Serum levels of chemokines CCL4 and CCL5 in cirrhotic patients indicate the presence of hepatocellular carcinoma.

BACKGROUND: Most hepatocellular carcinomas (HCCs) are diagnosed at an advanced stage. The prognostic value of serum tumour markers alpha-fetoprotein (AFP) and des-gamma-carboxy prothrombin (DCP) is limited. The aim of our study is to evaluate the diagnostic value of serum growth factors, apoptotic and inflammatory mediators of cirrhotic patients with and without HCC. METHODS: Serum samples were collected from cirrhotic potential liver transplant patients (LTx) with (n=61) and without HCC (n=78) as well as from healthy controls (HCs; n=39). Serum concentrations of CRP, neopterin and IL-6 as markers of inflammation and thrombopoietin (TPO), GCSF, FGF basic and VEGF, HMGB1, CK-18 (M65) and CK18 fragment (M30) and a panel of proinflammatory chemokines (CCL2, CCL3, CCL4, CCL5, CXCL5 and IL-8) were measured. Chi square, Fisher exact, Mann-Whitney U-tests, ROC curve analysis and forward stepwise logistic regression analyses were applied. RESULTS: Patients with HCC had higher serum TPO and chemokines (P<0.001 for TPO, CCL4, CCL5 and CXCL5) and lower CCL2 (P=0.008) levels than cirrhotic patients without HCC. Multivariate forward stepwise regression analysis for significant parameters showed that among the studied parameters CCL4 and CCL5 (P=0.001) are diagnostic markers of HCC. Serum levels of TPO and chemokines were lower, whereas M30 was significantly higher in cirrhotic patients than in HCs. CONCLUSIONS: High serum levels of inflammatory chemokines such as CCL4 and CCL5 in the serum of cirrhotic patients indicate the presence of HCC.

Association of pre- and early post-transplant serum amino acids and metabolites of amino acids and liver transplant outcome.

The aim of the present study was to investigate association of serum amino (AA) acids and metabolites of AAs with post-transplant outcome in liver transplant recipients. Eighty-nine patients with end-stage liver diseases and available pre- and early post-transplant serum were characterised as patients with (GI) and without one-year mortality (GII) and patients with and without early graft dysfunction (EAD). A panel of pre- and early post-transplant serum levels of AAs and early and metabolites of tryptophan were measured using tandem mass spectrometry. Patient groups had significantly higher pre-transplant serum levels of phenylalanine, tryptophan, and tryptophan metabolites than healthy controls (for all p<0.001). Pre-transplant serum levels of all these parameters were significantly higher in GI than in GII (for all p<0.001). GI had a higher MELD score and re-transplantation number than GII (p≤0.005 for both investigations). Serum bilirubin on day 5 and serum phenylalanine on day 10 post-transplant were associated parameters of mortality, whereas day 1post-transplant phenylalanine and kynurenine and female gender were associated parameters of EAD. Our results indicate that pre- and early post-transplant levels of phenylalanine, tryptophan and metabolites of tryptophan are increased in patients and are associated with EAD and one-year mortality in liver transplant recipients.

One-step single-chain Fv recombinant antibody-based purification of gp96 for vaccine development.

Heat shock proteins such as gp96 (grp94) isolated from tumor or infected cells are able to induce specific cytotoxic T-cell responses and protective immunity. To facilitate rapid and efficient isolation, we generated gp96-specific recombinant single-chain Fv (scFv) antibodies from a semisynthetic phage display library. When immobilized on Sepharose beads, these antibodies allow a high-yield, one-step purification of native gp96 molecules from both mouse and human tumor cell lysates. gp96 molecules eluted from these affinity columns under mild conditions are still capable of generating antigen-specific CTL responses in mice. Thus, scFv-purified gp96 is still associated with peptides; however, in contrast to conventionally purified gp96, scFv-isolated gp96 is free of contaminating material such as mitogenic concanavalin A and proteolytic cathepsins. With the help of these high-yield antibody columns, it is now possible to rapidly isolate immunogenic gp96-peptide complexes from small amounts of tumor material to a purity that allows their use in cancer immunotherapy protocols.

A new method for determining anti-B cell antibodies and their specificity using flow cytometry.

We describe a new flow cytometric B cell crossmatch method with improved sensitivity and specificity. It is based on the coating of B cell surface immunoglobulins with an unconjugated polyvalent anti-human immunoglobulin antibody. The method also provides a means for determining the specificity of anti-B cell antibodies and, potentially, for anti-HLA class II antibody specificity differentiation (DR, DQ or DP) in a binding inhibition test using mouse monoclonal antibodies directed against human HLA class II antigens.

Amino acid sequence based PCR primers for amplification of rearranged human heavy and light chain immunoglobulin variable region genes.

Previously described primers for PCR amplification of variable immunoglobulin (Ig) genes were based on gene sequences. To include the large number of amino acid sequences of antibodies whose DNA has not been sequenced and to ensure a maximal fit to rearranged human Ig variable region genes, we have made a comprehensive comparison of both protein and nucleotide sequences. The resulting set of 15 primers was able to amplify a wide range of rearranged antibody variable region genes. Restriction sites included in the primers facilitate cloning of the PCR products into various expression vectors. Sequence analyses of PCR-amplified cDNA derived from a polyclonal B cell population showed that maximal enrichment is obtained for highly represented variable Ig gene subgroups. Rarely occurring V kappa 4 and V lambda 5 subgroups were not detected. Rearranged Ig variable region genes from each of 19 human B cell lines were also amplified. Comparisons to germline sequences allowed the allocation of rearranged genes to the original Ig genes. This primer set should be very useful for generating large repertoires of rearranged V genes and for amplifying genes of individual B cell clones.

Monitoring of scFv selected by phage display using detection of scFv-pIII fusion proteins in a microtiter scale assay.

We describe here a method for the efficient and rapid analysis of antigen binding characteristics of recombinant antibodies (ab) selected by phage display. This novel approach combines the bacterial production of soluble single chain ab (scFv)-pIII fusion proteins on a microtiter scale with the detection of these fusion proteins via a pIII-specific ab. It facilitates the parallel analysis of large numbers of clones and is more efficient than current analysis protocols. Applying this technique, we analysed phage display selection of tetanus toxoid (TTX) specific scFv with respect to: (i) the productive expression of fusion proteins; (ii) the enrichment of specific scFv in subsequent rounds of phage display selection on a polyclonal level; (iii) the antigen specificity of individual scFv clones; (iv) the antigen binding affinity of a selected scFv. A TTX-specific scFv (clone 4.3) was further examined in a mono- and bivalent form by surface plasmon resonance analysis. ScFv 4.3 possesses a subnanomolar affinity and a low off rate constant.

Generation of a large complex antibody library from multiple donors.

We have generated a large complex library of single chain antibodies based on four individual libraries from each of 50 donors. DNA coding for the heavy and light chain variable domains of the IgM and IgG repertoires was amplified by PCR using two different sets of primers. Each individual library was composed of approximately 1-5x10(7) independent clones giving a final combined library of 4x10(9) members. Screening this library by phage display of single chain antibodies with small haptens, peptides and proteins yielded specific antibodies for each class of antigen.

Regulation of antibody response by an IgG-anti-Ig autoantibody occurring during alloimmunization. I. A few IgG molecules inactivate one B cell.

We have shown previously that alloimmunized rats develop a broadly reactive IgG-antiimmunoglobulin autoantibody in addition to antidonor antibodies. The findings presented herein demonstrate that this "physiological" antibody suppresses antigen receptor-induced IgM production of B cells derived from rats of the same strain. When affinity-purified IgG-anti-Ig was added to cell cultures, the antibody production of B cells was maximally inhibited at the minute concentration of 0.9 pg/10(6) cells. Higher or lower IgG-anti-Ig concentrations resulted in weaker suppression. The same result was obtained when spleen lymphocytes were used instead of purified B cells. Based on the molecular weight of IgG and Avogadro's number, our results indicate that a few molecules of IgG-anti-Ig are sufficient to inhibit the antibody production of a single B cell. Activity at this minuscule concentration demonstrates that IgG-anti-Ig antibodies are exquisitely active immunoregulatory molecules. In addition to the stimulatory effect of IgM-anti-Ig rheumatoid factors reported by others, our findings define the second component of an immunoregulatory mechanism: suppression of the B cell response by an IgG-anti-Ig autoantibody produced during alloimmunization.

Suppression of antibody response to transfusions in rats by preconditioning with antibody-coated cells.

In a Lewis-BN rat model, the antibody response to transfused blood cells from strongly histoincompatible donors was suppressed by preincubation of blood with specific antidonor antiserum or broadly reactive heterologous antilymphocyte serum (ALS). Suppression was achieved even when excess antiserum was removed by washing prior to injection. The suppressive effect was dose-dependent. Broadly reactive ALS was even more effective than specific antidonor antiserum in inhibiting the primary antibody response. Lewis animals pretreated three times with ALS-coated BN blood showed no secondary antibody response against subsequent BN-blood booster injections. Our experiments may be relevant for the prevention of undesired sensitization to donor-specific transfusions prior to related donor kidney transplantation.

Induction of a suppressive serum factor, prevention of sensitization, and prolongation of kidney graft survival in rats by transfusion with antibody-coated blood cells.

Transfusion with antibody-coated allogeneic blood cells suppresses the cytotoxic antidonor antibody response in a strongly incompatible rat combination (BN----LEW). Cell coating with homologous recipient antidonor antiserum, rat monoclonal antibodies against MHC class I donor antigens, or rabbit antirat lymphocyte serum all were effective. The suppression was not abrogated by repeated booster transfusions with untreated donor blood. Moreover, the suppression extended to antibody-uncoated antigens present on the same donor cell. Not only the antibody response but also the Graft-versus-host reaction against donor antigens was suppressed. The serum of pretreated animals contained suppressive activity. It suppressed the cytotoxic antibody response as well as the cellular immune response (GVH) when transferred into syngeneic recipients. A weaker suppression of antibody response was obtained by transferring spleen cells of pretreated animals into syngeneic recipients. The transfer data suggest that broadly reactive serum factor(s) were mainly responsible for the suppressive effect. Transfusion with LEW-anti-BN-coated donor cells before transplantation induced markedly prolonged kidney graft survival in the BN----LEW combination without additional immunosuppression (untreated controls: 8.4 +/- 0.4, pretreated recipients: 124 +/- 36 days, P less than 10(-4)).

Regulation of antibody response by an IgG-anti-Ig autoantibody occurring during alloimmunization. II. Selective inactivation of antigen receptor-occupied B cells.

Heterologous antiimmunoglobulin antibodies are efficient regulators of the B cell response. We have shown that during the immune response against allogeneic cells the immune system develops autologous IgG-antiimmunoglobulin. A few molecules of this "physiological" autoantibody suppress the IgM production of one B cell in vitro. In the current series of experiments we further define the regulation of antigen receptor-activated B cells by this autoantibody. To mimic the in vivo situation, where IgG-anti-Ig appears a few days after alloimmunization, the antibody's effect on an already ongoing B cell response was studied. Interestingly, we found that the IgG-anti-Ig loses its suppressive effect when added to the cell culture 1 or 2 days after B cell activation, but that suppression can be completely restored when the cells are restimulated via their antigen receptor. Thus, the IgG-anti-Ig antibody suppresses B cells only when their antigen receptor is occupied. Even restimulated B cells become refractory 8 hr after activation, and later (24 hr) regain their susceptibility to IgG-anti-Ig-induced suppression. The Fc receptor is involved in mediating suppression since the antibody's suppressive capacity is abolished after removal of its Fc region. Possible mechanisms of B cell suppression by IgG-anti-Ig are crosslinking of antigen receptor with Fc receptor, or cocapping and functional interaction of the two receptors as a result of their separate occupancy. Our experiments demonstrate that B cell regulation by IgG-anti-Ig produced during an immune response to allogeneic cells is governed by 3 restriction mechanisms: antigen receptor occupancy, activation stage dependency, and optimal antibody concentration.

A B cell-suppressive IgG-antiimmunoglobulin antibody induced by alloimmunization.

This study is an extension of our previous report that alloimmunization produces a serum factor that suppresses the lymphocytotoxic antibody response. The experiments presented herein show that: (1) serum and IgG of pretreated rats contain an antibody directed against the constant region of IgG; (2) the anti-IgG has strong anti-Fc-gamma and weak anti-Fab activity; (3) the antibody also reacts with IgM; (4) the antiimmunoglobulin is an autoantibody; and (5) the antiimmunoglobulin suppresses the primary B cell response in vitro.

The association of kidney graft outcome with pretransplant serum IgG-anti-F(ab')2 gamma activity.

Pretransplant sera of 474 kidney graft recipients were tested for IgG-anti-F(ab')2 gamma activity. The patients had significantly higher IgG-anti-F(ab')2 gamma activity than healthy controls (P = 0.0004). Serum lymphocytotoxic antibodies were correlated with IgG-anti-F(ab')2 gamma (P = 0.004), whereas CMV infection and blood transfusions were not. We found a significant association between pretransplant IgG-anti-F(ab')2 gamma activity and early and 1-year kidney graft outcome. This association was pronounced in recipients with no lymphocytotoxic antibodies. Recipients with immediately functioning grafts and a creatinine < 130 mumol/L at 1 year had strikingly higher pretransplant IgG-anti-F(ab')2 gamma activity than patients with graft failure (P < 0.0001).

Role of anti-IgG autoantibodies in kidney transplantation.

To demonstrate whether anti-IgG autoantibodies of different isotypes and specificities play a role in kidney transplantation, pretransplant sera of recipients (a) with well-functioning grafts (n = 40); (b) with reversible rejection episodes that were treated successfully (n = 63); and (c) with irreversible graft rejection (n = 40) were tested for four different anti-IgG autoantibody activities. A protective effect of IgG-anti-F(ab)2 gamma antibodies on graft survival was observed (p less than 0.01). High pretransplant IgG-anti-Fc gamma activity was found to be associated with a low kidney graft survival rate (p less than 0.02). Pretransplant IgM-anti-F(ab)2 gamma and IgM-anti-Fc gamma activities showed no effect on kidney graft outcome.

Long-lasting kidney graft survival after immunization with antibody-coated blood cells: mediation by immunosuppressive autoantibodies.

Treatment of LEW rats before transplantation with BN blood cells preincubated with LEW-anti-BN serum resulted in prolonged BN kidney graft survival (untreated, 8 +/- 0.4 days; pretreated, 124 +/- 36). The serum of pretreated animals contains a factor which suppresses T cell proliferation against donor antigens. This effect was not mediated by antiidiotypic antibodies because it was reproduced in a donor-unrelated system. The serum and IgG of pretreated animals also suppressed the humoral immune response. As for T cells, the antibody-suppressive effect was not donor-restricted. A broadly reactive anti-immunoglobulin (anti-B cell) autoantibody was found in the IgG fraction of immunized animals. The mediation of immunosuppression by broadly reactive anti-B and T cell autoantibodies induced by the immunization with antibody-coated blood cells is discussed.

Idiotypic vaccine for treatment of human B-cell lymphoma. Construction of IgG variable regions from single malignant B cells.

Immunoglobulin idiotypes (Id) of malignant B cells represent highly specific markers which can be used for vaccination. PCR-amplification of immunoglobulin genes enables the rapid production of large amounts of Id vaccines. However, the separate amplification and subsequent recombination of heavy and light chains can lead to a loss of the relevant Id. To preserve the original chain pairs, we used single malignant B cells derived from an immunocytoma patient. Cytoplasm was extracted and the mRNA transcribed into cDNA. The VH and VL genes were then amplified by PCR and cloned into a vector for expression in E. coli. Id production was checked using an anti-Id mouse monoclonal Ab raised against the patient's tumor-specific IgG. One out of 3 constructs expressed the relevant Id. Analysis of the first 31 light chain residues revealed an identical sequence for the malignant B cells' IgG and the recombinant Id construct. Exchange of either the heavy or light chain with an unrelated chain resulted in loss of the Id. An unrelated sequence derived from the c-myc protein is coupled to the Id vaccine. The lymphoma patient was shown to have Abs to the c-myc sequence. This sequence therefore, increases the Id+ Ab's antigenicity. CD spectroscopy showed an alpha-helical structure for the c-myc epitope. In conclusion, a B-cell lymphoma autovaccine was produced containing immunogenic sequences that do not alter the steric conformation of the tumor-specific Id.

The antigen binding domain of non-idiotypic human anti-F(ab')2 autoantibodies: study of their interaction with IgG hinge region epitopes.

In previous studies we described a natural human IgG-anti-F(ab')2 autoantibody family with immunoregulatory properties. Genes coding for the variable regions of the heavy and light chains of the Abs were isolated from a natural Ig gene library and scFv Abs were expressed in E. coli. The scFv Abs bound to F(ab')2 but not to Fab fragments. This points to an epitope located in the hinge region since Fab fragments are lacking most of the hinge. In order to verify our hypothesis, double chain peptides comprising the lower-, middle-, and part of the upper hinge subregion of IgG1-IgG4 were synthesized on cellulose membranes and tested for binding to the Abs. The results show binding of Abs to IgG1 and IgG4 hinge region peptides. In order to identify the key residues of the discontinuous epitopes we carried out complete substitutional analyses in which each amino acid of the wt peptides was substituted by all other amino acids except cysteine. The exchange of proline in the IgG1 or IgG4 middle hinge region abrogated the binding, revealing the importance of this subregion for epitope expression. No binding to the IgG2 or IgG3 hinge was detected. These results indicate that scFv anti-F(ab')2 Abs recognize the hinge region of IgG1 and IgG4 and that the expression of the epitope depends on an intact middle hinge subregion.

Restriction mechanisms of B cell regulation by a physiological IgG-anti-immunoglobulin autoantibody.

Immunization of LEW rats with strongly histoincompatible BN blood cells induces, in addition to anti-donor antibody, a broadly reactive IgG autoantibody which binds to IgG and IgM molecules (IgG anti-Ig). Minute amounts of affinity purified IgG anti-Ig (0.2 pg/10(6) cells) suppress the antibody production in vitro of antigen receptor (AgR)-stimulated B cells derived from rats of the same strain. The suppressive antibody is also active in the whole serum IgG fraction. Importantly, anti-Ig-induced suppression is governed by restriction mechanisms: only AgR-occupied B cells are affected, the suppression is cell cycle dependent, and maximum suppression is obtained at an optimum IgG concentration. Treatment of rats in vivo with 0.8 mg Ig-anti-Ig (whole IgG fraction) along with allogeneic cells resulted in nearly complete suppression of the anti-donor antibody response. Possible mechanisms of B cell suppression by IgG anti-Ig are crosslinking of AgR with FcR, or cocapping of the two receptors with sterical interaction as a consequence of their separate occupation. Both alternatives lead to the release of an inactivating signal.

Short-term T cell receptor directed immunotherapy induces organ specific peripheral tolerance in a strongly incompatible rat model.

We analysed the effect of selective alpha/beta-T cell elimination on allograft survival in a strongly histoincompatible DA-->LEWIS rat model by treatment of recipients with the mouse monoclonal antibody R73 two times before and seven times after transplantation. R73 induced virtually indefinite cardiac allograft survival in 44% of six-week-old LEWIS recipients, whereas donor-type skin allografts were rejected within 11 days. The remaining 56% of animals presented a mean cardiac survival time of 41 +/- 13 days. Graft prolongation was age dependent since in ten-week-old animals the survival time was only of 19 +/- 5 days (untreated controls: 7 +/- 1 days). R73 induced a rapid decrease of R73-positive T cells in the peripheral blood from 70% before treatment to 2%. From the fifth day of treatment a gradual T cell recovery was registered. The T cell marker CD5 decreased from 72% to 17% but recovered already from the second day of treatment. Determination of alpha/beta-TCR, CD3 and CD5 density on T cells during R73 therapy showed that the initial T cell decrease was due to T cell elimination, whereas modulation of alpha/beta-TCR was predominant during the following days. Anti-R73 antibodies appeared regularly during the first week of treatment and blocked R73 activity, indicating their anti-idiotypic nature. The present findings show that short-term R73 therapy is able to induce long-lasting allograft survival. This experimental model can be used to study the basis of peripheral organ specific T cell tolerance.

The T-cell suppressive effect of bufadienolides: structural requirements for their immunoregulatory activity.

Many studies indicate that substances similar to cardenolides and bufadienolides naturally occur in mammals. The majority of previous studies focused on their cardiovascular, renal, and central nervous action. We analyzed the immunoregulatory property of 52 bufadienolides. Human T-cells were stimulated "in vitro" with mitogens or alloantigens in the presence of bufadienolides. The most active compound totally inhibited T-cell activity at a concentration of 0.75 pmol/10(5) cells. This effect is 16,384 x stronger than that of cortisol and 256 x stronger than that of cyclosporin A or tacrolimus. Preactivated T cells were downregulated and, most importantly, suppressed viable T cells could not be restimulated. Lack of the 17 beta-lactone ring dramatically reduced the activity of bufadienolides. Substitution at C3 also affected their function: components with a 3-OH group were up to 1000 x stronger than those without. The replacement of 14 beta-OH with an epoxy-group slightly decreased the activity. Because there is evidence that the latter change abolishes the cardiac activity, this finding is relevant for therapeutic applications in which immunosuppression without the risk of cardiotoxicity is attempted. One of the substances analyzed in this study was Proscillaridin A. A similar bufadienolide occurs naturally in mammals. We speculate that bufadienolides represent an important bioregulatory link between the cardiovascular, nervous and immune systems.