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Background Airline crew members report adverse health effects during and after inhalation exposure to engine oil fumes sourced to the air supply system onboard commercial and military aircraft. Most investigations into the causal factors of their reported symptoms focus on specific chemical contaminants in the fumes. The adverse health effects reported in aircrew exposed to the aircraft air supply, bled unfiltered off the engine or Auxiliary Power Unit (APU) may be related to particulate exposures, which are widely known to effect health. While oil contaminates the aircraft air supply, some suggest that this will only occur when there is a bearing seal failure, others document that there is low level oil contamination of the air supply during normal engine operation. This brief pilot study explores whether particulate exposure may be associated with the normal engine/APU and air supply operation and to therefore increase the understanding that UFP exposures may have on crew and passengers. Methods An ultrafine particle counter was utilised by an experienced airline captain in the passenger cabin of four short-haul commercial passenger aircraft. All flights were under 90 min on aircraft from two different carriers ranging from 7 months to 14 years old. Results UFP concentrations showed maximum concentrations ranging from 31,300 to 97,800 particles/cm3 when APU was selected on as a source of air on the ground and with engine bleed air and the air conditioning packs selected on during the climb. In 2 of the 4 flights the peaks were associated with an engine oil smell. Increases in UFP particle concentrations occurred with changes in engine/APU power and air supply configuration changes. Conclusions This study identified increases in UFP concentrations associated with engine and APU power changes and changes in air supply configuration. These results correlated with times when engine and APU oil seals are known to be less effective, enabling oil leakage to occur. The concentrations reached in the passenger cabins exceeded those taken in other ground-based environments. UFP exposures in aircraft cabins during normal flight indicates there will be health consequences for long serving aircrew and some passengers.

A hypothesis is advanced in which life began from a Darwinian selection among a diversification of molecular species containing the phosphate moiety which broke the constraints implicit in the Second Law of Thermodynamics, discussed famously by Schrodinger, by obtaining energy from specific infrared frequencies located in the phosphorus-oxygen vibration at a frequency around 1000 cm-1. We propose the source of this energy was from the internal conversion of solar broadband energy by the phosphate mineral Apatite, present at the bottom of a primitive biogenesis pond. In this scenario, life is re-defined as being hotter than its environment and as using its excess energy, supplied by infra-red conversion, to react with its molecular environment and pump itself up the “entropy slope” thereby; replication is through breakages of increasingly large phosphate containing biopolymers. The idea has implications for modern explanations of living systems.

Summary The combination of noninvasive compositional analysis by Raman microspectrometry with high-resolution imaging in the scanning electron microscope greatly expands the analytical capabilities of the electron microscope. However, the chemical preparation of scanning electron microscope (SEM) specimens, although adequate for low-resolution imaging of superficial detail, is not the true representation of the chemistry and composition of the sample, as extraction and aggregation artefacts as a result of dehydrating and cross-linking agents are abundant. The original chemical composition and ultrastructure is only preserved using cryo preparation methods. Therefore, a complete cryo transfer flange was designed and built to add cryogenic control of specimens to the configuration of the EMRAM instrument, a combined Raman spectrometer and XL-30 ESEM instrument. The Raman spectra of two model specimen, polystyrene beads and 2.3M sucrose were studied at ambient and cryogenic temperatures as well as during a heating ramp. Comparing the fingerprint regions of polystyrene and sucrose, both measured at ambient and at cryogenic conditions, only small spectral differences were observed for the main peaks of both molecules. A pronounced sharpening of the bands occurred in the 800–400 cm−1 region, a result of the reduction of intermolecular interactions. The enhanced visibility of the lower frequency modes may offer interesting potential for more detailed interpretation of Raman spectra.

The vertical rotator is a design based stereological method for estimating object volume using measurements made on co-axial sections. Recently we used the method to estimate eye volume in a number of pathological studies. In the present note we apply an error prediction method for the vertical rotator which has recently been published and analyse the raw data from our earlier study on eye volume in juvenile Dover sole. Application of the new theory allows us to assess the variance contributions due to rotating plane and test line sampling and estimate the underlying biological variability in eye volume. In all cases the coefficient of error introduced by the rotating test was about 10 times greater than due to the test line contribution.

A combined scanning electron microscope and focussed ion beam instrument is suitable for micro- and nanopatterning, cross-sectioning and subsequent imaging, of specimens at room temperature as well as under cryo conditions. In order to reveal internal details, samples are conventionally milled with the ion beam positioned perpendicular to the sample surface. Using this approach certain limitations are frequently encountered, e.g. accumulation of redeposited material, shadowing effects, image distortion and a limited imaging area. Here we show an approach in which samples are pre-trimmed using a microtome to obtain a sample block face that is parallel to the ion beam. This new grazing incidence geometry eliminates the need for removal of bulk material with the ion beam and enables immediate fine polishing of a pre-selected area of interest. Many of the limitations previously described are avoided and in addition milling time is reduced, whilst creating larger cross-sectional areas. Another advantage is that electron imaging can be accomplished by tilting the sample surface perpendicular to the electron beam, providing a geometrically undistorted image. The proposed approach is suitable for materials that can be microtomed, both in ambient and cryogenic conditions, and proves to be of particular benefit for biological and food samples.

There is inadequate pediatric neurosurgical training to meet the growing burden of disease in low- and middle-income countries (LMIC). Subspecialty expertise in the management of hydrocephalus and spina bifida—two of the most common pediatric neurosurgical conditions—offers a high-yield opportunity to mitigate morbidity and avoid unnecessary death. The CURE Hydrocephalus and Spina Bifida (CHSB) fellowship offers an intensive subspecialty training program designed to equip surgeons from LMIC with the state-of-the-art surgical skills and equipment to most effectively manage common neurosurgical conditions of childhood. Prospective fellows and their home institution undergo a comprehensive evaluation before being accepted for the 8-week training period held at CURE Children’s Hospital of Uganda (CCHU) in Mbale, Uganda. The fellowship combines anatomy review, treatment paradigms, a flexible endoscopic simulation lab, daily ward and ICU rounds, radiology rounds, and clinic exposure. The cornerstone of the fellowship is the unique operative experience that includes a high volume of endoscopic third ventriculostomy with choroid plexus cauterization, myelomeningocele closure, and ventriculoperitoneal shunting, among many other procedures performed at CCHU. Upon completion, fellows return to their home institution to establish or rejuvenate a robust pediatric practice as part of a worldwide network of CHSB trainees committed to the care of underserved children. To date, the fellowship has graduated 33 surgeons from 20 different LMIC who are independently performing thousands of hydrocephalus and spina bifida operations each year.

Summary The stereological estimation of second-order descriptors of spatial architecture appears to be inherently more time-consuming and labour-intensive than the estimation of first-order quantities (total quantities or ratios). Therefore, far fewer researchers tend to make use of second-order approaches in their stereological research projects. In this paper, we use a tutorial approach to illustrate how a desire for practical simplicity has provided us with a data collection method that can be used to simultaneously estimate both first-order and second-order properties of the microstructure of a defined anatomical feature of an organ. The approach does not rely on new results from theory, but nevertheless allows either isotropic uniform random or vertical uniform random sections to be used to make estimates of a comprehensive list of 10 microstructural parameters using relationships that are well known in the literature. The probe used in all cases is an isotropically distributed Ruler and the data collection protocol is easy to learn and apply. We illustrate the method on brain tissue but emphasize that the approach can also be applied to non-biological material.

Epilepsy prevalence in the developing world is many fold that found in developed countries. For individuals whose conditions failed to respond to pharmacotherapy, surgery is the only opportunity for cure. In Uganda, we developed a center for treatment of intractable temporal lobe epilepsy (iTLE) that functions within the technologic and expertise constraints of a severely low resource area. Our model relies on partnership with epilepsy professionals and training of local staff. Patients were prescreened at regional clinics for iTLE. Individuals meeting inclusion criteria were referred to the treating Ugandan hospital (CURE Children's' Hospital of Uganda, CCHU) for video-EEG (electroencephalography), computed tomography (CT) imaging, and neuropsychological evaluation. Data were transferred to epilepsy experts for analysis and treatment recommendations. Ten patients were diagnosed with iTLE and surgically treated at CCHU. Six (60%) were seizure free, and there was no neurologic morbidity or mortality. Our model for surgical treatment of pharmacoresistant TLE has functioned successfully in a true developing world low resource setting.

The theory that increasing cancer incidence rates in developed countries are primarily the consequence of an expanding ageing population and improved diagnostic testing is widely held. In the United Kingdom the proportion of people aged 50 and over has increased by 45% since 1951 and this proportion is set to increase by a further 36% by the year 2031, so the United Kingdom does indeed have an expanding ageing population. However, the increase in cancer incidence affects people across the whole age spectrum. To test the hypothesis that the age of onset of cancer (overall and specific) in England and Wales is decreasing over time we have developed The Cancer Incidence Temporality Index (CITI), which gives a crude measurement of the portion of the population, in which cancer incidence is rising fastest over time: I=(SigmaO(a)/ SigmaE(a))/(SigmaO(a)/SigmaE(a)), where I is the CITI value, O is the observed number of cases and E is the expected number of cases; 'a' and 'b' refer to separate summation ranges for younger and older age groups. Population data and cancer incidence data in England and Wales, 1971-1999 were obtained from the UK Office for National Statistics. The trends in CITI values have been shown graphically for cancer overall and for specific tumour sites. The impact of diagnostic testing is also addressed. The results of this study suggest that the average age of onset of prostate, breast and cervical cancer is temporally decreasing. The study also suggests that for cancer overall the trend for the age of onset of cancer in males has stabilised since 1990 and has started to reverse in females from 1995 despite the expanding ageing population. A similar trend is observed for leukaemias. The CITI analysis for colon cancer shows that the age of onset in both males and females is increasing over time. The trend for ovarian cancer is similar to that for colon cancer. The CITI analysis for NHL in males is similar to that for colon cancer, however, in females the trend stabilised after 1990. The CITI may aid prediction of changes in the age of onset of cancer and thus aid targeted aetiological research. In addition, we suggest the need for a mathematical model, which may measure the changes in the age of onset of cancer in units of time.

During a 12-mo longitudinal study, bulk-tank milk was collected each month from organic (n = 17) and conventional (n = 19) dairy farms in the United Kingdom. All milk samples were analyzed for fatty acid (FA) content, with the farming system type, herd production level, and nutritional factors affecting the FA composition investigated by use of mixed model analyses. Models were constructed for saturated fatty acids, the ratio of polyunsaturated fatty acids (PUFA) to monounsaturated fatty acids, total n-3 FA, total n-6 FA, conjugated linoleic acid, and vaccenic acid. The ratio of n-6:n-3 FA in both organic and conventional milk was also compared. Organic milk had a higher proportion of PUFA to monounsaturated fatty acids and of n-3 FA than conventional milk, and contained a consistently lower n-6:n-3 FA ratio (which is considered beneficial) compared with conventional milk. There was no difference between organic and conventional milk with respect to the proportion of conjugated linoleic acid or vaccenic acid. A number of factors other than farming system were identified which affected milk FA content including month of year, herd average milk yield, breed type, use of a total mixed ration, and access to fresh grazing. Thus, organic dairy farms in the United Kingdom produce milk with a higher PUFA content, particularly n-3 FA, throughout the year. However, knowledge of the effects of season, access to fresh grazing, or use of specific silage types could be used by producers to enhance the content of beneficial FA in milk.

The effects of mild chronic dehydration on fermentation rate and mucosal surface area in the cecum, dorsa and ventral colon, and descending colon of the hindgut were investigated in South African donkeys (n = 11) in agricultural work. Dehydration representing a 6% drop in BW (n = 6) was associated with increased fermentation activity in the cecum (252 +/- 22.9 vs. 161 +/- 13.5 micromol/g of DM.h(-1), P < 0.01) and enhanced fluid retention in the ventral colon (0.81 +/- 0.026 vs. 0.73 +/- 0.034 mL/g gut, P < 0.05). Fermentation activity in the next segment of the hindgut, the ventral colon, of dehydrated donkeys was also greater numerically (92.5 +/- 22.60 vs. 77.9 +/- 10.40 micromol/g of DM.h(-1)), but this difference was not significant. Total mucosal and crypt surface area per unit volume of gut (Sv, microm2/microm3) was greater in dehydrated donkeys for the cecum (253 +/- 23.0 vs. 161 +/- 13.5, P < 0.01), the ventral colon (286 +/- 6.2 vs. 171 +/- 9.8, P < 0.01), the dorsal colon (276 +/- 18.2 vs. 256 +/- 11.0, P < 0.05), and the descending colon (260 +/- 20.3 vs. 191 +/- 15.2, P < 0.05). Enhanced fermentation activity and enhanced mucosal absorptive or secretory capacity within the hindgut during chronic dehydration was associated with an observed maintenance of appetite. These adaptations in the hindgut are valuable physiological attributes for working donkeys in semi-arid regions where they are frequently exposed to chronic dehydration.

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 µg/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 (P < 0.03/0.001). The weights of the acini and septal tissue were also increased by 39–46% in PNA and ABA fed animals, respectively (P < 0.002); 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.

Organophosphate (OP) pesticides are often used in combination with one another and with the components of formulations. Evidence already exists for interactions in the neurotoxic effects of OPs through interference with metabolism, but there is also potential for interactions related directly to cell damage. The purpose of this work was to investigate this possibility for OPs and the components of one of their common formulations in vitro. NB2a neuroblastoma cells were induced to differentiate in the presence of the OPs diazinon and chlorpyrifos, in combination with a commercial formulation (identified as Commercial Formulation 1) of the compounds and, independently, the components of that formulation. The compounds were tested in pairs in various proportions and the resulting inhibition of neurite outgrowth was measured by light microscopy and quantitative image analysis. Interactions were determined in terms of enhanced or reduced effects of the paired compounds in comparison with the expected additive effects estimated from the effects of each compound on its own. Synergism was detected between combinations of: 10 microM chlorpyrifos and 500 nM pyrethrum; chlorpyrifos and one of the solvents (regular spirit) found in Commercial Formulation 1. All other combinations of OPs and products were additive in their neurotoxicity. The data suggest that exposure to multiple OP-containing pesticide formulations may lead to synergistic neurotoxicity by a direct mechanism at the cellular level.

Aims To monitor pregnancies in women with pregestational Type 1 diabetes for pregnancy loss, congenital malformations and fetal growth parameters, in a geographically defined area in the north west of England. Methods Population cohort study of 547 pregnancies in women with Type 1 diabetes from maternity clinics in 10 centres over a 5-year period (1995–1999 inclusive). Main outcome measures were numbers and rates of miscarriages, stillbirths, neonatal and post-neonatal deaths; prevalence of congenital malformations; birth weight in relation to gestational age. Results Among 547 pregnancies, there were six (1.1%) pairs of liveborn twins, 439 (80.3%) liveborn singletons; 72 (13.2%) spontaneous abortions, 14 (2.6%) stillbirths and 16 (2.9%) terminations. Four of the terminations were performed because of congenital malformations. Both the stillbirth rate (30.1/1000 total births (95% confidence interval (CI) 16.6–50.0)), and prevalence of congenital malformations (84.3/1000 live births (95% CI 60.3–113.8)) were significantly higher than the local population (P

Previous research has demonstrated impaired renal development, particularly with respect to glomerular number, in victims of sudden infant death syndrome (SIDS). The present study used stereological principles to estimate the volume of the upper lobe of the right lung, total number of terminal bronchiolar duct endings (TBDE), and gas exchange surface area of this lobe within a group of human infants. The infants were classified according to cause of death (SIDS or non-SIDS), and further subdivided according to birth-weight: normal birth-weight (NBW) or low birth-weight (LBW). The results demonstrated that TBDE density was significantly reduced in SIDS compared to non-SIDS (P = 0.014), but only reduced from non-SIDS NBW values in the SIDS NBW group (P = 0.044). Total TBDE number was significantly reduced in SIDS from non-SIDS (P = 0.001), and was significantly reduced from non-SIDS NBW values in SIDS NBW (P = 0.023). Mean gas exchange surface area per TBDE was significantly increased in SIDS compared to non-SIDS cases (P = 0.049). The results of the present study indicate developmental delay of the lung in SIDS NBW infants who had previously not been considered growth retarded based on their normal body parameters. Pediatr Pulmonol. 2001; 31:339–343. © 2001 Wiley-Liss, Inc.

Organ development may be assessed by estimating the total number of functional units within an organ over time; the potential functional capacity of that organ may be represented by the total number of functional units present in the fully developed, mature organ. Relative development of the lung at birth is essential to provide sufficient oxygenation of body tissues and so maintain ex utero life. Estimation of the number of one type of functional unit of the lung – terminal bronchiolar duct endings – provides important information regarding development of the lung. This investigation used stereological techniques, specifically Cavalieri’s Principle and the “physical disector”, to estimate total number of terminal bronchiolar duct endings in the upper lobe of the right lung of a group of 14 control infants between 0 and 66 weeks post-natal age. Results demonstrate that total terminal bronchiolar duct ending number does not increase significantly over the first 24 weeks of post-natal life in normal infants ( P =0.997). The unbiased, design-based techniques used in this paper confirm previous model-based research that indicates that terminal bronchiolar duct ending development is completed before birth.

Confocal laser scanning rnicroscopy (CLSM) was used to examine living organisms within marine biofilms growing on microscope slides and on natural opaque and uneven substrata such as rocks and shells. The results were compared to images of identical fields observed under transmitted llght and phase contrast microscopy and scanning electron microscopy. Confocal microscopy proved superior in sevcrdl respects: (l) It gave clear images of organisms even if they were overlaid with a thick layer of mucus, other organlsms and debr~s. (2) It did not require the biofilm to be disrupted or dned and allowed samples to be re-examined at intervals. (3) It could also distinguish living (fluorescing) organisms from dead cells or inorganic matter. (4) Examining optical 'slices' of samples allowed the 3 dimensional structure of the biofdm to be v~sualised.H owever, species identification, particularly of diatoms, was much easier using scanning electron microscopy. Methods were developed for overlaying a fixed grid on samples so that specific sites or individual organisms could be accurately relocated for re-examination. Appropriate staining methods were also evaluated. Confocal microscopy will prove to be an invaluable aid for examining the structure and growth of living biofilrns in studies of shore ecology and marine fouling.

In multiphase systems consisting of ‘particles’ embedded in a matrix the three-dimensional spatial distribution of the particles may represent important structural information. In systems where the matrix is transparent or translucent recent developments in microscopy allow the three-dimensional location of particles to be recorded. Using these data a spatial statistical, or second-order stereological, analysis can be carried out. In second-order stereology functions of interparticle distances are used as summary statistics of the spatial distributions. These statistics show whether the particles are randomly arranged or, more commonly, either clustered together or inhibited from close approach to each other. This paper focuses on the estimation of one of these spatial statistics, the nearest-neighbour distance distribution function or G-function. In practice, estimation of the G-function is plagued by an ‘edge-effect’ bias introduced by the sampling process itself. There exist a number of G-function estimators that tackle this edge effect problem; for single sample ‘bricks’ it can be shown that these estimators become increasingly accurate as the brick size increases, i.e. they are consistent. However, in many practical cases the size of a sampling brick is fixed by experimental constraints and in these circumstances the only way to increase sample size is to take replicated sampling regions. In this paper we review a number of existing G-function estimators and propose a new estimator. These estimators are compared using the criterion of how well they overcome the edge-effect when they are applied to replicated samples of a fixed size of brick. These comparisons were made using Monte-Carlo simulation methods; the results show that three existing estimators are clearly unsuitable for estimating the G-function from replicated sample bricks. Of the other estimators the recommended estimator depends upon the number of replicates taken; however, we conclude that if a total of more than about 800 points are analysed then the bias in the pooled estimate of the G-function can be reduced to tolerable levels.

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.

SUMMARYAs a major morphological feature in establishing the form of the nervous system, it is recognized that neurons are initially overproduced, then naturally occurring cell death reduces the neuron number to the functional requirement. However, the mechanisms controlling the selective elimination of certain neurons during a general phase of cell death are not fully understood. One event that seems to be pivotal is the establishment of neural connections, the degree of which may be influential regarding the fate of specific neurons. However, little quantitative evidence is available to either support or refute this theory. In this current study, a Stereological measurement of gap junction per neuron was carried out within the invertebrate model system of the tapeworm metacestode Mesocestoides corti, which has previously been shown to overproduce neurons during the asexual reproduction stage of its life-cycle. Novel Stereological estimation methods with ‘ vertical sections’ indicated that prior to asexual division the cerebral ganglion possessed approximately 268 neurons, each with a gap junction surface area of 250 μm2. As division progressed, the neuron number increased to approximately 700, while the total surface area of gap junction remained statistically unchanged. As a result the surface area of gap junction per neuron decreased to 106 μm2, less than half that in the undividing stage. These results provide the first non-biased quantitative data regarding changes in the mean surface area of gap junction per neuron in a developing cerebral ganglion.

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.

The aim of this study was to make unbiased quantitative estimates of the number of T8 cytotoxic cells and T4 helper cells and macrophages in the head and tail of rat epididymides, using newly developed design-based stereological methods in fertile control animals, and to compare these with results from vasectomized animals. The location of these cells within the intra- and interepithelial compartments of the epididymis was also studied. In control animals, both the T4 helper cells and macrophages and T8 cytotoxic cells were found in significantly higher numbers (P < 0.01) in the head interepithelial location than in the intraepithelial site. In addition, there were significantly more (P < 0.01) T8 cytotoxic cells in the tail interepithelial region. Within the head region of vasectomized rats, there was a significant increase in T8 cytotoxic cells in the intraepithelial location (P < 0.01), and a decrease in T4 helper cells and macrophages in the interepithelial region (P < 0.05). Serum samples from vasectomized animals, when compared with those from control animals, showed a significant increase in cytotoxic antisperm antibody. This suggests that, in response to the increased spermatozoal antigenic absorption that takes place in the epididymis after vasectomy, the immune system redistributes its T cell subsets to modify the immune tolerance that may occur.

SUMMARY The area of an individual bounded surface (e.g. the boundary of a properly sampled cell) can be estimated from an isotropic uniform random stack of parallel sections, or of non-invasive planar scans, using the well-known spatial grid. A standing problem was to estimate the area of an individual bounded surface with an arbitrary degree of accuracy from a vertical (i.e. not isotropic) stack of sections or scans. A new tool to do this, called the ‘vertical spatial grid’, is presented.

Summary Experimental infection of rhesus monkeys (Macaca mulatta) with a virulent (W1) strain of Plasmodium knowlesi resulted in cerebral malaria. Electron microscopical examination of the brain revealed large numbers of intracytoplasmic lipofuscin pigment deposits in cerebellar Purkinje neurones and choroid plexus epithelium of the lateral ventricle. This lesion may be part of the nervous system response to ischaemic hypoxia.

Summary A rat-specific mouse monoclonal antibody, exclusively reacting with a blood-brain and blood-nerve barrier protein, was employed to assess the post-natal, age-related changes in morphology and intraparenchymal distribution of barrier competent microvessels in normal rat forebrain. Antibody binding first appeared in single cells over the external surfaces of the brain between days 3 and 6 post-partum and increased progressively with age. In mature rats, the cerebral grey matter showed higher vascularization by barrier competent microvessels than white matter. Microvessels in the hippocampus were immunocytochemically more avid but less “networked” than the rest of the neocortex. These features may be related to morphological, metabolic and haemodynamic changes associated with brain growth and development.

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.

Bioconjugated gold nanoparticles (Au NPs) are a promising tool for pharmaceutical applications. However, the ecotoxicity of these types of NPs has hardly been studied. We investigated the ecotoxicity and uptake of 4–5 nm Au NPs to which two types of polymer coatings were attached. One coating was an amphiphilic polymer only and the other an amphiphilic coating to which 10 kDa polyethylene glycol chains were attached. In both 72 h algal growth inhibition tests with the alga Pseudokirchneriella subcapitata and in 24 h resazurin cytotoxicity tests with the rainbow trout gill cell line RTGill-W1, the pegylated Au NPs were found less toxic compared to the amphiphilic coated particles. No uptake or direct interaction between particles and algal cells was observed. However, uptake/adsorption in fish gill cells reached up to >10 6 particles/ cell after 1 h and particles were eliminated for ‡96% after 24 h depuration. Both particle types were found within membrane enclosed vesicles in the cytoplasm of RTgill-W1 cells.

The combination of noninvasive compositional analysis by Raman microspectrometry with high-resolution imaging in the scanning electron microscope greatly expands the analytical capabilities of the electron microscope. However, the chemical preparation of scanning electron microscope (SEM) specimens, although adequate for low-resolution imaging of superficial detail, is not the true representation of the chemistry and composition of the sample, as extraction and aggregation artefacts as a result of dehydrating and cross-linking agents are abundant. The original chemical composition and ultrastructure is only preserved using cryo preparation methods. Therefore, a complete cryo transfer flange was designed and built to add cryogenic control of specimens to the configuration of the EMRAM instrument, a combined Raman spectrometer and XL-30 ESEM instrument. The Raman spectra of two model specimen, polystyrene beads and 2.3M sucrose were studied at ambient and cryogenic temperatures as well as during a heating ramp. Comparing the fingerprint regions of polystyrene and sucrose, both measured at ambient and at cryogenic conditions, only small spectral differences were observed for the main peaks of both molecules. A pronounced sharpening of the bands occurred in the 800-400 cm(-1) region, a result of the reduction of intermolecular interactions. The enhanced visibility of the lower frequency modes may offer interesting potential for more detailed interpretation of Raman spectra.

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.

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.

SUMMARYFifty tetrathyridia of Mesocestoides corti showing various degrees of scolex fission during asexual multiplication were subjected to estimation of the total number of neurons in the central ganglia, using a ‘fractionator’ designed sampling scheme on every 10th Historesin section through the specimen. Neurons were stained with Cresyl fast violet and viewed by transmission light microscopy. Cells were only counted if a nucleolus was present. The animals were divided into groups according to their stage in the asexual reproduction process indicated by head shape and sucker number. Animals which had a narrow head with 4 suckers contained about 250 neurons. As their heads became wider, in preparation for fission, neurons were overproduced by up to 5-fold. As additional suckers were developed (8 in total), neurons were reduced to about 500. Finally, when the animals had 2 clearly discernible heads, with 4 suckers on each, the total number of neurons (per head) was again approximately 250. As the heads became wider again, neuron over-production recommenced. Exceptionally shaped animals with an X- or T-shaped single head were observed. These results suggest that there is a preparatory stage for neuronal regeneration, during which the basic population of neurons is about 250. This is followed by neuron over-production followed by a selective cell death of neurons which occurred during asexual multiplication. Another cycle for neuronal regeneration can occur before an animal completes its fission process.

A combined scanning electron microscope and focussed ion beam instrument is suitable for micro- and nanopatterning, cross-sectioning and subsequent imaging, of specimens at room temperature as well as under cryo conditions. In order to reveal internal details, samples are conventionally milled with the ion beam positioned perpendicular to the sample surface. Using this approach certain limitations are frequently encountered, e.g. accumulation of redeposited material, shadowing effects, image distortion and a limited imaging area. Here we show an approach in which samples are pre-trimmed using a microtome to obtain a sample block face that is parallel to the ion beam. This new grazing incidence geometry eliminates the need for removal of bulk material with the ion beam and enables immediate fine polishing of a pre-selected area of interest. Many of the limitations previously described are avoided and in addition milling time is reduced, whilst creating larger cross-sectional areas. Another advantage is that electron imaging can be accomplished by tilting the sample surface perpendicular to the electron beam, providing a geometrically undistorted image. The proposed approach is suitable for materials that can be microtomed, both in ambient and cryogenic conditions, and proves to be of particular benefit for biological and food samples.

The stereological estimation of second-order descriptors of spatial architecture appears to be inherently more time-consuming and labour-intensive than the estimation of first-order quantities (total quantities or ratios). Therefore, far fewer researchers tend to make use of second-order approaches in their stereological research projects. In this paper, we use a tutorial approach to illustrate how a desire for practical simplicity has provided us with a data collection method that can be used to simultaneously estimate both first-order and second-order properties of the microstructure of a defined anatomical feature of an organ. The approach does not rely on new results from theory, but nevertheless allows either isotropic uniform random or vertical uniform random sections to be used to make estimates of a comprehensive list of 10 microstructural parameters using relationships that are well known in the literature. The probe used in all cases is an isotropically distributed Ruler and the data collection protocol is easy to learn and apply. We illustrate the method on brain tissue but emphasize that the approach can also be applied to non-biological material.

Three-dimensional (3-D) images of osteocyte lacunae were examined on a confocal microscope. Both geostatistical and morphological processing techniques were used to improve and to analyse them. By a geostastical approach, this study aims at improving 3-D confocal images before any further image processing. Optimized linear filters, which take account of the second-order statistics and the 3-D structure of the data, allow for the removal of imperfections such as noise and/or blur due to the axial convolution, and interpolate voxels on a face-centred cubic grid from an initial cubic grid. An application of this technique to 3-D biological images is demonstrated. In a second step, a 3-D binary image is digitized and cleaned with 3-D morphological filters. The standard 3-D measurements cannot be applied in this case, since all osteocytes cut the border of the field. For this reason a 3-D Boolean model has been adjusted, from which it is possible to derive all useful information on the repartition and the morphology of the osteocytes.

OBJECTIVE To investigate the effect of Type II (asymmetrical) intrauterine growth retardation (IUGR) on renal development. DESIGN A prospective descriptive study. SETTING Department of Fetal and Infant Pathology, Liverpool Children's Hospital. SUBJECTS Six (severely) affected IUGR stillbirths of known gestational age with a control group of stillbirths with birthweight greater than 10th centile, and eight liveborn IUGR infants who died within a year of birth with a control group of appropriately grown infants who died within a year of birth (postnatal groups). TECHNIQUES The kidneys from all the groups studied were analysed using unbiased, reproducible and objective design-based stereological techniques. MAIN OUTCOME MEASURES Total renal nephron (glomerular) numbers and average volumes of total nephron and cortical and medullary nephron segments. RESULTS Nephron number estimates lay below the control group's 5% prediction limit in five out of the six growth-retarded stillbirths, and were significantly (P less than 0.005, IUGR at 65% of the control mean) reduced in the postnatal group. Estimates of nephron (segment) volume did not differ between control and IUGR groups. CONCLUSIONS Type II intrauterine growth retardation may exert a profound effect on renal development. The reduced nephron number at birth, together with the lack of any early postnatal compensation in either nephron number or nephron size, emphasizes the need for vigorous antenatal surveillance for IUGR and consideration of elective preterm delivery of affected fetuses. A systematic review of other organs, which develop in a similarly rapid fashion during the late intrauterine period, is indicated by this work. With one exception, all birthweights in the growth-retarded groups were below the third centile, thus the precise quantitative relation between progressive IUGR and renal function requires further assessment.

The recently developed method of total vertical projections is illustrated to estimate the total dendritic length of a human Substantia Nigra neuron. Next, the length of the different orders of dendritic branches, and the mean segment length for each order--commonly regarded as important parameters in neuron physiology--are also estimated. Finally, it is shown how to estimate the mean dendritic length in a population of neurons from vertical slices of arbitrary and unknown thickness. Being unbiased and highly efficient, the proposed methods offer interesting alternatives to current procedures used for the metric analysis of neuron arborizations.

SUMMARY The spatial grid is a method for estimating the surface area of particles. A stack of perfectly registered sections is the essential prerequisite for its use. The confocal scanning light microscope provides such a stack by optical sectioning. The spatial grid method is briefly described and applied to an osteocyte lacuna in dry mineralized human mandible. This type of cell was chosen because of its very complex shape. The variance of the area estimate is studied and compared with the results of a simulation.

The issue of function-property correlation studies in quantitative microstructure analysis is introduced. Modern digital stereological techniques are described and their role in such studies is discussed. Surface and volume estimation algorithms are presented in relation to the corresponding geometrical sampling probes utilized for analysis. The procedures are illustrated in both biomedical and metallurgical samples and the potential in further studies is discussed.

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 Children's 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.

The prudent use of the atomic force microscope as a supra-vital live cell imaging tool requires that cell viability must be determined before and after scanning. Complementary optical techniques in conjunction with the fluorescent dyes rhodamine-123 and ethidium homodimer have been used within this study to determine cell viability after increasing loads are applied in contact mode. Guideline force ranges for five commonly cultured cell lines, human squamous carcinoma (A431), fibroblast, HeLa, Potorous tridactylis (PtK2) and rat intestinal epithelial (RIE) cells are given.

Preface.- Acknowledgments.- List of Contributors.- List of Figures.- List of Tables.- Introduction: Environmental Impact on Reproductive Health, Recent Trends and Developments. Section 1: The Link Between Reproductive Health and Environment. Epidemiological Studies on the Relationship between Semen Quality and Environmental Chemicals: Historic and Contemporary Compounds. Environment and Fertility. Section 2: Reproductive Health Effects. Male Reproductive Status and its Relationship with Man-Made, Hormone-Disrupting Substances: Studies In Flanders, Belgium.- Potential Impact of the Environment on the Male Reproductive Function: The Example Of Cryptorchidism.- Advanced Glycation End Products in Polycystic Ovarian Syndrome.- Oocytes and Ovarian Follicles as Targets of Endocrine Disrupters: Consequences for Reproductive Health.- Section 3: Pollutants And Exposure. Non-Pesticide Endocrine Disrupters and Reproductive Health.-Medical Drugs Impairing Fertility.- Impact of Air Pollution on Reproductive Health in Northern Bohemia.- Section 4: Policy Instruments And Issues. Pesticides as Endocrine Disrupters: Identification of Hazards for Female Reproductive Function.- Assessment of Fertility and Other Health Impacts for Selected Pollutants in Flanders: Opportunities and Limitations.- Problems in Assessing Low Dose Effects of Endocrine Disrupters.- Getting the Message Across - WWF's and ISDE's Perspectives on Communication Strategies to Reduce Exposures to Hazardous Chemicals.- Environmental Ethics and Fertility in Early Cultures and The Industrial Age.- Section 5: Regional Aspects. Reproductive Health Problems in the Greater Athens Area.-Environment and Reproductive Health in Bulgaria.- List of Abbreviations.- List of Units.- Index

Preface. Acknowledgments. List Of Contributors. List Of Figures. List Of Tables. List Of Boxes. Introduction: Concepts In The Relationship Of Congenital Diseases With The Environment. Section 1: Methods: Endpoints For Prenatal Exposures In Toxicological Studies. Congenital Defects Or Adverse Developmental Effects In Vertebrate Wildlife: The Wildlife-Human Connection. Epidemiological Methods. Eurocat: Surveillance Of Environmental Impact. Clinical Teratology.- Section 2: Teratogens: Dioxins And Congenital Malformations. Links Between In Utero Exposure To Pesticides And Effects On The Human Progeny. Does European Pesticide Policy Protect Health?. Association Of Intra-Uterine Exposure To Drugs With Congenital Defects: The Thalidomide Effect.- Section 3: Congenital Diseases: Endocrine Disrupter Exposure And Male Congenital Malformations. Testicular Dysgenesis Syndrome As A Congenital Disease. Endocrine Disrupters, Steroidogenesis And Inflammation. Environmental Impact On Congenital Diseases: The Case Of Cryptorchidism. Where Are We Now, And Where Are We Going? Environmental Risk And Sex Ratio In Newborns.- Section 4: Country Reports:Congenital Abnormalities In Greece: Functional Evaluation Of Statistical Data 1981 - 1995. Congenital Anomalies In Bulgaria. Congenital Anomalies In The British Isles. European Union-Funded Research On Endocrine Disrupters And Underlying Policy.- Section 5: Conclusions: Environmental Impacts On Congenital Anomalies - Information For The Non-Expert Professional. List Of Abbreviations. List Of Units. Index.

Foreword. List Of Contributors. List Of Figures. List Of Tables. List Of Boxes. Effects Of Mobility On Health.- Traffic, Noise And Health.-Environmental Noise, Annoyance And Sleep Disturbance.- Psychological And Social Aspects Of 'Transport And Health'.- Air Pollution And Health: An Overview With Some Case Studies.- The Respiratory Effects Of Air Pollution.- Photochemical Activity Of Importance To Health In The Aegean.-Monitoring Of Suspended Aerosol Particles And Tropospheric Ozone By The Laser Remote Sensing (Lidar) Technique: A Contribution To Develop Tools Assisting Decision-Makers.- Health Impact And Control Of Particle Matter.- Endocrine Problems Related To Mobility.- Sedentarism.- Mobility And Transport In The Johannesburg Plan Of Implementation And National Environmental Action Plans.- Integrating Health Concerns Into Transport Policies: From The Charter On Transport, Environment And Health To The Transport, Health And Environment Pan-European Programme.- Mobility And Health In Bulgaria.- Health And Mobility In Flanders (Belgium).- Country Report Austria On Mobility, Environmental Issues, And Health.- Health And Mobility In The UK.- Effects Of Mobility On Health - An Overview.- List Of Abbreviations. List Of Units. Index.

The feto-maternal relationships within the bovine placentome at 135 days gestation were investigated using unbiased stereology. This provides an estimate of the feto-maternal interface without physically separating these. The functional surface area, the volume of the fetal and maternal components, volumetric tissue shrinkage due to processing, and the feto-maternal surface amplification factor were estimated and quantified in standard units. In this healthy bovine pregnancy there were 105 placentomes with a total volume of 900 ml. The volumetric tissue shrinkage was 24% and the maternal: fetal tissue ratio overall was 0.92. The total feto-maternal exchange surface area was 18.5 m2 with a surface amplification factor of 108.1. Analysis of the coefficient of error from counting different numbers of points within the reference area suggested that 50% and 75% fewer points could be counted to achieve the result obtained by counting approximately 6000 points used in this study.

Preface and acknowledgements. List of contributors. List of figures. List of tables. List of boxes. Introduction: cancer and the environment - revisiting traditional views of involuntary exposure to carcinogens P. Nicolopoulou-Stamati, M.A. Pitsos. Summary. 1. Introduction. 2. Cancer and the environment. 3. Competing theories of aetiological mechanisms in cancer. 4. Exposure to carcinogens in early life - a strong indication that environmental influences are important. 5. Goal of this book. 6. Conclusions. References. Incorporating the environmental context in the study of cancer: issues and implications A. Novogradec, S. Harris Ali. Summary. 1. Introduction. 2. Evidence for the environmental basis of cancer. 3. Studying the cancer and environment relationship. 4. Implications of risk assessment for cancer policy and intervention. 5. Conclusions. References. Could the increase in cancer incidence be related to recent environmental changes? C.V. Howard, J.A. Newby. Summary 1. Introduction. 2. Cancer in pre-industrial society. 3. The increase in average life expectancy. 4. Cancer incidence vs. mortality rate. 5. Human epidemiological evidence that environmental factors are paramount in the aetiology of cancer. 6. Could chronic low dose exposure to carcinogens lead to cancer. 7. Changes in patterns of human development. 8. Temporal patterns of change. 9. Changes in the patterns of human exposure to ionising radiation. 10. Public perceptions. 11. Conclusions. Acknowledgements. References. The role of DNA damage and DNA-damaging environmental chemicals in carcinogenesis K. Peltonen. Summary 1. Introduction. 2. Metabolism. 3. Chemical carcinogenesis. 4. Biomarkers of exposure. 5. Methods of detection. 6. Conclusions. Acknowledgements. References. Gene-environment interaction in environmental carcinogens H. Autrup. Summary 1. Introduction. 2. Metabolism of chemical carcinogens. 3. Gen-gene interaction. 4. Defence against oxidative stress. 5. Conclusions. References. Health impact assessment of accidents with environmental carcinogens: a case study of the Belgian PCB/dioxin incident in 1999 L. Hens. Summary 1. Introduction. 2. Phenomenology of the Belgian PCB/dioxin incident in 1999. 3. Hazard identification. 4. Dose-response assessment. 5. Exposure assessment. 6. Risk assessment. 7. Conclusion. References. Anti-oxidants and chemopreventive agents as cancer enhancing agent: the other side of the coin S.Z. Abdel-Rahman, M. Paolini, M.S. Legator. Summary. 1. Introduction. 2. Co-carcinogenic properties of bCT. 3. The co-carcinogenicity of b-carotene enhances the transformation potential of benzo[a]pyrene and cigarette smoke condensate. 4. Cruciferae vegetables, glucosinolates and chemoprevention. 5. Co-carcinogenic properties of glucoraphanin, the natural bioprecursor of sulforaphane. 6. Conclusions. Acknowledgements. References. Legislative proposals for reversing the cancer epidemic and controlling run-away industrial technologies