Episodic Src activation in uveal melanoma revealed by kinase activity profiling.

BACKGROUND: The RAS/RAF/MEK/ERK pathway is involved in the balance between melanocyte proliferation and differentiation. The same pathway is constitutively activated in cutaneous and uveal melanoma (UM) and related to tumour growth and survival. Whereas mutant BRAF and NRAS are responsible for the activation of the RAS/RAF/MEK/ERK pathway in most cutaneous melanoma, mutations in these genes are usually absent in UM. METHODS: We set out to explore the RAS/RAF/MEK/ERK pathway and used mitogen-activated protein kinase profiling and tyrosine kinase arrays. RESULTS: We identified Src as a kinase that is associated with ERK1/2 activation in UM. However, low Src levels and reduced ERK1/2 activation in metastatic cell lines suggest that proliferation in metastases can become independent of Src and RAS/RAF/MEK/ERK signalling. Inhibition of Src led to the growth reduction of primary UM cultures and cell lines, whereas metastatic cell line growth was only slightly reduced. CONCLUSION: We identified Src as an important kinase and a potential target for treatment in primary UM. Metastasis cell lines seemed largely resistant to Src inhibition and indicate that in metastases treatment, a different approach may be required.

Effect of secondary structure on single nucleotide polymorphism detection with a porous microarray matrix; implications for probe selection.

Oligonucleotide arrays capable of detecting single nucleotide polymorphisms (SNPs) from amplified nucleic acid have many applications. The expected SNP is usually placed approximately in the center of the probe to ensure the maximum shift in Tm between complementary and SNP sequences. Unfortunately, different short probes (< 30 bases) selected using widely accepted criteria do not perform consistently in this type of assay. Here we present a systematic study on the effect of secondary structure on the ability of oligonucleotide probes to detect an SNP, using real-time array monitoring of a porous microarray substrate that incorporates a novel intra-array mixing system. These results demonstrate that, although positioning of an SNP in the middle of the probe is highly destabilizing, the effect of stable secondary structure on the signal obtained is so dramatic that such probes may be very insensitive. Therefore, if the SNP flanking sequence contains significant secondary structure, then more sensitive probes with good specificity may be obtained by positioning the mutation towards one end of the probe.

Vernier zone residue 4 of mouse subgroup II kappa light chains is a critical determinant for antigen recognition.

BACKGROUND: During the conversion of murine monoclonal antibodies directed against the human chorionic gonadotropin (hCG) into bacterially expressed single chain fragments (scFv), we found a major reduction of binding activity upon introduction of a primer encoded mutation. OBJECTIVES: In this study we tried to determine which mutation was responsible and on what manner this mutation affected antigen binding (structural effect versus direct involvement of the residue in binding). RESULTS: No binding could be detected, when the wild type residue methionine at position 4 within the Framework region 1 of the Vkappa light chain was substituted by serine in two antibodies with a subgroup II kappa light chain. However, a similar replacement within an anti-hCG antibody with a subgroup IV kappa light chain and thereby having leucine as wild type residue, did not affect the binding characteristics. The mutant scFv's derived from both AB-s sensitive for substitution by serine never reacted with antigen in ELISA. Analysis with surface plasmon resonance revealed a residual binding only on a sensorchip with a high density coating of antigen; however, an increased dissociation, relative to that of the wild type scFv and the absence of reactivity in ELISA suggest a drastically altered affinity. CONCLUSION: A structural explanation for the changed binding characteristics can be the influence of the position 4 residue, as being a constituent of the Vernier zone, on the position of the CDR1 loop of Vkappa, which might harbour residues that directly bind to antigen, or indirectly positions other variable loops of the binding pocket. An increased sensitivity for trypsin digestion supported the hypothesis of a local conformational change in the serine mutant of the subgroup II kappa containing antibody.

Absolute conservation of residue 6 of immunoglobulin heavy chain variable regions of class IIA is required for correct folding.

While studying the expression of single-chain antibodies (scFv) derived from several murine monoclonal antibodies, we found that residue 6 in Framework region 1 of the heavy chain variable domain plays a crucial role in antibody folding. Binding activity of three murine antibodies with a heavy chain variable region (VH) subgroup IIA was completely lost when at this position the wild-type residue glutamine (Q) was substituted by glutamate (E). Increased sensitivity towards trypsin digestion of soluble scFv suggested that the lack of binding activity was caused by incorrect folding of Q6E mutants. Grafting of the three additional class IA derived FR1 residues, based upon the comparison between both classes of VH sequences, on to the 'defect' subgroup IIA sequence, partially restored the antigen binding activity of the Q6E-containing scFv. Our results suggest that residue 6 of the heavy chain may be part of a folding nucleus, involving the first two beta-strands of Framework region 1. The evolutionary conservation of either glutamine or glutamate at position 6 in different antibody families may well indicate that within immunoglobulin VH domains, different family specific folding nuclei have evolved.

Selection of human anti-human immunodeficiency virus type 1 envelope single-chain antibodies from a peripheral blood cell-based phage repertoire.

Monoclonal antibodies play an important role in the development of diagnostic assays. Instead of using hybridoma technology to isolate human immunodeficiency virus type 1-specific antibodies, a phage-displayed antibody library was generated from a small number (10(7)) of peripheral blood lymphocytes from a seropositive donor. Two families of single-chain antibodies (scFvs) were selected by biopanning with the envelope precursor gp160. ELISA and competition in the BIAcore system revealed that one antibody family recognized a conformation-sensitive epitope within gp120, while the other antibody family was gp41-specific. The latter group had sequence similarity to antibodies recognizing the cluster III epitope of gp41. Binding of scFvs to gp160 could be inhibited with the donor's serum antibodies, indicating that antibodies with a similar specificity were circulating in the donor's blood. Competition experiments suggested that the epitope of the anti-gp41 antibodies was recognized by a broad range of patients' sera: 21 out of 22 sera from North American and all 20 sera from African seropositive patients inhibited binding of scFvs. In contrast, three sera from this panel did not react with the epitope of the anti-gp120 antibodies. These data indicate that, because of the conserved nature of its epitope, the anti-gp41 antibody will be suitable for diagnostic applications.

Determination of active single chain antibody concentrations in crude periplasmic fractions.

We have measured active single chain antibody (scFv) concentrations under mass transfer limitation conditions using surface plasmon resonance on the BIAcore. For the creation of a standard curve scFv 4Dwt, derived from monoclonal antibody (mAb) 4D, directed against human chorionic gonadotropin (hCG), was purified by positive affinity chromatography. Determination of the active antibody fraction after purification was performed using anti-FLAG, reactive against a tag sequence C-terminally fused to the scFv. Two independent experiments showed that the activity remaining represented over 75% of the total amount of purified protein. Calibration curves on high density antigen surfaces showed a linear relationship between antibody concentration and binding rates. Periplasmic fractions of six mutant scFvs, also derived from mAb 4D, revealed a clear difference in the amount of soluble active scFv expressed in the periplasm of E. coli compared with the total amount of antibody present, indicating the necessity of measuring active antibody concentrations. This rapid concentration determination method will be particularly useful for accurately comparing affinity constants, using antibody concentrations determined with the BIAcore, of the many different scFv fragments routinely isolated from phage display libraries.

Significance of the immune response to a major, conformational B-cell epitope on the hepatitis C virus NS3 region defined by a human monoclonal antibody.

The nonstructural protein NS3 of hepatitis C virus (HCV) possesses two enzymatic domains which are thought to be essential for the virus life cycle: an N-terminal serine-type proteinase, responsible for the processing of nonstructural polypeptides, and a C-terminal nucleoside triphosphatase/helicase, presumably involved in the unwinding of the viral genome. The human antibody response to NS3 usually appears early in the course of HCV infection and is predominantly directed against the carboxyl-terminal portion; however, its fine specificity and clinical significance are largely unknown. We have generated a human monoclonal antibody (hMAb), designated CM3.B6, from a cloned B-cell line obtained from the peripheral blood of a patient with chronic HCV infection, which selectively recognized the purified NS3 protein expressed in bacteria or in eukaryotic cells transfected with full-length or NS3 cDNA. Fine-specificity studies revealed that CM3.B6 recognized a 92-amino-acid sequence (clone 8, amino acids 1363 to 1454) selected from an NS3 DNase fragment library but failed to bind to 12-mer peptides synthesized from the same region, suggesting recognition of a conformational B-cell epitope. Experiments using deletion mutants of clone 8 and competitive inhibition studies using a panel of NS3 peptide-specific murine MAbs indicated that limited N-terminal and C-terminal deletions resulted in a significant reduction of hMAb binding to clone 8, thus identifying a minimal antibody binding domain within clone 8. Competition experiments showed that binding of CM3.B6 to the NS3 protein was efficiently inhibited by 39 of 44 (89%) sera from HCV-infected patients, suggesting that the hMAb recognized an immunodominant epitope within the NS3 region. More importantly, recognition of the sequence defined by CM3.B6 appeared to accurately discriminate between viremic and nonviremic anti-HCV positive sera, suggesting potentially relevant clinical applications in the diagnosis and treatment of HCV infection.

A human monoclonal antibody specific for the N terminus of the hepatitis C virus nucleocapsid protein.

PBMC from a patient with chronic hepatitis C virus (HCV) infection were immortalized with EBV and plated by limiting dilution. Cultures secreting antibodies reactive in a commercial HCV II generation ELISA, which incorporates Ag derived from the nucleocapsid, NS3, and NS4 regions, were repeatedly cloned in the presence of feeder cells and growth factors. Of 23 initially immunoreactive cultures, only one cloned line, designated B12.F8, secreted HCV nucleoprotein-specific IgG1(kappa), whereas no reaction with recombinant polypeptides derived from NS3, NS4, and NS5 regions were documented. Human mAb (hmAb) B12.F8 was shown to recognize the native HCV nucleoprotein expressed in eukaryotic cells transfected with a core cDNA construct by immunofluorescence. The fine specificity of this hmAb was evaluated using synthetic oligopeptides covering the entire HCV nucleocapsid region. A weak but consistent reactivity was observed by PEPSCAN using a 12-mer encompassing residues 34-45 of the HCV-deduced amino acid sequence. Such weak reactivity is indicative for conformational epitopes and, in concurrence with this assumption, we found that longer peptides from the region containing residues 27-59 were more efficiently recognized and effectively inhibited binding of hmAb B12.F8 to recombinant nucleocapsid protein. Several overlapping immunoreactive fragments from the nucleocapsid region were selected from a random cDNA library consisting of DNase I fragments of recombinant core Ag. Best reactive recombinants were identified within residues 1-78 of the HCV sequence, in agreement with the results obtained using synthetic peptides. Comparative experiments on the fine specificity of sera from HCV-infected patients with anticore antibodies invariably showed recognition of peptides 8-40 and 27-59, as well as recombinant fragments spanning from residues 1 to 73, suggesting that hmAb B12.F8 identifies a major B cell epitope within the immunodominant nucleoprotein amino terminal subregion.

Nucleosomal fragments in serum may directly reflect cell-mediated cytotoxic activity in vivo.

In in vitro experiments the activity of cytotoxic T-cells and natural killer cells has been shown to cause nuclear DNA fragmentation leading to the release of nucleosomal multimers from the target cells. These multimers form a ladder-like pattern with a periodicity of approximately 200 bp during gel electrophoresis. The objective of the present study was to show the relevance of the presence of these nucleosomal multimers in vivo during diseases that show cell-mediated cytotoxicity. Nucleosomal multimers (n greater than 5) could be detected using nick translation followed by electrophoresis in a series of sera of a chimpanzee infected with hepatitis A virus and in sera drawn from several hepatitis B patients. The multimers were present during periods expected to show an increased activity of cell-mediated cytotoxicity in the liver. During these periods the injury of the liver cells was also mirrored by the classical parameter, the release of a specific liver enzyme into the serum. The liver enzyme activity in the serum and the detection of the nucleosomal multimers did not completely overlap, however. It is postulated that the proposed nick translation assay is useful as a simple diagnostic test for cell-mediated cytotoxicity since it reflects this activity under different in vivo situations.

Inhibition of human hemopoiesis by non-A, non-B hepatitis virus.

All etiologies of acute viral hepatitis are associated with a transient suppression of hemopoiesis and, rarely, with the development of aplastic anemia. Both hepatitis A and hepatitis B viruses directly inhibit the growth and differentiation of human bone marrow progenitor cells in vitro. We now report a similar effect of a non-A, non-B (NANB) hepatitis agent on human bone marrow progenitor cells. Three chimpanzees were inoculated with a putative NANB agent. Coded sera were blindly evaluated for their ability to affect human bone marrow colony formation in vitro. Sera obtained during the acute phase of NANB hepatitis inhibited the in vitro growth of human erythroid (CFU-E, BFU-E) and granulocyte-macrophage (CFU-GM) progenitor cells, compared with sera obtained before inoculation. Sera obtained after remission of both the biochemical and histological hepatitis and sera obtained from a chimpanzee who underwent biochemical but not histological remission did not inhibit the stem cell assays as much as the acute phase sera. These results suggest an approach to identifying the viremic phase of NANB hepatitis. Inhibition of human bone marrow proliferation appears to be a common property of all known hepatitis viruses.

Detection of integration during active replication of hepatitis B virus in the liver.

A method is described that enables the unequivocal detection of the integration of hepatitis B virus DNA (HBV-DNA) into the genomic DNA of the host cell, while at the same time the virus exhibits active replication. This detection was achieved by separating the low molecular weight replicating HBV-DNA from the high molecular weight genomic DNA of the host by electrophoresis of the total undigested cellular DNA through low melting agarose. The high molecular weight DNA was isolated from this gel and electrophoresed after digestion with restriction enzyme(s) on a second agarose gel. Transfer of the DNA content of both gels and hybridization of these blots with 32P-labelled HBV-DNA will reveal whether any integrated and/or actively replicating DNA is present. By using this method the presence of a single copy of HBV-DNA integrated in host DNA was demonstrated in hepatocellular carcinoma tissue of a patient with active HBV replication.