Daniel Jupiter

Bcl-xL antisense oligonucleotide and cisplatin combination therapy extends survival in SCID mice with established mesothelioma xenografts

Bcl‐xL functions as a dominant regulator of apoptotic cell death and is implicated in chemotherapeutic resistance of malignant pleural mesothelioma (MPM). Mesothelioma cell lines demonstrate increasing levels of Bcl‐xL as resistant clones are selected invitro. Moreover, upon introduction of antisense oligonucleotides specific to Bcl‐xL mRNA, MPM cells are sensitized to chemotherapeutic agents. Here we describe the therapeutic effects of a novel combination therapy, Bcl‐xL antisense oligonucleotide (ASO 15999) and cisplatin, on mesothelioma cell lines in vitro and invivo; in addition, efficacy of ASO 15999 in decreasing tumor load as well as its effect on survival in an animal model. Finally, we initiated preliminary toxicity studies involved with intraperitoneal (IP) injections of ASO 15999 into mice. This novel combination, with doses of cisplatin four times below established IC50 levels, significantly decreased viability of MPM cell lines after 48 hr. The growth of established mouse flank human tumor xenografts was reduced with intra‐tumor administration of ASO 15999. Local spread and development of IP xenografts was reduced with treatments of ASO alone, and survival of mice afflicted with these xenografts was prolonged after administration of ASO alone and ASO 15999 + cisplatin combination therapy. These findings suggest that ASO 15999 sensitizes MPM cell lines to the toxic effects of cisplatin. ASO 15999 induced reduction of Bcl‐xL is effective in slowing the progression of human mesothelioma cell lines both in vitro and in vivo. More notably, the combination of Bcl‐xL ASO and cisplatin extends survival in an orthotopic tumor xenograft model.

Marrow stimulation improves meniscal healing at early endpoints in a rabbit meniscal injury model

PURPOSEnTo critically evaluate the effect of marrow stimulation (MS) on the extent of healing and the local biological environment after meniscal injury in ligamentously stable knees in a rabbit model.nnnMETHODSnA reproducible 1.5-mm cylindrical defect was created in the avascular portion of the anterior horn of the medial meniscus bilaterally in 18 New Zealand White rabbits (36 knees). In right knees (MS knees), a 2.4-mm Steinman pin was drilled into the apex of the femoral intercondylar notch and marrow contents were observed spilling into the joint. Left knees served as controls. Rabbits were killed in 3 groups (n = 6 rabbits each) at 1, 4, and 12 weeks with meniscal harvest and blinded histomorphometric and histologic evaluation using an established 3-component tissue quality score (range, 0 to 6). One-week specimens were also evaluated for the presence of proregenerative cytokines using immunohistochemistry.nnnRESULTSnThe mean proportion of the avascular zone defect bridged by reparative tissue was greater in MS knees than in controls at each endpoint (1 week, 55% v 30%, P = .02; 4 weeks, 71% v 53%, P = .047; 12 weeks, 96% v 77%, P = .16). Similarly, there was a consistent trend toward superior tissue quality scores in knees treated with MS compared with controls (1 week, 1.8 v 0.3, P = .03; 4 weeks, 4.3 v 2.8, P = .08; 12 weeks, 5.9 v 4.5, P = .21). No statistically significant differences, however, were observed at the 12-week endpoint. Increased staining for insulin-like growth factor I, transforming growth factor-β, and platelet-derived growth factor was observed in regenerated tissue, compared with native meniscal tissue, in all specimens at 1 week. Staining density for all growth factors was similar, however, in reparative tissue of MS and control knees.nnnCONCLUSIONSnThe results of this study suggest that marrow stimulation leads to modest improvements in quality and quantity of reparative tissue bridging a meniscal defect, particularly during the early recovery period.nnnCLINICAL RELEVANCEnClinical evaluation of marrow stimulation techniques designed to enhance healing in isolated meniscus repair surgery may be indicated.

Up-regulation of Bcl-xl by hepatocyte growth factor in human mesothelioma cells involves ETS transcription factors

Bcl-xl and the hepatocyte growth factor (HGF) receptor c-Met are both highly expressed in mesotheliomas, where they protect cells from apoptosis and can confer resistance to conventional therapeutic agents. In our current study, we investigate a model for the transcriptional control of Bcl-xl that involves ETS transcription factors and the HGF/Met axis. In addition, the effects of activated c-Met on the phosphorylation of the ETS family transcriptional factors were examined. The transient expression of ETS-2 and PU.1 cDNAs in mesothelioma cell lines resulted in an increase in the promoter activity of Bcl-xl and consequently in its mRNA and protein expression levels, whereas the transcriptional repressor Tel suppressed Bcl-xl transcription. The activation of the HGF/Met axis led to rapid phosphorylation of ETS family transcription factors in mesothelioma cells through the mitogen-activated protein kinase pathway and via nuclear accumulation of ETS-2 and PU.1. A chromatin immunoprecipitation assay further demonstrated that the activation of c-Met enhanced the binding of ETS transcriptional factors to the Bcl-x promoter. Finally, we determined the Bcl-xl and phosphorylated c-Met expression levels in mesothelioma patient samples; these data suggest a strong correlation between Bcl-xl and phosphorylated c-Met levels. Taken together, these findings support a role for c-Met as an inhibitor of apoptosis and an activator of Bcl-xl.

Lymphatic muscle cells in rat mesenteric lymphatic vessels of various ages.

BACKGROUNDnRecent studies on aging-associated changes in mesenteric lymph flow in situ demonstrated predominance of the severe negative chronotropic effect of aging on the contractility of aged mesenteric lymphatic vessels (MLV). At the same time, contraction amplitude of the aged vessels was only slightly diminished by aging and can be rapidly stimulated within 5-15 minutes. However, the detailed quantitative evaluation of potential aging-associated changes in muscle cells investiture in MLV has never been performed.nnnMETHODS AND RESULTSnIn this study we, for the first time, performed detailed evaluation of muscle cells investiture in MLV in reference to the position of lymphatic valve in different zones of lymphangion within various age groups (3-mo, 9-mo and 24-mo Fischer-344 rats). Using visual and quantitative analyses of the images of MLV immunohistochemically labeled for actin, we confirmed that the zones located close upstream (pre-valve zones) and above lymphatic valves (valve zones) possess the lowest investiture of lymphatic muscle cells. Most of the high muscle cells investiture zones exist downstream to the lymphatic valve (post-valve zones). The muscle cells investiture of these zones is not affected by aging, while pre-valve and valve zones demonstrate significant aging-associated decrease in muscle cells investiture.nnnCONCLUSIONSnThe low muscle cells investiture zones in lymphatic vessels consist of predominantly longitudinally oriented muscle cells which are positioned in pre-valve and valve zones and connect adjacent lymphangions. These cells may provide important functional impact on the biomechanics of the lymphatic valve gating and electrical coupling between lymphangions, while their aging-associated changes may delimit adaptive reserves of aged lymphatic vessels.

Doxycycline attenuates burn-induced microvascular hyperpermeability.

BACKGROUND Burns induce systemic microvascular hyperpermeability resulting in shock, and if untreated, cardiovascular collapse. Damage to the endothelial cell adherens junctional complex plays an integral role in the pathophysiology of microvascular hyperpermeability. We hypothesized that doxycycline, a known inhibitor of matrix metalloproteinases (MMPs), could attenuate burn-induced adherens junction damage and microvascular hyperpermeability. METHODS Male Sprague-Dawley rats were divided into sham, burn, and burn + doxycycline (n = 5). The experimental groups underwent a 30% total body surface area full-thickness burn. Fluorescein isothiocyanate–albumin was administered intravenously. Mesenteric postcapillary venules were examined with intravital microscopy to determine flux of albumin from the intravascular space to the interstitium. Fluorescence intensity was compared between the intravascular space to the interstitium at 30, 60, 80, 100, 120, 140, 160, and 180 minutes after burn. Parallel experiments were performed in which rat lung microvascular endothelial cells were treated with sera from sham or burn animals as well as separate groups pretreated with either doxycycline or a specific inhibitor of MMP-9. Monolayer permeability was determined by fluorescein isothiocyanate albumin-flux across Transwell plates and immunofluorescense staining for the adherens junction protein &bgr;-catenin was performed. Western blot and gelatin zymography were performed to assess MMP-9 level and activity. RESULTS MMP-9 levels were increased after burn. Monolayer permeability was significantly increased with burn serum treatment; this was attenuated with doxycycline as well as the specific MMP-9 inhibitor (p < 0.05). Damage of the endothelial cell adherens junction complex was induced by serum from burned rats, and doxycycline restored the integrity of the adherens junction similar to the MMP-9 inhibitor. Intravital microscopy revealed microvascular hyperpermeability after burn; this was attenuated with doxycycline (p < 0.05). CONCLUSION Burns induce microvascular hyperpermeability via endothelial adherens junction disruption associated with MMP-9, and this is attenuated with doxycycline.

An Immunological Fingerprint Differentiates Muscular Lymphatics from Arteries and Veins

The principal function of the lymphatic system is to transport lymph from the interstitium to the nodes and then from the nodes to the blood. In doing so lymphatics play important roles in fluid homeostasis, macromolecular/antigen transport and immune cell trafficking. To better understand the genes that contribute to their unique physiology, we compared the transcriptional profile of muscular lymphatics (prenodal mesenteric microlymphatics and large, postnodal thoracic duct) to axillary and mesenteric arteries and veins isolated from rats. Clustering of the differentially expressed genes demonstrated that the lymph versus blood vessel differences were more profound than between blood vessels, particularly the microvessels. Gene ontology functional category analysis indicated that microlymphatics were enriched in antigen processing/presentation, IgE receptor signaling, catabolic processes, translation and ribosome; while they were diminished in oxygen transport, regulation of cell proliferation, glycolysis and inhibition of adenylate cyclase activity by G-proteins. We evaluated the differentially expressed microarray genes/products by qPCR and/or immunofluorescence. Immunofluorescence documented that multiple MHC class II antigen presentation proteins were highly expressed by an antigen-presenting cell (APC) type found resident within the lymphatic wall. These APCs also expressed CD86, a co-stimulatory protein necessary for T-cell activation. We evaluated the distribution and phenotype of APCs within the pre and postnodal lymphatic network. This study documents a novel population of APCs resident within the walls of muscular, prenodal lymphatics that indicates novel roles in antigen sampling and immune responses. In conclusion, these prenodal lymphatics exhibit a unique profile that distinguishes them from blood vessels and highlights the role of the lymphatic system as an immunovascular system linking the parenchymal interstitium, lymph nodes and the blood.

Prevalence of Podiatric Medical Problems in Veterans versus Nonveterans

BACKGROUNDnLower-extremity pathologic abnormalities have been common in military recruits for many years. Many of these conditions can become chronic and persist even after retiring from military service. We hypothesized that certain foot abnormalities are more prevalent in veterans versus nonveterans. The purpose of this study was to evaluate what foot and ankle disorders are associated with veteran status while controlling for other demographic factors.nnnMETHODSnThe National Health Interview Survey (Podiatry Supplement) from 1990 was used for this secondary data analysis. The data were divided into veterans and nonveterans, and the prevalence of podiatric medical problems, including callus, flatfoot deformity, bunion deformity, hammer toe deformity, arthritis, and sprain, was evaluated for each group.nnnRESULTSnFlatfoot deformity and arthritis were significantly more prevalent in veterans versus nonveterans in the United States. Bunion deformity was significantly more prevalent in male veterans than in male nonveterans. Male veterans were less likely than male nonveterans to have sprains, and female veterans were more likely than their nonveteran counterparts to have sprains.nnnCONCLUSIONSnThese results may help us understand the potential risk factors for podiatric medical problems and may be used for formulating prevention programs.

Global approximation of CR functions on bloom-graham model graphs in ℂ n

We define a class of generic CR submanifolds of C of real codimension d, 1 < d ≤ n, called the Bloom-Graham model graphs, whose graphing functions are partially decoupled in their dependence on the variables in the real directions. We prove a global version of the Baouendi-Treves CR approximation theorem for Bloom-Graham model graphs with a polynomial growth assumption on their graphing functions.