Assessment of anastomotic leakage: A Novel System for the Training of Surgeons to Perform a Tubular Anastomosis and Objectively Evaluate Anastomotic Leak

Introduction: Dehiscence of the suture line of an anastomosis can lead to reoperation, temporary or permanent stoma, and even sepsis or death. Few techniques for the laboratory training of tubular anastomosis use ex-vivo animal tissues. We describe a novel model that can be used in the laboratory for the training of anastomosis in tubular tissues and objectively assess any anastomotic leak. [See PDF for complete abstract]

Characterization of Optically Stimulated Luminescent Detectors in Photon & Proton Beams for Use in Anthropomorphic Phantoms

This study investigated characteristics of optically stimulated luminescent detectors (OSLDs) in protons, allowing comparison to thermoluminescent detectors, and to be implemented into the Radiological Physics Center’s (RPC) remote audit quality assurance program for protons, and for remote anthropomorphic phantom irradiations. The OSLDs used were aluminum oxide (Al2O3:C) nanoDots from Landauer, Inc. (Glenwood, Ill.) measuring 10x10x2 mm3. A square, 20(L)x20(W)x0.5(H) cm3 piece of solid water was fabricated with pockets to allow OSLDs and TLDs to be irradiated simultaneously and perpendicular to the beam. Irradiations were performed at 5cm depth in photons, and in the center of a 10 cm SOBP in a 200MeV proton beam. Additionally, the Radiological Physics Center’s anthropomorphic pelvic phantom was used to test the angular dependence of OSLDs in photons and protons. A cylindrical insert in the phantom allows the dosimeters to be rotated to any angle with a fixed gantry angle. OSLDs were irradiated at 12 angles between 0 and 360 degrees. The OSLDs were read out with a MicroStar reader from Landauer, Inc. Dose response indicates that at angles where the dosimeter is near parallel with the radiation beam response is reduced slightly. Measurements in proton beams do not show significant angular dependence. Post-irradiation fading of OSLDs was studied in proton beams to determine if the fading was different than that of photons. The fading results showed no significant difference from results in photon beams. OSLDs and TLDs are comparable within 3% in photon beams and a correction factor can be posited for proton beams. With angular dependence characteristics defined, OSLDs can be implemented into multiple-field treatment plans in photons and protons and used in the RPC’s quality assurance program.

Development and Implementation of the Use of Optically Stimulated Luminescent Detectors in the Radiological Physics Center Anthropomorphic Quality Assurance Phantoms

The Radiological Physics Center (RPC) uses both on-site and remote reviews to credential institutions for participation in clinical trials. Anthropomorphic quality assurance (QA) phantoms are one tool the RPC uses to remotely audit institutions, which include thermoluminescent dosimeters (TLDs) and radiochromic film. The RPC desires to switch from TLD as the absolute dosimeter in the phantoms, to optically stimulated luminescent dosimeters (OSLDs), but a problem lies in the angular dependence exhibited by the OSLD. The purpose of this study was to characterize the angular dependence of OSLD and establish a correction factor if necessary, to provide accurate dosimetric measurements as a replacement for TLD in the QA phantoms. A 10 cm diameter high-impact polystyrene spherical phantom was designed and constructed to hold an OSLD to study the angular response of the dosimeter under the simplest of circumstances for both coplanar and non-coplanar treatment deliveries. OSLD were irradiated in the spherical phantom, and the responses of the dosimeter from edge-on angles were normalized to the response when irradiated with the beam incident normally on the surface of the dosimeter. The average normalized response was used to establish an angular correction factor for 6 MV and 18 coplanar treatments, and for 6 MV non-coplanar treatments specific to CyberKnife. The RPC pelvic phantom dosimetry insert was modified to hold OSLD, in addition to the TLD, adjacent to the planes of film. Treatment plans of increasing angular beam delivery were developed, three in Pinnacle v9.0 (4-field box, IMRT, and VMAT) and one in Accuray’s MultiPlan v3.5.3 (CyberKnife). The plans were delivered to the pelvic phantom containing both TLD and OSLD in the target volume. The pelvic phantom was also sent to two institutions to be irradiated as trials, one delivering IMRT, and the other a CyberKnife treatment. For the IMRT deliveries and the two institution trials, the phantom also included film in the sagittal and coronal planes. The doses measured from the TLD and OSLD were calculated for each irradiation, and the angular correction factors established from the spherical phantom irradiations were applied to the OSLD dose. The ratio of the TLD dose to the angular corrected OSLD dose was calculated for each irradiation. The corrected OSLD dose was found to be within 1% of the TLD measured dose for all irradiations, with the exception of the in-house CyberKnife deliveries. The films were normalized to both TLD measured dose and the corrected OSLD dose. Dose profiles were obtained and gamma analysis was performed using a 7%/4 mm criteria, to compare the ability of the OSLD, when corrected for the angular dependence, to provide equivalent results to TLD. The results of this study indicate that the OSLD can effectively be used as a replacement for TLD in the RPC’s anthropomorphic QA phantoms for coplanar treatment deliveries when a correction is applied for the dosimeter’s angular dependence.

Reduced neutrophil count in people of African descent is due to a regulatory variant in the Duffy antigen receptor for chemokines gene.

Persistently low white blood cell count (WBC) and neutrophil count is a well-described phenomenon in persons of African ancestry, whose etiology remains unknown. We recently used admixture mapping to identify an approximately 1-megabase region on chromosome 1, where ancestry status (African or European) almost entirely accounted for the difference in WBC between African Americans and European Americans. To identify the specific genetic change responsible for this association, we analyzed genotype and phenotype data from 6,005 African Americans from the Jackson Heart Study (JHS), the Health, Aging and Body Composition (Health ABC) Study, and the Atherosclerosis Risk in Communities (ARIC) Study. We demonstrate that the causal variant must be at least 91% different in frequency between West Africans and European Americans. An excellent candidate is the Duffy Null polymorphism (SNP rs2814778 at chromosome 1q23.2), which is the only polymorphism in the region known to be so differentiated in frequency and is already known to protect against Plasmodium vivax malaria. We confirm that rs2814778 is predictive of WBC and neutrophil count in African Americans above beyond the previously described admixture association (P = 3.8 x 10(-5)), establishing a novel phenotype for this genetic variant.

Physical and functional interaction between the dopamine transporter and the synaptic vesicle protein synaptogyrin-3.

Uptake through the dopamine transporter (DAT) represents the primary mechanism used to terminate dopaminergic transmission in brain. Although it is well known that dopamine (DA) taken up by the transporter is used to replenish synaptic vesicle stores for subsequent release, the molecular details of this mechanism are not completely understood. Here, we identified the synaptic vesicle protein synaptogyrin-3 as a DAT interacting protein using the split ubiquitin system. This interaction was confirmed through coimmunoprecipitation experiments using heterologous cell lines and mouse brain. DAT and synaptogyrin-3 colocalized at presynaptic terminals from mouse striatum. Using fluorescence resonance energy transfer microscopy, we show that both proteins interact in live neurons. Pull-down assays with GST (glutathione S-transferase) proteins revealed that the cytoplasmic N termini of both DAT and synaptogyrin-3 are sufficient for this interaction. Furthermore, the N terminus of DAT is capable of binding purified synaptic vesicles from brain tissue. Functional assays revealed that synaptogyrin-3 expression correlated with DAT activity in PC12 and MN9D cells, but not in the non-neuronal HEK-293 cells. These changes were not attributed to changes in transporter cell surface levels or to direct effect of the protein-protein interaction. Instead, the synaptogyrin-3 effect on DAT activity was abolished in the presence of the vesicular monoamine transporter-2 (VMAT2) inhibitor reserpine, suggesting a dependence on the vesicular DA storage system. Finally, we provide evidence for a biochemical complex involving DAT, synaptogyrin-3, and VMAT2. Collectively, our data identify a novel interaction between DAT and synaptogyrin-3 and suggest a physical and functional link between DAT and the vesicular DA system.

EVALUATION OF THE EFFECTIVENESS OF ANISOTROPIC ANALYTICAL ALGORITHM IN FLATTENED AND FLATTENING-FILTER-FREE BEAMS FOR HIGH ENERGY LUNG DOSE DELIVERY USING THE RADIOLOGICAL PHYSICS CENTER LUNG PHANTOM

The effectiveness of the Anisotropic Analytical Algorithm (AAA) implemented in the Eclipse treatment planning system (TPS) was evaluated using theRadiologicalPhysicsCenteranthropomorphic lung phantom using both flattened and flattening-filter-free high energy beams. Radiation treatment plans were developed following the Radiation Therapy Oncology Group and theRadiologicalPhysicsCenterguidelines for lung treatment using Stereotactic Radiation Body Therapy. The tumor was covered such that at least 95% of Planning Target Volume (PTV) received 100% of the prescribed dose while ensuring that normal tissue constraints were followed as well. Calculated doses were exported from the Eclipse TPS and compared with the experimental data as measured using thermoluminescence detectors (TLD) and radiochromic films that were placed inside the phantom. The results demonstrate that the AAA superposition-convolution algorithm is able to calculate SBRT treatment plans with all clinically used photon beams in the range from 6 MV to 18 MV. The measured dose distribution showed a good agreement with the calculated distribution using clinically acceptable criteria of ±5% dose or 3mm distance to agreement. These results show that in a heterogeneous environment a 3D pencil beam superposition-convolution algorithms with Monte Carlo pre-calculated scatter kernels, such as AAA, are able to reliably calculate dose, accounting for increased lateral scattering due to the loss of electronic equilibrium in low density medium. The data for high energy plans (15 MV and 18 MV) showed very good tumor coverage in contrast to findings by other investigators for less sophisticated dose calculation algorithms, which demonstrated less than expected tumor doses and generally worse tumor coverage for high energy plans compared to 6MV plans. This demonstrates that the modern superposition-convolution AAA algorithm is a significant improvement over previous algorithms and is able to calculate doses accurately for SBRT treatment plans in the highly heterogeneous environment of the thorax for both lower (≤12 MV) and higher (greater than 12 MV) beam energies.

INDUCTION OF STRONGER AND LONG LASTING VACCINE IMMUNITY TO TUBERCULOSIS

Tuberculosis is a major cause of death due to an infection in mankind. BCG vaccine protects against childhood tuberculosis although, it fails to protect against adult tuberculosis. BCG vaccine localizes to immature phagosomes of macrophages, and avoids lysosomal fusion, which decreases peptide antigen production. Peptides are essential for macrophage-mediated priming of CD4 and CD8 T cells respectively through MHC-II and MHC-I pathways. Furthermore, BCG reduces the expression of MHC-II in macrophages of mice after infection, through Toll-like receptor-1/2 (TLR-1/2) mediated signaling. In my first aim, I hypothesized that BCG-induced reduction of MHC-II levels in macrophages can decrease CD4 T cell function, while activation of other surface Toll-like receptors (TLR) can enhance CD4 T cell function. An in vitro antigen presentation model was used where, TLR activated macrophages presented an epitope of Ag85B, a major immunogen of BCG to CD4 T cells, and T cell derived IL-2 was quantitated as a measure of antigen presentation. Macrophages with BCG were poor presenters of Ag85B while, TLR-7/9/5/4 and 1/2 activation led to an enhanced antigen presentation. Furthermore, TLR-7/9 activation was found to down-regulate the degradation of MHC-II through ubiquitin ligase MARCH1, and also stimulate MHC-II expression through activation of AP-1 and CREB transcription elements via p38 and ERK1/2 MAP kinases. I conclude from Aim-I studies that TLR-7/9 ligands can be used as more effective ‘adjuvants’ for BCG vaccine. In Aim-II, I evaluated the poor CD8 T cell function in BCG vaccinated mice thought to be due to a decreased leak of antigens into cytosol from immature phagosomes, which reduces the MHC-I mediated activation of CD8 T cells. I hypothesized that rapamycin co-treatment could boost CD8 T cell function since it was known to sort BCG vaccine into lysosomes increasing peptide generation, and it also enhanced the longevity of CD8 T cells. Since CD8 T cell function is a dynamic event better measurable in vivo, mice were given BCG vaccine with or without rapamycin injections and challenged with virulent Mycobacterium tuberculosis. Organs were analysed for tetramer or surface marker stained CD8 T cells using flow cytometry, and bacterial counts of organisms for evaluation of BCG-induced protection. Co-administration of rapamycin with BCG significantly increased the numbers of CD8 T cells in mice which developed into both short living effector- SLEC type of CD8 T cells, and memory precursor effector-MPEC type of longer-living CD8 T cells. Increased levels of tetramer specific-CD8 T cells correlated with a better protection against tuberculosis in rapamycin-BCG group compared to BCG vaccinated mice. When rapamycin-BCG mice were rested and re-challenged with M.tuberculosis, MPECs underwent stronger recall expansion and protected better against re-infection than mice vaccinated with BCG alone. Since BCG induced immunity wanes with time in humans, we made two novel observations in this study that adjuvant activation of BCG vaccine and rapamycin co-treatment both lead to a stronger and longer vaccine-mediated immunity to tuberculosis.