Loss of CDX2 and high COX2 (PTGS2) expression in metastatic colorectal cancer.

Lack of expression of the tumour suppressor gene caudal-type homeobox 2 (CDX2) associates with poor outcomes in early stage colorectal cancer (CRC). Yet its prognostic value in the context of other prognostic biomarkers in metastatic CRC (mCRC) is unknown. Overexpressed cyclooxygenase-2 (COX2) has been reported in advanced CRC. However, CDX2 and COX2 relationship in mCRC remains undetermined. We aimed to assess their expression in mCRC tumours from a clinically characterised cohort and their influence on overall survival (OS) and progression-free survival (PFS) in first line. Among 720 consecutive mCRC patients, 346 had tumour samples appropriate for tissue microarray assembly and immunohistochemistry analyses. Clinical and survival data were retrospectively assessed. Loss of CDX2 expression was detected in 27 (7.8%) samples, enriched in poorly differentiated tumours (20%; p < 0.01) and in those with the BRAF p.V600E variant (40%; p < 0.01). Most tumours (93.4%) expressed COX2. COX2-negative samples were enriched in poorly differentiated mCRC. In unadjusted analyses, median OS (p < 0.001) and median PFS (p < 0.05) were inferior for patients with CDX2-negative versus CDX2-positive tumours. In conclusion, loss of CDX2 was significantly associated with poorly differentiated mCRC and BRAF p.V600E allele and a prognostic marker of worse OS.

Lysosomal responses to different gold forms (nanoparticles, aqueous, bulk) in mussel digestive cells: a trade-off between the toxicity of the capping agent and form, size and exposure concentration.

Gold nanoparticles (NPs) are increasingly used in technological materials and consumer products and may have toxicological characteristics distinct from bulk and aqueous gold. The aim of this work was to understand the effects of Au NPs especially, how the form, the size and the coating influence bioaccumulation/biodistribution and toxicity of NPs in mussels, Mytilus galloprovincialis. Mussels were exposed for 3 d to concentrations of Au (0.75, 75 and 750 µg Au/l) supplied as Au-Cit NPs (5 and 40 nm; Au5-Cit and Au40-Cit), bulk and aqueous Au (HAu(III)Cl4), and to the capping agent (Na-citrate) in doses used in the formulation of NPs (0.005, 0.5, 5 mg/l). Citrate-stabilised NPs formed stable suspensions of aggregates in seawater (SW) available for mussels. Au accumulation in soft tissues was similar in Au40-Cit and aqueous Au exposed mussels, lower in Au5-Cit and negligible after bulk exposure. Au NPs were identified (X-ray microanalysis) in different compartments of the endolysosomal system in digestive cells, and small size NPs (5 nm) were more accumulated than 40 nm NPs, aqueous and bulk. The degree of lysosomal membrane destabilisation was related with intralysosomal metal accumulation and depended on the form, NP size (Au5-Cit > Au40-Cit > aqueous > bulk) and concentration. Citrate alone provoked extreme reduction in lysosomal membrane stability. Toxicopathic alterations were recorded in digestive gland cells (vacuolisation, swollen RER, connective tissue disruption and cell death) especially in mussels exposed to 40 nm NPs. Deleterious effects resulted from digestive tract obliteration (agglomerates) and digestion malfunction. The toxic effect of Au-Cit NPs was influenced both by NP size, capping agent composition and the dose of capping agent carried by NPs, which was size dependent.

Development and comparison of the methods for quantitative electron probe X-ray microanalysis analysis of thin specimens and their application to biological material.

In recent years, there has been a return to the use of electron probe X-ray microanalysis for biological studies but this has occurred at a time when the Hall programme which acted as the mainstay for biological microanalysis is no longer easily available. Commercial quantitative routines rely on the Cliff-Lorimer method that was originally developed for materials science applications. Here, the development of these two main routines for obtaining quantitative data from thin specimens is outlined and the limitations that are likely to be met when the Cliff-Lorimer routine is applied to biological specimens is discussed. The effects of specimen preparation on element content is briefly summarized and the problems encountered when using quantitative analysis on resin-embedded materials emphasized.

Immunolocalization of duodenal cytochrome B: a relationship with circulating markers of iron status.

BACKGROUND: The brush border ferric reductase (Dcytb) is critical for the absorption of dietary iron and appears to be expressed on the duodenal enterocyte brush border. The Dcytb expression is increased in severe iron-deficient anaemia, but the situation in a more typical mild iron deficiency is unclear. This study investigated Dcytb expression in patients with normal iron status or mild iron deficiency and its relationships with enterocyte iron status. MATERIALS AND METHODS: Duodenal biopsy specimens and blood samples were obtained from 32 patients undergoing routine upper gastrointestinal endoscopy. Twenty-three specimens (six iron-deficient and 17 iron-replete) were processed for light-microscopy (LM) and for immunohistochemistry with antibodies against Dcytb and heavy/light chain ferritin subunits. The nine remaining biopsies (three iron-deficient and six iron-replete) were processed for electron microscopy (EM). Immunolocalization of Dcytb and intracellular ferritin was performed with appropriate primary antibodies followed by 10-nm gold conjugate labels. RESULTS: The LM process showed a strong negative correlation between immunolabelling intensity of Dcytb on the enterocyte brush border and serum iron saturation (P < 0.001), but only a weak negative correlation between this antigen and haemoglobin (P = 0.08) or serum ferritin concentrations (P = 0.4). EM confirmed anti-Dcytb preferential labelling of microvilli rather than enterocyte cytoplasm (P = 0.001), but preferential antiferritin labelling of cytoplasm (P < 0.02). There was no correlation with enterocyte cytoplasmic ferritin labelling (i.e. enterocyte iron status and Dcytb expression). CONCLUSIONS: Enterocyte Dcytb brush border expression is increased even in mild iron deficiency and may be related to serum iron saturation. The lack of correlation with enterocyte ferritin expression deserves further study with direct measurement of intracellular iron.

Electron beam damage studies of synthetic 6-line ferrihydrite and ferritin molecule cores within a human liver biopsy.

In order to achieve an accurate understanding of the crystal structure of 6-line ferrihydrite (6LFh) and ferritin molecule cores within a human liver biopsy using transmission electron microscopy (TEM), electron beam damage should be considered. For the case of 6LFh, the electron energy loss near-edge structure (ELNES) of core ionisation edges in the electron energy loss spectrum (EELS) combined with multiple linear least-square (MLLS) fitting of reference spectra together with analysis of selected area electron diffraction (SAED) patterns suggests that the iron in 6LFh is solely octahedrally coordinated Fe3+. With increasing electron dose, an increasing percentage of this octahedrally coordinated Fe3+ migrates to tetrahedral sites. When the dose exceeds 3 x 10(8) electrons/nm2, Fe2+ is found to be present in the material. This method also indicates that the iron in ferritin molecule cores within a human liver biopsy is the same as in 6LFh, entirely Fe3+ in octahedral coordination with oxygen. Again the percentage of octahedrally coordinated Fe3+ decreases as the accumulated electron dose increases and Fe2+ is produced in the liver biopsies when the electron dose exceeds 10(6)electrons/nm2.

Changes in intracellular electrolyte concentrations during apoptosis induced by UV irradiation of human myeloblastic cells.

Decreases in the intracellular concentrations of both K(+) and Cl(-) have been implicated in playing a major role in the progression of apoptosis, but little is known about the temporal relationship between decreases in electrolyte concentration and the key events in apoptosis, and there is no information about how such decreases affect different intracellular compartments. Electron probe X-ray microanalysis was used to determine changes in element concentrations (Na, P, Cl, and K) in nucleus, cytoplasm, and mitochondria in U937 cells undergoing UV-induced apoptosis. In all compartments, the initial stages of apoptosis were characterized by decreases in [K] and [Cl]. The largest decreases in these elements were in the mitochondria and occurred before the release of cytochrome c. Initial decreases in [K] and [Cl] also preceded apoptotic changes in the nucleus. In the later stages of apoptosis, the [K] continued to decrease, whereas that of Cl began to increase toward control levels and was accompanied by an increase in [Na]. In the nucleus, these increases coincided with poly(ADP-ribose) polymerase cleavage, chromatin condensation, and DNA laddering. The cytoplasm was the compartment least affected and the pattern of change of Cl was similar to those in other compartments, but the decrease in [K] was not significant until after active caspase-3 was detected. Our results support the concept that normotonic cell shrinkage occurs early in apoptosis, and demonstrate that changes in the intracellular concentrations of K and Cl precede apoptotic changes in the cell compartments studied.

Biphasic behavior of changes in elemental composition during staurosporine-induced apoptosis.

Although the identification of events that occur during apoptosis is a fundamental goal of apoptotic cell death research, little is know about the precise sequence of changes in total elemental composition during apoptosis. We evaluated total elemental composition (Na, Mg, P, Cl, S, and K) in relation to molecular and morphological features in human U937 cells induced to undergo apoptosis with staurosporine, an intrinsic pathway activator. To evaluate total elemental content we used electron probe X-ray microanalysis to measure simultaneously all elements from single, individual cells. We observed two phases in the changes in elemental composition (mainly Na, Cl and K). The early phase was characterized by a decrease in intracellular K (P<0.001) and Cl (P<0.001) content concomitant with cell shrinkage, and preceded the increase in proteolytic activity associated with the activation of caspase-3. The later phase started with caspase-3 activation, and was characterized by a decrease in the K/Na ratio (P<0.001) as a consequence of a significant decrease in K and increase in Na content. The inversion of intracellular K and Na content was related with the inhibition of Na+/K+ ATPase. This later phase was also characterized by a significant increase (P<0.001) in intracellular Cl with respect to the early phase. In addition, we found a decrease in S content and an increase in the P/S ratio. These distinctive changes coincided with chromatin condensation and DNA fragmentation. Together, these findings support the concept that changes in total elemental composition take place in two phases related with molecular and morphological features during staurosporine-induced apoptosis.

Tuberculosis and HIV seroprevalence in Lambeth, Southwark and Lewisham, an area of South London.

Since the mid-1980s the number of cases of TB notified within the U.K. has continued to rise although the contribution of HIV to this rise remains unclear. A 12-month prospective cohort study was conducted at chest and HIV clinics in four hospitals in Lambeth, Southwark and Lewisham (LSL), an area of South London, to determine the proportion of patients with culture-proven TB infected with HIV. Secondary aims were to determine the proportion of patients with TB and undiagnosed HIV at first presentation to chest clinics, to determine the proportion of patients presenting with TB as an AIDS defining illness (ADI) and to identify risk factors for co-infection with TB and HIV. In chest clinics, demographic data and left-over blood from patients aged 16 or over with culture-proven TB was collected, anonymised and HIV tested. In HIV clinics, demographic data on patients with TB already known to be HIV seropositive were also obtained. Twenty-one patients (13%, 95% CI-8-19%) of 159 with culture-proven TB were infected with HIV Four (3%) of 133 patients at first presentation to chest clinics had undiagnosed HIV; two were subsequently diagnosed. Of the 21 patients withTB and HIV, nine (43%) presented with TB as an ADI. Patients with TB and HIV were significantly more likely to be aged between 35 and 55 years compared to HIV seronegative patients [12/21 (57%) vs. 38/138 (28%), P=0.006]. None of the patients from the Indian Subcontinent were HIV seropositive [0/21 vs. 25/138 (18%), P=0.047]. At the present time, universal HIV testing of patients with culture-provenTB in chest clinics within the U.K. is unlikely to significantly reduce the number of patients with undiagnosed HIV.

Potassium concentration is reduced in cultured rabbit tracheal smooth muscle cells after withdrawal of serum.

Comparison of elemental concentrations in growth-arrested airway smooth muscle cells with those in their proliferating counterpart showed that potassium (K(+)) was significantly reduced, whereas concentrations of other elements sodium (Na(+)), magnesium (Mg(2+)), phosphorus (P), and chlorine (Cl(-)) remained unchanged. Reduced K(+)concentration was associated with a change in the cells from a spindle shape to a flattened form.

Long freeze-drying times are not necessary during the preparation of thin sections for X-ray microanalysis.

The temperature profile that occurs when a brass block warms up in a vacuum evaporation unit was determined. Freshly drawn human blood was concentrated by centrifugation, and the pellet was cryofixed and cryosectioned. The cryosections were subject to different freeze-drying protocols, using a freeze-drier with a temperature-controlled stage, to determine the effect of freeze-drying time on element distribution. Spectra were collected by spot analyses at various distances across the interface between red cells and plasma. Concentrations of sodium were high and variable outside the cell and low in the cell interior, with potassium showing the reverse distribution. The number of counts under the iron peak closely followed the potassium distribution. The concentration of sodium was higher than expected at 40 nm inside the cell membrane. This was attributed to the formation of ice crystals at the interface between the cells and plasma during cryofixation and the use of a wide probe size.

Changes in elemental content during apoptotic cell death studied by electron probe X-ray microanalysis.

Recent data suggest that changes in ionic content, primarily potassium, play a pivotal role in the progression of apoptosis. However, the changes in total element content, i.e., sodium (Na), magnesium (Mg), phosphorous (P), chlorine (Cl), potassium (K), and calcium (Ca), during apoptosis have not been evaluated. Electron probe X-ray microanalysis (EPXMA) was used to measure total element content in U937 cells before and after the induction of apoptosis. As an experimental model we used U937 cells irradiated with ultraviolet (UV) light. Apoptosis was evaluated with phase-contrast microscopy, with scanning and transmission electron microscopy, and with the fluorescent dye bisbenzimide (Hoechst 33342). Plasma membrane permeability as a measure of cell death was determined by trypan blue dye exclusion. To investigate element content with EPXMA, cells were cryoprepared, i.e., cryofixed and freeze-dried, and analyzed as whole cells using a scanning electron microscope. We found that the UV irradiation induced rapid (within 2 h) morphological changes associated with apoptosis, such as plasma membrane blebbing, condensation of the chromatin, and the formation of membrane-bound apoptotic bodies. At this time, 95% of the apoptotic cells excluded trypan blue dye. EPXMA results demonstrated that UV light-irradiated apoptotic cells (cells with membrane-bound apoptotic bodies) had a lower Cl content (P < 0.001) and K content (P < 0.001) and a higher Na content (P < 0.001) in comparison with nonirradiated control cells. Also, P and Ca content was higher in apoptotic cells than in control cells, but this difference did not reach statistical significance. No differences were found in Mg. These data indicated that morphological changes characteristic of apoptotic cell death are related with significant changes in sodium, chlorine, and potassium content. In addition, we demonstrated that these changes in elemental composition were not associated with loss of cell membrane integrity.

A procedure to prepare cultured cells in suspension for electron probe X-ray microanalysis: application to scanning and transmission electron microscopy.

We describe a simple procedure to prepare cultured cells in suspension to analyse elemental content at the cellular level by electron probe X-ray microanalysis. Cells cultured in suspension were deposited onto polycarbonate tissue, culture plate well inserts, centrifuged at low g, washed to remove the extracellular medium, cryofixed and freeze-dried, and analysed in the scanning mode of a scanning electron microscope. We tested the effect of different washing solutions (150 mM ammonium acetate, 300 mM sucrose, and distilled water) on the elemental content of cultured cells in suspension. The results demonstrated that distilled water was the best washing solution to prepare cultured cells. In addition, the low Na content, high K content and high K/Na ratio of the cells indicated that this procedure, based on the centrifugation at low g followed by cryopreparation, constitutes a satisfactory method to prepare cultured cells in suspension. We also investigated the effects of different accelerating voltages on X-ray signal collection. The results showed that moderate accelerating voltages, i.e. 10-11 kV, should be used to analyse whole cells in the scanning mode of the scanning electron microscope. We show that this method of preparation makes it possible to prepare cryosections of the cultured cells, thus permitting analysis of the elemental content at the subcellular level, i.e. nucleus, cytoplasm and mitochondria, using a scanning transmission electron microscope.

Changes in elemental concentrations are associated with early stages of apoptosis in human monocyte-macrophages exposed to oxidized low-density lipoprotein: an X-ray microanalytical study.

This study examines ion homeostasis in monocyte-macrophages committed to death by apoptosis. X-ray microanalysis has been used to demonstrate that intracellular concentrations of potassium decreased whilst those of sodium increased following 3 h of exposure to 100 microg/ml of oxidized low-density lipoprotein (LDL) in vitro. In contrast, the maximal incidence of cell death, as determined by the inability to exclude trypan blue, was not seen until 24 h of exposure. At 12 h, less than 1 per cent of cells were stained using terminal transferase-mediated DNA nick-end labelling, which is generally accepted as a marker of late stages in the apoptotic pathway. This is the first demonstration of early perturbations of ion homeostasis in monocyte-macrophages exposed to concentrations of oxidized LDL known to cause apoptosis.

A cryoclamp for the rapid cryofixation of the isolated blood-perfused rabbit cardiac papillary muscle preparation at predefined times during the contraction cycle.

There is increasing evidence that the distribution of monovalent cations in cardiac cells may be non-uniform, particularly in the region immediately beneath the sarcolemma, and we have proposed that a build-up of sodium in this region could be an important factor in the development of ischaemia-reperfusion injury. Electron probe X-ray microanalysis is ideal for the study of such changes in distribution but the application of the technique to this problem imposes severe requirements on the specimen and on the method for cryofixation. The specimen must be perfused through its vasculature so that it can be made truly ischaemic and be successfully reperfused. It is necessary to be able to cryofix the specimen without disturbance of its blood supply, electrical stimulation or temperature. It is also important to know the time in the contraction cycle when cryofixation occurs. Here we describe the design of an automated cryofixation device which can be used to cryofix a blood perfused papillary muscle preparation at predetermined time points in the contraction cycle. Preliminary data obtained from the analysis of rabbit papillary muscles subjected to varying periods of ischaemia are included as an example of the use of the cryoclamp.

Capillary surface area is reduced and tissue thickness from capillaries to myocytes is increased in the left ventricle of streptozotocin-diabetic rats.

The left ventricles of normal and diabetic rats, fixed by vascular perfusion were examined using modern stereological techniques to quantify changes in the morphology accompanying streptozotocin-induced diabetes. The heart weight to body weight ratio increased in diabetic rats whilst left ventricular volume remained unchanged. Papillary muscles from the diabetic animals showed prolonged time to peak tension and relaxation, and altered sensitivity to adrenalin and calcium. The apparent cardiomyopathy observed when body weight loss exceeds heart weight loss in experimental diabetes was accompanied by specific pathological changes in the composition of the left ventricle. In the diabetic animals the volume of extracellular components increased threefold and the volume of capillaries fell. The surface density and total surface area of capillaries was reduced, and oxygen diffusion distance to myocyte mitochondria increased. The volume fraction of myocyte mitochondria was reduced during streptozotocin-induced diabetes.

Cells are not just bags of water: a personal appreciation of the work of Dr. Brij L. Gupta and his contribution to biological X-ray microanalysis.

This paper surveys the contribution made by the work of Dr. B. L. Gupta to the science of biological X-ray microanalysis. A brief biographical sketch of Brij's early years is given, this is followed by considerations about the models for water transport across epithelia. The ultrastructural and histochemical studies carried out by Brij Gupta and colleagues are reviewed to introduce the historical need for the use of EPXMA for the study of ion and water transport in epithelia. The outstanding contribution made by Brij Gupta's work in this field is outlined, and his understanding of the subcellular distribution ions in other cell types and in the pericellular environment is acknowledged.

The effect of nebulized salbutamol therapy on the incidence of postoperative chest infection in high risk patients.

Patients who smoke heavily and those with pre-existing airflow obstruction are at particular risk of postoperative respiratory infection following upper abdominal surgery. This invariably prolongs hospital stay and increases morbidity. In order to determine whether high dose bronchodilator therapy in the perioperative period reduced the risk of infection, all patients undergoing elective upper abdominal surgery were assessed for risk of developing postoperative infection. Fifty-three patients were identified as high risk according to previously published criteria and were randomly allocated to receive nebulized salbutamol (5 mg) or saline placebo 6 hourly for 48 h beginning 1 h preoperatively. There was no difference in rates of postoperative chest infection in the two groups and this study, therefore, provides no support for the routine preoperative use of bronchodilators in these patients.