eScholarship@UMMS

PURPOSE: To demonstrate the feasibility of using a myeloperoxidase (MPO)-specific paramagnetic magnetic resonance (MR) contrast agent to identify active inflammation in an animal model of common carotid artery (CCA) aneurysm.

MATERIALS AND METHODS: All animal experiments were approved by the institutional animal care and use committee. Elastase-induced saccular aneurysms were created at the root of the right CCA in 16 New Zealand white rabbits. Intramural and perivascular injection of Escherichia coli lipopolysaccharide (LPS) was performed with an endovascular approach to induce aneurysm inflammation. After intraarterial injection of an MPO-specific (di-5-hydroxytryptamide of gadopentetate dimeglumine, 0.1 mmol per kilogram of bodyweight) or a non-MPO-specific (di-tyrosine of gadopentetate dimeglumine, 0.1 mmol/kg) contrast agent, animals underwent 3-T MR imaging. Intramural presence of MPO in aneurysms in which LPS had been injected was confirmed at immunohistologic analysis. Active MPO activity was verified by measuring the spectrophotometric oxidation of guaiacol.

RESULTS: Endovascular injection of LPS resulted in inflammatory cell infiltration into the aneurysm wall, and there was a difference in active MPO expression between aneurysms in which LPS had been injected and control aneurysms (20.3 ng of MPO per milligram of tissue vs 0.12 ng of MPO per milligram of tissue, respectively; P < .002). MR imaging with di-5-hydroxytryptamide of gadopentetate dimeglumine revealed a difference in enhancement ratio between inflamed aneurysms in which LPS had been injected and control aneurysms (1.55 +/- 0.05 vs 1.16 +/- 0.10, respectively; P < .02). In inflamed aneurysms, di-5-hydroxytryptamide of gadopentetate dimeglumine exhibited delayed washout kinetics compared with the kinetics of di-tyrosine of gadopentetate dimeglumine. This finding enabled the verification of MPO specificity.

CONCLUSION: The findings of this pilot study established the feasibility of an animal model of saccular aneurysm inflammation that can be seen with clinical-field-strength MR imaging and use of the enzyme-sensitive MR contrast agent di-5-hydroxytryptamide of gadopentetate dimeglumine, which is a paramagnetic MPO substrate that specifically enhances MR signal.

In 2008 we witnessed a rapid advancement in stent technology, which is reflected in the high number of case reports, publications of case series, and randomized trials. Stents not only served for a combined intrasaccular and extrasaccular treatment of challenging aneurysms but also assisted the revascularization in acute and chronic ischemic conditions of the neurovascular system. Although a self-expanding nitinol semiopen cell stent is currently used for intracranial occlusive disease, a new retrievable closed-cell designed stent is widely used for aneurysms because of its easy delivery through a microcatheter in frequently tortuous head and neck as well as cerebrovascular circulation (Figure 1). However, despite numerous publications in the field, the widespread acceptance of the use of stents to routinely treat carotid stenosis awaits the results of the multicenter randomized clinical trials that should be available in 2009. The role of interventional neuroradiology in the treatment of acute ischemic stroke continues to expand and excite interest.

Epidermal growth factor receptor (EGFR) imaging in brain tumors is essential to visualize overexpression of EGFRvIII variants as a signature of highly aggressive gliomas and to identify patients that would benefit from anti-EGFR therapy. Seeking imaging improvements, we tested a novel pretargeting approach that relies on initial administration of enzyme-linked anti-EGFR monoclonal antibodies (mAb; EMD72000) followed by administration of a low-molecular-weight paramagnetic molecule (diTyr-GdDTPA) retained at the site of EGFR mAb accumulation. We hypothesized that diTyr-GdDTPA would become enzyme activated and retained on cells due to binding to tissue proteins. In support of this hypothesis, mAb-enzyme conjugates reacted with both membrane-isolated wild-type (wt) EGFR and EGFRvIII, but they bound primarily to EGFRvIII-expressing cells and not to EGFRwt-expressing cells. In vivo analysis of magnetic resonance (MR) tumor signal revealed differences in MR signal decay following diTyr-GdDTPA substrate administration. These differences were significant in that they suggested differences in substrate elimination from the tissue which relied on the specificity of the initial mAb binding: a biexponential signal decay was observed in tumors only upon preinjection with EGFR-targeted conjugates. Endpoint MRI in this setting revealed detailed images of tumors which correlated with immunohistochemical detection of EGFR expression. Together, our findings suggest an improved method to identify EGFRvIII-expressing gliomas in vivo that are best suited for treatment with therapeutic EGFR antibodies.

BACKGROUND AND PURPOSE: Thrombus formation during endovascular embolization of intracranial aneurysms occurs in 2.9%-6% of patients. Use of IIb/IIIA inhibitors such as abciximab or eptifibatide intravenously has been reported in management of this complication. Because the intra-arterial infusion of IIb/IIIA inhibitors may require lower doses to achieve thrombolysis, it may reduce the risk of haemorrhage. Therefore, we retrospectively analyze our database and review the literature.

METHODS: This is a retrospective analysis of a prospectively acquired database of patients with ruptured or unruptured aneurysm treated intra-arterially for thrombus formation during endovascular coil embolization between July 2005 and August 2008. Patient demographics, aneurysmal characteristics, procedural, clinical outcome and complications were recorded.

RESULTS: From July 2005 to August 2008, out of 184 patients who underwent coil embolization, 19 patients (15 smokers, 14 female, mean age 52) developed intraprocedural thrombus formation and received intra-arterial abciximab treatment. Mean aneurysm size was 6.6 mm+/-4.9 mm; neck size was 3.8 mm+/-2.1 mm. Eight (42.1%) aneurysms were ruptured. Most aneurysms (63.1%) were in anterior communicating and middle cerebral arteries. Thrombus was visualized in all cases by angiogram and treated intra-arterially with a mean dose of 10.5 mg+/-4.2. There were no periprocedural hemorrhagic complications. No deaths or other complications occurred during follow-up.

CONCLUSION: Thrombus formation during coil embolization of intracranial aneurysms occurred more in women and smokers. Low doses of intra-arterial abciximab may be effective in the thromboembolic complications occurring during endovascular embolization of intracranial aneurysms.

Emerging evidence in both vertebrates and invertebrates is redefining glia as active and mobile players in synapse formation, maturation and function. However, the molecular mechanisms through which neurons and glia interact with each other to regulate these processes is not well known. My thesis work begins to understand how glia use secreted factors to modulate synaptic function. We use Drosophila melanogaster, a simple and genetically tractable model system, to understand the molecular mechanisms by which glia communicate with neurons at glutamatergic neuromuscular junctions (NMJs). We previously showed that a specific subtype of glia, subperineurial peripheral glia cells (SPGs), establish dynamic transient interactions with synaptic boutons of the NMJ and is required for synaptic growth. I identified a number of potential functional targets of the glial transcription factor, reverse polarity (repo) using ChIP-chip. I found that one novel target of Repo, Wg, is expressed in SPGs and is regulated by repo in vivo. Wnt/Wg signaling plays a pivotal role during synapse development and plasticity, including the coordinated development of the molecular architecture of the synapse. While previous studies demonstrated that Wg is secreted by motor neurons, herein I provide evidence that a significant amount of Wg at the NMJ is additionally provided by glia. I found that Wg derived from SPGs is required for proper GluR distribution and electrophysiological responses at the NMJ. In summary, my results show that Wg expression is regulated by Repo in SPGs and that glial-derived Wg, together with motor neuron-derived Wg, orchestrate different aspects of synapse development. My thesis work identifies synapse stabilization and/or assembly as a new role for SPGs and demonstrates that glial secreted factors such as Wg regulate synaptic function at the Drosophila NMJ.

Replication of HIV-1 requires the assembly and release of mature and infectious viral particles. In order to accomplish this goal, HIV-1 has evolved multiple methods to interact with the host cell. HIV-1 recruits the host cell ESCRT machinery to facilitate the release of nascent viral particles from the host cell membrane. Recruitment of these cellular factors is dependent on the presence of short motifs in Gag referred to as Late-domains. Deletion or mutation of these domains results in substantial decrease in the release of infectious virions. However, previously published work has indicated that over-expression of the E3 ubiquitin ligase, NEDD4.2s is able to robustly rescue release of otherwise budding-defective HIV-1 particles. This rescue is specific to the NEDD4.2s isoform as related E3 ubiquitin ligases display no ability to rescue particle release. In addition, rescue of particle release is dependent on the presence of the partial C2 domain and a catalytically active HECT domain of NEDD4.2s. Here I provide evidence supporting the hypothesis that a partial C2 domain of NEDD4.2s constitutes a Gag interacting module capable of targeting the HECT domains of other E3 ubiquitin ligases to HIV-1 Gag. Also, by generating chimeras between HECT domains shown to form poly-ubiquitin chains linked through either K48 or K63 of ubiquitin, I demonstrate that the ability of NEDD4.2s to catalyze the formation of K63-polyubiquitin chains is required for its stimulation of HIV-1 L-domain mutant particle release. In addition, I present findings from on-going research into the role of the HIV-1 accessory protein Nef during viral replication using the culture T-cell line, MOLT3. My current findings indicate that downregulation of CD4 from the host cell membrane does not solely account for the dramatic dependence of HIV-1 replication on Nef expression in this system. In addition, I present evidence indicating that Nef proteins from diverse HIV-1 Groups and strains are capable of enhancing HIV-1 replication in this system. Analysis of a range of mutations in Nef known to impact interaction with cellular proteins suggest that the observed replication enhancement requires Nef targeting to the host cell membrane and may also require the ability to interact with select Src-kinases. Lastly, we find that the ability of Nef to enhance replication in this system is separate from any increase in viral particle infectivity, in agreement with current literature.

During heart valve remodeling, and in many disease states, valvular interstitial cells (VICs) shift to an activated myofibroblast phenotype which is characterized by enhanced synthetic and contractile activity. Pronounced alpha smooth muscle actin (αSMA)-containing stress fibers, the hallmark of activated myofibroblasts, are also observed when VICs are placed under tension due to altered mechanical loading in vivo or during in vitro culture on stiff substrates or under high mechanical loads and in the presence of transforming growth factor-beta1 (TGF-β1). The work presented herein describes three distinct model systems for application of controlled mechanical environment to VICs cultured in vitro. The first system uses polyacrylamide (PA) gels of defined stiffness to evaluate the response of VICs over a large range of stiffness levels and TGF-β1 concentration. The second system controls the boundary stiffness of cell-populated gels using springs of defined stiffness. The third system cyclically stretches soft or stiff two-dimensional (2D) gels while cells are cultured on the gel surface as it is deformed. Through the use of these model systems, we have found that the level of 2D stiffness required to maintain the quiescent VIC phenotype is potentially too low for a material to both act as matrix to support cell growth in the non-activated state and also to withstand the mechanical loading that occurs during the cardiac cycle. Further, we found that increasing the boundary stiffness on a three-dimensional (3D) cell populated collagen gel resulted in increased cellular contractile forces, αSMA expression, and collagen gel (material) stiffness. Finally, VIC morphology is significantly altered in response to stiffness and stretch. On soft 2D substrates, VICs cultured statically exhibit a small rounded morphology, significantly smaller than on stiff substrates. Following equibiaxial cyclic stretch, VICs spread to the extent of cells cultured on stiff substrates, but did not reorient in response to uniaxial stretch to the extent of cells stretched on stiff substrates. These studies provide critical information for characterizing how VICs respond to mechanical stimuli. Characterization of these responses is important for the development of tissue engineered heart valves and contributes to the understanding of the role of mechanical cues on valve pathology and disease onset and progression. While this work is focused on valvular interstitial cells, the culture conditions and methods for applying mechanical stimulation could be applied to numerous other adherent cell types providing information on the response to mechanical stimuli relevant for optimizing cell culture, engineered tissues or fundamental research of disease states.

Purpose: Spirituality has been associated with better cardiac autonomic balance, but its association with cardiovascular risk is not well studied. We examined whether more frequent private spiritual activity was associated with reduced cardiovascular risk in postmenopausal women enrolled in the Women’s Health Initiative Observational Study.

Methods: Frequency of private spiritual activity (prayer, Bible reading, and meditation) was selfreported at year 5 of follow-up. Cardiovascular outcomes were centrally adjudicated, and cardiovascular risk was estimated from proportional hazards models.

Results: Final models included 43,708 women (mean age: 68.9±7.3; median follow-up: 7.0 years) free of cardiac disease through year 5 of follow-up. In age-adjusted models private spiritual activity was associated with increased cardiovascular risk (HR: 1.16; CI 1.02, 1.31, weekly vs. never; 1.25; CI 1.11, 1.40, daily vs. never). In multivariate models adjusted for demographics, lifestyle, risk factors, and psychosocial factors, such association remained significant only in the group with daily activity (HR 1.16; CI: 1.03, 1.30). Subgroup analyses indicate this association may be driven by the presence of severe chronic diseases.

Conclusion: In aging women, higher frequency of private spiritual activity was associated with increased cardiovascular risk, likely reflecting a mobilization of spiritual resources in order to cope with aging and illness.

Last year the Office of Inspector General of the US Department of Health and Human Services published detailed regulations regarding the appropriate interactions between physicians and the pharmaceutical industry. Fortunately, the American Medical Association (AMA), through an ethics subcommittee, has developed a succinct opinion statement for physicians to use to determine appropriate gifts and continuing medical education sponsored by industry. This article clarifies the regulations, reviews the history behind the governmental rules, lists the seven guidelines published by the AMA, and directs physicians to additional information on this topic.

The c-myc protooncogene and its polypeptide product are important regulators of cell proliferation and differentiation, and ovarian steroids are believed to stimulate growth of various uterine cell types through altered expression of the c-myc gene. To determine whether c-myc expression may also be involved in the growth and development of endometriosis, we assessed c-myc expression in eutopic and ectopic endometrial tissue obtained from women undergoing surgery for endometriosis. Immunocytochemistry using a monoclonal antibody to the c-myc protein demonstrated positive staining of glandular and stromal cell nuclei, and cytoplasmic staining of glandular but not stromal cells in both eutopic and ectopic endometrium. These findings suggest that c-myc expression may be an important regulator of cell proliferation in endometriotic tissue.

A desire for improved worklife balance has prompted more faculty to work less than one full-time equivalent. While not exclusive, women faculty are especially to make this request. This chapter explores the hidden value of part-time faculty and the perceptions of department chairs and their department managers about these special faculty members.

Identifies the basic cost-effective diagnostic testing for couples with infertility, evaluation, and treatment options.

The evolution of the mammary gland from a sweat gland-like skin organ occurred almost 100 million years ago. This unique development was in a large part responsible for the rapid proliferation of the class of vertebrates that we now know as mammals. The advantage of the mammary gland was that it provided a ready source of nutrition for the still-developing infant, obviating the need for prolonged incubation in a fragile and vulnerable egg encasement. From a functional point of view, there are those who would contend that the human mammary gland has outlived its usefulness; however, the discovery of the multiple valuable and unique properties of human milk for humankind and the devastating results of attempting nonlactational nutrition for infants in many of the developing nations of the world would argue strongly against this point. We have just begun to appreciate the potential long-range sequelae of breast-feeding biologically and behaviorally.