C V Howard

The effect of intrauterine growth retardation on the development of renal nephrons

Objective To investigate the effect of Type II (asymmetrical) intrauterine growth retardation (IUGR) on renal development.

Cerebral malaria in the Rhesus monkey (Macaca mulatta): observations on host pathology

Infection was induced in five rhesus monkeys (Macaca mulatta) by intravenous inoculation with a virulent strain of Plasmodium knowlesi. Approximately one week after inoculation, four of the exposed animals developed acute malaria and died or were killed when moribund after varying periods of illness. Post-mortem and light microscopical examination showed marked cerebral vascular congestion and widespread plugging of the brain capillaries and venules (microvessels) by heavily parasitized erythrocytes mixed with uninfected erythrocytes. Electronmicroscopically, the major changes seen were adherence of large numbers of parasitized erythrocytes and macrophages to swollen microvascular endothelial cells; increased numbers of fibroblasts and deposition of collagen bundles in the extracellular matrix around damaged and parasite-packed microvessels were also found in many areas. This animal model may prove useful for further investigation of the pathogenesis of cerebral malaria.

Nerve fibres in urothelium and submucosa of neuropathic urinary bladder: an immunohistochemical study with S-100 and neurofilament.

Intravesical administration of drugs has been used commonly in spinal cord injury patients to suppress detrusor hyperreflexia (eg oxybutynin, verapamil, terodiline) or, to initiate a micturition reflex (eg 15S 15-methyl prostaglandin F2 alpha, protaglandin E2); however, the response has been variable and sometimes, unpredictable. This prompted us to study the presence of nerve fibres in the vesical urothelium and submucosa in mucosal biopsies taken from the dome and trigone (obtained prior to performing a therapeutic procedure eg, vesical lithotripsy, or a diagnostic cystoscopy) in 47 consecutive, unselected paraplegic/tetraplegic patients with a neuropathic urinary bladder. Nerve fibres were demonstrated by routine immunohistochemical technique using commercially available monoclonal and polyvalent antibodies against S-100 (DAKO A/S, Glostrup, Denmark) and Neurofilament (MILAB, Malmo, Sweden). Biopsy specimens were graded for the presence of nerve fibres on a 0-3 scale for urothelium, and superficial/deep submucosa separately in a blind and randomised manner. Virtually no fibre presence was found in one paraplegic patient and no superficial or single fibres were noted in a tétraplégie patient. Absence of C-fibre hyperplasia (Grade 0) was found in nine cases (paraplegic: 4; tétraplégie: 5); Grade 1 hyperplasia was observed in 17 cases (paraplegic: 4; tétraplégie: 13); Grade 2 hyperplasia was seen in 11 cases (paraplegic: 7; tétraplégie 4); and Grade 3 hyperplasia was noticed in eight cases (paraplegic 3: tétraplégie: 5). The magnitude of C-fibre hyperplasia was not significantly different between paraplegic and tétraplégie patients (x2= 4.64; P = 0.3262). The relationship, if any, between the degree of C-fibre hyperplasia and duration of paralysis was studied by categorising patients as < 5 years, and > 5 years of paralysis. No evidence of single fibre or fibre bundle hyperplasia (Grade 0) was seen in five and six cases, grade 1 hyperplasia in six and 11 cases, grade 2 hyperplasia in two and nine cases, and grade 3 hyperplasia in three and five cases respectively in these two categories of patients. (x2=1.92; P = 0.58). The possible relationship between C-fibre hyperplasia in the vesical mucosa/submucosa and (i) the vesical response to intravesical drug administration; (ii) the vesical urothelial proliferation arrest; (iii) the electrical stimulation of urinary bladder by implanted electrodes (sacral anterior root stimulator); and (iv) long-term indwelling urethral Foley catheter drainage, are discussed with illustrative case reports. In conclusion, mucosal biopsy and study of nerve fibres in urothelium and submucosa of neuropathic bladder has helped to generate hypotheses on the association between C-fibre hyperplasia and response to intravesical pharmacotherapy and the predictive value of such a study in identifying those patients who are likely to respond to intravesical pharmacotherapy.

PCNA independence of Ki‐67 expression in HPV infection.

12 Infectious Wart lesions were stained using the streptavidin‐biotin immunoperoxidase method for PCNA10, MIB‐1 (Ki‐67 equivalent antigen) and Human Papilloma Virus antigen to study the effect of HPV presence on epidermal proliferation. Using strict methods to avoid observer bias, Ki‐67 antigen was found in a high proportion of nuclei in the suprabasal layers together with HPV antigens, in the absence of PCNA staining. This finding indicates that DNA synthesis related, Ki‐67 antigen bearing structures can be raised in the human nucleus in the absence of induction of PCNA bearing structures, suggesting also structural independence between the antigen bearing molecules.

Comparative pathology of dome and trigone of urinary bladder mucosa in paraplegics and tetraplegics

Paraplegic/tetraplegic individuals are prone to develop chronic urinary tract infection, urinary calculi and bladder outlet obstruction, and have a 16 to 28 times higher risk for squamous cell bladder cancer. The preferable method of monitoring those patients who are at high risk of developing vesical neoplasia has been an annual check-up inclusive of cystoscopy and cold cup bladder biopsy of all suspicious areas as well as predetermined random sites. It may be desirable to take a biopsy from one site (when there is no suspicious lesion) with a flexible cystoscope while the patient is sitting in the wheelchair itself in the outpatient clinic instead of multiple biopsies from the dome, trigone and both lateral walls of the urinary bladder taken in the operation theatre set-up using a rigid cystoscope with the patient positioned in lithotomy. Before adopting such a cost-saving and more convenient procedure routinely, we evaluated whether any significant additional histopathological findings are obtained by taking bladder biopsies from the dome and the trigone of the urinary bladder instead of just one, be it dome or trigone in the absence of any visible urothelial lesion in the bladder. In forty consecutive tetraplegic/paraplegic patients who did not have any cystoscopically distinguishable urothelial neoplastic lesion such as papilloma, cold cup biopsies of the dome and the trigone were taken randomly before carrying out any diagnostic or therapeutic procedure, eg electrohydraulic lithotripsy of vesical calculi. All the biopsy specimens were evaluated by a pathologist who was unaware of the clinical details and not involved with the primary diagnosis. In 15 cases, significant additional histopathological finding(s) were recorded in the trigone biopsy which were not seen in the dome biopsy (follicular cystitis: n = 4; squamous metaplasia: n = 4; extensive squamous metaplasia with focal atypia: n = 1; limited focal atypia: n = 1; extensive glandular metaplasia: n = 1; intestinal metaplasia and possibly follicular cystitis: n = 1; and follicular cystitis and intestinal metaplasia: n = 1; mild atypia: n = 1; extensive calcification of epithelial denudation: n = 1). Histopathology of dome biopsies revealed significant additional histopathological finding(s) in nine cases (follicular cystitis: n = 2; squamous metaplasia: n = 2; intestinal metaplasia: n = 1; squamous metaplasia and adenomatoid metaplasia and mild atypia: n = 1; features of interstitial cystitis: n = 1; mild dysplasia: n = 1; mild crypt hyperplasia of urothelium with mild atypia: n = 1). Thus in twenty cases (50%), significant additional findings were obtained by taking cold cup random biopsy of the dome as well as the trigone in the absence of any visible morphological changes. Although single site biopsy may be less traumatic, more convenient to the patient as well as to the staff, and cost-saving, in the spinal cord injury patients with neuropathic bladder, it may not be diagnostically adequate even in those patients who do not have any cystoscopically distinguishable lesion in the urinary bladder.

Vesical urothelium proliferation in spinal cord injured persons: an immunohistochemical study of PCNA and MIB.1 labelling.

Urinary tract infection occurs more commonly, is more virulent and proves more difficult to eradicate in spinal cord injury persons than in the neurologically intact. In order to find out the peculiarities of the neuropathic bladder which make it vulnerable to recurrent cystitis, we studied the proliferation status of the urothelium in spinal cord injured persons. Eleven consecutive, unselected male spinal cord injury patients (aged 18–73 years) were included in the study. Those with, or undergoing treatment for acute urinary tract infection were excluded. All patients underwent cystoscopy and cold cup bladder biopsy from the trigone and bladder dome. Immunocytochemical analysis was performed using defined, commercially available antibodies for PCNA (PCNA 10, DAKO) and MIB-1 (raised against recombinant DNA defined segment of Ki-67 antigen DAKO) streptavidin/biotin and alkaline phosphatase immunocytochemistry (for MIB-1 with microwave-enhanced antigen retrieval) were used to demonstrate the presence of cell cycling-associated nuclear proteins. Foci of lymphocytic aggregations present in the sections served as in-section controls for antigen preservation. Ten patients showed labelling of 20–70% of cells for PCNA in basal cell layers of dome lining. Higher urothelial layers showed a variable, but generally reduced degree of labelling. Of these 10 patients, three showed complete absence of MIB-1 activity in the basal and other layers of dome urothelium and two demonstrated only a very occasional positive nucleus. MIB-1 labelling was < 5% in four others and it was between 5% and 10% in one. In the trigone, PCNA labelling was 20–70% in nine and < 20% in two others. Of the former nine, MIB-1 labelling was < 5% in five; patchy staining was observed in one; and MIB-1 labelling was completely absent in three. The absence of proliferation was not correlated with urothelium thickness. Absence of MIB-1 expression, with maintained PCNA expression suggests effective arrest due to external factors. The dissociation between MIB-1 and PCNA expression may be an effect of defective innervation of the bladder in spinal cord injured persons. Absence of proliferation would be expected to alter the relative maturity of bladder lining cells. Alterations in the surface structure of urothelium may contribute to changes in the expression of cell adhesion molecules relevant to bacterial adherence and colonisation.

A quantitative immunocytochemical analysis of total surface area of blood-brain barrier in developing rat brain

A mouse monoclonal antibody which specifically reacts with putative blood-brain barrier (BBB) competent endothelial cells of rat cerebral capillaries was used to identify barrier competent cells in the central nervous system (CNS). The development of the cerebral capillaries and the BBB was examined and quantified, from day 6 to day 40 postpartum, using immunocytochemical and unbiased stereological techniques.There was a progressive increase in capillary formation postnatally, with collateral branching observed with progressive age. BBB development was confined to individual endothelial cells located at the periphery of the cortex until day 10 postpartum. Antibody binding progressively increased postnatally, contributing 30% of the total capillary surface area by day 20. There was a rapid elevation of reactivity from day 20 to day 40, with a mean of 83% by day 40.The BBB constitutes minimal amounts of brain vascular capillaries before day 10 of life in the rat. There is a slower increase in BBB than in total capillaries between days 10 and 20. There is a reversal of this trend between days 20 and 40.

Cerebral malaria in the rhesus monkey (Macaca mulatta): Light and electron microscopic changes in blood cells and cerebrovascular endothelia

To assess the interaction between the cellular elements of the blood and neurovascular endothelia in cerebral malaria, brain tissue from adult rhesus moneys (Macaca mulatta) infected with a virulent (W1) strain of Plasmodium knowlesi were studied by light and electron microscopical techniques. Light microscopical examination showed sequestration of macrophages and margination of erythrocytes containing late stages of the parasite in the capillaries and venules throughout the brains of the infected monkeys. Brain microvascular lesions (associated with parasitised erythrocytes and macrophage attachment to vascular walls) seen with the electron microscope, were swelling of the endothelial cells, formation of pseudopodia, increased numbers of pinocytotic vesicles and disorganisation of the mitochondria. Parasitised mature erythrocytes and macrophages adhered to the vascular endothelial lining in equal proportions. The endothelial ultrastructural alterations were similar to those described in experimental rodent and in clinical human cerebral malaria.

An Assessment of Volume-weighted Mean Nuclear Volume Estimates as a Prognostic Index for Neuroblastoma

This study evaluates an objective, unbiased, stereologic parameter (volume-weighted mean nuclear volume vV) as a prognostic indicator for survival of neuroblastoma in comparison with three histopathologic grading systems. In this retrospective study 24 consecutive, nonselected patients from the registry of Royal Liverpool Childrens Hospital, Alder Hey, England were analyzed. Only primary lesions obtained before chemotherapy were used. Follow-up time in surviving patients (n = 10) was 4.5 to 20 years. All lesions were regraded blind and twice by two pathologists. vV was estimated on routinely processed, hematoxylin and eosin-stained, 5-microns sections, requiring on average less than 20 min per patient. Results show an absolute cut-off point for survival at vV = 270 microns 3. No patient with vV less than this value is alive at present (n = 7). In addition, actuarial survival curves for nonsurvivors show a bimodal pattern of survival time, separating patients with vV greater than (n = 7) and less than (n = 7) 270 microns 3. In comparison with the same analysis for the results of regrading by means of the Hughes or Beckwith system, results of vV estimation were superior. In comparison with the Shimada system the results confirmed the strong dichotomy for survivors and nonsurvivors, although with more overlap. vV has the advantage of predicting length of survival in nonsurvivors. The combination of Shimada grading and vV measurement, on the basis of the material studied, seems to offer useful prediction of biologic behavior. vV was always estimated on the small cell population, reducing the problems of biopsy representability.

Stereological Analysis of the Neocortex With Respect To Total Neuronal Number and Mean Nuclear Volume in Sudden Infant Death Syndrome (SIDS)

Stereological Analysis of the Neocortex With Respect To Total Neuronal Number and Mean Nuclear Volume in Sudden Infant Death Syndrome (SIDS)