Myofibroblast activation in colorectal cancer lymph node metastases.

BACKGROUND: Myofibroblasts have an important role in regulating the normal colorectal stem cell niche. While the activation of myofibroblasts in primary colorectal cancers has been previously described, myofibroblast activation in lymph node metastases has not been described before. METHODS: Paraffin-embedded lymph node sections from patients with macrometastases, micrometastases and isolated tumour cells were stained to identify myofibroblasts and to characterise the distribution of different cell types in tumour-containing lymph nodes. The extent of myofibroblast presence was quantified and compared with the size of the metastasis and degree of proliferation and differentiation of the cancer cells. RESULTS: We show substantial activation of myofibroblasts in the presence of colorectal metastases in lymph nodes, which is intimately associated with glandular structures, both in micro- and macrometastases. The degree of activation is positively associated with the size of the metastases and the proportion of Ki67+ve cancer cells, and negatively associated with the degree of enterocyte differentiation as measured by CK20 expression. CONCLUSION: The substantial activation of myofibroblasts in tumour-containing lymph nodes strongly suggests that these metastatic cancer cells are still significantly dependent on their microenvironment. Further understanding of these epithelial-mesenchymal interactions could lead to the development of new therapies in metastatic disease.

Some Thoughts on AI Stimulated by Michael Wooldridge's Book "The Road to Conscious Machines. The Story of AI".

Following a brief over view of the contents of Michael Wooldridge's book I give an account of my own background in computing and AI. I then cover a range of topics stimulated by reading the book including machine learning's relationship to AI, applications to medical areas, the need to consider probabilistic effects on decisions, the importance of self-reproduction and whether AI can be made moral. I finish with a discussion of the mind-brain relationship and what makes us human.

Synthesis of HLA antigens from membrane-associated messenger RNA.

Proteins encoded by genes in the human major histocompatibility region, HLA, are present on the plasma membranes in the cells of most tissues. We have isolated messenger RNA (mRNA) from lymphoblastoid cells and studied its templating activity for HLA polypeptides by using an mRNA-dependent cell-free translation system and immunorecipitation with specific antisera. We estimate the sizes of the mRNA to be 1,700, 1,300, and 900 nucleotides for HLA-A, -B, and -C (HLA-ABC), HLA-DR, and beta 2-microglobulin, respectively. These sizes are consistent with our inability to detect larger polypeptide precursor for more than one HLA-ABC or HLA-DR antigen. Subcellular fractionation into cytosol and membrane fractions indicated that mRNA for the HLA-ABC and HLA-ABC and HLA-DR polypeptides was found predominantly in the membrane fraction. The beta 2-microglobulin mRNA appeared to be divided into roughly equal portions between both fractions. Nevertheless, we believe that in the cell, beta 2-microglobulin is synthesized in the rough endoplasmic reticulum, and that, because the protein is relatively small, the mRNA is released from the polyribosomes during the isolation procedure. We conclude that the properties of the HLA region and the beta 2-microglobulin mRNA are consistent with current models for the synthesis and membrane insertion of eukaryotic cell surface proteins. Our findings do not suggest the existence of multigenic precursor polypeptides for HLA antigens in contrast to those for several viral membrane glycoproteins.

Expression of HLA-A, -B, and -C and beta 2-microglobulin antigens in human choriocarcinoma cell lines.

Immunoprecipitation of [35S]methionine-labeled cell extracts and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that cells from two human choriocarcinomas JaR and BeWo contained beta 2-microglobulin. The JaR cells, and one subline of BeWo, did not express surface HLA-A, -B, and -C (HLA-ABC) antigens, nor did JaR contain the antigens when cell-free extracts were measured in a sensitive radioimmunoassay. It may be concluded that the lack of expression of HLA-ABC antigens in choriocarcinoma (and in the trophoblast) is not a result of lack of beta 2-microglobulin, as is the case for the Burkitt's lymphoma cell line, Daudi.

5-Fluorouracil response in a large panel of colorectal cancer cell lines is associated with mismatch repair deficiency.

BACKGROUND: Colorectal cancer is (CRC) one of the commonest cancers and its therapy is still based on few drugs. Currently, no biological criteria are used to choose the most effective of the established drugs for treatment. METHODS: A panel of 77 CRC cell lines was tested for sensitivity to 5-fluorouracil (5FU) using the SRB assay. The responses were grouped into three categories and correlated with genetic changes in the cell lines. RESULTS: The strongest and most clearcut correlation was between 5-fluorouracil response and replication error status (mismatch repair deficiency). All the other significant correlations (loss of heterozygosity for DCC and mutations in TGFbIIR) are secondary to the association with replication error status. INTERPRETATION AND CONCLUSION: Our findings validate previous analyses based mainly on clinical data, and indicate that replication error status could be a useful guide to 5-fluorouracil-based CRC therapy. Essentially, all previously described correlations with 5FU response are secondary to the association with replication error status.

Humanised IgG1 antibody variants targeting membrane-bound carcinoembryonic antigen by antibody-dependent cellular cytotoxicity and phagocytosis.

BACKGROUND: The effect of glycoengineering a membrane specific anti-carcinoembryonic antigen (CEA) (this paper uses the original term CEA for the formally designated CEACAM5) antibody (PR1A3) on its ability to enhance killing of colorectal cancer (CRC) cell lines by human immune effector cells was assessed. In vivo efficacy of the antibody was also tested. METHODS: The antibody was modified using EBNA cells cotransfected with beta-1,4-N-acetylglucosaminyltransferase III and the humanised hPR1A3 antibody genes. RESULTS: The resulting alteration of the Fc segment glycosylation pattern enhances the antibody's binding affinity to the FcgammaRIIIa receptor on human immune effector cells but does not alter the antibody's binding capacity. Antibody-dependent cellular cytotoxicity (ADCC) is inhibited in the presence of anti-FcgammaRIII blocking antibodies. This glycovariant of hPR1A3 enhances ADCC 10-fold relative to the parent unmodified antibody using either unfractionated peripheral blood mononuclear or natural killer (NK) cells and CEA-positive CRC cells as targets. NK cells are far more potent in eliciting ADCC than either freshly isolated monocytes or granulocytes. Flow cytometry and automated fluorescent microscopy have been used to show that both versions of hPR1A3 can induce antibody-dependent cellular phagocytosis (ADCP) by monocyte-derived macrophages. However, the glycovariant antibody did not mediate enhanced ADCP. This may be explained by the relatively low expression of FcgammaRIIIa on cultured macrophages. In vivo studies show the efficacy of glycoengineered humanised IgG1 PR1A3 in significantly improving survival in a CRC metastatic murine model. CONCLUSION: The greatly enhanced in vitro ADCC activity of the glycoengineered version of hPR1A3 is likely to be clinically beneficial.

Genetic characteristics of the HeLa cell.

The genotype of the patient Henrietta Lacks from whose cervical carcinoma the HeLa cell was derived was deduced from the phenotypes of her husband and children, and from studies of the HeLa cell. Hemizygous expression of glucose-6-phosphate dehydrogenase in HeLa, together with the deduced heterozygosity of Mrs. Lacks, is consistent with clonal origin of her neoplasm.

Polyclonal origin of colonic adenomas in an XO/XY patient with FAP.

It is widely accepted that tumors are monoclonal in origin, arising from a mutation or series of mutations in a single cell and its descendants. The clonal origin of colonic adenomas and uninvolved intestinal mucosa from an XO/XY mosaic individual with familial adenomatous polyposis (FAP) was examined directly by in situ hybridization with Y chromosome probes. In this patient, the crypts of the small and large intestine were clonal, but at least 76 percent of the microadenomas were polyclonal in origin.

Detection of circulating tumour cells in peripheral blood with an automated scanning fluorescence microscope.

We have developed an automated, highly sensitive and specific method for identifying and enumerating circulating tumour cells (CTCs) in the blood. Blood samples from 10 prostate, 25 colorectal and 4 ovarian cancer patients were analysed. Eleven healthy donors and seven men with elevated serum prostate-specific antigen (PSA) levels but no evidence of malignancy served as controls. Spiking experiments with cancer cell lines were performed to estimate recovery yield. Isolation was performed either by density gradient centrifugation or by filtration, and the CTCs were labelled with monoclonal antibodies against cytokeratins 7/8 and either AUA1 (against EpCam) or anti-PSA. The slides were analysed with the Ikoniscope robotic fluorescence microscope imaging system. Spiking experiments showed that less than one epithelial cell per millilitre of blood could be detected, and that fluorescence in situ hybridisation (FISH) could identify chromosomal abnormalities in these cells. No positive cells were detected in the 11 healthy control samples. Circulating tumour cells were detected in 23 out of 25 colorectal, 10 out of 10 prostate and 4 out of 4 ovarian cancer patients. Five samples (three colorectal and two ovarian) were analysed by FISH for chromosomes 7 and 8 combined and all had significantly more than four dots per cell. We have demonstrated an Ikoniscope based relatively simple and rapid procedure for the clear-cut identification of CTCs. The method has considerable promise for screening, early detection of recurrence and evaluation of treatment response for a wide variety of carcinomas.

Selection for beta 2-microglobulin mutation in mismatch repair-defective colorectal carcinomas.

Novel peptide antigens complexed with human leukocyte antigen (HLA) and beta 2-microglobulin (beta 2M) molecules are presented at the cell surface to cytotoxic T lymphocytes (CTLs), provoking lysis of the antigen-presenting cell [1]. In tumor cells, genetically altered or abnormally expressed proteins provide a source of peptides that can be presented to CTLs; the resulting anti-tumour CTL responses may provide part of the body's defence against cancer. Disabling mutations in the HLA and beta 2M proteins required for peptide presentation allow a tumour cell to escape destruction by CTLs. Cells with deficient DNA mismatch repair have high spontaneous mutation rates [2] and produce many altered proteins that are a potential source of numerous unique peptides. Mutator tumour cells might therefore be particularly vulnerable to immune surveillance and CTL attack. Mutator phenotypes [3,4] and loss of beta 2M (or HLA) expression [5,6] are both relatively common among sporadic colorectal tumours. We have compared the frequency of beta 2M mutations in sporadic colorectal and other tumours with and without a mutator phenotype. Mutations were more frequent among colorectal tumours with the microsatellite instability indicative of a defect in DNA mismatch repair. The inactivating beta 2M mutations were predominantly frameshifts, which is consistent with the underlying mismatch repair defects. Evasion of immune surveillance by acquiring beta 2M mutations therefore occurs at high frequency in tumour cells with a mutator phenotype due to defective DNA mismatch repair.

The HLA system and the analysis of multifactorial genetic disease.

The human leukocyte antigen (HLA) system comprises closely linked genes controlling highly polymorphic proteins involved in the presentation of peptides to the T-cell receptor. Specific alleles at HLA loci are associated with diseases, often those suspected to be of autoimmune aetiology. Many of these associations result from linkage disequilibrium between the HLA gene studied and other HLA genes or non-HLA genes close by. Owing to its high level of polymorphism and its candidate role in many diseases, HLA was the first system used in many techniques of genetic mapping, such as affected-sib-pair analysis and association (linkage disequilibrium) studies. Much remains unknown about the reasons why diseases are associated with HLA. Experience gained from HLA has, however, shown how other loci involved in complex traits can be identified by studying families with multiple affected cases or sib pairs, followed by linkage-disequilibrium mapping and then analysis of candidate genes.

MHC antigens and cancer: implications for T-cell surveillance.

Evidence for a direct involvement of cell-mediated immune mechanisms in the control of human tumours remains sketchy. The past year, however, has seen advances in our understanding of the relationship between the immune system and tumour cells, and has given rise to new prospects for immunological treatment and the prevention of cancers.

EpCAM and gpA33 are markers of Barrett's metaplasia.

AIMS: To characterise a specific and sensitive marker of Barrett's metaplasia (BM). METHODS: Cases of normal oesophageal squamous mucosa (11 fresh endoscopic biopsies and 10 formalin fixed, paraffin embedded tissue blocks), BM (11 biopsies and 11 tissue blocks), and normal gastric body mucosa (five biopsies and five tissue blocks) were analysed using reverse transcriptase PCR, Western blotting, and immunohistochemistry for EpCAM, and reverse transcriptase PCR for gpA33. RESULTS: Strong EpCAM mRNA expression was detected in all the BM cases, in contrast to weak expression in all the normal gastric mucosal samples and no expression in any of the normal oesophageal mucosal samples tested. Strong gpA33 mRNA expression was detected in all the BM cases, in contrast to weak expression in a quarter of the normal gastric mucosal samples and no expression in any of the normal oesophageal mucosal samples tested. Western blotting showed EpCAM protein expression in all the BM cases and in none of the normal gastric or oesophageal mucosal samples tested. Immunohistochemistry showed strong EpCAM protein expression in BM and complete absence of expression in normal oesophageal squamous epithelium. Scattered EpCAM expressing cells were found in the gland bases of normal gastric body mucosa. CONCLUSIONS: EpCAM protein and gpA33 mRNA expressions are specific and sensitive markers of BM.

Applicability of carcinoembryonic antigen-specific monoclonal antibodies to radioimmunoguided surgery for human colorectal carcinoma.

Two carcinoembryonic antigen (CEA)-specific monoclonal antibodies (MAbs), PR1A3 and T84.66, were tested to determine whether they could accurately localize colorectal carcinoma and therefore be applicable in radioimmunoguided surgery (RIGS). Twenty-one tumors by three human colorectal carcinoma cell lines with various levels of CEA expression (KM-12c, C75, and Clone A) were successfully implanted in the intra-abdominal organs of 15 nude mice. The tumors was localized using a portable radioisotope detector (Neoprobe 1000) 48 h after injection of radiolabeled MAbs (10 mCi/mouse) when the precordial counts were <20 per 2 s. Histopathological identification of radiolabeled MAbs were also performed using immunohistochemistry and microautoradiography. Radioactivity counted on a portable radioisotope detector correlated well with that on a gamma counter. The distribution in the blood was significantly greater than in other organs (P < 0.001). Localization indices of the tumor in various organs was from 1.1 to 8.5 in the PR1A3-pretreated mice and 3.0 to 8.6 in the T84.66-pretreated mice. Silver grains and immune staining were distributed in the tumor cells of the PR1A3-pretreated mice, whereas they were in the necrotic debris as well as the tumor cells of the T84.66-pretreated mice. There were significantly more silver grains in the liver in the T84.66-pretreated mice than in the PR1A3-pretreated mice (P = 0.004). The sensitivity and specificity of tumor localization by RIGS were 71.4 and 91.4% in the PR1A3-pretreated mice, whereas they were 60 and 76% in the T84.66-pretreated mice. A study using specific anti-CEA MAbs suggested PR1A3 as an efficient immune probe for RIGS in colorectal carcinoma with a low rate of false-positive detection.

Exclusion of PTEN and 10q22-24 as the susceptibility locus for juvenile polyposis syndrome.

Juvenile polyposis syndrome (JPS; MIM 174900) is an autosomal dominant condition with incomplete penetrance characterized by hamartomatous polyps of the gastrointestinal tract and a risk of gastrointestinal cancer. Gastrointestinal hamartomatous polyps are also present in Cowden syndrome (CS; MIM 158350) and Bannayan-Zonana syndrome (BZS; also called Ruvalcaba-Myhre-Smith syndrome; MIM 153480). The susceptibility locus for both CS and BZS has recently been identified as the novel tumor suppressor gene PTEN, encoding a dual specificity phosphatase, located at 10q23.3. A putative JPS locus, JP1, which most likely functions as a tumor suppressor, had previously been mapped to 10q22-24 in both familial and sporadic juvenile polyps. Given the shared clinical features of gastrointestinal hamartomatous polyps among the three syndromes and the coincident mapping of JP1 to the region of PTEN, we sought to determine whether JPS was allelic to CS and BZS by mutation analysis of PTEN and linkage approaches. Microsatellite markers spanning the CS/BZS locus (D10S219, D10S551, D10S579, and D10S541) were used to compute multipoint lod scores in eight informative families with JPS. Lod scores of < -2.0 were generated for the entire region, thus excluding PTEN and any genes within the flanking 20-cM interval as candidate loci for familial JPS under our statistical models. In addition, analysis of PTEN using a combination of denaturing gradient gel electrophoresis and direct sequencing was unable to identify a germline mutation in 14 families with JPS and 11 sporadic cases. Therefore, at least a proportion of JPS cases are not caused by germline PTEN alteration or by an alternative locus at 10q22-24.

Integration of deoxyribonuclease-treated DNA in bacillus subtilis transformation.

Normal preparations of B. subtilis DNA have weight average native molecular weights of 10 to 30 x 10(6). For any given preparation the upper and lower 95% size limits may differ by a factor of ten or more. Single-stranded molecular weights indicate an average of 1 to 4 breaks per single strand of the native DNA. The reduction in transforming activity and viscosity following DNAase I digestion can be accounted for by a direct relationship between the transforming activity of a DNA and its single-stranded molecular weight. Uptake studies with DNAase I treated heavy ((2)H(15)N (3)H) DNA show that single strand breaks inhibit integration less than transformation. A provisional estimate of the size of the integrated region based on correlating the single strand size of the donor-recipient complex with the donor-recipient density differences following alkali denaturation came to 1530 nucleotides. Using a competent, nonleaky thymine-requiring strain of B. subtilis grown in 5-BU medium before and after transformation, it was shown that (a) No detectable amount of DNA synthesis is necessary for the initial stages of integration, (b) Cells which have recently been replicating DNA are not competent. (c) Cells containing donor DNA show a lag in DNA replication following transformation, (d) When donor DNA is replicated it initially appears in a density region between light and hybrid. This indicates that it includes the transition point formed at the time of reinitiation of DNA synthesis in the presence of 5-BU following transformation. A model is proposed in which donor DNA is integrated at the stationary growing point of the competent cell, which is in a state of suspended DNA synthesis.

Evolution of HL-A and other major histocompatibility systems.

The HL-A region is a complex highly polymorphic system containing many genes with interrelated functions on the cell surface. Population data suggest the action of natural selection, perhaps via linked immune-response genes. Major histocompatibility systems, such as HL-A, are presumed to have their counterparts in at least all mammalian species. The complexity of the region suggests evolution by gene duplication. The integrity of the region may even extend to the human equivalent of the complex T locus of the mouse.