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Editor,—In their recent article Hopwoodet al (Gut 1997;41:156–63) conclude that only freshly acquired specimens cultured for less than six hours are suitable for studying stress responses ex vivo. This statement arises from the observation that cells from unheated oesophageal biopsy samples cultured for 22 hours (in unsupplemented HAMS-F10 media in an incubator containing room air with 5% CO2 at 37°C) exhibited a “stressed” state. The maintenance ex vivo of gastrointestinal biopsy samples has posed formidable problems in the past; however, successful organ culture of oesophageal biopsy specimens for up to 48 hours has been reported by Browning and Trier using Trowell’s technique,1 2 and in some cases oesophageal tissue has been cultured for up to seven months.3 For successful organ culture the conditions are critical, particularly the addition of calf serum and insulin to the media, and incubation in a gassed mixture of 95% O2 and 5% CO2.1 Under these conditions, ultrastructural integrity, proliferative activity and protein synthesis are maintained. In Hopwood et al’s study, standard tissue culture rather than organ culture conditions were used and this may account for the finding that prolonged culture mimicked thermal stress. Although the tissue may appear intact by light microscopic examination, the integrity of the cultures can be readily assessed by lactate dehydrogenase release into the media and by the electron microscopic appearance of the tissue.4
It has recently been shown that acid has a dynamic effect on the cell phenotype of Barrett’s oesophagus depending on the pattern of acid exposure. However, these phenotypic effects were only apparent after 24 hours of organ culture.4 Therefore, more prolonged organ culture may be necessary in order to investigate fully the oesophageal stress protein response to environmental factors such as oesophago-gastroduodenal refluxate. To conclude that analysis of stress responses using human oesophageal tissue is only feasible following short term culture using the methods described in this study is premature.
Editor,—Stress proteins comprise a wide class of proteins including drug metabolising enzymes, protein kinases/phosphatases, molecular chaperones, metabolic enzymes, DNA repair enzymes, and transcription factors, all of which coordinately respond to subtle or dramatic changes in the extracellular milieu.1-1 Most studies in the field have centred on regulation of the stress proteins using microbes, invertebrates, and human tumour cell lines, and the data acquired do not necessarily reflect in vivo regulation of the same pathways in mammalian tissue. Developing ex vivo cell type specific models will be required to obtain a more physiologically relevant understanding of the effects of stress protein function on normal human tissue integrity and repair.
However, ex vivo organ culture studies will, by virtue of removing the cells from their normal environment, acutely stress and alter some pathways being investigated. Rapid changes in primary/secondary metabolites and signal transduction cascades are well documented following cellular perturbation.1-2-1-5 Such perturbations can stem, in part, from transiently lowered oxygen concentrations during tissue extraction and/or from protein/lipid/DNA damage resulting from oxidative stress.1-6 Activation of these transduction cascades changing gene expression may prevent the physiological study of a stress response system ex vivo.
For example, the AMP activated protein kinase cascade is rapidly perturbed in rat liver ex vivo and the tissue needs to be snap frozen in situ prior to animal death to prevent such hypoxic induced changes in metabolism from altering the system.1-7 1-8 In addition, protein kinase C activity in rat brain has a half-life of minutes ex vivo and very rapid manipulation is required to preserve enzyme activity.1-9 Induction of acute stress can be prolonged; during short term ischaemia and repurfusion in cardiac tissue, HSP70 induction occurs for many hours after injury.1-10 As such, before setting up an ex vivo system to study the heat shock response in normal human oesophageal epithelium, I suspected that metabolic stress would be imposed on the tissue after biopsy and we first set out to determine whether there was, in fact, a biochemical stress imposed by the act of sample collection. Ex vivo culture methods used were those previously optimised to show differences in endocytosis between normal and inflamed oesophageal tissue.1-11 Using these methods to allow direct comparison between the two pathways, we demonstrated that even before organ culture, changes in the steady state level of protein synthesis could occur, compared with rapidly frozen tissue (Gut 1997;41:156–63; fig 1), thus any conclusions we formed on ex vivo heat shock may be compromised by the underlying stress to the cells.
Despite these caveats, successful analysis of the heat shock response ex vivo has been fortuitously possible and we detected two major stress proteins synthesised after heat shock. We have since purified and generated antibodies to one of the heat shock proteins and showed, by immunohistochemistry, unique localisation in vivo (Vojtesek B, Moitra S, Johnston D, et al, manuscript submitted). Nevertheless, we feel that further basic and clinical research in this area will be required to determine whether these and other stress pathways are similarly regulated in vivo. One approach, based on the use and success of in vivo NMR to study metabolic flux in a non-invasive manner,1-12 overcomes the biochemical stresses that are imposed on cells when studying biological processes ex vivo, but this method obviously has its limitations for many enzymatic pathways.
It is my opinion that freshly acquired biopsy samples are preferred to study stress responsive systems and, despite the well documented fact that addition of serum and growth factors can control the survival of cells ex vivo,1-13 the relevance of ex vivo metabolic state to in vivo metabolism is not yet clear. Before premature claims are made that ex vivo organ culture does not induce metabolic stress, I suggest sensitive assays are developed to detect possible perturbations, such as changes in metabolites (such as ATP concentrations), changes in intracellular pH, or activation of well recognised regulators of cell stress, such as JNK or MAP kinases1-3 1-14 and NF-κB or p53 transcription factors.1-15 1-16 Markers of stress, including morphological integrity, proliferative activity, protein synthesis, and lactate dehydrogenase release, are not sufficiently sensitive nor specific to detect subtle changes in metabolism. In addition, the use of radiolabelled thymidine to measure proliferative capacity and the use of radiolabelled methionine to measure protein synthesis can activate cellular damage response pathways1-17 1-18 and are therefore inherently perturbing to the system under study. As such, it would not be accurate to claim that ex vivo organ culture of oesophageal epithelium does not activate interfering stress responses until more sensitive assays are developed.
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