Search

This chapter addresses the evidence available for assessing the incidence of cancer in pre-industrial societies. We conclude that cancer rates must have been very low in pre-industrial societies living traditional lifestyles, and this is considered in the light of the increasing average life expectancy experienced in industrialised societies during recent times. Cancer incidence rates are compared with the more widely reported 5-year mortality rates. Recent epidemiological evidence, from twinning studies, for the connection between human cancer and environmental factors is discussed.

Cancer is a complex issue that has been the subject of observation and research for many disciplines which have investigated the different aspects of cancer aetiology. The predominant theory for the past 50 years has been that cancer is the result of cumulative mutations that alter specific locations in a cell’s DNA and which alter the proteins encoded by cancer-related genes. Susceptibility to mutation has been mainly researched with respect to people’s genetic make up and their lifestyles, which are largely dictated by behavioural patterns. However, a growing body of scientific evidence strongly implicates the environment in the causation of cancer. Findings from studies of wildlife, cancer trends, human migration, childhood cancer, twinning and industrial accidents suggest that concentrating solely on genetic origin and behavioural pattern as causes of DNA defects, and consequently the main causes of cancer, needs to be re-evaluated. There is now clear scientific evidence implicating exposure to radiation and man-made chemicals as major causes of mutation. In addition there is evidence that non-genotoxic mechanisms causing tissue dysgenesis might be a significant aetiological mechanism during development and this must become an element of any future re-evaluation.

Speculation about potential neurotoxicity due to chronic exposure to low doses of organophosphate (OP) pesticides is not yet supported by experimental evidence. The objective of this work was to use a cell culture model of chronic OP exposure to determine if such exposure can alter the sensitivity of nerve cells to subsequent acute exposure to OPs or other compounds. NB2a neuroblastoma cells were grown in the presence of 25 microM diazinon for 8 weeks. The OP was then withdrawn and the cells were induced to differentiate in the presence of various other pesticides or herbicides, including OPs and OP-containing formulations. The resulting outgrowth of neurite-like structures was measured by light microscopy and quantitative image analysis and the IC(50) for each OP or formulation was calculated. The IC(50) values in diazinon-pre-exposed cells were compared with the equivalent values in cells not pre-exposed to diazinon. The IC(50) for inhibition of neurite outgrowth by acute application of diazinon, pyrethrum, glyphosate or a commercial formulation of glyphosate was decreased by between 20 and 90% after pre-treatment with diazinon. In contrast, the IC(50) for pirimiphos methyl was unaffected and those for phosmet or chlorpyrifos were increased by between 1.5- and 3-fold. Treatment of cells with chlorpyrifos or with a second glyphosate-containing formulation led to the formation of abnormal neurite-like structures in diazinon-pre-exposed cells. The data support the view that chronic exposure to an OP may reduce the threshold for toxicity of some, but by no means all, environmental agents.

Lectins are proteins or glycoproteins of nonimmune origin, which bind specifically to carbohydrate structures. They are widespread in the human diet, and many are resistant to digestion. High doses of lectins have been shown to stimulate intestinal and pancreatic growth. The aim of the present study was to investigate the long-term actions of low doses of lectins on the rat intestine and pancreas. A long-term carcinogenesis study was performed using low levels (40 micro g/rat/day) of peanut (PNA) or mushroom lectin (ABA) which bind to O-linked (mucin-type) oligosaccharides in the gut. While this was primarily designed as a colon carcinogenesis study, the pancreas was also investigated. No significant changes in colon carcinogenesis were seen, however, the colons were slightly heavier in the lectin treated groups. The weight of the pancreas was significantly greater (by 18 and 23%) in both lectin treated groups (P0.03/0.001). The weights of the acini and septal tissue were also increased by 39-46% in PNA and ABA fed animals, respectively (P0.002); there was no significant change in the endocrine pancreas. In conclusion, long-term feeding of low doses of lectin can influence pancreatic growth, and this trophic action may have potential adverse implications for the development of pancreatic cancer in humans.

Entire reproductive tracts were removed from seven normal healthy heifers and qualitative unbiased estimates made of endometrial gland volume density and glandular surface density. After examining approximately 55 microscopic fields of endometrium from each tract, a mean glandular surface density value of 10.2 mm(2)/mm(3) (CE 3.1%) was obtained. The stereological method was then employed in optimising the design of the main study. The endometrial height was measured for 17 healthy cycling heifers and 19 similar cows. Subsequently, unbiased estimates were made of intercaruncular endometrial gland volume per unit surface for all cattle were investigated; differences between heifers and cows generally, and the possible effect of the follicular and luteal phases of the oestrous cycle were compared. The mean surface area of glands per unit area of endometrium at the intercaruncular site in heifers and cows was approximately 18 mm(2)/mm(2) in the follicular phase and 26 mm(2)/mm(2) in the luteal phase, figures similar to the gland area found in women. The intercaruncular gland volume increased significantly, by about 30% during the luteal phase of the bovine oestrous cycle in heifers, from 0.01 to 0.13 per mm(3). The differences in endometrial anatomy between site of sampling and either follicular or luteal phases of the oestrous cycle were always more significant in heifers than cows. The endometrial thickness in cows was always greater than for heifers, irrespective of the site of sampling. It was concluded that the intercaruncular endometrium of cattle was far more active physiologically than recognised previously.

Stereological sampling regimes, in particular volume and number estimation, often require systematic uniformly random sections throughout a specimen. A method has been developed to increase the efficiency of preparing fish larvae for sectioning prior to histological or stereological analysis. Embedding a group of larvae in a resin block using this technique greatly reduces the quantity of sections produced and allows easy assessment of sample groups. Saving time in this way therefore makes stereology a more viable research tool.

At present, no information is available with regards to either neocortical neuronal mean nuclear volume or maturation (functional or morphological) in abnormal paediatric brains. Using the nucleator estimator technique, the mean neocortical nuclear volume was estimated in sudden infant death syndrome (SIDS) cases [10 normal birth weight (NBW) and 10 low birth weight (LBW) cases classified by birth weight for gestational age] and compared to 10 NBW control cases. Both the control and SIDS LBW cases showed an increase in mean nuclear volume with age; the SIDS NBW cases showed no increase. At 8 months, the SIDS NBW cases showed a reduced mean nuclear volume (p = 0.02) when compared to controls. The SIDS LBW cases showed no statistically significant difference (p = 0.10) when compared with controls. A deficiency in mean neuronal nuclear volume may represent a deficiency in neuronal function.

The estimation of object volume from rotationally randomised sections relies on an ancient geometrical principle due to the Alexandrian mathematician Pappus. In this paper we describe two studies that make estimates of eye volume using a stereological version of the Pappus principle. The first study uses a design-based version of the Pappus theorem to make estimates of eye volume in Sprague–Dawley rats and the second uses a model-based implementation for estimating eye volume in juvenile Dover sole. In the sole study we compare the estimates of volume with estimates made using the Cavalieri method and show that the Pappus method gives identical volume estimates with a markedly lower coefficient of variation (2.5%) than the Cavalieri method (13.5%).

To comprehend the complexity of the environmental hormone disrupter problem, it is important to have a conceptual framework within which to interpret detailed scientific information. This chapter attempts to provide such a framework, with the intention of making the more technical chapters and their references understandable by decision-makers, journalists, lawyers and other people who have an interest but who do not necessarily come from a scientific background.

This chapter concludes the contributions provided in these proceedings. Current understanding of the health effects of endocrine disrupters on humans does not provide proof. However, it strongly suggests that endocrine disrupters are causing important adverse health effects including reduced sperm counts, increases in congenital malformations (cryptorchidism, hypospadias), an increase in testicular cancer, and altered immune responses, to list some of them. At the same time, there is a lack of knowledge concerning the testing and monitoring methods which will lead to improved risk assessment.

This chapter introduces key themes from the book, reflecting the topics of the ‘Awareness of the Health Impacts of Waste Management Policies’ Seminar, in Kos, Greece, November, 1998. The waste management and health scene is introduced by an outline of concerns relating to persistent organic pollutants, as well as through perspectives from Less Developed Countries and from industry. An overview of policy lines for waste management includes an assessment of recent proposals by the European Commission, as well as selected examples from European countries and the USA. Descriptions of developments in research, tools and conceptual approaches for waste management and health issues are provided. Research into health effects of dioxins and PCBs is outlined, as are reviews of technological options for waste management, proposed developments in health impact assessment, environmental taxes as a waste management tool, and integrated regional waste management approaches. A series of case studies provide real-world examples of research and policy development including a review of the effects of waste management on wildlife and domestic animals. In conclusion, important crossover themes and challenges are outlined. Topical issues include differences between technological capacity and actual performance, burden of proof and the precautionary principle, hazard versus risk assessment, and societal dimensions of awareness and attitudes. Time-lag, intergenerational effects and the introduction of the chemical hygiene concept are highlighted as important considerations, as well as the general need for prioritisation of the child and infant in all regulatory procedures. Such themes highlight the need for both scientific and policy advance in response to the links between waste management and public health.

Humans are at the top of the food web, and are therefore particularly vulnerable to exposure to those pollutants that are persistent and which bioconcentrate. The major route of human intake of these compounds is food of animal origin. Release and cycling of these compounds in the human food chain is an important problem.

The organic chemical industry, in the space of half a century, has produced millions of tons of persistent carbon-based compounds which can now be found in every part of the globe and in the body fat of every living creature. These compounds can be either intentionally released products (such as DDT), or unintentionally released as industrial chemical by-products (such as the dioxin-like substances). The majority of the dioxins and furans that have been introduced into the environment have come about as a result of the incineration of municipal and clinical waste, which contains high levels of halogenated compounds and heavy metals. Persistent organic pollutants (POPs) are lipophilic and difficult to metabolise, and therefore they bioconcentrate in the body over time and biomagnify up the food web.

This chapter concludes the information provided in these proceedings. The state of the art of the knowledge on health hazards associated with waste deposit sites and incinerators is overviewed. In particular cancers, congenital malformations, genetic damage, neurological and behavioural changes, immunological impairment, reproductive failure and interference with hormones seem to be the main health end points of the pollutants involved. These data also point to the fragmentary and ad hoc character of this information. Both elements are crucial in the scientific uncertainty which is manifest.

Even the most modern municipal waste incinerators (MWIs) emit an aerosol of ultrafine particles which current bag filter technology cannot abate. Collection efficiencies for particles < 2.5 μm are between 5 and 30 per cent. For particles of < 1 μm, collection efficiency, most particles will pass unabated. Ultrafine particles have been found to be chemically highly reactive, even when originating from a relatively unreactive bulk material. The massive surface area associated with a small mass of nanometre-sized particles can act as a catalytic surface for the secondary formation of organic compounds.

Classical stereology is capable of quantifying the total amount or ‘density’ of a geometrical feature from sampled information, but gives no information about the local spatial arrangement of the feature. However, stereological methods also exist for quantifying the ‘local’ spatial architecture of a 3D microstructure from sampled information. These methods are capable of quantifying, in a statistical manner, the spatial interaction in a structure over a range of distances. One of the key quantities used in a second-order analysis of a volumetric feature is the set covariance. Previous applications of covariance analysis have been ‘model-based’ and relied upon computerized image analysis. In this paper we describe a new ‘design-based’ manual method, known as linear dipole probes, that is suitable for estimating covariance from microscopic images. The approach is illustrated in practice on vertically sectioned lung tissue. We find that only relatively sparse sampling per animal is required to obtain estimates of covariance that have low inter-animal variability.

Publisher Summary The chapter uses a series of ocular tissues to demonstrate how intact or thick specimens can be prepared and analyzed using confocal microscopy and computerized three-dimensional (3-D) reconstruction. The chapter describes protocols for the use of histochemical and immunocytochemical reagents for analysis of specimens by confocal microscopy. The descriptions given in tables relate to analysis of ophthalmic tissue, a particular area of research interest. The chapter describes the use of picrosirius red (PSR) for the analysis of connective tissue in thick sections of the optic nerve. Connective tissue, such as collagen and elastin, is known to be present along the length of the optic nerve, where it provides structural support to the axon bundles that transmit visual impulses from the retina to the brain. Accurate analysis of the 3-D structure of biological specimens using confocal microscopy and computerized 3-D reconstruction first requires that the original 3-D data sets are optimally collected and processed. The combined use of these stereological tools with confocal microscopy and computerized 3-D reconstruction will improve the understanding of structural features and relationships that occur within normal and diseased tissues.

The commercial fungicide methyl 1-[(butylamino) carbonyl]-1H-benzimidazol-2-ylcarbamate (benomyl) is teratogenic in rats. Its mode of action is believed to be related to its ability to inhibit the polymerization of brain tubulin. In this study its effects were studied in cultured neuronal cells during differentiation and neurite outgrowth. Mouse NB2a and human SH-SY5Y neuroblastoma cells were induced to differentiate by addition of dibutyryl cyclic AMP and at the same time were exposed to various concentrations of benomyl. Benomyl significantly inhibited neurite outgrowth in both cell lines at concentrations of 10(-8) M and above with IC50 values of 5.9 x 10(-7) M and 1.0 x 10(-6) M in the NB2a and SH-SY5Y cells respectively. The results show that benomyl inhibits neuronal cell differentiation at concentrations likely to be achieved during the development of fetal abnormalities in rats in vivo.

The estimation of ‘size’ for an interconnected nonconvex phase is an ongoing problem in materials science and biomedicine. Examples of this type of phase include the pore space in a porous rock such as sandstone or the marrow space found in cancellous bone. A reasonable definition of size for this type of phase is provided by the mean volume-weighted star volume. This is defined as the average volume of the phase of interest seen unobscured from a typical point within the phase. One practical advantage of this definition of size is that it is easy to estimate stereologically. In this paper we extend the concept of star volume to consider the mean surface-weighted star volume. The mean surface-weighted star volume is the volume of a continuous phase seen unobscured from a typical point of the interface. We expect that this parameter could be useful in biological problems involving mass transfer across an interface. The stereological estimation of mean surface-weighted star volume is described and the method is illustrated on piglet lung tissue.

In the past, the detection of fetal damage has tended to be restricted to the naked eye identification of major malformations, with the period of organ maturation being relatively neglected. Increasingly, however, unbiased design-based stereology is being used in developmental toxicological studies. In the field of intrauterine growth retardation, such methods are capable of providing new insights into fetal vulnerability during critical periods in organogenesis, with consequences for both post-natal and adult disease.

Objective: To monitor pregnancies in women with pre-existent insulin dependent diabetes for pregnancy loss, congenital malformations, and fetal growth in a geographically defined area of north west England. Design: Population cohort study. Setting: 10 maternity units in Cheshire, Lancashire, and Merseyside which had no regional guidelines for the management of pregnancy in diabetic women. Subjects: 462 pregnancies in 355 women with insulin dependent diabetes from the 10 centres over five years (1990-4 inclusive). Main outcome measures: Numbers and rates of miscarriages, stillbirths, and neonatal and postneonatal deaths; prevalence of congenital malformations; birth weight in relation to gestational age. Results: Among 462 pregnancies, 351 (76%) resulted in a liveborn infant, 78 (17%) aborted spontaneously, nine (2%) resulted in stillbirth, and 24 (5%) were terminated. Of the terminations, nine were for congenital malformation. The stillbirth rate was 25.0/1000 total births (95% confidence interval 8.9 to 41.1) compared with a population rate of 5.0/1000, and infant mortality was 19.9/1000 live births (5.3 to 34.6) compared with 6.8/1000. The prevalence of congenital malformations was 94.0/1000 live births (63.5 to 124.5) compared with 9.7/1000 in the general population. When corrected for gestational age, mean birth weight in the sample was 1.3 standard deviations greater than that of infants of non-diabetic mothers. Infants with congenital malformations weighed less than those without. Conclusion: In an unselected population the infants of women with pre-existent insulin dependent diabetes mellitus have a 10-fold greater risk of a congenital malformation and a fivefold greater risk of being stillborn than infants in the general population. Further improvements in the management of pregnancy in diabetic women are needed if target of the St Vincent declaration of 1989 is to be met. Key messages Infants of women with established insulin dependent diabetes mellitus have 10 times the population risk of congenital malformations and five times the stillbirth rate Excess mortality among infants of women with pre-existent insulin dependent diabetes mellitus is predominantly due to congenital malformations The birth prevalence of congenital malformations can be reduced by good periconceptional glycaemic control, but the challenge remains to implement this on a population basis Macrosomia remains a problem among infants of women with established insulin dependent diabetes mellitus

Using a specially designed algorithm for the measurement of the surface area of shapes with highly irregular contours, growth curves were developed for post-natal septal growth in humans using post-mortem specimens of a study population of 30 cases, distributed over the age range from birth to 62 years. From the results a rapid growth phase for the total septum is evident immediately after birth, lasting until the second year of life. Then, a gradual deceleration of growth is recognized with a plateau eventually being reached at the age of 36 years. Mathematical analysis of the growth curve shows that the curve for the total septum is the sum of two separate mathematical equations, representing the cartilaginous and bony contribution, respectively. It is demonstrated that the cartilaginous septum reaches adult dimensions (lateral surface area) at the age of two years. Subsequent growth of the septum is due to expansion of the perpendicular plate, i.e. the bony parts of the septum.

An association between the arrest of renal development and intra-uterine growth retardation (IUGR) has been demonstrated in human beings and it has been suggested that the same defect may occur in the kidneys of lambs affected by IUGR. Using design-based stereological methods, the physical disector and Cavalieri's principle, smaller absolute numbers of glomeruli were found in all six IUGR lambs studied with a low birthweight and in two of six control lambs studied with a normal birthweight than in other lambs with a normal birthweight. There was no difference in absolute numbers of glomeruli between twin births and singletons. The absolute numbers of glomeruli in three stillborn lambs were distributed among results obtained from the normal and IUGR lambs in accordance with their individual bodyweights. IUGR had a profound detrimental effect on the renal development of the lambs.

Replication of human papillomavirus (HPV) is thought to require the expression of components of the host cell DNA replication apparatus. The expression of the mRNA encoding the DNA replication-dependent histones H2B, H3, and H4 was investigated in a series of verrucae using an FITC-labelled oligonucleotide cocktail for in situ hybridization, to explore the possibility of HPV inducing the transcription of host cell histones. In contrast to normal epidermis, in which the majority of histone mRNA labelling was confined to basal cells, a bimodal distribution of histone positivity was observed in all verrucae. In addition to basal hyperproliferation-associated labelling, numerous positive suprabasal cells were found within the stratum spinosum. By contrast, in another hyperproliferative lesion, psoriasis, the labelling pattern was similar to that found in the normal controls, with characteristic basal hyperproliferation but no evidence of suprabasal labelling. The presence of HPV replication in every verruca and its absence from all controls of normal and psoriatic skin were demonstrated immunohistochemically, using a polyclonal antibody to the viral capsid proteins. It is proposed that the presence of histone mRNAs in differentiated suprabasal keratinocytes of verrucae is due to the virus turning on parts of the cellular apparatus in order for it to replicate in the absence of cellular proliferation. The results also emphasize that when assessing proliferation with in situ hybridization for histone mRNA, the possibility of HPV causing the labelling of non-proliferating cells should be considered.

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 tetraplegic patient. Absence of C-fibre hyperplasia (Grade 0) was found in nine cases (paraplegic: 4; tetraplegic: 5); Grade 1 hyperplasia was observed in 17 cases (paraplegic: 4; tetraplegic: 13); Grade 2 hyperplasia was seen in 11 cases (paraplegic: 7; tetraplegic 4); and Grade 3 hyperplasia was noticed in eight cases (paraplegic 3: tetraplegic: 5). The magnitude of C-fibre hyperplasia was not significantly different between paraplegic and tetraplegic patients (chi(2) = 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 as5 years, and5 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. (chi(2) = 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.

Although the occurrence of fibrosing colonopathy is temporally associated with the introduction of high-strength pancreatic enzyme supplements, its pathogenesis remains uncertain. The UK case-control study showed fibrosing colonopathy to be associated with high doses of high-strength pancreatic enzyme supplements and with a group of brands which occupy only 30% of the market. Two alternative hypotheses were proposed to explain the aetiology of fibrosing colonopathy: exposure to high levels of enzymes or to as yet unidentified components of the formulation. Comparison of the anatomical pathology of fibrosing colonopathy with that of previously encountered forms of obstructive gastrointestinal pathology, such as stricturing lesions due to potassium chloride preparations and nonsteroidal anti-inflammatory drugs, confirmed it to be a previously unencountered, long-segment lesion of the colon. Thus the use of the descriptive term 'stricture' is a misnomer leading to much clinical confusion when discussing obstructive bowel pathology in cystic fibrosis patients. Gavage studies in the rat with one of the two monomers (ethyl acrylate) forming the methacrylic acid copolymer (Eudragit L30D55) used for the enteric coating of the high-strength pancreatic enzyme supplements, have shown pathology comparable to fibrosing colonopathy. These findings prompted a series of exploratory studies in adolescent pigs. After seven days caecal gavage of Eudragit L30D55 at doses of 10, 50 or 500 mg/kg/day (comparable to human intake), extensive fibrosing colonopathy-like changes, inclusive of dense submucosal fibrosis, were noted at all dose levels in seven out of nine animals. Similar studies of the monomer components of the Eudragit L30D55 copolymer, at dose levels of 0.015 to 50 mg/kg/day, representing possible residues in Eudragit L30D55, did not produce comparable changes. The conclusion is that, although the precise mechanisms have not been elucidated, the role of enteric coatings containing Eudragit L30D55 in the pathogenesis of fibrosing colonopathy requires urgent further study.

Immunofluorescent labeling and neural tracing techniques were employed in conjunction with confocal scanning laser microscopy to study the intact neuroanatomy of the central nervous system of the trypanorhynch tapeworm Grillotia erinaceus. Immunocytochemical labeling for the general nerve fibre marker PGP 9.5 showed a pattern of extensive labeling that paralleled findings obtained with the neural tracer DiI. In contrast, immunocytochemical labeling for 5-hydroxytryptamine (5-HT) was localised to cell bodies lying on the periphery of the ganglion, with fine immunoreactive fibres radiating out towards the bothridia. Following the retrograde transport of the fluorescent molecule DiI through axotomised nerve cords, it successfully labeled both the cerebral ganglion and associated nerve fibres within the scolex. The cerebral ganglion was shown to give rise to posterior nerve cords, an array of radial fibres that pass out to the bothridia, and to contain a centrally disposed group of cell bodies thought to be involved in efferent functions.

Summary A method has been developed, using a silicon-rubber-based sealant, which allows 2–3-mm-thick specimens to be maintained in a protected fluid environment for a number of months, without risk of dehydration. Following this, the specimen can be retrieved, stained, embedded and sectioned further. For example, 2-mm-thick sections of fixed unstained bone are easily examined by means of epi-illuminated polarized light and fluorescence microscopies using either conventional or confocal optics. The method could easily be extended to other tissues, for example brain tissue.

Cell proliferation rates of diagnostic marrow aspirate cells of 21 children with Acute Lymphoblastic Leukaemia and 16 controls were compared using immunocytochemical labelling of PCNA and Ki-67 antigen as assessed by Confocal Laser Scanning Microscopy. The results showed an unexpected, highly significant degree of dissociation between PCNA and Ki-67 expression in ALL blasts. The PCNA labelling indices of ALL patients were significantly increased (mean 44, range 24-77) compared with normal reactive marrow cells (mean 13.8, range 4-26) (p0.000001, Mann Whitney U two tailed test), suggesting an abnormal commitment to proliferation. Ki-67 expression was raised to a lesser extent in ALL cells (mean 14.8, range 1.2-35) when compared to non-malignant proliferations (mean 6.6, range 1.7-25) (p0.02). PCNA/Ki-67 LI ratios in ALL (mean 7, range 1.1-35) were higher than in controls (mean 2.7, range 1.04-6.5, p0.09). As cell proliferation rates actually achieved in the bone marrow do not differ as strongly as suggested by the extreme difference in PCNA labelling, a pathological dissociation of PCNA/Ki-67 expression exists, suggesting immortalisation.

Flow-cytometric analysis of proliferation index (PI) has potential use in predicting prognosis in malignancy. Its relevance to heterogeneous tumors has not been conclusively studied. In nephroblastoma, where the epithelial components are considered more differentiated than others, potentially different PIs may exist within a single lesion based on the inverse relation between differentiation and proliferation. Proliferating cell nuclear antigen (PCNA) and MIB-1 (Ki-67 equivalent antigen) demonstration in histologic sections by immunoperoxidase methods may allow for determination of PI in relation to tissue type.A consecutive unselected series of 8 pediatric nephroblastoma patients was used to study the relation between PI and histologic differentiation as established by flow-cytometric analysis of nuclear suspensions prepared from formalin-fixed and paraffin-embedded tissue and by PCNA/MIB1 staining of parallel histologic sections. PI by PCNA/MIB1 was established using 5-microns paraffin sections, immunoperoxidase, and quantification procedures detailed in the literature. The mitotic index (MI) of tissue components was separately assessed using 5-microns hematoxylin and eosin-stained sections and counting procedures detailed in the literature.The 8 lesions showed a PI of 4 to 20% as determined by flow cytometry. Using PCNA staining, the epithelium showed a mean PI of 55.5% (range 40 to 80%), that was significantly higher (p0.001, Wilcoxon's two-tailed rank sum test) than blastema (mean PI: 34.1%, range 17.5 to 76.5%) and stroma (mean PI of 14.9%, range 5 to 24%, p0.001, Wilcoxon's two-tailed rank sum test). Although, probably due to tissue antigen preservation, acceptable MIB-1 staining was not achieved in all lesions (5 of 8), the results, although generally with lower labeling indices, confirmed the PCNA findings. The relative MI of epithelial components was higher than that of stroma and blastema in keeping with the immunocytochemical findings. In 6 of 8 cases, the PI by flow-cytometric analysis was lower than the lowest value for the PI (labeling index) of an individual tissue type found by PCNA or MIB staining.The differences found between PI of the different tissue components in nephroblastoma are difficult to understand if the epithelial components (with the highest PI values) are considered as differentiation products from the other components of the lesion. The relation between PIs as determined by PCNA/MIB-1 analysis/mitotic index, for the three components and the PI as established by flow cytometry is not simply explained by the relative volume of the tissue components.

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.

Summary Renal weight (left-right combined), as a parameter of renal development, is required to be less than half the normal value for age for a statistically confident diagnosis of hypoplasia. “Medullary ray glomerular counting” (MRGC), counting cortical glomerular generations, has been proposed as a simple technique of possibly greater sensitivity. Recent development of the Disector method for the unbiased stereological estimation of total glomerular number has provided a, hitherto unavailable, “golden standard” with which to determine the diagnostic potential of MRCG. Both “true” (actual number of generations seen) and “assumed” (a subjective “guess” of the total number of generations) MRGC counts were determined in 11 pairs of kidneys from spontaneously aborted, normally developed, non-malformed fetuses (gestational age: 15–40 weeks). Each kidney was randomly analysed blind and on two separate occasions by two paediatric pathologists using a written protocol. Results were compared with unbiased stereological estimates of glomerular number. Intra- and inter-observer and intra- and inter-(left-right) renal reproducibility were analysed. In conclusion, MRGC, using “real” counts, is a highly reproducible parameter of renal development from 15–36 weeks' gestation. Sensitivity for detection of both hypoplasia and maturation delay increases with gestational age and generally exceeds that of renal weight.

The disector method, a stereologic procedure unbiased by feature size, shape, or tissue-processing methods, for the estimation of total glomerular number was performed on pairs of human kidneys from 11 normal spontaneous second trimester abortions and stillbirths (15 to 40 weeks gestation). In addition, gestational age-dependent patterns of change in the average volume of the nephron and its cortical and medullary segments were analyzed. Mean glomerular number, plateauing at 36 weeks, increased from 15,000 at 15 weeks to 740,000 at 40 weeks. Average volume of the medullary nephron segment (Henle's loop) increased throughout pregnancy. Average volume of the cortical nephron segment (Tubuli Contorti) decreased from 15 weeks to 25 weeks, then increased after 36 weeks. Fractional volume of the renal cortex decreased from 15 weeks to 40 weeks. Three to 4 hours of microscopic analysis time were required per kidney on routinely processed 5-microns hematoxylin and eosin-stained paraffin sections. Average coefficient of error for number estimation was 8.02%. Average intra- and interobserver reproducibilities were 96.8 and 93.7%, respectively. The demonstrated temporal differences in the development of the cortical and medullary nephron components may result in a dissociation of function, which may explain the increased incidence of fetal hydrops in the second trimester of pregnancy, and which must be taken into account in the treatment of (very) premature infants. Although the number of kidneys included in this study is limited, as they reflect the whole period of antenatal development relevant to neonatal intensive care, the disector method of glomerular number estimation shows significant potential for the analysis and increased understanding of the development of renal function. The method appears to be more sensitive in detecting small and early deviations from normal renal growth and development than previously available parameters e.g., renal weight and (cortical) volume.

An efficient method is presented for obtaining, in under 4 h, an unbiased estimate of the total number of neurons in the human neocortex, with a coefficient of error on the estimate of approximately 5%. The novel sampling scheme used in this study is unbiased and was designed so that only a small amount of neocortical grey matter had to be removed. Hence, the majority of the cerebral grey matter and all the internal grey matter was left intact for further resampling and analysis. Each cerebral hemisphere was subdivided into the four major neocortical regions, sliced coronally at 7-mm intervals and the volume of the neocortex determined using Cavalieri's principle. Uniform sampling of neocortex was performed in the hemisphere followed by regional subsampling with a varying sampling fraction being taken from each region. Neuronal density estimates were made in thick plastic sections using optical disectors. Shrinkage estimates were made in parallel with the number estimates and found to be negligible. The total number of neocortical neurons in the right hemisphere of five normal 80-year-old men was found to be 13.7 x 10(9) with an inter-individual coefficient of variation of 12%.

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

Centralisation of care leads to better outcome Editor—The finding of a persistently poor outcome of pregnancy in women with insu› lin dependent diabetes in two (northern) English regions is an important statement of the problem. 12 Both studies provide figures and show outcomes that are no different from those widely reported in the past. Unfortunately, neither give evidence of any degree of centralisation of obstetric or diabetic care, with on›site neonatal intensive care, although this is a proved means of improving the outcome of pregnancy for diabetic mothers. 3 The St Vincent declara›

Background: Having established a glomerular number deficit in fetal growth retardation(1), human fetal kidneys were studied with the aim of determining periods of specific vulnerability related to proliferative activity. Materials and Methods: 24 Fetus, evenly distributed between 13 and 22 weeks gestational age were used. Tissue was routinely fixed and embedded. Monoclonal antibodies (DAKO, PC10, MIB-1) were used (2,3) to separately assess proliferation of renal blastema, stroma and glomeruli and epithelial components such as tubuli, Henle's loops and collecting ducts. Results, Discussion: Proliferative activity remains high in the epithelial components of the nephrogenic zone, with glomerular and blastematous tissue showing reduced activity after week 18. A period of significantly increased activity for collecting ducts, thick and thin segements of Henle's loops and stroma was found between weeks 16 and 18 for both cortex and medulla. Conclusion: Proliferation related vulnerability of glomerulogenesis is not limited to a specific period of intrauterine development in contrast to the development of the remainder of the nephrons, which may be especially vulnerable between 16 and 18 weeks of gestation. Ref.: 1: Br J Obst Gynaecol 1992; 99:296-301, 2. J Pathel 1990; 162 : 285-294, 3. J Pathol 1992; 168:357-363

SUMMARY Techniques for analysing three-dimensional spatial point patterns are demonstrated on data from a confocal microscope recording the locations of cells in three dimensions. New computational techniques are proposed for edge corrections and empty space measurement. A novel feature of the data is replication and nesting in a sampling design: multiple spatial patterns were observed from each of several animals. For this we develop a ratio regression approach.

SUMMARY A semi-automatic image analysis system based on the stereological approach of point-sampled intercepts for mean volume estimation is described. The method used requires no assumptions about particle geometries and is thus general in its application. To ease implementation in this application, the assumption of particle convexity is made, making the measurement of intercepts easier for the user. The system was tested with computer-generated sample sections to determine the accuracy and precision of the estimates obtained. The results were found to be within 10% of expected values in all cases. When tested with ‘real’ samples a typical result gave a coefficient of variation of 9% from eight independent estimates of one sample.