine (2-AP), an inhibitor of the dsRNA-dependent protein kinase (PKR), thus indicating that PKR has no effect on mRNA expression directly. However, when cells were preincubated with Poly I:C in the presence of 2-AP, the levels of N mRNA were restored suggesting that Poly I:C can limit viral transcription through an antiviral mechanism dependent of PKR. The effect of VHSV on the expression of transcripts for different immune genes was determined, but significant induction was found only for genes related to the type I interferon (IFN) response, such as IFN-1 and -2 and the three Mx isoforms. Heat-inactivated virus failed to induce IFN-1 and -2, suggesting that early events in the VHSV life cycle were necessary for the type I IFN response. Poly I:C alone also induced transcripts for the antiviral Mx proteins. Prior exposure of RTS11 to VHSV did not prevent Poly I:C from inducing transcripts for Mx1, Mx2 and Mx3. Perhaps the failure of VHSV to disable antiviral mechanisms in RTS11 accounts for the aborted infections.
The yeast Malassezia sympodialis, which is an integral part of the normal cutaneous flora, has been shown to elicit specific IgE- and T-cell reactivity in atopic eczema (AE) patients. The M. sympodialis allergen Mala s 11 has a high degree of amino acid sequence homology to manganese superoxide dismutase (MnSOD) from Homo sapiens (50%) and Aspergillus fumigatus (56%). Humoral and cell-mediated cross-reactivity between MnSOD from H. sapiens and A. fumigatus has been demonstrated. Taken together with the recent finding that human MnSOD (hMnSOD) can act as an autoallergen in AE patients sensitised to M. sympodialis, we hypothesized that cross-reactivity could also occur between hMnSOD and Mala s 11, endogenous hMnSOD thus being capable of stimulating an immune response through molecular mimicry. Herein we demonstrate that recombinant Mala s 11 (rMala s 11) is able to inhibit IgE-binding to recombinant hMnSOD and vice versa, indicating that these two homologues share common IgE-binding epitopes and providing an explanation at a molecular level for the autoreactivity to hMnSOD observed in AE patients sensitised to Mala s 11. Using molecular modelling and mapping of identical amino acids exposed on the surface of both Mala s 11 and hMnSOD we identified four regions each composed of 4-5 residues which are potentially involved in IgE-mediated cross-reactivity. Mutated rMala s 11 molecules were produced in which these residues were altered. Native-like folding was verified by enzymatic activity tests and circular dichroism. The rMala s 11 mutants displayed lower IgE-binding in comparison to wild-type rMala s 11 using plasma from AE patients. In particular, mutation of the residues E29, P30, E122 and K125 lowered the IgE-binding to Mala s 11. The results of this study provide new insights in the molecular basis underlying the cross-reactivity between Mala s 11 and hMnSOD.
The anti-inflammatory cytokine IL-10 is a key modulator of immune responses. A better understanding of the regulation of this cytokine offers the possibility of tipping the balance of the immune response toward either tolerance, or enhanced immune responses. Histone deacetylases (HDACs) have been widely described as negative regulators of transcriptional regulation, and in this context, the primarily nuclear protein HDAC11 was shown to repress il-10 gene transcriptional activity in antigen-presenting cells (APCs). Here we report that another HDAC, HDAC6, primarily a cytoplasmic protein, associates with HDAC11 and modulates the expression of IL-10 as a transcriptional activator. To our knowledge, this is the first demonstration of two different HDACs being recruited to the same gene promoter to dictate divergent transcriptional responses. This dynamic interaction results in dynamic changes in the expression of IL-10 and might help to explain the intrinsic plasticity of the APC to determine T-cell activation versus T-cell tolerance.