Biomedical subjects
Publications and source records attributed to R M Taylor.
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Three stable carcinoma cell lines, designated RM22-F5, RM17-5R, and RM1-4, were established from spontaneously occurring mammary carcinomas in old, outbred, female Wistar rats. The RM22-F5 and RM17-5R cells were keratin-positive and formed epithelial monolayers, whereas RM1-4 cells exhibited a spindle-like morphology and intense vimentin staining. When injected into nude mice, RM22-F5, RM17-5R and RM1-4 cells formed well-differentiated, poorly differentiated and undifferentiated carcinomas, respectively. The relative growth rates of the tumor cells in vitro were RM1-4 > RM22-F5 > RM17-5R. The growth of RM22-F5, but not of RM17-5R and RM1-4 cells, was significantly stimulated by insulin, epidermal growth factor, dexamethasone, 17 beta-estradiol and progesterone in vitro. Ovariectomy reduced the growth of RM22-F5 cells in vivo and these cells (but not RM1-4 or RM17-5R) were estrogen-receptor (ER)-positive. None of the lines were positive for the progesterone receptor (PR). Spontaneous lung and lymph-node metastases were observed in nude mice injected with RM22-F5 or RM17-5R cells, respectively. In contrast, RM1-4 cells were non-metastatic but invasive. Karyotype analysis revealed that RM22-F5 cells were hyperdiploid, RM17-5R were hypotetraploid, and RM1-4 were diploid with a sizeable insertion in chromosome 1. A point mutation in codon 12 (G to A transition) and loss of the normal allele of the H-ras-1 gene was detected in the DNA from RM22-F5 cells. No p53 mutations were apparent in any of the cell lines. The results indicate that RM22-F5 cells are hormone-dependent with an ER+/PR- phenotype, while the RM17-5R and RM1-4 lines are hormone-independent and ER-/PR-. These cell lines exhibit the spectrum of biological properties and genetic alterations observed in human breast cancers and may, therefore, be novel and useful models for understanding sporadic breast cancer in post-menopausal women.
An in vitro model for cross-correction of lysosomal storage disorders from genetically modified cells was developed to approximate the physiological conditions needed for gene therapy in vivo. beta-Glucuronidase (GUSB)-deficient mucopolysaccharidosis (MPS) type VII (Sly disease) cells were studied to determine the amount and stability of enzyme transfer. Gene transfer by retroviral vectors encoding rat or human GUSB corrected the enzymatic deficiency in cultured MPS VII fibroblasts from humans, dogs, and mice. The vector-transduced cells released GUSB into the culture supernatant in amounts proportional to the amounts of cellular GUSB activity. The activity of the exported GUSB was stable in tissue culture medium for at least two weeks. Uninfected MPS VII target cells incorporated GUSB from the medium when the vector-infected donor cells were separated from the deficient target cells by a fluid-permeable membrane. The high level of GUSB in the cross-corrected target cells decreased rapidly after the enzyme donor cells were removed.
Over the past few years there has been increasing awareness of the importance of humoral mechanisms in the rejection of renal transplants. In this study we have monitored the development of antibodies directed against donor T and B lymphocytes using the sensitive flow cytometric technique. Forty-two cadaveric renal transplants were studied both before and for a maximum of 14 days after transplantation. Donor cells were separated from spleen on the day of transplantation and stored in liquid nitrogen until required. The dual colour flow cytometric assay was used to detect IgG or IgM directed against donor T or B lymphocytes. Using AB sera as controls, results were expressed as relative median fluorescence (RMF) and then correlated with the clinical performance of the grafts. Significant associations were found between the incidence of donor-directed antibodies and the development of clinical rejection. The magnitude of the rise in antibody levels was also related to graft performance. In patients showing severe graft rejection, high levels of antibodies of the IgG class developed before the clinical diagnosis of rejection was made. The routine use of this test allows the prediction of impending severe rejection to be made and may have important implications for immunosuppressive therapy.
To understand the events involved in selective eosinophil migration into allergic inflammatory sites, we studied the expression of vascular endothelial adhesion molecules in the nasal mucosa. Ten subjects with asymptomatic seasonal allergic rhinitis and 13 nonallergic subjects underwent localized allergen challenge of one inferior turbinate. Twenty-four hours later, biopsy specimens were obtained from the inferior turbinates, bilaterally in the seasonally allergic subjects and unilaterally in the nonallergic control subjects. The specimens were divided, sectioned, and either stained for identification of eosinophils or analyzed immunohistochemically for intercellular adhesion molecule-1, E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and von Willebrand's factor. Intercellular adhesion molecule-1 expression was observed in all mucosal specimens, with no significant difference among groups. E-selectin showed minimal baseline expression, and low levels were significantly induced on the challenged mucosa of the allergic compared with nonallergic subjects (p < 0.05). VCAM-1 was expressed basally and was significantly upregulated by allergen challenge, compared with the nonchallenged side and nonallergic control subjects (p < 0.05). Submucosal eosinophils increased significantly in allergic subjects 24 hours after antigen challenge, compared with nonallergic control subjects and weakly correlated with VCAM-1 expression (rs = 0.33, p = 0.06). Our results suggest that endothelial activation accompanies allergic inflammation. Furthermore, because the counterligand for VCAM-1, very late activation antigen-4, is present on eosinophils, VCAM-1 upregulation may contribute to the selective recruitment of these cells to the nasal mucosa.
A rapid and robust limiting dilution assay was developed to measure the frequency of donor-reactive, IL-2 (interleukin 2) producing, helper T lymphocytes in the peripheral T cell population of organ allograft recipients. The IL-2 bioassay was performed using two methodologies to assess the response of CTLL-2 indicator cells. The first depended on spectrophotometric detection of bioreduced XTT whilst the second involved measurement of [3H]thymidine incorporation. The radioisotopic method was slightly more sensitive but both assays could be used for analysis of limiting dilution culture supernatants after primary incubation of recipient lymphocytes with donor splenic cells for 48 hours. All the assays produced results which conformed to single hit kinetics, indicating that IL-2 was production was dependent on a single limiting cell type. The frequency of allospecific helper lymphocytes in the peripheral T cell population of normal volunteers did not vary significantly during a 28-day period. It was found that immunosuppressed allograft recipients had a significantly reduced proportion of T cells in their peripheral blood mononuclear cell population. However, it was possible to measure the frequency of donor-reactive helper cells in the T cell population of transplant patients. These frequency values were very low in two renal allograft recipients who were HLA-DR matched to their donor organs. Three of four HLA-DR mismatched cardiac recipients showed a significant decrease in the frequency of their donor-reactive helper lymphocytes during the period of monitoring. The fourth patient, who received antilymphocyte antibodies for the first three days after transplantation, showed significant fluctuations in the frequency of these cells. The four cardiac recipients showed little histopathological evidence of acute graft rejection with only one patient experiencing a brief episode of moderate rejection; this patient showed a high frequency of donor-reactive helper cells when assayed immediately after this episode but the frequency subsequently declined.
Specific adhesion molecules stabilize the binding between lymphocytes and antigen bearing cells; this intercellular adhesion is vital to both the affector and effector phases of an immune response. It is not known whether adhesion molecules and their counter-receptors can form the cross-species interactions that will facilitate human T cell recognition of xenogeneic porcine target cells. In this report it is demonstrated that a higher proportion of mitogen-activated than of resting human lymphocytes adhere to cultured porcine renal epithelial cells. Furthermore, antibody blocking experiments demonstrated that at least part of this cell-cell binding is stabilized by the human adhesion molecules LFA-1 (lymphocyte function-associated antigen-1) and the alpha 4-containing integrins. It is possible that this capacity for cross-species adhesion will play a role during the cell-mediated rejection of clinical porcine xenografts.
In order to study the immunogenicity of parenchymal cells within an allograft, renal tubular cells were propagated from both PVG and DA strain rats. These cells were induced to express class II major histocompatibility (MHC) antigens by stimulation for 4 days with interferon-gamma (IFN-gamma). It was found that resting lymphoid cells derived from Lewis rats responded vigorously after stimulation with irradiated splenic cells from PVG rats. However, stimulation with renal cells from PVG rats did not result in interleukin (IL-2) production or lymphoproliferation. Furthermore, lymphocytes from this mixture failed to respond to secondary stimulation by PVG splenic cells; lymphocytes primed by mixture with DA renal cells responded normally to secondary stimulation by PVG splenic cells. These results indicate that renal epithelial cells can specifically anergise allogeneic lymphocytes.
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A total of 1016 consecutive renal transplants performed between 1976 and 1990 were analysed retrospectively to determine the incidence of urological complications and possible predisposing factors. Some 189 episodes of ureteric obstruction and/or urinary leak occurred in 143 patients (overall incidence 14.1%). The median annual rate of urinary leak was 5.1%; that of ureteric obstruction was 4.5% pre-1986 and 16.1% post-1986. Sixty-three episodes of urinary leak occurred in 54 patients from 1 day to 3 months post-transplant and 60% involved the distal ureter. Thirty were treated primarily by reconstructive surgery, ten required nephrectomy and three died of associated sepsis. A total of 126 episodes of ureteric obstruction occurred in 104 patients from 1 day to 12 years post-transplant and 86% involved the distal ureter. Prior to 1986, 10/11 patients with ureteric obstruction were treated by reconstructive surgery, but since then 88 (95%) have been treated by percutaneous nephrostomy, with or without stenting, with only one graft lost and no deaths. Children had a significantly increased incidence of ureteric obstruction (P < 0.001) whilst male recipients had an increased incidence of urinary leak (P = 0.04). More patients with ureteric obstruction than those without had two or more episodes of rejection (P = 0.03). No single cause for the increased incidence of ureteric obstruction since 1986 has been identified. Continued attention to technical detail and further study of this trend is warranted.
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The 'Em' polypeptide is the most abundant cytosolic polypeptide in mature wheat embryos. It is selectively and completely degraded within the first 24 h of germination. Extracts from germinated embryos contain endopeptidase activities which degrade the Em polypeptide. These are separable into a major and minor component by ion-exchange chromatography and the use of inhibitors shows the major component to be a cysteine proteinase. This activity shows a strong preference for the Em polypeptide as a substrate, being inactive against polypeptides which are not developmentally regulated and showing only low activity towards developmentally related, but otherwise nonhomologous 'dehydrin' polypeptides.
Germinating wheat embryos contain two endoproteolytic activities which digest the prominent 'Em' polypeptide. These are easily assayed in clarified embryonic homogenates and are distinguishable by the pattern of their peptide products and by their different pH optima. One activity has a pH optimum of 4.0; the second activity is a cysteine endoproteinase with a preference for the 'Em' protein as its substrate. It is maximally active between pH 5.5 and 6 at 25 degrees C. Analysis of the early cleavage products of the cysteine proteinase indicates scissile bonds between residues Glu32-Ala33 and Asn36-Leu37 in the 'Em' polypeptide. This endoproteinase has been purified and identified as a single polypeptide species of ca. 38,000 kDa.
In renal transplantation, treatment of steroid-resistant rejection (SRR) with antithymocyte globulin (ATG) has been widely reported but over-immunosuppression remains a common problem. In the first ten patients (group 1) treated for SRR with rabbit ATG, three developed serious viral infections and two deaths occurred due to CMV pneumonitis. ATG was only omitted if thrombocytopenia or neutropenia occurred. In the next 17 patients (group 2) with SRR, ATG was administered according to the absolute T lymphocyte count. T lymphocytes were measured by flow cytometric analysis of CD3-labelled lymphocytes. ATG dosage was adjusted on a daily basis to keep the absolute T lymphocyte count under 50 cells/microliters. Administration of ATG according to the absolute T lymphocyte count resulted in a significant reduction in the mean dose of ATG given to the group 2 patients (P < 0.001). A significant decrease in the incidence of serious viral infections (P = 0.04) was achieved without reducing the ability of ATG to reverse the SRR (P = 0.29) or increasing the number of grafts lost at 1 year in the group 2 patients (P = 0.23).