Scanning laser-induced endothelial injury: a standardized and reproducible thrombosis model for intravital microscopy.

Vascular injury models are indispensable for studying thrombotic processes in vivo. Amongst the available methods for inducing thrombosis, laser-induced endothelial injury (LIEI) has several unique advantages. However, a lack of methodological standardization and expensive instrumentation remain significant problems decreasing reproducibility and impeding the adoption of LIEI in the wider scientific community. In this, study, we developed a standardized protocol for scanning laser-induced endothelial injury (scanning-LIEI) of murine mesenteric veins using the intrinsic 405 nm laser of a conventional laser scanning confocal microscope. We show that our model produces thrombi with prominent core-shell architectures and minimal radiation-related fluorescence artefacts. In comparison with previous methods, the scanning-LIEI model exhibits reduced experimental variability, enabling the demonstration of dose-response effects for anti-thrombotic drugs using small animal cohorts. Scanning-LIEI using the intrinsic 405 nm laser of a confocal laser scanning microscope represents a new method to induce standardized vascular injury with improved reproducibility of thrombus formation. The reduced need for instrument customisation and user experience means that this model could be more readily adopted in the research community.

A simple method allowing DIC imaging in conjunction with confocal microscopy.

Current optical methods to collect Nomarski differential interference contrast (DIC) or phase images with a transmitted light detector (TLD) in conjunction with confocal laser scanning microscopy (CLSM) can be technically challenging and inefficient. We describe for the first time a simple method that combines the use of the commercial product QPm (Iatia, Melbourne Australia) with brightfield images collected with the TLD of a CLSM, generating DIC, phase, Zernike phase, dark-field or Hoffman modulation contrast images. The brightfield images may be collected at the same time as the confocal images. This method also allows the calculation of contrast-enhanced images from archival data. The technique described here allows for the creation of contrast-enhanced images such as DIC or phase, without compromising the intensity or quality of confocal images collected simultaneously. Provided the confocal microscope is equipped with a motorized z-drive and a TLD, no hardware or optical modifications are required. The contrast-enhanced images are calculated with software using the quantitative phase-amplitude microscopy technique (Barone-Nugent et al., 2002). This technique, being far simpler during image collection, allows the microscopist to concentrate on their confocal imaging and experimental procedures. Unlike conventional DIC, this technique may be used to calculate DIC images when cells are imaged through plastic, and without the use of expensive strain-free objective lenses.

Specific localization, gamma camera imaging, and intracellular trafficking of radiolabelled chimeric anti-G(D3) ganglioside monoclonal antibody KM871 in SK-MEL-28 melanoma xenografts.

The chimeric monoclonal antibody KM871, directed against the G(D3) antigen, is under evaluation for its potential to target melanoma. To facilitate the in vivo evaluation of biodistribution properties and measurement of pharmacokinetics, KM871 was radiolabeled with (125)I via tyrosine residues and with (111)In via the bifunctional metal ion chelator C-functionalized trans-cyclohexyl diethylenetriaminepentaacetic acid (CHX-A"-DTPA) to lysine residues. Using antigen-positive SK-MEL-28 melanoma cells, immunoreactivities of 42 and 40% cell binding were obtained, respectively, for the two radioconjugates. Binding was enhanced in the presence of added unlabeled antibody. A humanized A33 antibody was similarly labeled with the two isotopes and used as a control. To determine and compare in vivo biodistribution characteristics of KM871 radiolabeled with (111)In or (125)I, mixtures of the radioconjugates were injected i.v. into BALB/c nude mice bearing G(D3)-positive-SK-MEL-28 melanoma xenografts. Gamma camera images were acquired; groups of five mice were sacrificed at various time intervals, and tumors, blood, and tissues were analyzed. (111)In-labeled CHX-A"-DTPA-KM871 showed a maximum tumor uptake of 41.9 +/- 7.0% injected dose/g at 72 h with prolonged retention over a 15-day period. The tumor:blood ratio was 3:1 by 72 h, and higher ratios were observed at later time points. No abnormal accumulation of (111)In-labeled conjugate was found in normal tissues. In contrast, there was little accumulation of (125)I-labeled KM871 in the same tumors. The specificity of antibody localization was confirmed by the low tumor uptake values for radiolabeled control antibody. Gamma camera imaging demonstrated excellent uptake of (111)In-labeled CHX-A"-DTPA-KM871 in the xenografts. Chromatographic analyses of xenograft cytosolic extracts demonstrated tumor internalization and catabolism of radiolabeled KM871 with the formation of small molecular weight metabolites. Laser scanning confocal microscopy demonstrated that the majority of intracellular KM871 is localized to lysosomes. Despite the catabolism of the radioconjugate, a dose-dependent increase in KM871 tumor localization was shown through immunohistochemical examination of xenograft biopsies. This study demonstrates for the first time the in vivo localization of a radiolabeled anti-G(D3) monoclonal antibody to G(D3)-expressing xenografts using gamma camera scanning techniques and tumor cell internalization of KM871 tagged with a green fluorescent dye, Alexa Fluor 488, through confocal microscopy. KM871 has potential for targeting tumors in patients with melanoma.

Risk of infection from needle reuse at a phlebotomy center.

OBJECTIVES: This study determined infection risk for HIV, hepatitis B virus (HBV), and hepatitis C virus (HCV) from needle reuse at a phlebotomy center that possibly exposed 3810 patients to infection. METHODS: We used a model for the risk of infection per blood draw, supplemented by subsequent testing results from 1699 patients. RESULTS: The highest risk of transmission was for HBV infection: 1.1 x 10(-6) in the best case and 1.2 x 10(-3) in the (unlikely) worst case. Subsequent testing yielded prevalence rates of 0.12%, 0.41%, and 0.88% for HIV, HBV, and HCV, respectively, lower than National Health and Nutrition Examination Survey III prevalence estimates. CONCLUSIONS: The infection risk was very low; few, if any, transmissions are likely to have occurred.

Azithromycin prophylaxis during a hospitalwide outbreak of a pertussis-like illness.

A questionnaire regarding tolerability and adherence was administered for 5 days to hospital employees who received azithromycin prophylaxis during a hospitalwide outbreak of a pertussis-like illness. Analysis of the 239 responses from those having received prophylactic azithromycin determined that it was well tolerated and accounted for a minimal loss of days worked; 81.5% were fully adherent with the regimen.

Characterization of mouse A33 antigen, a definitive marker for basolateral surfaces of intestinal epithelial cells.

The murine A33 antigen is emerging as a definitive marker of intestinal epithelial cells. Cloning and sequence determination of cDNAs encoding mA33 antigen predict a novel type 1 transmembrane protein of 298 amino acids, comprising an extracellular domain with two immunoglobulin-like domains, a single-span transmembrane domain, and a highly acidic cytoplasmic domain. On the basis of conservation of amino acid sequence and genomic structure, the mA33 antigen is a member of a growing subfamily within the immunoglobulin superfamily, which includes transmembrane proteins CTX/ChT1, CTM/CTH, and CAR. During embryonic development, mA33 antigen expression is first observed in the inner cell mass of blastocysts before implantation. Intestinal expression of mA33 antigen is initiated in the hindgut at E14.5 and increases steadily throughout late embryonic and postnatal life into adulthood. The protein is specifically expressed on the basolateral surfaces of intestinal epithelial cells of all lineages, independent of their position along the rostrocaudal and crypt-villus axes. Thus the mA33 antigen appears to be a novel marker for both proliferating and differentiating intestinal epithelial cells.

Two outbreaks of multidrug-resistant Salmonella serotype typhimurium DT104 infections linked to raw-milk cheese in Northern California.

CONTEXT: Salmonella serotype Typhimurium definitive type 104 (DT104), with resistance to 5 drugs (ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline), has emerged as the most common multidrug-resistant Salmonella strain in the United States. However, illnesses resulting from this strain have not been associated definitively with a source in this country. OBJECTIVE: To determine the source of 2 outbreaks of Salmonella Typhimurium DT104. DESIGN: Matched case-control study conducted between March 24 and April 5, 1997 (outbreak 1), enhanced surveillance for new cases dating from February 1, 1997 (outbreak 2), and environmental and laboratory investigations. SETTING AND PARTICIPANTS: The case-control study included residents of 2 adjacent counties in northern California infected with the outbreak strain of Salmonella Typhimurium var Copenhagen and age-matched controls. For enhanced surveillance, a case was defined as Salmonella Typhimurium infection in a person exposed to fresh Mexican-style cheese. MAIN OUTCOME MEASURES: Risk factors for infection and source of implicated food. RESULTS: Outbreak 1 peaked in February 1997; 31 patients were confirmed by culture as having Salmonella Typhimurium var Copenhagen infection, isolates of which showed indistinguishable pulsed-field gel electrophoresis (PFGE) patterns. The outbreak strain was phage type DT104 with the 5-drug resistance pattern. Sixteen cases and 25 controls were enrolled in the case-control study; 15 of 16 Salmonella Typhimurium var Copenhagen cases compared with 14 of 24 matched controls reported eating unpasteurized Mexican-style cheese, (matched odds ratio, 7.9; 95% confidence interval, 1.1-354.9). Enhanced surveillance uncovered outbreak 2, which peaked in April 1997 and was caused by a non-Copenhagen variant of Salmonella Typhimurium. During outbreak 2, Salmonella Typhimurium was isolated from 79 persons who ate fresh Mexican-style cheese from street vendors and from cheese samples and raw milk. The PFGE pattern of the milk isolate matched 1 of the 3 patterns recovered from patients; all strains were phage type DT104b with the 5-drug resistance pattern. CONCLUSION: Raw-milk products pose a risk for multidrug-resistant Salmonella Typhimurium DT104 infections.

An outbreak of Escherichia coli O157:H7 infection from unpasteurized commercial apple juice.

BACKGROUND: Escherichia coli O157:H7 infections have traditionally been associated with animal products, but outbreaks associated with produce have been reported with increasing frequency. In fall 1996, a small cluster of E. coli O157:H7 infections was epidemiologically linked to a particular brand (brand A) of unpasteurized apple juice. OBJECTIVE: To define the extent of the outbreak, confirm the source, and determine how the apple juice became contaminated. DESIGN: Descriptive epidemiologic study and traceback investigation. SETTING: Western United States and British Columbia, Canada. PATIENTS: Patients with E. coli O157:H7 infection who were exposed to brand A apple juice. MEASUREMENTS: Clinical outcome and juice exposure histories of case-patients, pulsed-field gel electrophoresis of case and juice isolates, and juice production practices. RESULTS: Seventy persons with E. coli O157:H7 infection and exposure to brand A unpasteurized apple juice were identified. Of these persons, 25 (36%) were hospitalized, 14 (20%) developed the hemolytic uremic syndrome, and 1 (1%) died. Recalled apple juice that was produced on 7 October 1996 grew E. coli O157:H7 with a pulsed-field gel electrophoresis pattern indistinguishable from that of case isolates. Apple juice produced on 7 October 1996 accounted for almost all of the cases, and the source of contamination was suspected to be incoming apples. Three lots of apples could explain contamination of the juice: Two lots originated from an orchard frequented by deer that were subsequently shown to carry E. coli O157:H7, and one lot contained decayed apples that had been waxed. CONCLUSIONS: Standard procedures at a state-of-the-art plant that produced unpasteurized juices were inadequate to eliminate contamination with E. coli O157:H7. This outbreak demonstrated that unpasteurized juices must be considered a potentially hazardous food and led to widespread changes in the fresh juice industry.

Immunocytochemical detection of endothelin receptors in rat cultured airway nerves.

Endothelin-1 (ET-1) has been shown to potentiate cholinergic neurotransmission in human bronchus as well as in airways from a variety of animal species, suggesting that ET receptors exist prejunctionally on airway cholinergic nerves. We have successfully isolated and maintained rat tracheal para-sympathetic neurons in culture. Most cultured cells were associated with specific fluorescence for the nerve cell marker protein gene product 9.5 (PGP 9.5). These cultures contained a high proportion of parasympathetic neurons. Importantly, specific immunofluorescent antibodies for ETB receptors were colocalized with those for PGP 9.5. Therefore, for the first time, ETB receptors have been shown to exist on airway parasympathetic neurons in culture.

Recording of mitochondrial transmembrane potential and volume in cultured rat osteoclasts by confocal laser scanning microscopy.

Osteoclasts are multinuclear bone-resorbing cells which contain abundant mitochondria. Morphological studies have suggested that a correlation may exist between mitochondrial concentration and bone resorption by osteoclasts. However, investigation of mitochondrial transmembrane potential (delta psi) and volume has been hampered by the difficulty in obtaining a sufficient number of osteoclasts for assessing these characteristics by flow cytometric analysis. In this study, we have used confocal laser scanning microscopy after loading the cells with Rhodamine 123 and 10-nonyl Acridine Orange to record mitochondrial delta psi and volume, respectively, in isolated rat osteoclasts cultured on bovine bone slices. Optimal staining conditions were found to be 10 micrograms ml-1 for 40 min for Rhodamine, and 1 microM for 10 min for the 10-nonyl Acridine Orange derivative. Two osteoclast populations, whose shape seemed to reflect bone resorption and migratory functions, were identified depending on their shape and on the distribution of the two dye probes. 'Round-shaped' osteoclasts had significantly higher mitochondrial delta psi and volume in the apical regions than in the basolateral portions (p < 0.00001). In contrast, mitochondrial delta psi and volume in 'irregular-shaped' osteoclasts were rather evenly distributed in both these regions (p > 0.05). Our results indicate that there is an apical polarization of mitochondria in osteoclasts corresponding to the energy demands associated with bone resorption.

A rapid method for the assessment of bone architecture by confocal microscopy.

Conventional ways of demonstrating and analysing the components of osseous tissue have always been hampered by the difficulty of physically sectioning bone. In this study, we have used Acridine Orange staining of 100-micron-thick unembedded bone slices and then assessed the cellular and tissue architecture by confocal microscopy. The result showed the Acridine Orange, by differential staining of the cellular nucleic acids, permits ready assessment of cell shape and cell organization as well as variations in growth patterns. Our studies have provided a new and relatively easy way of assessing the morphology of bone specimens by rendering unnecessary the need for embedding, decalcification and thin sectioning of the osseous tissue.

In situ visualization of spontaneous calcium waves within perfused whole rat heart by confocal imaging.

We describe the first direct visualization of Ca2+ oscillations in the perfused whole rat heart. Dye loading at a low temperature and enhanced optical-section techniques of confocal microscopy by elimination of the refractive index mismatch with use of saline-immersible objective lens enabled us to image multiple Ca2+ waves in the subepicardial myocardium of the fluo 3-loaded heart. These Ca2+ waves were sporadically seen even with a physiological extracellular Ca2+ perfusion in either a paced or an arrested heart and propagated beyond cellular boundaries within the three-dimensional structures of cardiac muscle. Under these conditions, the velocity of wave propagation was 60-100 microns/s and the frequency of initiation was relatively low (< 2 Hz). With an increase in extracellular Ca2+ concentration, however, the waves became more prevalent and tended to be multifocal, and an increasing fraction of the waves exhibited faster propagation velocities and higher frequencies. These results suggest that perfused rat hearts exhibit spontaneous Ca2+ waves in an apparently resting state and that under Ca(2+)-overload conditions the multifocal and high-frequency waves become more widespread in the heart syncytium, which may provide an understanding of the ionic basis for the summation of afterdepolarizations and triggering of arrhythmias seen under pathological conditions.

Spontaneous and propagated calcium release in isolated cardiac myocytes viewed by confocal microscopy.

Laser scanning confocal microscopy of the Ca(2+)-sensitive fluorophore fluo-3 has been used to investigate spontaneous and propagated calcium release at high temporal and spatial resolution in enzymatically dispersed rat cardiomyocytes. Waves of fluorescence which propagated throughout the cytosol were evident in spontaneously contracting cardiac cells containing fluo-3, but not in cells containing Ca(2+)-insensitive fluorophores [2',7'-bis (carboxyethyl)-5,6-carboxyfluorescein, SNARF-1, rhodamine-123, or tetramethylrhodamine-labeled dextran]. These waves represent localized areas of elevated [Ca2+] [975 +/- 13 (SE) nM, range 800-1,500 nM; n = 16 cells]. Ca2+ waves were initiated by the spontaneous release of Ca2+ from the sarcoplasmic reticulum (SR) and propagated through cells at rates of 50-150 microns/s. Ca2+ waves were usually initiated at the cell ends, but multiple and variable initiation foci were observed in some cells. Where waves intersected within a single cell there was extinction of wave propagation, confirming the SR as the direct source of Ca2+ and revealing a refractory period in SR Ca2+ release. In some cells high-frequency Ca2+ waves lead to synchronized elevation of [Ca2+] throughout the entire cytosol and within the time period associated with cell depolarization. These observations support the hypothesis that some cardiac arrhythmias are initiated by spontaneous and propagated Ca2+ release and involve subsequent depolarization, global elevation of intracellular [Ca2+], and cell contraction.

Phototropism and geotropism in maize coleoptiles are spatially correlated with increases in cytosolic free calcium.

Phototropism and gravitropism in the shoots and roots of higher plants are the result of asymmetric growth. This is explained by the redistribution of growth regulators following exposure to gravity or unilateral light (the Cholodny-Went hypothesis). The positive phototropism and the negative geotropism of grass seedling coleoptiles are believed to result from lateral movement of auxin from the irradiated to the shaded side and from the upper to the lower side, respectively. Many physiological processes in plants, including auxin-induced cell elongation, are reported to be under the control of calcium. Added auxin triggers oscillations in cytosolic free calcium ([Ca2+]cyt) and cytosolic pH (pHcyt) in epidermal cells of maize coleoptiles. Until recently, it has not been possible to visualize these changes spatially with the commonly used fluorescent cation indicators. Using a scanning laser confocal microscope, a new visible wavelength Ca2+ probe fluo-3 and the fluorescent pH indicator BCECF, we have recorded rapid light-induced increases in [Ca2+]cyt and a lowering of pHcyt of cells on the shaded side of maize coleoptiles. In horizontally orientated coleoptiles, [Ca2+]cyt increases and pHcyt decreases in the more rapidly elongating cells on the lower side. For the first time, rapid changes in [Ca2+]cyt and pHcyt are correlated directly with increases in cell elongation stimulated by light and gravity.

Confocal imaging of ionised calcium in living plant cells.

Laser-scanning confocal microscopy has been used in conjunction with Fluo-3, a highly fluorescent visible wavelength probe for Ca2+, to visualize Ca2(+)-dynamics in the function of living plant cells. This combination has overcome many of the problems that have limited the use of fluorescence imaging techniques in the study of the role of cations (Ca2+ and H+) in plant cell physiology and enables these processes to be studied in single cells within intact plant tissue preparations. Maize coleoptiles respond to application of ionophores and plant growth hormones with elevations in cytosolic Ca2+ that can be resolved with a high degree of spatial resolution and can be interpreted quantitatively.