Transmission electron microscopic demonstration of vimentin in rat osteoblast and osteocyte cell bodies and processes using the immunogold technique

Background: The immunogold labeling technique and transmission electron microscopy were used to demonstrate the expression and position of the intermediate filament vimentin in rat osteoblast and osteocyte cell bodies and cell processes. Conventional light and transmission electron microscopic studies of bone cells demonstrated adjacent cell linkage to be mediated by osteoblast and osteocyte processes present within the canalicular system traversing the bone matrix. The cell processes were filled with densely packed filaments, many of which have been shown previously to be actin microfilaments. The appearance, however, of 10 nm diameter filaments in some cell processes and the fact that the intermediate filament vimentin has been defined in many cells of mesenchymal origin raised the possibility that some of these filaments might be vimentin. The ultrastructural colloidal gold immunochemical technique allowed for demonstration in situ of the expression of vimentin filaments plus accurate definition of their position. Methods: The studies were performed in newborn rat femoral and tibial diaphyseal cortical bone and in 1-week-old repair bone from 2.4 mm diameter defects made through the lateral cortex in 6-week-old rat femurs and tibias. The bone tissues for the immunochemical study were fixed in 1% glutaraldehyde, 4% paraformaldehyde, and 0.1 M phosphate buffer (pH 7.4) for 2 days. Decalcification was performed in 6% EDTA for 2–3 days. Infiltration involved use of Lowicryl resin K4M, and the embedding and curing processes were performed in a cryostat with temperatures ?30°C. An antivimentin monoclonal antibody was used for labeling using the postembedding technique. Effective antibody dilutions ranged from 1:10 to 1:200, with the dilutions of 1:25 and 1:100 showing the best combination of filament labeling with the least matrix background. The grids were exposed to 10 nanometer gold colloid conjugated goat anti-mouse IgM for demonstration of binding. Results: Vimentin immunolabeling was defined clearly in relation to filaments within the osteoblast and osteocyte cell body cytoplasm, throughout the entire length of the osteoblast and osteocyte cell processes, and in close relationship to the intercellular gap junctions which were present within the cell processes both close to the cell bodies and within the canaliculi well away from them. Conclusions: Immunogold labeling demonstrates the presence of the intermediate filament vimentin in osteoblast and osteocyte cell bodies and processes of rat bone. Vimentin distribution is not concentrated to specific areas, is present throughout the extent of the bodies and processes, and is seen immediately adjacent to gap junctions. © 1995 Wiley-Liss, Inc.     

What role does decreased ovarian reserve play in the aetiology of infertility related to endometriosis? Reply

Dear Sir, Thank you for the opportunity to respond to Dr Check’sletter. One of the expectations of writing these Debate articlesis that some response will be elicited. In     

Opsonophagocytosis of fluorescent polystyrene beads coupled to Neisseria meningitidis serogroup A, C, Y, or W135 polysaccharide correlates with serum bactericidal activity

We developed a polysaccharide-specific flow cytometric opsonophagocytic assay (OPA) for the simultaneous measurement of functional antibody to Neisseria meningitidis serogroups A, C, Y, and W135. OPA titers significantly correlated with serum bactericidal assay titers for all serogroups tested (mean r = 0.96; P < 0.001). OPA could be used in meningococcal vaccine evaluation.     

Cardiovascular patterns associated with threat and challenge appraisals: a within-subjects analysis

Previous studies demonstrated distinct cardiovascular patterns associated with threat and challenge appraisals for groups of participants. We extend these results by assessing whether appraisals continue to be associated with these cardiovascular response patterns within an individual as appraisals change. Participants completed four verbal mental arithmetic tasks for which they made appraisals before and after each task. Cardiac reactivity and total peripheral resistance (TPR) were calculated for the first and last minutes of each task, and the number of responses and percent correct were measured for each task. In line with our prediction, pretask appraisals were related to some task-related cardiac responses across the four tasks. In addition, task-related cardiovascular reactivity and behaviors both influenced appraisals following the task. Our findings suggest that an idiographic analysis of appraisals, cardiovascular physiology, and task-related behaviors provides a richer understanding of the appraisal process and reveals sex differences deserving further assessment.     

Small-cell carcinoma of the ovary in peritoneal fluid

Two cases of small-cell carcinoma of the ovary in the ascitic fluid and peritoneal/pelvic washings of a 30- and 28-yr-old woman, respectively, are presented and discussed. Smear preparations from the ascitic fluid showed loose clusters and single malignant cells with scant cytoplasm and nuclei with smooth to irregular nuclear membranes, granular chromatin, and small nucleoli. In the second case peritoneal/pelvic washing specimens contained clusters and single malignant cells with a moderate amount of cytoplasm and nuclei with smooth nuclear membranes, granular, clumped chromatin, and prominent nucleoli. Histology confirmed the diagnosis of small-cell carcinoma of the ovary. These are the first reported cases of this rare ovarian neoplasm present on fluid cytology. Its differentiation from other small-cell neoplasms on peritoneal fluid cytology from young women is discussed. Diagn Cytopathol 1994; 11:266–270. © 1994 Wiley-Liss, Inc.     

Comparison of an Adherence Domain and a Structural Region of Streptococcus mutans Antigen I/II in Protective Immunity against Dental Caries in Rats after Intranasal Immunization

Previous studies have identified an N-terminal saliva-binding region (SBR) on Streptococcus mutans surface antigen I/II (AgI/II) and suggested its importance in the initial adherence of S. mutans to saliva-coated tooth surfaces and subsequent development of dental caries. In this study, we compared the SBR with a C-terminal structural region of AgI/II (AgII) in their abilities to induce protective immunity against caries in rats. When SBR, AgII, or the whole AgI/II molecule was administered intranasally as a conjugate with the B subunit of cholera toxin (CT), in the presence of CT adjuvant, substantial levels of salivary immunoglobulin A anti-AgI/II antibodies were induced. Evaluation of caries activity showed that the SBR, though not as protective as the parent molecule, was superior to AgII and thus can be further considered as a component in a multivalent caries vaccine.     

Changes in thymic export of L-selectin+ γδ and αβ T cells during fetal and postnatal development

We have used the technique of in situ intrathymic injection of fluorescein isothiocyanate to examine L-selectin expression on γδ and αβ T cells immediately after emigrating from the thymus of fetal and postnatal animals. We found that the percentage of L-selectin⁺ thymocytes exported per day decreased by half after birth and that the export of T cells from the thymus does not rely on expression of the peripheral lymph node homing receptor, L-selectin. Analysis of L-selectin on emigrant and mature T cell subsets revealed a remarkable heterogeneity of expression, both in terms of the numbers of cells expressing this molecule as well as the level of expression. γδ T cells, reportedly not having a propensity for homing to lymph nodes, not only contained the highest proportion of L-selectin⁺ cells, but also expressed far more of this molecule than either CD4⁺CD8^? or CD4^?CD8⁺ αβ T cells. Furthermore, those emigrant T cells expressing L-selectin are somewhat immature in their expression of this molecule. Subsequent maturation resulted in up-regulation of L-selectin on mature peripheral blood T cells, maturation that was clearly independent of extrinsic antigen. This antigen-independent post-thymic maturation appeared to occur as part of the normal progression from immature thymocyte to mature peripheral T cell in both fetal and postnatal animals.