Focussed ion beam milling at grazing incidence angles.

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.

Obatoclax induces Atg7-dependent autophagy independent of beclin-1 and BAX/BAK.

Direct pharmacological targeting of the anti-apoptotic B-cell lymphoma-2 (BCL-2) family is an attractive therapeutic strategy for treating cancer. Obatoclax is a pan-BCL-2 family inhibitor currently in clinical development. Here we show that, although obatoclax can induce mitochondrial apoptosis dependent on BCL-2 associated x protein/BCL-2 antagonist killer (BAX/BAK) consistent with its on-target pharmacodynamics, simultaneous silencing of both BAX and BAK did not abolish acute toxicity or loss of clonogenicity. This is despite complete inhibition of apoptosis. Obatoclax dramatically reduced viability without inducing loss of plasma membrane integrity. This was associated with rapid processing of light chain-3 (LC3) and reduction of S6 kinase phosphorylation, consistent with autophagy. Dramatic ultrastructural vacuolation, not typical of autophagy, was also induced. Silencing of beclin-1 failed to prevent LC3 processing, whereas knockout of autophagy-related (Atg)7 abolished LC3 processing but failed to prevent obatoclax-induced loss of clonogenicity or ultrastructural changes. siRNA silencing of Atg7 in BAX/BAK knockout mouse embryonic fibroblasts did not prevent obatoclax-induced loss of viability. Cells selected for obatoclax resistance evaded apoptosis independent of changes in BCL-2 family expression and displayed reduced LC3 processing. In summary, obatoclax exhibits BAX- and BAK-dependent and -independent mechanisms of toxicity and activation of autophagy. Mechanisms other than autophagy and apoptosis are blocked in obatoclax resistant cells and contribute significantly to obatoclax's anticancer efficacy.

Verification of cell viability at progressively higher scanning forces using a hybrid atomic force and fluorescence microscope.

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.

Detection of replicative integrity in small colonic biopsies using the BrdUrd comet assay.

The alkaline single-cell gel electrophoresis or comet assay is a relatively simple method of measuring DNA single-strand breaks and alkali-labile sites in individual cells. Previously, we have used a combination of this with bromodeoxyuridine labelling of DNA and immunolocalisation of the BrdUrd to show that DNA replicative integrity can be assessed in single cultured cells. This study demonstrates the application of the technique to single cells derived from small human colonic biopsies isolated at routine endoscopy. A high level of reproducibility within replicate comet slides and between comet slides prepared from various colonic sites within a single patient is shown. Preliminary results demonstrate that defects in replication can be detected in tumour and premalignant colonic tissue adjacent to the tumour, suggesting that alterations in replicative integrity are an early event in neoplasia, appearing in premalignant mucosal cells. This development deems the BrdUrd comet assay suitable as an ex vivo molecular end point that can be measured easily in tissue collected by biopsy at routine colonic endoscopy. Thus, the BrdUrd comet assay has the potential to facilitate trial investigations of diet- or environment-related factors that may affect replicative integrity in the colon and provides a novel biomarker for colon carcinogenesis.

Expression of oestrogen and progesterone receptors by mast cells alone, but not lymphocytes, macrophages or other immune cells in human upper airways.

BACKGROUND: Nasal polyposis often coexists with asthma in airway inflammatory conditions characterised by the infiltration of a range of immune cells. A potentially important role for ovarian hormones has been implicated in airway inflammation but the cellular target for such action is not known. METHODS: Expression of oestrogen receptors (ER) and progesterone receptors (PR) was examined using immunohistochemistry in formalin fixed nasal polyp tissues from 47 subjects. The cells positive for ER or PR were confirmed by spatial location, dual immunolabelling, and histochemical staining. RESULTS: Consistent with the known features of nasal polyps, CD4+ (T helper/inducer), CD8+ (cytotoxic/suppressor), CD68+ (macrophages), mast cells, eosinophils and neutrophils were all clearly detected by their relevant monoclonal antibodies or appropriate histochemical staining, but only mast cells tested positive for ER/PR labelling with their polyclonal and monoclonal antibodies. The frequencies for expression were 61.7% for ER positive and 59.6% for PR positive cells. The expression of ER/PR was independent of patient sex and age but was highly correlated with the numbers of mast cells (r = 0.973, p<0.001 for ER; r = 0.955, p<0.001 for PR). Fewer than 5% of mast cells were found to be negative for ER/PR expression. CONCLUSIONS: Mast cells alone, but not lymphocytes, macrophages, or other immune cells, express ER/PR in human upper airways. Numerous ER/PR positive mast cells exist in nasal polyps, indicating that this may be a major route for the involvement of sex hormones in airway inflammation when exposed to the higher and varying concentration of oestrogen and progesterone characteristic of females.

Tritiated thymidine ([3H]-TdR) and immunocytochemical tracing of cellular fate within the asexually dividing cestode Mesocestoides vogae (syn. M. corti).

This report documents the presence of an active thymidine kinase (TK) system within Mesocestoides vogae tetrathyridia as quantified by tritiated thymidine ([3H]-TdR) incorporation using liquid scintillation counting. A 100-fold increase in [3H]-TdR incorporation was observed at 37 degrees C when compared with its incorporation at 0 degrees C. Thymidine's competitive analogue, BrdU, competed for sites within newly replicated DNA. Immunohistochemical trials performed here using antibodies against BrdU identified cells that have entered and passed through S-phase. Positively stained nuclei were most numerous at the anterior tip of tetrathyridia especially within the ganglia, lesser numbers of these cells occurred along the growing commissure and amongst surface tegumental cytons suggesting that stem cells do not exist in one region but are found throughout the entire body. As M. vogae has no internal organ systems the major sites for cell proliferation are those exhibiting maximal cell recruitment and undergoing tissue repair. These results show that it is possible to monitor changes in the cell recruitment pattern within this cestode. Thus use of BrdU and immunohistochemistry demonstrates how spatial arrangement and cellular reorganization can be successfully traced within M. vogae.

The bromodeoxyuridine comet assay: detection of maturation of recently replicated DNA in individual cells.

The single-cell gel electrophoresis (Comet) assay is a relatively simple method of measuring DNA single strand breaks and alkali-labile sites in individual cells. We have combined this with bromodeoxyuridine (BrdUrd) labeling of DNA and immunolocalization of the BrdUrd to assess DNA replicative integrity on a single-cell basis. We show that the existence of strand discontinuities in recently replicated domains of DNA, caused during semiconservative replication or exacerbated by the arrest of replicative polymerases at UV irradiation- or chemical-induced lesions, can be detected in individual cells. Data obtained from BrdUrd-Comets are consistent with biochemical data derived with a range of techniques showing that DNA replication involves the creation of strand breaks or gaps adjacent to recently replicated material, and that DNA damage prolongs the duration of such discontinuities where DNA polymerases are stalled opposite lesions (R. T. Johnson et al, The Legacy of Cell Fusion, pp. 50-67, Oxford: Science Publications, 1994; R. B. Painter, J. Mol. Biol., 143: 289-301, 1980.). Compared with standard biochemical techniques, the BrdUrd-Comet assay is simple and suitable for the accurate and automatable assessment of replicative integrity in very small numbers of mammalian cells, such as may be obtained by biopsy.

The spatial organisation of the central nervous system of Mesocestoides corti as revealed by microinjection of carbocyanine dye.

The carbocyanine dyes DiI, DiA and DiO were microinjected into the cerebral ganglion of intact Mesocestoides corti tetrathyridia to determine the spatial organisation and connectivity patterns of the CNS. Of the dyes tested, DiI proved to be the most effective, giving highly fluorescent and persistent staining of even very fine calibre afferent and efferent nerve fibres. DiI labelling, in conjunction with transmission electron microscopy, revealed the nervous system to consist of sensory endings, directly connected to the cerebral ganglion by elongated cellular tracts, efferent nerve fibres which innervated the suckers, and longitudinal nerve cords which travelled along the remainder of the body.

Macrophages and apoptotic cells in human colorectal tumours.

The numerical relationship between tumour associated macrophages (TAM) and apoptotic cells in 12 human colorectal tumours was evaluated. TAM were labelled immunohistochemically and apoptotic cells were visualized by counterstaining with haematoxylin and eosin (H&E). The stereological techniques, Cavalieri's estimator of volume and the Disector were used to estimate both tumour volume and numerical density of both cell types. The occurrence of TAM per unit volume of tissue increased with increasing tumour volume to a maximum in a tumour of 110.5 cm3, after which numbers declined. Levels of apoptosis also increased with tumour volume though more erratically than levels of TAM and declined for tumour volumes greater than 80 cm3. This is the first report of an attempt to assess the relationship between apoptotic cells and TAM in human tumours.

Are retroviruses involved in the aetiology of human breast cancer?

To further investigate the possibility for retroviral involvement in the etiology of human breast cancer we processed peripheral blood monocytes and malignant breast tissue biopsies from 10 patients with breast cancer (infiltrating ductal carcinoma or infiltrating lobular carcinoma; ages 40-80 years) and 20 normal healthy women (with no evidence or family history of breast cancer. 10 age-matched controls and 10 women age 22-27 years) for the assay of the retroviral enzyme, reverse transcriptase, using an ELISA and for election microscopy examination for the detection of retroviral-like particles. Reverse transcriptase activity was detected in 5 out of 10 samples of monocyte culture medium and in 1 out of 10 of malignant tissue biopsies from the patients with breast cancer. In contrast, reverse transcriptase was not detected in the culture medium of the monocytes from any of the control subjects. Electron microscopy did not reveal the presence of any retroviral-like particles in any sample of monocyte culture medium or in any of the malignant or normal breast tissue biopsies. Despite evidence for the presence of reverse transcriptase in a subsample of the monocyte culture medium and breast tissue biopsies from the cohort of breast cancer patients who participated in this study, the role of retroviruses in human breast cancer remains unclear.

Use of real-time confocal laser scanning microscopy to study immediate effects of photodynamic activation on photosensitized erythrocytes.

With a view towards the design of systems capable of combining the use of chemotherapy and photodynamic therapy in the treatment of cancer and other disorders, it has been proposed that photosensitized erythrocytes might be employed as carriers/vehicles for agents such as cancer chemotherapeutics. In studying the light dependent release of entrapped agents from such a system, the efficacy of light induced release is usually studied by measuring release of an entrapped component into centrifugation supernatants following photoactivation. It has hitherto been extremely difficult to examine what occurs upon immediate irradiation at the microscopic level in real-time. In this study we demonstrate that, using real-time confocal laser scanning microscopy, it is possible to directly observe immediate short-term events occurring during direct irradiation with the visualizing beam. Following irradiation of photosensitized erythrocytes with the visualizing beam form the confocal scanning system, it was noticed that some from of cell-disruptive event occurred. In this study we demonstrate a dose dependent response between this relatively immediate, light induced disruptive event with respect to both irradiation exposure and photosensitizer concentration. We suggest that this system may provide a novel means of observing, at a microscopic level, events occurring in real-time during photodynamic therapy.

The effects of copper deficiency on human lymphoid and myeloid cells: an in vitro model.

Cu has long been known to influence immune responses. An in vitro model system was established in which human myeloid (HL-60), B-lymphoid (Raji) and T-lymphoid (Molt-3) cell lines could be grown in culture media of varying Cu levels. Initially Cu was removed from the medium by dialysis of fetal calf serum against a metal-ion chelator, minor depletion of other trace metals being obviated by repletion with appropriate metal salts. The growth rate of HL-60 was significantly (P < 0.05) inhibited by 72 h Cu depletion. Molt-3 cells required a longer period, up to 144 h, in Cu-depleted medium before growth was impaired. Raji-cell growth was not affected. These results confirmed clinical observations that T-cell functions were more sensitive to Cu deprivation than B cells. Analysis of intracellular metal levels in Molt-3 cells showed that Cu levels had been significantly lowered (P < 0.05) although Ca2+ levels were raised. Intracellular activity of the antioxidant enzyme superoxide dismutase (EC 1.15.1.1) was significantly impaired (P < 0.05) in Molt-3 cells grown in Cu-depleted medium. Activity of the mitochondrial enzyme cytochrome c oxidase (EC 1.9.3.1) was also significantly impaired (P < 0.05) by Cu depletion. Each of these findings indicates an increase in the potential for cellular damage by reduced antioxidant activity, impairment of normal mitochondrial activity and excessive Ca2+ influx. A major consequence of the type of damage occurring under these circumstances is membrane disruption. This was confirmed by scanning electron microscopy of Molt-3 cells grown under varying Cu levels.

The central nervous system of Grillotia erinaceus (Cestoda: Trypanorhyncha) as revealed by immunocytochemistry and neural tracing.

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.

Structure and ultrastructure of muscle systems within Grillotia erinaceus metacestodes (Cestoda: Trypanorhyncha).

Three distinct muscle types have been identified within the metacestode of Grillotia erinaceus. These consist of peripheral somatic myofibres plus two muscle systems directly involved in parasite attachment to the host, i.e. the tentacular bulb and its antagonistic retractor muscle. In common with other cestodes the somatic muscle consists of smooth-type fibres running longitudinally and obliquely to the main body axis. The retractor muscle consists of myofibres with centrally displaced nuclei. Upon contraction these latter fibres become spirally orientated causing the muscle to coil and lateral membranes to become elevated as spikes. Definitive nerve processes have not been identified within somatic or retractor muscle. Individual tentacular bulbs form the proximal terminus for a closed hydraulic system. Each bulb consists of overlapping, contrarotating myofibres which display obvious striations; the striations appear in alternate fibres to be in transverse and oblique planes. Adjacent myofibres are separated by approximately 0.5 micron, possess abundant mitochondria and have shallow t-tubules plus associated vesicles of sarcoplasmic reticulum at each Z line. Thick myofilaments are surrounded by 13, shared, thin myofilaments. Close neuronal control for the bulb muscle is suggested by the presence of obvious motor end-plates which contain both lucent and dense neurovesicles.