A. R. Morley

Cell kinetics in flat (avillous) mucosa of the human small intestine

A new method for the analysis of small-intestinal crypt-cell kinetics using routine peroral diagnostic biopsies is described. Untreated patients with childhood and adult coeliac disease and adults with the gluten-sensitive enteropathy of dermatitis herpetiformis were studied, together with groups of adult and childhood controls. In the classical flat avillous mucosae the increase in crypt size was found to be three-dimensional. The number of proliferating cells per crypt was shown to be markedly increased, and an even greater rise in the crypt-cell production rate was demonstrated. A significant increase in the mitotic index was also confirmed in the avillous mucosae. On the basis of these findings it is suggested that the characteristic crypt morphology in glutensensitive enteropathy can be explained as an adjustment to accommodate the expanded mass of proliferating and maturing cells necessary to support the augmented cell production rate. We may speculate that this in turn is a response to a pathologically rapid loss of cells from the mucosal surface.

VARIATION IN THE DURATION OF MITOSIS IN THE CRYPTS OF LIEBERKUHN OF THE RAT; A CYTOKINETIC STUDY USING VINCRISTINE

The variation in the duration of mitosis (tm) with cell position in the small intestinal crypts of the adult rat has been measured by a stathmokinetic technique using vincristine. The value for the whole crypt column was 0.43 hr, or 26 min. At the bottom of the crypt tm was in excess of 1 hr, but rapidly decreased and throughout the greater part of the proliferative compartment was between 0.40 and 0.50 hr. At the top of the proliferative compartment an increase in tm was demonstrated.

The cell cycle time in the flat (avillous) mucosa of the human small intestine

A hyperproductive mucosal state in gluten-sensitive enteropathy has been proposed on the basis of an elevated mitotic index, but this parameter is dependent on the mitotic duration when used as an index of proliferative status. The mitotic duration was therefore measured in two control patients with normal villous mucosae and in two patients with the flat avillous mucosa of untreated gluten-sensitive enteropathy, using two different stathmokinetic techniques with vincristine. No significant difference in mitotic duration was found but values obtained for cell cycle time showed a halving in the flat mucosae. An increased rate of cell production in the small bowel mucosa of untreated gluten-sensitive enteropathy is thus confirmed.

Renal allograft rejection--in situ demonstration of cytotoxic intratubular cells.

A nonisotopic in situ hybridization method to detect perforin mRNA was developed in cytospin preparations of IL-2-stimulated normal human lymphocytes and applied to formalin-fixed acutely rejected renal transplant material. Individual cells expressing perforin mRNA were localized in severely damaged tubular areas, and a number of these cells appeared to be located inside the tubular basement membrane in close association with tubular epithelial cells. Immunoperoxidase staining in acetone-fixed cryostat sections of acutely rejected kidney confirmed that a considerable proportion of infiltrating cells was CD8+; many of these were in an intratubular location. In addition, perforin protein was identified in individual cells in similar locations to perforin mRNA-positive cells. Again, some intratubular cells were identified. Our findings illustrate that these cells can be fully activated with definite cytotoxic potential. Previously we have demonstrated that T lymphocytes proliferate within the tubular compartment during tubulitis, a characteristic condition in acute renal allograft rejection, and that there is associated tubular epithelial cell proliferation. In this study we think that we have further clarified the consequences of invasion of tubules by lymphoid cells. Our in situ hybridization method in rapid and convenient and may be applied to archival material.

Cell proliferation in the castrate mouse seminal vesicle in response to testosterone propionate. I. Experimental observations.

The cell proliferation kinetics following induced DNA synthesis in the mouse seminal vesicle were measured after treatment with testosterone propionate. Fraction labelled mitosis curves at 24, 48 and 72 hr after injection gave t2 values of 1·5, 2·0 and 1·8 hr respectively, and ts values of 10·5, 8·0 and 8·0 hr. Tc measured 48 hr after stimulation was 17·5 hr. Growth fraction rose from 0·14 at 24 hr to 0·64 at 48 hr, and fell to 0·32 by 72 hr. A simple model is proposed in which the rise and fall of mitotic index and labelled index is determined by the ‘cell distribution ratio’.

In situ lymphoproliferation in renal transplant biopsies

A double immunohistochemical labelling procedure in paraffin-embedded renal tissue is reported in which CD3 was targeted as a T cell marker and Ki67 as a marker of cell proliferation. Proliferating and quiescent T cells were unequivocally identified in situ, and their precise location within the kidney was clarified by the use of periodic acid-Schiff counterstaining to outline the basement membranes. Proliferating tubular epithelial cells were also clearly identified. The results showed that T lymphocytes proliferate within the tubular compartment during acute renal allograft rejection. Preliminary evaluation of the method in routine transplant biopsies indicated significant correlations between histologically defined rejection grade and mean intratubular T lymphocytes per tubular cross section and between proliferation of tubular epithelial cells and of intratubular T lymphocytes. The associated tubular epithelial cell proliferation may be a response to local damage.

The measurement of cell production rates in the crypts of lieberkuhn

A method is described of assessing the proliferative state in the small bowel mucosa employing the metaphase-arresting (stathmokinetic) properties of vincristine, applicable to both animals and man. In the rat a value for the migration rate of 1.78 cell positions per hour was obtained by the stathmokinetic method, in agreement with a further measurement of the migration rate made using tritiated thymidine. Values for the transit time through the crypt (34 hours) and for the cell production rate (39 cells per crypt per hour), are in good agreement with previously published values. A control patient and a patient with the flat mucosa of gluten-sensitive enteropathy were also studied by the vincristine technique. The migration and cell production rates were markedly increased in the flat mucosae. A hyperproductive proliferative state is therefore confirmed in the flat mucosa of untreated gluten-sensitive enteropathy. The results show the vincristine technique to be eminently applicable to problems involving intestinal cell kinetics.

The associations of HLA and other genetic markers with glomerulonephritis

SummaryOne hundred and seventy-nine patients with various forms of glomerulonephritis confirmed histologically were tested for HLA A and B antigens: Thirty-four with membranous glomerulonephritis were also typed for DR antigens. One hundred and forty-one of these patients were further tested for blood group, red cell enzyme, and plasma protein systems. The minimal-change and the mesangio-capillary glomerulonephritis showed a significant association with B8 and Bw44 antigens respectively, whereas the membranous nephritis in addition to B8 was also found to be associated with DR3 antigen. Previously described associations with Henoch-Schönlein and Bergers nephritis were not proved. A large group with nonspecific proliferative glomerulonephritis did not show any association with HLA. Among the other single-gene characters studied, a significant association was found with Bf (Factor B or C3 proactivator) and adenosine deaminase, both markers thought to be involved in the immune response.The closc association of the markers located on chromosome 6 and glomerulonephritis indicates that there may be an immunological component in the aetiology of the disease. The significance of the various associations found is discussed.

Tissue distribution of amyloid P component as defined by a monoclonal antibody produced by immunization with human glomerular basement membranes

SummaryA monoclonal antibody reactive against amyloid P component (NCL-AMP) has been developed following immunization of mice with partially-purified human glomerular basement membranes (GBM) and standard hybridization and cloning techniques. The antibody reactivity was evaluated by enzyme-linked immunosorbent assay (ELISA) and by the indirect immunoperoxidase technique on sections of frozen and fixed human kidney and other tissues. The distribution of amyloid P component in various normal tissues is described and the possible co-localization with the Goodpasture antigen is discussed. In addition, the suitability of the antibody for detection of amyloid deposits in renal amyloidosis is demonstrated and its potential for use in other pathological conditions is considered.

In vivo bromodeoxyuridine incorporation in normal mouse kidney: Immunohistochemical detection and measurement of labelling indices

SummaryA method is reported forin vivo bromodeoxyuridine incorporation in mice and its subsequent visualization in kidney by immunohistochemistry.Following formal-sublimate fixation of the kidney, bromodeoxyuridine labelled nuclei were detected in paraffin sections with a monoclonal antibody and visualized by an immunoperoxidase technique. This rapid and unequivocal method was used to measure labelling indices in tubules, glomerular tuft and Bowmans capsule in normal male T70 (Beige) mice, at intervals up to 72 h after labelling. Significant differences were found between the labelling indices of these three populations of cells, which appeared to show different cell kinetic behaviour.