Biomedical subjects
Publications and source records attributed to H Chong.
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Activin A is a dimeric protein hormone that regulates numerous cellular functions. A clear physiological role for this molecule in pregnancy is suggested by previous studies in the human, wherein activin A rises dramatically as women approach parturition. To determine whether the rodent is a suitable animal model for further studies of activin action during pregnancy, the serum concentration of activin A was measured in pregnant rats. Activin A was detected in the serum of pregnant rats, beginning on day 12, and the serum concentration rose progressively through gestation (22-fold) and dramatically (140-fold) in labor. The potential target tissues for circulating activin were then identified in two ways. First, iodinated activin was injected into pregnant rats, and the tissues targeted by labeled ligand were identified in vivo. A tissue targeted by activin A in the pregnant rat was the uterine myometrium. To determine the ligand specificity of the uterine myometrial cells, the uteri of pregnant rats were collected and analyzed by in situ ligand binding. 125I-activin A binding was specific for the uterus myometrium, and the ligand binding was competed by unlabeled activin A but not by inhibin A. This result suggests that the receptor in this tissue compartment is an activin-specific receptor. The production of abundant activin A and the ability of exogenous ligand to target the myometrium of the uterus provides a pathway by which activin could regulate uterine function during pregnancy.
Many tumor cells do not express co-stimulatory molecules, and this may account, in part, for their poor ability to stimulate T cells directly. One strategy to enhance immune recognition would be to express such molecules on the tumor cell. Here, we show that expression of a member of the B7 family of co-stimulatory molecules by CMT93 murine colorectal tumor or 1735 murine melanoma cells resulted in a local antitumor response in immunocompetent mice. The antitumor effect was diminished in athymic nude mice, indicating that T cells played an important part in this response. The ability of the B7-expressing tumor cells to generate systemic protective immunity was investigated by excision of tumors that developed from the initial inoculation followed by rechallenge with parental tumor cells. CMT93 is a poorly immunogenic tumor and no significant systemic immunity was elicited by the expression of B7-1 in these cells. 1735 melanoma is a mildly immunogenic tumor. Unexpectedly, the systemic immunity obtained with 1735 tumors expressing B7-1 or B7-2 was weaker than that generated by parental 1735 cells (p < 0.001, stratified logrank test), even when coexpression of interferon-gamma in the B7-1 cells produced high levels of surface MHC class I expression. These results suggest that some caution is appropriate when considering the use of these molecules in the gene therapy of cancer.
Animal models have clearly shown that tumor cells may be amenable to molecular manipulation which can result in immune activation and rejection of unmodified cells (Chapters 4 and 5). The challenge now is to design clinical trials which have a realistic chance of success, (although the definition of 'success' is itself an important issue [see Chapter 9]. How should such a strategy be formulated? A review of the previous fifteen years since the first (immune) gene transfer studies were reported, encompasses a great wealth of data. Unfortunately, far from crystallising a set of unifying principles, these diverse reports shroud us in a fog of uncertainty as to how best to proceed. However, if this technology is to have practical, widespread application in the treatment of cancer patients, it is necessary to identify certain critical immunological goals which any protocols should achieve. Clear elucidation of these goals, by unifying the huge amount of disparate experimental data, must eventually be accomplished. In this chapter, we have reviewed the literature covering the era of molecular immunotherapy. We propose four general goals around which widely applicable clinical protocols, not necessarily dependent upon tumour type or experimental bias, might be based and suggest how they may be achieved in the context of gene transfer.
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We report the discovery, on routine screening, of a replication-competent retrovirus (RCR) produced from a third generation amphotropic packaging cell line, GP + envAM12. In this line, the gag-pol and env helper genes are located on separate plasmids to minimise the chances of recombination events that may lead to RCR formation. Plasmid pBabeNeo was transfected into GP + envAM12 to obtain a producer line. Supernatant from this line was used to infect two lines, B16 and 1735-puro, and stable colonies which were obtained were pooled. Supernatants from both these lines were able to transfer the neo gene to fresh cells through several passages, indicating the presence of helper virus. Rescue of puro from 1735-puro cells, which contain the pBabePuro provirus, was also achieved. Mobilisation of lacZ from TELCeB6 cells, which contain the MFGnlslacZ provirus, was also possible. The RCR was able to transfer lacZ to ampli-GPE cells, an ecotropic packaging cell line, but transfer to fresh GP + envAM12 cells was minimal, demonstrating receptor interference of the RCR by the amphotropic envelope. These observations strongly suggest that a RCR with an amphotropic envelope arose in the GP + envAM12-pBabeNeo producer line. To our knowledge, this is the first report of RCR arising from routine use of GP + envAM12 cells or any similar third generation packaging line and has important implications for the use of such lines in human gene therapy protocols.
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Previously we have demonstrated safe and effective transfer of the HSVtk cytotoxic gene to primary murine melanoma tumors by direct injection of plasmid and retroviral vectors in which the HSVtk gene is driven by the tissue-specific tyrosinase promoter. However, for general clinical application such forms of therapy should, ideally, be effective against disseminated metastases. We report here that the number of recently established lung metastases of B16 melanoma in C57BL mice treated with ganciclovir is reduced compared to controls after multiple i.v. administrations of high titer retroviral supernatant encoding the HSVtk gene, but not after administration of liposome-complexed plasmid DNA. Using polymerase chain reaction analysis, integration of the provirus was observed in metastasis-bearing lungs (4 of 6 mice) and in the spleens of some ganciclovir-treated animals (2 of 6 mice) but not in the testes, brain, heart, liver, or kidney. The reduction in the number of experimental metastases in C57BL mice exceeded the anticipated extent of transduction of tumor cells, which is indicative of a marked bystander effect. This magnitude of reduction was not observed in immunodeficient athymic mice, suggesting that the immune system plays some part in the bystander effect. In support of these data, we show that, whereas the parental tumor cells are only poorly immunogenic, an effective antitumor immune response is generated following the killing of neoplastic cells in vivo as a result of treatment with ganciclovir. These effects may be responsible for augmenting the efficacy of retroviral infection. The combination of local cell killing by the HSVtk/ganciclovir system and the induction of antitumor immunity suggests new opportunities for the design of vectors for the gene therapy of cancer.
Novel antibodies have been generated by immunizing with bacterially expressed fragments of the repetitive motif of the Ki-67 gene. One such antibody, MIB1, recognizes a fixation and embedding resistant epitope on the Ki-67 protein if sections are previously microwaved in a citrate buffer. We have investigated the utility of this antibody as a marker of cell proliferation in archival material. The microwave technique is simple but requires careful monitoring since different tissues and fixatives require different irradiation times. Strong nuclear immunoreactivity was detected with all fixatives studied. Cytoplasmic staining was not identified. In a wide range of normal tissues the distribution and number of MIB1 immunoreactive cells matched that of cryostat sections stained with Ki-67. In nude mouse xenografts in which the growth fraction had been defined using a fraction of labelled mitosis method, the labelling index with MIB1 matched that previously determined for Ki-67 and correlated well with the growth fraction. Other markers of proliferation (e.g. proliferating cell nuclear antigen) have been shown to be expressed in DNA repair, thus we investigated expression of MIB1 immunoreactivity in situations of DNA repair in vivo--ultraviolet irradiated human skin. MIB1 staining correlated with semi-conservative DNA synthesis rather than excision repair DNA synthesis. Finally, the morphological and cell cycle distribution of MIB1 expression is identical to that of Ki-67. Thus, MIB1 represents a new anti-Ki-67 antibody which appears to be a robust marker of cell proliferation easily applicable to archival material.
Variants of one strain of Neisseria gonorrhoeae, grown in vivo or in vitro, that have been previously shown to differ in infectivity, serum resistance, and capsule production were compared with use of monoclonal antibodies and lectins. Monoclonal antibodies to virulent gonococci recognized an antigenic site of the lipopolysaccharide (LPS) produced in large amounts by gonococci grown in vivo but present only in a small proportion of in vitro-grown gonococci. This antigen (C-LPS) was found in all 85 different gonococcal isolates studied but not among nonpathogenic neisseriae. It was shared by group B and C meningococci but not by groups A and D. Enzyme-linked immunosorbent assay and Western blot analysis showed that N-acetylglucosamine and N-acetylgalactosamine form part of the epitope. The C-LPS antigen was shown by immunofluorescence to be present on the surface of the gonococci and also free as slime. This antigen appears to confer resistance to killing by normal sera.
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The purpose of the present single-case experimental investigation was to examine the efficacy of paradoxical instruction in the treatment of exacerbation-based discontinuous encopresis and chronic constipation. The subject, a 9-yr old male, complained of infrequent bowel movements and daily soiling. In addition, toileting and related activities appeared to cause considerable anxiety. An ABAB reversal design with 1-yr follow-up was used as a means of evaluating treatment effectiveness. Dependent measures included weekly parental records of soiling and appropriate bowel movements. Results indicated a clear demonstration of functional control and consistent improvement in the encopretic disturbance. These findings were maintained at 1-yr follow-up.
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