A model for de novo synthesis and assembly of tight intercellular junctions. Ultrastructural correlates and experimental verification of the model revealed by freeze-fracture

The structure and function of intercellular tight (occluding) junctions, which constitute the anatomical basis for highly regulated interfaces between tissue compartments such as the blood-testis and blood-brain barriers, are well known. Details of the synthesis and assembly of tight junctions, however, have been difficult to determine primarily because no model for study of these processes has been recognized. Primary cultures of brain capillary endothelial cells are proposed as a model in which events of the synthesis and assembly of tight junctions can be examined by monitoring morphological features of each step in freeze-fracture replicas of the endothelial cell plasma membrane. Examination of replicas of non-confluent monolayers of endothelial cells reveals the following intramembrane structures proposed as `markers' for the sequential events of synthesis and assembly of zonulae occludentes: (1) development of surface contours consisting of elongate terraces and furrows (valleys) orientated parallel to the axis of cytoplasmic extensions of spreading endothelial cells, (2) appearance of small circular PF face depressions (or volcano-like protrusions on the EF face) that represent cytoplasmic vesicle-plasma membrane fusion sites, which are positioned in linear arrays along the contour furrows, (3) appearance of 13-15 nm intramembrane particles at the perimeter of the vesicle fusion sites, and (4) alignment of these intramembrane particles into the long, parallel, anastomosed strands characteristic of mature tight junctions. These structural features of brain endothelial cells in monolayer culture constitute the morphological expression of: (1) reshaping the cell surface to align future junction-containing regions with those of adjacent cells, (2) delivery and insertion of newly synthesized junctional intramembrane particles into regions of the plasma membrane where tight junctions will form, and (3) aggregation and alignment of tight junction intramembrane particles into the complex interconnected strands of mature zonulae occludentes. The distribution of filipin-sterol complex-free regions on the PF intramembrane fracture face of junction-forming endothelial plasmalemmae corresponds precisely to the furrows, aligned vesicle fusion sites and anastomosed strands of tight junctional elements.To test the functional significance of these morphological features of junction-forming cells and to validate the interpretation that they are reliable indicators of the stages of tight junction genesis, primary cultures of bovine brain capillary endothelium were treated with 25 [mu]g/ml of Cytochalasin-D or 0.25 mg/ml of n-ethylmaleimide (Sigma Chemical Co.) in order to prevent cytoskeletal mediation of surface contouring (step 1) or to inhibit vesicle fusion with the plasmalemma (step 2) and thereby prevent junction formation as a consequence of failure of the vesicle fusions to insert tight junctional intramembrane particles into the plasma membrane. Examination of platinum replicas of freeze-fractured control and treated endothelial monolayer cultures confirmed the absence of surface contours in Cytochalasin-D-treated cells, which exhibited no zonulae occludentes, and also clearly showed that n-ethylmaleimide-treated cells, which lacked tight junctions, did not have the rich endowment of vesicle fusion sites (and IMPs) which were conspicuous in control cells. Demonstration of the failure of MDCK cells to form tight junctions when cultured in the presence of 5-10 [mu]g/ml of cycloheximide (Griepp et al., 1983) lends further support for the schemata proposed above.Advantages of this model include: (1) all stages of de novo tight junction formation are present in each monolayer culture, and (2) cultures possess vast areas of tight junction-containing membrane which are easily sampled by freeze-fracture. This model will provide the basis for future attempts to identify the signals that regulate tight junction formation, and will facilitate studies to characterize the protein(s) of the endothelial tight junctions, the messages (m-RNA) that code for them, and ultimately, the genes bearing their blueprint.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25900/1/0000463.pd

Isolated rat brain capillarie spossess intact, structurally complex, interendothelial tight junctions; freeze-fracture verification of tight junction integrity

Populations of isolated brai capillaries have been proposed as useful models for in vitro studies of the blood-brain barrier. Preliminary investigations of barrier properties using such preparations of brain microvessels have suggested that the tight interendothelial junctions (zonulae occludentes) are intact and retain the impermeability to the protein tracer horseradish peroxidase, exhibited by them in vivo. The endothelial junctions of isolated capillaries are therefore assumed to be functionally `tight' in vitro. In order to determine the precise structural organization of these occluding junctions, including an estimate of their tightness (complexity), and to demonstrate a method for simple but precise assessment of junctional integrity, pellets of isolated rat brain capillaries were freeze-fractured and then replicated with platinum and carbon. The freeze-fracture images of interendothelial zonulae occludentes revealed complex arrays of intramembrane ridges and grooves characteristics of tight junctions. Longitudinal fractures of the cellular lining of capillaries exposed vast expanses of interendothelial plasma membrane interfaces and the junctional complexes situated between the cells. From such arrays, the elaborate and complex architecture of the zonulae occludentes could bre readily appreciated. Situated on the PF fracture faces are 6-8 parallel ridges which display a high degree of anastomosing between adjacent strands. The EF facture face contains grooves complementary to the PF face ridges. The zonulae occludentes of these capillary endothelial cells are similar in complexity to those reported in the literature for reptilian brain capillaries and therefore can be presumed `very tight'. This study demonstrate that freeze-fracture of pellets of brain capillaries alleviates problems inherent in whole tissue preparations and, in addition, demonstrates the usefulness of freeze-fracture as a tool to monitor junction structure during in vitro investigation of the blood-brain barrier.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/24599/1/0000007.pd

The calculation of enthalpy-entropy diagrams for and the specific impulse of rocket fuel systems

http://deepblue.lib.umich.edu/bitstream/2027.42/3712/5/bad5839.0001.001.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/3712/4/bad5839.0001.001.tx

Baseline Immune Biomarkers as Predictors of MBSR(BC) Treatment Success in Off-Treatment Breast Cancer Patients

Researchers focused on patient-centered medicine are increasingly trying to identify baseline factors that predict treatment success. Because the quantity and function of lymphocyte subsets change during stress, we hypothesized that these subsets would serve as stress markers and therefore predict which breast cancer patients would benefit most from mindfulness-based stress reduction (MBSR)-facilitated stress relief. The purpose of this study was to assess whether baseline biomarker levels predicted symptom improvement following an MBSR intervention for breast cancer survivors (MBSR[BC]). This randomized controlled trial involved 41 patients assigned to either an MBSR(BC) intervention group or a no-treatment control group. Biomarkers were assessed at baseline, and symptom change was assessed 6 weeks later. Biomarkers included common lymphocyte subsets in the peripheral blood as well as the ability of T cells to become activated and secrete cytokines in response to stimulation with mitogens. Spearman correlations were used to identify univariate relationships between baseline biomarkers and 6-week improvement of symptoms. Next, backward elimination regression models were used to identify the strongest predictors from the univariate analyses. Multiple baseline biomarkers were significantly positively related to 6-week symptom improvement. The regression models identified B-lymphocytes and interferon-γ as the strongest predictors of gastrointestinal improvement (p \u3c .01), +CD4+CD8 as the strongest predictor of cognitive/psychological (CP) improvement (p = .02), and lymphocytes and interleukin (IL)-4 as the strongest predictors of fatigue improvement (p \u3c .01). These results provide preliminary evidence of the potential to use baseline biomarkers as predictors to identify the patients likely to benefit from this intervention

A Randomized Controlled Trial of the Effects of Mindfulness-Based Stress Reduction (MBSR[BC]) on Levels of Inflammatory Biomarkers Among Recovering Breast Cancer Survivors

Purpose: The purpose of this substudy of a large randomized controlled trial was to evaluate the efficacy of the Mindfulness-Based Stress Reduction (Breast Cancer) (MBSR[BC]) program compared to usual care (UC) in normalizing blood levels of pro-inflammatory cytokines among breast cancer survivors (BCS). Method: A total of 322 BCS were randomized to either a 6-week MBSR(BC) program or a UC. At baseline and 6 and 12 weeks, 10 ml of venous blood and demographic and clinical data were collected and/or updated. Plasma cytokines (interleukin [IL]-1β, IL-6, IL-10, tumor necrosis factor [TNF] α, transforming growth factor [TGF] β1, soluble tumor necrosis factor receptor [sTNFR] 1) were assayed. Linear mixed models were used to assess cytokine levels across three time points (baseline and 6 and 12 weeks) by group (MBSR[BC] vs. UC). Results: Of the six measured cytokines, three were nondetectable at rates greater than 50% (IL-10, IL-1β, TGF-β1) and, because of overall low prevalence, were not analyzed further. For the remaining cytokines (TNFα, IL-6, sTNFR1), results showed that TNFα and IL-6 increased during the follow-up period (between 6 and 12 weeks) rather than during the MBSR(BC) training period (between baseline and 6 weeks), while sTNFR1 levels did not change significantly across the 12-week period. Conclusions: Study results suggest that MBSR(BC) affects cytokine levels in BCS, mainly with increases in TNFα and IL-6. The data further suggest that B-cell modulation may be a part of immune recovery during breast cancer management and that increases in TNFα and IL-6 may be markers for MBSR(BC)-related recovery