Correlation of extended RAS and PIK3CA gene mutation status with outcomes from the phase III AGITG MAX STUDY involving capecitabine alone or in combination with bevacizumab plus or minus mitomycin C in advanced colorectal cancer.

BACKGROUND: Mutations affecting RAS genes are now established predictive markers of nonresponse to anti-EGFR antibodies in advanced CRC. This analysis assessed the prognostic and predictive impact of extended RAS and PIK3CA gene mutation status in patients receiving capecitabine plus or minus bevacizumab (±mitomycin C) in the randomised phase III MAX study. METHODS: DNA was extracted from archival macrodissected formalin-fixed paraffin-embedded tumour tissue. Mutation status was determined using pyrosequencing, confirmed with Sanger sequencing (for equivocal RAS) and correlated with efficacy outcomes. Predictive analyses were undertaken using a test for interaction involving both C vs CB+CBM. RESULTS: Of the available 280 of the 471 (59.4%) patients, mutations in KRAS exons 2, 3 and 4 and NRAS 2, 3 and 4 were as follows: 32%, 2.9%, 2.2%, 1.4%, 0.7% and 0% (total RAS MT 39%). The PIK3CA MT rate was 7.5% exon 9 and 3.6% exon 20. Extended RAS gene mutation status (WT vs MT) had no prognostic impact for PFS (HR 0.91 (0.71-1.17)) or OS (HR 0.95 (0.71-1.25)). The RAS gene mutation status was not predictive of the effectiveness of bevacizumab for PFS (HR 0.56 (0.37-0.85) for RAS MT and HR 0.69 (0.5-0.97) for RAS WT; P for interaction 0.50). The PIK3CA mutation was neither predictive for bevacizumab effect nor prognostic. CONCLUSION: Of KRAS exon 2 WT patients, 10% had additional RAS mutations. Neither all RAS gene mutation status nor PIK3CA mutation status was prognostic for PFS or OS, or predictive of bevacizumab outcome in patients with advanced CRC.

TGF-beta-induced p38 activation is mediated by Rac1-regulated generation of reactive oxygen species in cultured human keratinocytes.

p38 has been shown to be involved in TGF-beta-induced gene expression, but the upstream of the signaling pathway leading to the activation of p38 is left undefined. We investigated the pathway in cultured human keratinocytes (HaCat cells). Western blot analysis revealed that TGF-beta induced the activation of p38 within 1 h post TGF-beta treatment. H2O2 also strongly induced p38 activation in a time dependent manner. We also observed that TGF-beta-induced p38 activation was inhibited by PDTC, pyrrolidinedithiocarbamate, a known antioxidant, and DPI, diphenylene iodonium chloride, one of the known NADPH oxidase inhibitors. In contrast, TGF-beta-induced Smad2 phosphorylation was not affected. To test whether reactive oxygen species (ROS) is involved in TGF-beta-induced p38 activation, we examined the generation of ROS and activation of NADPH oxidase. FACS analysis showed that TGF-beta induced generation of ROS in time-dependent manner. DPI, an inhibitor of NADPH oxidase, inhibited TGF-beta-induced ROS production. Lucigenin-based NADPH oxidase assay indicated that TGF-beta-induced NADPH oxidase activity started as early as 5 min following treatment and peaked at about 15 min with induction of about 2-folds. The activity remained elevated up to 1 h. Immunofluorescence microscopy study showed that Rac1, one of the subunits of NADPH oxidase, translocated from cytoplasm to the membrane within 5 min. Pretreatment with DPI dramatically reduced TGF-beta-induced NADPH oxidase activity. Collectively, our data suggest that TGF-beta-induced p38 activation is mediated by Rac1-regulated generation of reactive oxygen species in cultured human keratinocytes.

Fast association rates suggest a conformational change in the MHC class I molecule H-2Db upon peptide binding.

Major histocompatibility complex (MHC) class I molecules bind peptides in the endoplasmic reticulum (ER). For this binding reaction, when performed in vitro, widely differing association rates have been reported. We have expressed empty soluble H-2Db class I molecules in Chinese hamster ovary (CHO) cells and generated complete sets of association, dissociation, and equilibrium constants of unmodified peptides using tritium-labeled peptides and stopped-flow fluorescence spectroscopy. We find that (i) the transition midpoint of temperature denaturation (Tm) of the protein is shifted from 30.5 to 56 degrees C upon the binding of a high-affinity peptide. (ii) With the peptide SV-324-332 (sequence FAPGNYPAL) at 4 degrees C, the dissociation rate constant of 1.02 x 10(-5) s-1 and an equilibrium constant of 8.5 x 10(7) M-1 predict an association rate constant of 870 M-1 s-1 for a simple one-step model of binding. (iii) In contrast, binding of this peptide proceeds much faster, with 1.4 x 10(6) M-1 s-1. These "mismatch kinetics" suggest that peptide binding occurs in several steps, most likely via a conformational rearrangement of the peptide binding groove. The structure of the peptide-class I complex at the time-point of peptide recognition may therefore be different from the equilibrium crystal structures. (iv) Association of modified peptides, in the presence of detergent, or above the Tm of the empty molecule is considerably slower. This might explain why fast on-rates have not been observed in previous studies.

Kupffer cell staining by an HFE-specific monoclonal antibody: implications for hereditary haemochromatosis.

Hereditary haemochromatosis is an inherited disorder of iron absorption that leads to excessive iron storage in the liver and other organs. A candidate disease gene HFE has been identified that encodes a novel MHC class I like protein. We report the development of a monoclonal antibody (HFE-JB1) specific for recombinant refolded HFE protein. The antibody immunoprecipitates a 49 kD protein from the cell line U937, a histiocytic lymphoma. It binds HFE but does not recognize other recombinant non-classic MHC class I proteins (HLA-E, F and G), nor does it react with a variety of recombinant classic class I MHC molecules. COS cells transfected with HFE in culture are stained specifically. The immunohistochemical staining pattern in human tissues is unique and can be defined as a subset of the transferrin receptor positive cells. In the liver HFE protein was shown to be present on Kupffer cells and endothelium (sinusoidal lining cells), but absent from the parenchyma. Kupffer cells from an untreated C282Y HH patient failed to stain with the antibody. In the normal gut scattered cells in the crypts are stained. HFE was also present on capillary endothelium in the brain (a site of high levels of transferrin receptor) and on scattered cells in the cerebellum and cortex. These results raise interesting questions concerning the function of HFE in the control of body iron content and distribution.

Vaccinia virus serpins B13R and B22R do not inhibit antigen presentation to class I-restricted cytotoxic T lymphocytes.

Vaccinia virus (VV) inhibits the presentation of certain epitopes from influenza virus nucleoprotein (NP), haemagglutinin (HA) and non-structural 1 (NS1) proteins to CD8+ cytotoxic T lymphocytes (CTL) by an unknown mechanism. We have investigated whether VV genes B13R and B22R, which encode proteins with amino acid similarity to serine protease inhibitors (serpins), are involved in this process. Recombinant VVs were constructed which express influenza virus proteins HA, NP or NS1 and which lack serpin gene B13R or both B13R and B22R. The lysis of cells infected with these viruses by influenza virus-specific CD8+ CTL was compared to the lysis of cells infected with viruses expressing both the influenza proteins and the serpin genes. Cytotoxicity assays showed that deletion of the VV serpin genes B13R and B22R and other genes between B13R and B24R did not increase the level of lysis, indicating that these genes are not involved in inhibition of antigen presentation of the epitopes tested.

Spontaneous rupture of bladder in pregnancy. A case report.

A case of spontaneous rupture of bladder in pregnancy is reported. Due to its rarity, the diagnosis of this condition is difficult.

Positive and negative selection in transgenic mice expressing a T-cell receptor specific for influenza nucleoprotein and endogenous superantigen.

A transgenic mouse was generated expressing on most (> 80%) of thymocytes and peripheral T cells a T-cell receptor isolated from a cytotoxic T-cell clone (F5). This clone is CD8+ and recognizes alpha alpha 366-374 of the nucleoprotein (NP 366-374) of influenza virus (A/NT/60/68), in the context of Class I MHC Db (Townsend et al., 1986). The receptor utilizes the V beta 11 and V alpha 4 gene segments for the beta chain and alpha chain, respectively (Palmer et al., 1989). The usage of V beta 11 makes this TcR reactive to Class II IE molecules and an endogenous ligand recently identified as a product of the endogenous mammary tumour viruses (Mtv) 8, 9, and 11 (Dyson et al., 1991). Here we report the development of F5 transgenic T cells and their function in mice of the appropriate MHC (C57BL/10 H-2b, IE-) or in mice expressing Class II MHC IE (e.g., CBA/Ca H-2k and BALB/c H-2d) and the endogenous Mtv ligands. Positive selection of CD8+ T cells expressing the V beta 11 is seen in C57BL/10 transgenic mice (H-2b). Peripheral T cells from these mice are capable of killing target cells in an antigen-dependent manner after a period of in vitro culture with IL-2. In the presence of Class II MHC IE molecules and the endogenous Mtv ligand, most of the single-positive cells carrying the transgenic T-cell receptor are absent in the thymus. Unexpectedly, CD8+ peripheral T-cells in these (H-2k or H-2d) F5 mice are predominantly V beta 11 positive and also have the capacity to kill targets in an antigen-dependent manner. This is true even following backcrossing of the F5 TcR transgene to H-2d scid/scid mice, in which functional rearrangement of endogenous TcR alpha- and beta-chain genes is impaired.

Molecular analysis of the association of HLA-B53 and resistance to severe malaria.

The protective association between the human leukocyte antigen HLA-B53 and severe malaria was investigated by sequencing of peptides eluted from this molecule followed by screening of candidate epitopes from pre-erythrocytic-stage antigens of Plasmodium falciparum in biochemical and cellular assays. Among malaria-immune Africans, HLA-B53-restricted cytotoxic T lymphocytes recognized a conserved nonamer peptide from liver-stage-specific antigen-1 (LSA-1), but no HLA-B53-restricted epitopes were identified in other antigens. These findings indicate a possible molecular basis for this HLA-disease association and support the candidacy of liver-stage-specific antigen-1 as a malaria vaccine component.

Thymic depletion and peripheral activation of class I major histocompatibility complex-restricted T cells by soluble peptide in T-cell receptor transgenic mice.

Injection of mice transgenic for a class I major histocompatibility complex-restricted T-cell receptor with a soluble peptide antigen from influenza virus nucleoprotein results in clonal depletion of double-positive immature thymocytes in the thymus and activation of mature T cells in the periphery, accompanied by a transient up-regulation of the T-cell receptor and CD3 and CD8 coreceptor molecules.

Restoration of antigen presentation to the mutant cell line RMA-S by an MHC-linked transporter.

In mammalian cells, short peptides derived from intracellular proteins are displayed on the cell membrane associated with class I molecules of the major histocompatibility complex (MHC). The surface presentation of class I-peptide complexes presumably alerts the immune system to intracellular viral protein synthesis. Peptides derived from the cytosol must reach the cisternae of the endoplasmic reticulum where they are required for the assembly of stable class I molecules, and it has been proposed that the products of the two MHC-encoded ATP-binding cassette (ABC) transporter genes function to deliver the peptides across the membrane of the endoplasmic reticulum. This idea is supported by experiments in which transfection of a human cell line defective in class I expression with a complementary DNA of one of these genes restored cell surface expression levels. Here we show that the complete phenotype of the mouse mutant cell line RMA-S, in which lack of surface expression of stable class I molecules correlates with an inability to present viral peptides originating in the cytosol, is repaired by the cDNA of the other transporter gene. These results are consistent with the possibility that the two transporter polypeptides form a heterodimer.

Restoration of H-2b expression and processing of endogenous antigens in the MHC class I pathway by fusion of a lymphoma mutant to L cells of the H-2k haplotype.

The RMA-S lymphoma mutant cannot process and present antigens to H-2-restricted cytotoxic T lymphocytes. It synthesizes major histocompatibility complex class I heavy (H-2KbDb) and light beta 2-microglobulin (beta 2mb) chains of normal size and charge, but only a fraction of these assemble and reach the cell surface. As a first step investigating the genetic defect of this line, we have fused it to a L cell fibroblast line (H-2KkDk/beta 2ma). The fusion restored H-2Kb, Db and beta 2mb expression as well as the ability to process and present internally derived (minor histocompatibility and influenza virus nucleoprotein) antigens in RMA-S. This shows that the mutation(s) responsible for the phenotype of RMA-S is (are) not located within the MHC class I heavy and light chain genes. Other cellular factors, derived from the L cell fusion partner, can control antigen processing and transport of MHC class I molecules. These findings are discussed in relation to the observation that assembly and transport of MHC class I molecules can be induced in the mutant by H-2b-restricted peptides. The recessive nature of the defect and its independence of MHC class I genes in the mutant has important implications for future transfection studies, of this and similar mutants, aiming at establishing cells containing non-assembled MHC class I molecules of different alleles and identifying the gene(s) controlling processing of endogenous antigens.

Resistance to H-2-restricted but not to allo-H2-specific graft and cytotoxic T lymphocyte responses in lymphoma mutant.

The lymphoma mutant RMA-S escaped graft rejection after transplantation over a minor histocompatibility barrier, whereas it was rejected in H-2 allogeneic mice. The parental control line was rejected in both situations. The mutant, which had been selected against MHC class I molecules retained 5 to 10% of the wild-type H-2Db, Kb, and beta 2-microglobulin expression on the cell surface. It remained sensitive to allo-H-2b CTL in vitro, but was completely resistant to minor histocompatibility antigen-specific, H-2b-restricted CTL. It was equally resistant to other H-2b-restricted responses against internally derived Ag, such as tumor-specific CTL or a CTL clone specific for the influenza virus nucleoprotein. The results indicate a target cell defect that selectively abolishes the sensitivity to H-2-restricted CTL directed against internally processed Ag. This appears sufficient to shift the transplantation response over a minor histocompatibility Ag barrier from rejection to acceptance. There are two possible explanations for the results: 1) a block in the MHC class I-directed pathway for internal Ag processing, and 2) subthreshold H-2/Ag ligand density in relation to triggering requirements of restricted CTL. Regardless of the type of defect, the results demonstrate a difference between allo-H-2-specific and H-2-restricted CTL recognition at the level of the target cell.

Influenza-specific cytotoxic T-cell recognition is inhibited by peptides unrelated in both sequence and MHC restriction.

Two related peptides from the nucleoprotein (NP) sequence 365-380, derived from influenza virus isolates A/PR/8/34 and A/NT/60/68, are recognized by mutually exclusive sets of Db (Class I)-restricted cytotoxic T-lymphocyte (CTL) clones. These peptides compete with each other for presentation on Db-bearing target cells in vitro. A Kk-restricted nucleoprotein epitope (NP 50-63), which is unrelated in sequence, competes more efficiently on H-2b target cells but is not itself recognized by virus-specific CTL from influenza-infected H-2b mice. A peptide sequence from the class I molecules Cw3 and Db can also compete, but additional unrelated peptides do not do so at equimolar concentrations. Our results show that competition is at the level of the target cell and imply that the binding specificity of the class I molecule Db is broader than indicated by the immune response phenotype of the C57BL (H-2b) mouse.

HIV-1 gag-specific cytotoxic T lymphocytes defined with recombinant vaccinia virus and synthetic peptides.

Current candidate vaccines fail to protect primates against challenge with human immunodeficiency virus (HIV) in the presence of antibody responses; this underlines the importance of studying cell-mediated immunity to HIV and identifying specific epitopes that stimulate cytotoxic T lymphocytes (CTL). Using a recombinant vaccinia virus to express the gag protein of HIV-1 we found HLA class-I-restricted gag-specific CTL in thirteen out of fifteen healthy HIV seropositive patients. We then used short synthetic peptides in the lysis assay to screen for gag CTL epitopes. In one patient we have identified a peptide in p24 that is recognized by CTL in association with HLA-B27. This peptide, and further peptide sequences defined by these methods, could be incorporated in vaccines designed to induce cell-mediated immunity against HIV.

Specific recognition of influenza virus polymerase protein (PB1) by a murine cytotoxic T-cell clone.

Cytotoxic T-cell clones were raised in CBA mice that recognised both A/X31 and A/JAP/305/1957 influenza virus. Here, we describe one CTL clone that recognises target cells infected with a recombinant vaccinia virus expressing influenza PB1.

Mouse H-2k-restricted cytotoxic T cells recognize antigenic determinants in both the HA1 and HA2 subunits of the influenza A/PR/8/34 hemagglutinin.

We have constructed two chimeric influenza hemagglutinin (HA) genes in which the HA1 and HA2 subunits of the HA molecule have been interchanged between influenza A/PR/8/34 (H1 subtype) and A/NT/60/68 (H3 subtype). These genes were used to construct recombinant vaccinia viruses that expressed intact chimeric HA. These recombinant viruses were used to test whether murine CTL recognize antigenic determinants in either the HA1, HA2, or both subunits. We found that both subunits of the HA molecule contain determinants for CTL. This implies that CTL have, at least in part, separate antigenic determinants from B lymphocytes, which recognize mainly epitopes within the HA1 subunit.