Microarray analysis of healing rat Achilles tendon: evidence for glutamate signaling mechanisms and embryonic gene expression in healing tendon tissue.

Tendon healing is a complex process consisting of a large number of intricate pathways roughly divided into the phases of inflammation, proliferation, and remodeling. Although these processes have been extensively studied at a variety of levels in recent years, there is still much that remains unknown. This study used microarray analyses to investigate the process at a genetic level in healing rat Achilles tendon at 1, 7, and 21 days postinjury, roughly representing the inflammation, proliferation, and remodeling phases. An interesting temporal expression profile was demonstrated, identifying both known and novel genes and pathways involved in the progression of tendon healing. Both inflammatory response and pro-proliferative genes were shown to be significantly upregulated from 24 h postinjury through to 21 days. Day 7 showed the largest increase in genetic activity, particularly with the expression of collagens and other extracellular matrix genes. Interestingly, there was also evidence of central nervous system-like glutamate-based signaling machinery present in tendon cells, as has recently been shown in bone. This type of signaling mechanism has not previously been shown to exist in tendon. Another novel finding from these analyses is that there appears to be several genes upregulated during healing which have exclusively or primarily been characterized as key modulators of proliferation and patterning during embryonic development. This may suggest that similar pathways are employed in wound healing as in the tightly regulated progression of growth and development in the embryo. These results could be of use in designing novel gene-based therapies to increase the efficacy and efficiency of tendon healing.     

Effect of amyloid peptides on the increase in TrkA receptor expression induced by nicotine in vitro and in vivo

The ability of nicotine to induce a cytoprotective or neuroprotective action occurs through several down-stream mechanisms. One possibility is that the drug increases the expression of tyrosine kinase A (TrkA) nerve growth factor (NGF) receptors. Certain β-amyloid peptides (e.g., Aβ1–42) have been shown to bind with high affinity to α7 nicotinic receptors and thus interfere with a potentially neurotrophic influence. Treatment of differentiated PC-12 cells with nicotine produced a concentration-dependent increase in cell-surface TrkA receptors that occurred concomitantly with cytoprotection. The effect of nicotine was blocked by either of the α7 receptor antagonists α-bungarotoxin (α-BTX) or methyllycaconatine. The cytoprotective action of nicotine also was inhibited by pretreatment with 10–100 nM Aβ1–42. Nicotine also was administered (four injections of 30 μg, spaced evenly over 24 h) to rats by direct injection into a lateral cerebral ventricle. Brain TrkA expression was increased significantly in hippocampus and entorhinal cortex (up to 32% above control), with no changes found in cerebral cortex or hypothalamus. The nicotine-induced increases in TrKA expression in hippocampus and entorhinal cortex were significantly inhibited by 10 μg α-BTX or by 10 nmol Aβ1–42. Therefore, physiologically relevant concentrations of Aβ1–42 can prevent nicotine-induced TrkA receptor expression in brain regions containing cholinergic neurons susceptible to the neurotoxicity associated with Alzheimer’s disease.     

Additive effects of CD59 expression in Gal knockout mice in vitro but not in an ex vivo model

Abstract: Transgenic expression of the human complement regulatory molecule CD59 in mice and genetic deletion of the major xenoantigen galactose α 1,3 galactose (Gal KO) each resulted in partial protection of spleen cells from lysis by human serum. These protective effects were additive when the two genetic modifications were combined. However, when the effects of these genetic modifications were examined in an ex vivo model in which mouse hearts were perfused with human plasma, it was Gal KO which was the modification which determined protection. CD59 expression alone was not protective and CD59 expression in combination with Gal knockout did not result in a significant additional increase in protection over and above that provided by Gal knockout alone. The likely explanation for this discrepancy between the in vitro and ex vivo data is that the H2-K^b promoter used to drive CD59 expression results I in substantially less expression on endothelium than on spleen cells.     

Frequency of T-Cell Progenitors in Nude Mice

The hypothesis that prothymocytes are distinct from and regulated independently of multilineage hemopoietic progenitors was tested by enumeration of these two cell populations in normal versus congenitally athymic (nude) mice. The absence of a thymus and of peripheral T cells in nude mice had no effect on the frequency of either multilineage progenitors (day 12 CFU-S) or prothymocytes (CFU-T), suggesting that there is no feedback regulation of CFU-T frequency. Thymus seeding from the bone marrow is therefore likely to be regulated by the availability of niches for prothymocyte maturation, rather than by feedback control of prothymocyte production.     

Effects of topical anti-inflammatory drugs on eosinophil survival primed by epithelial cells. Additive effect of glucocorticoids and nedocromil sodium

Background Eosinophil infiltration is a hallmark of the inflammatory response in rhinitis and in nasal polypcsis. Objective We studied the effect of steroids and nedocromil sodium on eosinophil survival primed by epithelial cells from healthy (nasal mucosa) and inflamed (nasal polyp) respiratory tissue. Methods Blood eosinophils were incubated with increasing concentrations (10^-11 10^-5 M) of topical steroids (fiuticasone propionate, budesonide, triamcinolone acetonide and beclomethasone dipropionate) and/or nedocromil sodium prior to the addition of human epithelial cell conditioned media (HECM), eosinophil viability was measured and IC₅₀ for each drug was calculated. Results All four steroids and nedocromil sodium caused a dose-related inhibition of HECM-induced eosinophil survival. The IC₅₀ of steroids were lower in eosinophils primed by mucosa HECM than on those primed by polyp HECM (fluticasone, 4nM vs 114nM: budesonide, 21 nM vs 280 nM; triamcinolone, 7 nM vs 853 nM; and beclomethasone, 171 nM vs 181 nM). The combined inhibitory effect of 10^-7M budesonide plus 10^-5M nedocromil (43.8 ± 10.8%, P < 0.03) was significantly higher than budesonide (28.5 ± 9.2%) or nedocromil (16.7 ± 5.4%) alone and close to 10^-5M budesonide (52.3 ± 11%). No differences were found in cytokine (IL-8, IL-6, GM-CSF, TNFα, IL-lβ and RANTES) concentrations between HECM from mucosa and polyps. Conclusion These results suggest that topical anti-inflammatory drugs may diminish airway eosinophilic infiltration by decreasing eosinophil viability, that nasal polyp epithelial cell secretions may induce steroid resistance in eosinophils, and that nedocromil sodium has additive effects with steroids.     

Immunoelectron microscopic studies on centromere-kinetochore complexes detached from chromosomes

The centromere-kinetochore complexes of Chinese hamster ovary (CHO) cells were detached and separated from the condensed chromatin by treatment with hydroxyurea and caffeine. By labelling the complex for immunoelectron microscopy (immuno-EM) with a mixture of antibodies against centromere proteins (anti-CENP-A,-B, -C) in some cells, we could demonstrate complete detachment of the complexes. No remnants were left at the bulk of condensed chromatin in these cells. In some mitotic cells complex and chromatin were found side by side. It could be shown that the fine structure of the separated material of the complex differs significantly from that of the rest of chromatin. The complex consists of proteins and DNA. This leads us to suppose that the organization of chromatin in the centromere-kinetochore complex is different.     

Comparison of the use of bulk to micro culture of cell preparations for lymphokine-activated cytotoxicity assays.

Different assay systems have been used to quantitate lymphokine-induced natural cytotoxic activity as a measure of immune status. This study compares the effects of inducing cytotoxicity in a bulk culture system, where effector cells are transferred to a micro culture well for assay, to a micro culture system where the effector cells are not transferred. The effector/target ratio for both the bulk and micro culture systems was calculated using the number of viable effector cells present at the time of target cell addition. After overnight incubation with interleukin-2 (IL-2), the lytic activity of murine spleen cells to targets using a micro culture system was increased two-fold over the bulk culture method. This increase was amplified further after 5 days of activation with IL-2, in that the micro culture system resulted in a four-fold increase in cytotoxic activity. The loss of some adherent cells in the bulk culture system did not explain the overall decrease in recovered cytotoxicity. The difference appeared to be related to cell loss during centrifugation. Therefore, the E/T ratios are different in the two systems if not corrected for the number of viable cells.