Changes in the extents of viable and necrotic tissue, interstitial fluid pressure, and proliferation kinetics in clone A human colon tumour xenografts as a function of tumour size

Abstract. Total, viable and necrotic tumour tissue, tumour cell yields, and colony forming efficiencies were measured in clone A human colon tumour xenografts as neoplasms grew from about 100 mm³ to about 6000 mm³. The volumes of the total, viable and necrotic compartments were fit using the Verhulst equation to obtain estimates of growth rates and maximal sizes of the various compartments (carrying capacities). Additionally, at four discrete tumour volumes (250, 850, 2500 and 5500 mm³), hypoxic percentages, proportions of parenchymal tumour and host cells, interstitial fluid pressures, and proliferation kinetics including measurements of apoptosis were determined. There were interesting relationships between the shapes of the curves for total, viable and necrotic tissue to some of the other endpoints measured. Specifically, the volumetric growth curves for the total and viable tumour tissue compartments were identical to a volume of approximately 1000 mm³, but diverged at larger sizes, with the viable cell compartment exhibiting a smaller carrying capacity. The shape of the growth curve for the necrotic compartment exactly mimicked that for the total volume compartment, but was delayed in time by about 21 days. Similarity in shape to that of the overall tumour volume/necrotic volume curves was also seen for the curve for the increase in interstitial fluid pressure, and for the increase in the size of the host cell compartment. In contrast, the growth of the hypoxic compartment and of the parenchymal tumour cell compartment were similar in shape to that of the viable compartment. These data indicate that these compartments are functionally linked. Marked changes in cell kinetic parameters occurred as tumour size increased from 250–5500 mm³. The labelling index and growth fractions decreased from 0.256–0.125, and 0.77–0.40 respectively, and the cell loss factor increased from 0.52–0.74. The volumetric and potential doubling times increased from 4.3–17.6 and 2.1–4.6 days respectively. The cell kinetic changes could not be clearly related to the changes in shape of either the overall tumour volume or the viable tumour volume.     

Quantitative studies of superior cervical sympathetic ganglia in a variety of primates including man. II. Neuronal packing density

The volumes of a sample of primate superior cervical sympathetic ganglia were measured and related to body weight and to the number of ganglionic neurons. Estimates of volumes of the ganglia varied between 1.956 mm³ in squirrel monkey and 173.530 mm³ in a human specimen. Average cell densities for the ganglia ranged from 4,455 cells/mm³ in a human ganglion to 32,528 cells/mm³ in a squirrel monkey ganglion. Mean cell territories varied from 0.0000307 mm³ in a squirrel monkey ganglion to 0.0002245 mm³ in a human ganglion. Analysis of the data reveals striking trends of correlation between body size, volume of ganglia, and average cell territories. Since similar correlations have been described for other types of neuronal cell aggregates, it is suggested that for any given nucleus, ganglion or cortical area, the neuronal packing density varies as a function of body size.     

THE CELL PROLIFERATION KINETICS OF THE EMT6/M/AC MOUSE TUMOUR AT FOUR VOLUMES DURING UNPERTURBED GROWTH IN VIVO

The cell proliferation kinetics of the EMT6/M/AC mouse tumour were determined at four different volumes between 1–5 mm³ and 175 mm³. The decrease in the growth rate between these volumes was mainly due to a decrease in the rate constant for cell production. A small increase in the rate constant for cell loss occurred, but this was thought to be insignificant. The cell loss factor increased from 40% at 1–5 mm³ to over 70% in the 175 mm³ tumours. An increase in the median cell cycle time, from 14-1 hr to 18-5 hr was also found between these same volumes. Results obtained for the NCTC fibrosarcoma and the R-l rhabdomyosarcoma indicate that there may be a threshold volume in these sarcomas below which little or no cell loss takes place. This was not found in the EMT6/ M/AC tumour.     

A Distinct Class of Antibodies May Be an Indicator of Gray Matter Autoimmunity in Early and Established Relapsing Remitting Multiple Sclerosis Patients

We have previously identified a distinct class of antibodies expressed by B cells in the cerebrospinal fluid (CSF) of early and established relapsing remitting multiple sclerosis (RRMS) patients that is not observed in healthy donors. These antibodies contain a unique pattern of mutations in six codons along V_H4 antibody genes that we termed the antibody gene signature (AGS). In fact, patients who have such B cells in their CSF are identified as either having RRMS or developing RRMS in the future. As mutations in antibody genes increase antibody affinity for particular antigens, the goal for this study was to investigate whether AGS⁺ antibodies bind to brain tissue antigens. Single B cells were isolated from the CSF of 10 patients with early or established RRMS. We chose 32 of these B cells that expressed antibodies enriched for the AGS for further study. We generated monoclonal full-length recombinant human antibodies (rhAbs) and used both immunological assays and immunohistochemistry to investigate the capacity of these AGS⁺ rhAbs to bind brain tissue antigens. AGS⁺ rhAbs did not recognize myelin tracts in the corpus callosum. Instead, AGS⁺ rhAbs recognized neuronal nuclei and/or astrocytes, which are prevalent in the cortical gray matter. This pattern was unique to the AGS⁺ antibodies from early and established RRMS patients, as AGS⁺ antibodies from an early neuromyelitis optica patient did not display the same reactivity. Prevalence of CSF-derived B cells expressing AGS⁺ antibodies that bind to these cell types may be an indicator of gray matter-directed autoimmunity in early and established RRMS patients.     

Correlation of beta-camera imaging and immunohistochemistry in radioimmunotherapy using90Y-labeled monoclonal antibodies in ovarian cancer animal models

Tumor stroma contains much fibrin and monoclonal antifibrin antibody targeting is possible in tumors. In this study, nude mouse human ovarian carcinoma xenograft specimens were investigated after treatment with⁹⁰Y-labeled monoclonal antifibrin antibody Fab fragment or with⁹⁰Y-labeled OC125-monoclonal antibody F(ab′)₂ fragments. The mice received the radioimmunotherapy activity either intratumorally, intraperitoneally, or intravenously. Beta-camera imaging (BCI) is a novel device for studying activity distribution in tissue specimens and, together with immunohistochemistry (IHC) with OC125, antifibrin, anticarcinoembryonic antigen, anti-cytokeratin, and anti-placental alkaline phosphatase antibodies, was used for correlation of activity distribution of tissue specimens. These results were in concordance: Antigen distribution measured with IHC and radioactivity distribution were similar with the same antibodies, antifibrin, and OC125: However, these antigens demonstrated rather different distribution. Tissue studies revealed that activity was concentrated also in the necrotic tumor tissue, indicating that cell death was also caused by radiation. Differences in the tumor cell morphology were observed using different routes of administration. With BCI, it is possible to quantitate activities in frozen sections (microdosimetry), and these results were in concordance with absolute activities as measured by tissue sampling and well-counting. Three-dimensional reconstruction of tissue slices combined with radioactivity distribution measured with BCI allows estimation of total absorbed radiation dose in tumor after an appropriate dose planning.     

How Does B-Value Affect HARDI Reconstruction Using Clinical Diffusion MRI Data?

Background

A number of imaging factors can affect the orientation distribution function (ODF) reconstruction in high angular resolution diffusion imaging (HARDI). The aim of this study was to investigate the effect of the b-value on the HARDI reconstruction and to seek for the appropriate b-value for ODF reconstruction from clinical HARDI data.

Methods

Diffusion MRI data with various b-values were collected on a GE 3T MRI scanner. To reconstruct the diffusion ODF and fiber ODF, decomposition-based spherical polar Fourier imaging and deconvolution-based constrained spherical deconvolution approaches were applied separately. The full width at half maximum (FWHM) of the ODF and the angular difference of the peaks extracted from ODF were measured to investigate the effect of b-value on the ODF reconstruction. Visual inspection of the ODF was used to evaluate the reconstructions.

Results

The FWHM of the ODFs in the corpus callosum, which was chosen as the region of interest (ROI), decreased with increasing b-values. The differences in the FWHM for the diffusion ODF and the fiber ODF between the b-values of 2000 s/mm² and 2500 s/mm² were not significant. The angular differences of the ODF between 2000 s/mm² and 2500 s/mm² were lowest in both single-directional and two-directional situations. The ODFs became sharper and crossing-fiber situations were detected with an increase in b-value. B = 2000 s/mm² and above revealed most of the two-way or three-way crossing-fiber structures.

Conclusions

Considering both the signal-to-noise ratio and the acquisition time, b = 2000 s/mm² is the basic requirement for ODF reconstruction using current HARDI methods on clinical data. This study can provide a useful reference for researchers and clinicians attempting to set appropriate scan protocols for specific HARDI experiments.     

Fluorescent immunohistochemistry and in situ hybridization analysis of mouse pancreas using low-power antigen-retrieval technique.

To facilitate the immunological reaction of antibodies with antigens in fixed tissues, it is necessary to unmask or retrieve the antigens through pretreatment of the specimens. However, adjustment of heating-induced antigen retrieval is always required for different tissues and antigens. In this study, by using a low-power antigen-retrieval technique with appropriate dilution of antibodies, we successfully immunostained key antigens in pancreas such as insulin, PDX-1, glucagon, cytokeratin, and CD31, which have previously presented a particular challenge for investigators because of the rapid autodigestion and high nonspecific antibody binding in this tissue. Satisfactory results were obtained when immunohistochemistry and fluorescence in situ hybridization analysis were combined in the same slides.     

Multi-Shell Hybrid Diffusion Imaging (HYDI) at 7 Tesla in TgF344-AD Transgenic Alzheimer Rats

Diffusion weighted imaging (DWI) is widely used to study microstructural characteristics of the brain. Diffusion tensor imaging (DTI) and high-angular resolution imaging (HARDI) are frequently used in radiology and neuroscience research but can be limited in describing the signal behavior in composite nerve fiber structures. Here, we developed and assessed the benefit of a comprehensive diffusion encoding scheme, known as hybrid diffusion imaging (HYDI), composed of 300 DWI volumes acquired at 7-Tesla with diffusion weightings at b = 1000, 3000, 4000, 8000 and 12000 s/mm² and applied it in transgenic Alzheimer rats (line TgF344-AD) that model the full clinico-pathological spectrum of the human disease. We studied and visualized the effects of the multiple concentric “shells” when computing three distinct anisotropy maps–fractional anisotropy (FA), generalized fractional anisotropy (GFA) and normalized quantitative anisotropy (NQA). We tested the added value of the multi-shell q-space sampling scheme, when reconstructing neural pathways using mathematical frameworks from DTI and q-ball imaging (QBI). We show a range of properties of HYDI, including lower apparent anisotropy when using high b-value shells in DTI-based reconstructions, and increases in apparent anisotropy in QBI-based reconstructions. Regardless of the reconstruction scheme, HYDI improves FA-, GFA- and NQA-aided tractography. HYDI may be valuable in human connectome projects and clinical research, as well as magnetic resonance research in experimental animals.     

Accurate quantification of the influence of benthic macro- and meio-fauna on the geometric properties of estuarine muds by micro computer tomography

This paper describes the novel application of high resolution micro computer tomography (microCT) to the quantification of the properties of marine biogenic structures. CT scanning has been used to examine sediments in the past but the resolution of most previous techniques has been dependent upon commercial medical CT scanners which only have a slice thickness of ≥ 0.625 mm. In addition adequate software has not previously been available to rapidly quantify all the properties of biogenic structures. The microCT technique developed here used a standard core sample of estuarine sediment and new software was developed to calculate the axial variation of the following burrow parameters: number, diameter, volume, surface area and density. The increased resolution has resulted in the first quantification of meiofaunal burrow structures.A test core has shown, as an example, that the total volume of burrows created by macrofaunal organisms decreased from 827 mm³, within the top 15 mm of the core, to 204.2 mm³ at a depth 60 -75 mm within the core. Total burrow surface area decreased from 1883 mm² to 512 mm², for these depth ranges, respectively and burrow diameter ranged from 2.37-2.58 mm, remaining fairly constant between depths. Meiofaunal burrow structures decreased from 1.3-0.1 mm³ within the top 6 mm of the core with burrow surface area decreasing from 33.52-3.4 mm². Again, burrow diameter remained relatively constant, ranging from 0.23-0.25 mm.Quantification to this resolution is required to identify the impact of infaunal organisms on factors such as oxygen penetration, vertical and horizontal (across burrow walls) gradients in redox conditions and chemical/nutrient speciation and flux. The quantification of these burrow properties will improve the ability to examine the interrelationships between chemical, physical and biological processes and their role in ecological functioning. The present study indicates that there is potential for further development of this software to allow more detailed analysis of burrow structures and surface features including parameters such as burrow length, shape and sediment surface roughness.     

Immunohistochemical evaluation of ras oncogene expression in pulmonary and pleural neoplasms

We undertook an immunohistochemical analysis of human bronchopulmonary epithelial neoplasms and pleural mesotheliomas using a monoclonal antibody which recognizes ras oncogene products (p21ras). The monoclonal antibody, RAP-5, recognizes both unaltered and certain mutated p21ras. Formalin fixed and paraffin embedded tissue samples of 187 lung epithelial tumors and 27 pleural mesotheliomas were investigated; normal and bronchiectatic lungs were similarly studied. Normal lung and pleural tissue did not immunostain except for occasional type II pneumocytes. Reactive type II pneumocytes adjacent to carcinomas and bronchiectasis immunostained consistently. Twenty four/34 (71%) squamous carcinomas immunostained. Only 8/50 (16%) adenocarcinomas immunostained focally and weakly whereas 19/24 (79%) bronchioloalveolar carcinomas immunostained. Eleven/18 (61%) large cell carcinomas immunostained with variable intensity. Eleven/13 (85%) carcinoids, 6/7 (85%) well differentiated neuroendocrine carcinomas, and 18/21 (86%) intermediate cell neuroendocrine carcinomas immunostained while none of 20 small cell neuroendocrine carcinomas immunostained. Only a few mesotheliomas were immunostained focally. Two/14 (14%) epithelial type and 1/9 (11%) biphasic type mesotheliomas immunostained weakly; none of 4 spindle cell mesotheliomas immunostained. We conclude that while at least occasional cases of most types of pulmonary epithelial neoplasms express p21ras, the frequency and intensity of the expression are distinctly greater in certain tumor types such as squamous, bronchioloalveolar, and neuroendocrine neoplasm except for the small cell type. Contrary to these lung epithelial neoplasms, most mesotheliomas did not immunostain for p21ras. Whether the enhanced p21ras expression may point to a different mechanism of transformation or may merely reflect differentiation features remains undetermined.     

Human dendritic reticulum cells of lymphoid follicles: their antigenic profile and their identification as multinucleated giant cells

The B-dependent areas of human lymphoid tissue contain non-lymphoid, non-phagocytic cells known as dendritic reticulum cells (DRC). These cells can be detected only very occasionally in routinely stained histologic sections. Recently we were able to overcome this limitation by preparing a monoclonal antibody, termed R 4/23, that reacts selectively with DRC. Thus by using an optimized immunoperoxidase method applied to frozen sections, it is possible to detect DRC in situ. To determine the antigenic profile of DRC, serial frozen sections of human tonsils were immunostained with R 4/23 and a large panel of other monoclonal antibodies or conventional antisera. In addition, touch imprints of tonsils and cytocentrifuge slides of cell suspensions with increased concentrations of DRC were immunostained with these reagents. DRC proved to be positive for mu, gamma, alpha, kappa and lambda chains, complement component C3b, C3b receptors, C3d receptors, HLA-A,B,C antigens, human Ia-like antigens, common ALL antigen (cALLa), and antigens that are characteristic of the monocyte/macrophage lineages. DRC did not express delta chains, T cell antigens, or antigens that are expressed on interdigitating reticulum cells (IDC) and Langerhans cells. DRC in touch imprints and suspensions prepared from hyperplastic tonsils were found to be giant cells often with 10 or more nuclei. In certain cases of follicular hyperplasia and of centroblastic-centrocytic lymphoma, DRC with several nuclei were also detectable in situ. These results show that (1) the phenotype of DRC differs from that of all other cell types in lymphoid tissue, (2) this phenotype most nearly resembles that of cells of the monocyte/macrophage series, thus suggesting that DRC are related to these cell lineages, and (3) DRC are multinucleated giant cells.     

PERTURBED CELL PROLIFERATION KINETICS OF THE EMT6/M/AC TUMOUR IN MICE TREATED WITH ANTI-MOUSE LYMPHOCYTE SERUM

The cell proliferation kinetics of the EMT6/M/AC tumour were determined at two volumes, 6-3 mm³ and 180 mm³, in mice treated with anti-mouse lymphocyte serum, AMLS. Comparison of the growth curve with that obtained in non-AMLS treated animals showed a marked increase in the growth rate at all volumes in the treated group. In contrast, the cell cycle time and the intermitotic phase times were not significantly different in the treated and untreated groups at comparable volumes. The increase in the growth rate in AMLS treated mice was obtained in spite of decreases in both the rate constant for cell production and the growth fraction, and was due to a marked decrease in the rate constant for cell loss.     

Ki-67 (MIB 5) immunostaining of mouse lung tumors induced by 4-nitroquinoline 1-oxide

 We immunostained mouse lung tumors using a mouse monoclonal antibody against recombinant Ki-67 antigen (clone; MIB 5) to establish an MIB 5 immunostaining method and to determine the extent of MIB 5 labeling to monitor cell proliferation activity in mouse lung tumors. A/J mice, treated with 4-nitroquinoline 1-oxide, were killed after 18 months. One hour before killing, bromodeoxyuridine (BrdU) was injected intraperitoneally. Lung tissues including tumors were fixed with phosphate-buffered 4% paraformaldehyde and embedded in paraffin. For MIB 5 immunostaining, two antigen-retrieval buffers, citrate buffer pH 6 and TRIS-HCl buffer pH 9.5 containing 5% urea, were tested, and constant and reproducible staining was obtained only with the TRIS-HCl buffer. The mean values of the MIB 5-positive cell index (PCI), the BrdU labeling index (LI), and the mitotic cell count for adenocarcinomas were 4.6%, 2.3%, and 7/mm², and those for adenomas were 1.2%, 0.7%, and 1.3/mm², respectively. Each of these values was significantly higher for adenocarcinomas than for adenomas. A close correlation was seen between the MIB 5 PCI and the BrdU LI for adenocarcinomas and adenomas and between the MIB 5 PCI and the mitotic cell count in adenocarcinomas. Thus, MIB 5 immunostaining is a useful method for assessing the proliferative activity of mouse tumor tissues. Accepted: 23 June 1998     

Amplification methods for the immunolocalization of rare molecules in cells and tissues

The needs to precisely assign macromolecules to specific locations and domains within tissues and cells and to reveal antigens which are present in low or even in trace amounts, led to the elaboration of a wide spectrum of immunocytochemical amplification procedures. These arise from the successive improvements of tissue preparation techniques, of antigen retrieval procedures and of immunological or non-immunological detection systems. Improvement of detection systems may be the most active in the development of amplification techniques. Since the early work of Coons, in which by the introduction of the indirect technique has started amplifying the signal, different systems have succeeded in increasing the sensitivity of antigens detection. Indeed, amplification techniques such as the multiple antibody layers, the multiple bridges, the enzyme complexes, the avidin-biotin, the silver intensification, and the numerous variations and combinations among these have increased the sensitivity for the detection of scarce tissue antigens. However, as shown by the recent progress carried out with new approaches such as the catalyzed reporter deposition (CARD) and the enhanced polymer one-step staining (EPOS), more efficient methods are still needed. In electron microscopy, few techniques have reached the resolution afforded by the post-embedding immunogold approach. In spite of this and in order to further increase its sensitivity, new probes and novel approaches are allowing combination of the gold marker with the amplification capacity of enzymes afforded by the CARD technique. Immunogold amplification strategies, such as the multiple incubations with the primary antibody and the use of an anti-protein A antibody have also led to enhanced signals displaying the advantages in terms of resolution and possibilities of quantification inherent to the colloidal gold marker.     

Faisabilité et intérêt d’un nouvel algorithme de reconstruction des images TEP-18FDG (AW OSEM DR) pour la délimitation des volumes cibles en radiothérapie. À propos d’un cas de néoplasie ORL

ObjectiveWe compared two reconstruction methods for 18fluorodeoxyglucose (¹⁸F-FDG) positron emission tomography (PET) images with “attenuation weighted ordered subset expectation maximization” using either the manufacturer-provided (AW-OSEM) or a “Detector response” (AW-OSEM DR) tomographic operator. We looked at the feasibility of using the latter reconstruction for radiotherapy target volumes definition in cancers of the superior aero-digestive tract (VADS). In this preliminary study, we first assessed the spatial resolution of images obtained with AW-OSEM and AW-OSEM DR on a Biograph™ 6, and secondly target volumes of radiotherapy “Gross Tumor Volume” (GTV), “Clinical Target Volume” (CTV) and “Planning Target Volume” (PTV) obtained with each of these reconstruction methods.Material and methodsThe spatial resolution was measured on a test object containing 4 radioactive point sources. Furthermore, radiotherapy target volumes have been defined with the software Eclipse™ on injected scanner (CT IV) and PET/CT (PET AW-OSEM and PET AW-OSEM DR) images.ResultsSpatial resolution was improved with AW-OSEM DR algorithm reconstruction compared to images obtained with AW-OSEM reconstruction (from 7.5 mm down to 5.4 mm for the highest reduction). GTV from AW-OSEM DR reconstruction with 42 and 50% of the “Standard uptake value maximum” (SUVmax) semi-automatic threshold (1.2 and 0.7 cm³ respectively) were lower than those obtained with AW-OSEM (3.6 and 2.2 cm³ respectively). They were also lower than GTV defined with CT IV (5.5 cm³). It was the same for CTV and PTV.ConclusionThis study showed that AW-OSEM DR reconstruction method allows less impaired spatial resolution than AW-OSEM. In the case of radiotherapy target volumes delineation, AW-OSEM DR may decrease the GTV, CTV and PTV and therefore the risk of side effects associated with organs at risk.     

Cellular proliferation in the anterior pituitary of the rat during the postnatal period

Summary Cellular proliferation in the anterior pituitary of 2-, 8-, 15- and 30-day-old rats was examined by injection of bromodeoxyuridine 1 h before autopsy. Bromodeoxyuridine incorporated into DNA was detected immunohistochemically by use of a monoclonal antibody. The highest rate of cell proliferation was found in 2-day-old animals; it decreased thereafter during the postnatal period. Possible toxic effects of colchicine on cellular proliferation were examined. Colchicine treatment (10 mg/kg in 8- and 30-day-old animals) significantly decreased the number of bromodeoxyuridine-labelled cells/mm² in 8-day-old rats. Some sections were doubly immunostained for bromodeoxyuridine and various pituitary hormones. The proportion of doubly-immunostained cells to all proliferating cells was generally low, ranging from 23% at 2 days to 32% at 30 days of age.On leave from the Department of Human Anatomy and Histology, Faculty of Medicine, University of Salamanca, Salamanca, Spain     

Absence of pituitary prolactin epitopes in immunoreactive prolactin of rat brain.

Immunoreactive prolactin (ir-PRL) in rat brain has been consistently documented. However, the identity of this ir-PRL is controversial. Ir-PRL is defined by its ability to bind to PRL antibodies. All previous studies of brain ir-PRL have used polyclonal antibodies, at least one of which apparently crossreacts with a portion of the proopiomelanocortin molecule. To begin to define the epitopes comprising ir-PRL in the brain, we utilized two monoclonal antibodies (MAb) that recognize pituitary PRL in a variety of species, including rat. Immunocytochemistry was performed on rat brains and pituitary glands using two monoclonal and one polyclonal PRL antibody. Although both MAb immunostained lactotrophs of the rat pituitary gland, neither antibody immunostained cell bodies or neuronal processes in the brain. However, the polyclonal antiserum immunostained lactotrophs and a system of neuronal cell bodies and processes in the brain. Thus, epitopes found in pituitary PRL from several species are not found in ir-PRL in rat brain.     

The volume and area of the capillaries in the endocrine and exocrine pancreas of the rat

Summary The capillary volumes in the endocrine and exocrine parenchyma of the pancreas were compared with a point-sampling technique. The islets were found to have a capillary volume of approximately 3.5%, while the value for the exocrine pancreas was significantly (P<0.001) lower at 2%. When the capillary wall area was measured, however, both types of parenchyma had a similar value of approximately 20 mm²/mm³ tissue. The reason for the discrepancy between these parameters is probably the lack of lymphatic capillarics, with their relatively small lumen in the islets.     

Three-dimensional localization of ultrasmall immuno-gold labels by HAADF-STEM tomography

The localization of scarce antigens in thin sections of biological material can be accomplished by pre-embedment labeling with ultrasmall immuno-gold labels. Moreover, with this method, labeling is not restricted to the section surface but occurs throughout the section volume. Thus, when combined with electron tomography, antigens can be localized in three dimensions in relation to the 3D (three-dimensional) ultrastructure of the cell. However, for visualization in a transmission electron microscope, these labels need to be enlarged by silver or gold enhancement. The increase in particle size reduces the resolution of the antigen detection and the large particles obscure ultrastructural details in the tomogram. In this paper we show for the first time that these problems can be avoided and that ultrasmall gold labels can be localized in three dimensions without the need for gold or silver enhancement by using HAADF-STEM (high angular annular dark-field-scanning transmission electron microscopy) tomography. This method allowed us to three-dimensionally localize Aurion ultrasmall goat anti-rabbit immuno-gold labels on sections of Epon-embedded, osmium-uranium-lead-stained biological material. Calculations show that a 3D reconstruction obtained from HAADF-STEM projection images can be spatially aligned to one obtained from transmission electron microscopy (TEM) projections with subpixel accuracy. We conclude that it is possible to combine the high-fidelity structural information of TEM tomograms with the ultrasmall label localization ability of HAADF-STEM tomograms.     

Mid-term function and remodeling potential of tissue engineered tricuspid valve: Histology and biomechanics

ObjectiveTricuspid valve reconstruction using a small intestinal submucosal porcine extracellular matrix (ECM) tube graft is hypothesized to be durable for six months and show signs of recellularization and growth potential. The purpose was to histologically and biomechanically test ECM valves before and after six months of implantation in pigs for comparison with native valves.MethodsTen 60 kg pigs were included, which survived tricuspid valve tube graft insertion. Anterior and septal tricuspid leaflets were explanted from all animals surviving more than one month and examined histologically (n = 9). Endothelialization, collagen content, mineralization, neovascularization, burst strength and tensile strength were determined for native valves (n = 5), ECM before implantation (n = 5), and ECM after six months (n = 5).ResultsCollagen density was significantly larger in ECM at implantation (baseline) compared to native leaflet tissue (0.3 ± 0.02 mg/mm³ vs. 0.1 ± 0.03 mg/mm³, p < .0001), but collagen density decreased and reached native leaflet collagen content, six months after ECM implantation (native vs. ECM valve at six months: 0.1 ± 0.03 mg/mm³ vs. 0.2 ± 0.05 mg/mm³, p = .8).Histologically, ECM valves showed endothelialization, host cell infiltration and structural collagen organization together with elastin generation after six months, indicating tissue remodeling and -engineering together with gradual development of a close-to-native leaflet structure without foreign body response.ConclusionsECM tricuspid tube grafts were stronger than native leaflet tissue. Histologically, the acellular ECM tube grafts showed evidence of constructive tissue remodeling with endothelialization and connective tissue organization. These findings support the concept of tissue engineering and recellularization, which are prerequisites for growth.