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.

The influenza A virus nucleoprotein gene controls the induction of both subtype specific and cross-reactive cytotoxic T cells.

Using genetically typed recombinant influenza A viruses that differ only in their genes for nucleoprotein, we have demonstrated that repeated stimulation in vitro of C57BL/6 spleen cells primed in vivo with E61-13-H17 (H3N2) virus results in the selection of a population of cytotoxic T lymphocytes (CTL) whose recognition of infected target cells maps to the gene for nucleoprotein of the 1968 virus. Influenza A viruses isolated between 1934 and 1979 fall into two groups defined by their ability to sensitize target cells for lysis by these CTL: 1934-1943 form one group (A/PR/8/34 related) and 1946-1979 form the second group (A/HK/8/68 related). These findings complement and extend our previous results with an isolated CTL clone with specificity for the 1934 nucleoprotein (27, 28). It is also shown that the same spleen cells derived from mice primed with E61-13-H17 virus in vivo, but maintained in identical conditions by stimulation with X31 virus (which differs from the former only in the origin of its gene for NP) in vitro, results in the selection of CTL that cross-react on target cells infected with A/PR/8/1934 (H1N1) or A/Aichi/1968 (H3N2). These results show that the influenza A virus gene for NP can play a role in selecting CTL with different specificities and implicate the NP molecule as a candidate for a target structure recognized by both subtype-directed and cross-reactive influenza A-specific cytotoxic T cells.

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.

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.

The role of returned migrants in England's poorest region.

"Work on the growing international phenomenon of return migration requires a base of comparative knowledge on the less conspicuous process of inter-regional return migration. Three questionnaire studies in the North-East of England identified about one-quarter of respondents as 'returned migrants' to that region. This group [is] attracted to work in new factories, but their economic status has not been markedly improved by geographic mobility."

Cytotoxic T cells recognize fragments of the influenza nucleoprotein.

Recent work has shown that a major population of murine influenza A specific cytotoxic T lymphocytes (CTL) recognize the viral nucleoprotein. In order to investigate the mechanism by which this nonglycoprotein component of the virus is recognized by CTL, a series of deletion mutants of an A virus NP gene were studied. The results showed that CTL recognize three distinct epitopes of the NP molecule. Both N- and C-terminal fragments of the protein are transported, independently of each other, to the site of recognition by CTL. These findings imply that a mechanism may exist for transport to the cell surface and presentation to CTL, of viral proteins and protein fragments that lack defined signal sequences.

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.

Viral recognition by influenza A virus cross-reactive cytotoxic T (Tc) cells: the proportion of Tc cells that recognize nucleoprotein varies between individual mice.

L cells (H-2k) transfected with DNA coding for the A/NT/60/68 influenza nucleoprotein (NP) gene have been found previously to provide a target cell for cytotoxic T (Tc) cells. In this report we have studied the repertoire of cytotoxic memory cells in mice of different strains primed by infection with several influenza A subtypes. L cells transfected with NP and Db genes enabled us to estimate the proportion of A virus cross-reactive Tc cells specific for NP. The most striking feature of our results was variation in the frequency of NP-specific Tc cells between individuals of an inbred mouse strain. Occasional individuals, although showing a strong A virus cross-reactive Tc response, had no A virus cross-reactive Tc cells that recognized NP. The NP-directed Tc repertoire represented between 0-40% of lysis of target cells infected with a heterologous type A virus. Thus, a significant proportion of A virus cross-reactive Tc cells must have different specificities for type A influenza virus. In C3H-H-2o2 mice, Dk is a low responder allele for NP recognition by Tc cells.

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.

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.

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.

Recognition of influenza A virus nucleoprotein by an H-2-restricted cytotoxic T-cell clone.

Cytotoxic T-cell clones raised against X-31 (H3N2) influenza virus in C57BL/6 mice can be directed against an influenza A virus subtype specific determinant (1). A representative T-cell clone (A3.1) has been used in combination with a set of genetically typed recombinant viruses, to show that the A/PR/8/34 nucleoprotein can be responsible for cytotoxic T-lymphocyte recognition of infected target cells.

Recognition of Db and Kb gene products by influenza-specific cytotoxic T cells.

A series of 16 H-2b-restricted, A influenza virus-specific cytotoxic T-cell clones are described and characterized. One is Kb restricted, the others Db restricted. The factors governing Kb or Db restriction patterns seen in the mixed populations from which clones are derived are investigated. The Kb-restricted clone does not recognize Kb mutant bml and influenza and all 15 Db-restricted clones do not recognize Db mutant bml4 and A influenza virus; these results are discussed in the light of findings in a variety of other viral systems. Representative Kb- and Db-restricted clones were used to assess the functional properties of cloned cosmids containing either Kb or Db genes expressed in transformed L-cells (k haplotype). The expression products of both cosmids functioned efficiently as mutually exclusive restriction elements for A influenza virus recognition.

Cytotoxic T lymphocytes recognize influenza haemagglutinin that lacks a signal sequence.

A surprising feature of most cytotoxic T lymphocytes (CTL) responding to influenza infection is that they recognize the unglycosylated (non-transmembrane) proteins of the virus, including the nucleoprotein. Recognition of cells that express nucleoprotein by CTL does not depend on a definite signal sequence within the protein, and the epitopes recognized can be defined with short synthetic peptides in vitro. Haemagglutinin (HA), the major transmembrane protein of the virus, is recognized by a minor population of CTL from infected mice. We have deleted the sequence coding for the N-terminal signal peptide from a complementary DNA encoding HA of the H1 subtype. The signal-deleted HA is detected with antibodies as a short-lived, unglycosylated, intracellular protein. However, CTL raised to the complete molecule recognize cells expressing the signal-deleted HA and vice versa. These results cast doubts on the assumption that CTL recognize the HA molecule only after its insertion into the plasma membrane.

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.

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.

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.