Serum Levels of Alpha-1 Antitrypsin following Vascular Limb or Intra-Muscular Delivery of AAV1 or AAV8 Gene Therapy Vectors in Rhesus Macaques
Alpha-one antitrypsin (AAT) deficiency is a genetic disease that results in both lung disease and potentially liver failure in affected patients. In un-affected people AAT is produced in the liver and secreted to act as an anti-protease (primarily counteracting the effects of neutrophil elastase) in the lung. On-going human clinical trials have focused on intra-muscular delivery of adeno-associated virus (AAV1) to patients. The goal of delivery to the muscle is to have the myocytes serve as bio-factories to produce normal AAT protein and secrete it into the blood where it can exert its normal function in the lung. In the last Phase II trial patients in the highest dose cohort were given 100 intra-muscular (IM) injections with serum AAT levels still below therapeutic thresholds.
Previous work has shown that delivering AAV vector to the musculature of the limb via the vasculature, while blood flow is obstructed using a tourniquet, leads to wide-spread gene expression in myocytes. We hypothesize that local delivery via IM injection results in saturated AAT expression within the myocytes surrounding the injection sight and that a more widespread delivery would result in an overall increase in serum AAT levels with the same dose of AAV gene therapy vector due to production by a larger overall number of myocytes. We have been able to show that we can attain similar or slightly higher (573.0 ng/ml versus 562.5 ng/nl) serum AAT levels using a vascular delivery method in rhesus macaques when compared to IM delivery. These results have been obtained using AAV1. Animals receiving either AAV1 or AAV8 show a decrease in muscle immune cell infiltrates following intra-vascular delivery versus IM delivery, which may improve long-term expression. Serum AAT data from animals dosed using AAV8, a serotype shown to better target muscle following vascular delivery, are currently being processed.
Obesity is the most prevalent chronic disease of childhood. Obese adolescents are likely to become obese adults with significant associated co-morbidities and early mortality. In Massachusetts, 30% of children ages 10-17 are overweight or obese. It is projected that 48% of Massachusetts’ adults will be obese by 2030. In March of 2015, the Good Fit Adolescent Weight and Wellness Center opened, with a goal of addressing this issue with a proven multidisciplinary approach.
Mobile technology continues to develop at a rapid pace. Adolescent access to mobile technology on smart phones and tablets continues to increase. Mobile fitness tracker applications are numerous and easy to use for today’s tech savvy teens. Successful weight loss and health maintenance is variable and has been difficult to validate with this technology so far.
The purpose of this study is to evaluate existing mobile applications to be used by adolescent patients in the Good Fit Center. Our aim is to determine whether adolescent patients will be compliant with diet and exercise challenges sent through a mobile application and social media platform. We will work closely with dietitians, physicians and surgeons to evaluate feasibility and compliance within the first year of this project. In the second year, we will then test the functionality of this mobile application as it relates to patient success in the Good Fit program.
The proposed research is a novel multimodal study combining behavioral sciences research, clinical outcomes research, and mobile technology to help to better understand the fitness management of adolescents struggling with morbid obesity. The findings of our research may have a number of important implications. These include the refinement of existing fitness strategies, as well as the development of a new useful piece of technology to combat obesity and improve the health and clinical outcomes of our nation’s children.
Melanoma, the leading cause of skin cancer death in the U.S., is increasing in incidence. Targeted therapies have been approved for treatment of advanced melanoma, but few patients experience extended survival benefit. In order to combat poor outcomes, new therapeutic targets are needed. Using cross-species oncogenomic analyses, our lab has identified a novel melanoma driver, Growth differentiation factor 6 (GDF6), a secreted bone morphogenetic protein (BMP) ligand that is amplified and overexpressed in human melanomas. Functional analyses show GDF6 acts via the BMP-SMAD1 pathway as a pro-survival factor in melanomas. Inhibiting GDF6 or the BMP pathway using shRNAs or the small molecule inhibitor, DMH1, induces melanoma cell death thereby abrogating melanoma growth in mouse xenografts. These results suggest GDF6 is an optimal target melanoma therapy. In order to better understand the dynamics of GDF6 signaling in melanoma cells, we are currently investigating the effect of exogenous GDF6 on cells with inhibited GDF6 expression to determine the required concentration to activate SMAD1 signaling and rescue viability. As GDF6 is a secreted ligand, we proposed developing antibodies to block the GDF6 interaction at its receptor, thereby inhibiting signaling. In collaboration with MassBiologics, we have generated a panel of monoclonal antibodies targeting GDF6. To identify antibodies capable of blocking GDF6 activity, we have devised a series of assays to eliminate antibodies from the panel. First, candidates are screened for affinity to GDF6. Second, candidates are screened for ability to block interaction between GDF6 and its receptor. Third, candidates are evaluated for ability to inhibit downstream signaling via SMAD1 pathway. After selection of final candidates, we will use a xenograft model to determine ability to inhibit melanoma growth in vivo. Currently, we have identified antibodies that are able to recognize GDF6 via western blot, and are proceeding to screen these antibodies for anti-GDF6 activity.
Burden of Adverse Metabolic Factors Is Associated With Increased Left Ventricular Concentricity in Adults With Normal-Range Body Mass Index: The Framingham Heart Study
Introduction: Persons with normal-range body mass index (BMI) but adverse metabolic characteristics associated with obesity have been described as metabolically-obese normal weight (MONW). We sought to determine whether adverse metabolic profile is associated with alterations in left ventricular (LV) structure or function among adults with normal BMI. Methods: From the 1794 Framingham Heart Study Offspring cohort adults who underwent cardiac magnetic resonance imaging (CMRI) , we identified 446 free of non-skin cancer and prevalent clinical cardiovascular disease (CVD) who had 18.5≤BMI<25.0 kg/m2 and complete covariates. We calculated a metabolic score (MS) where 1 point was assigned for each of: a) fasting glucose≥100 mg/dL or diabetes; b) SBP≥140 or DBP≥90 mmHg or antihypertensive treatment; c) TG≥150 or HDL_C <40(M)/<50(W) mg/dL or lipid treatment; d) HOMA-IR≥2.5; e) waist circumference ≥102/88cm for M/W. Participants were classified as MS0 (no points), MS1 (exactly 1 point), or MS2+ (≥2 points). LV mass (LVM), end-diastolic volume (EDV), ejection fraction (EF), and concentricity (LVM/EDV) were measured from breathhold cine SSFP CMR scans; we calculated LVM/BSA. Analysis of covariance (ANCOVA) was used to compare MS1 and MS2+ groups to the MS0 group. CMRI variables were adjusted for sex, age, heart rate (HR) and body size (BSA); LVM/BSA was adjusted for sex, age, HR only. We also tested for linear trend across metabolic groups. Results: LV concentricity increased with worsening metabolic status. This was driven by lower LV EDV, not increased LVM. LVM did not differ across (trend) or between MS-groups. LVEDV decreased across groups but only MS2 differed significantly from MS0. LVEF increased slightly but significantly across MS-groups. Conclusions: In a community-dwelling cohort, among participants who were free of cancer and clinical CVD and had normal BMI, worsening metabolic profile was associated with adverse remodeling of the left ventricle, reflected by greater LV concentricity.
Sex differences in cognition, emotional reactivity, and motor ability in gonadally-intact middle-aged marmosets (Callithrix jacchus)
Sex differences in cognition are well documented. Women outperform men on measures of perceptual speed and verbal abilities, while men outperform women on tests of spatial processing. Robust sex differences also exist in stress responses. However, it is unclear how these sex differences change over time and whether males and females follow different trajectories of age-related cognitive decline. Studies in nonhuman primate models can help resolve this issue. The common marmoset (Callithrix jacchus) is a New World primate with a short lifespan that can perform complex cognitive tasks in computerized settings that are comparable to those used with humans. The present study is part of a longitudinal project aimed at determining whether males and females follow different trajectories of cognitive aging. This report focuses on sex differences at study entry. Thirteen marmosets (7 females), aged 4-6 years were tested on a comprehensive battery of tasks assessing cognitive function, motor skills and emotional reactivity. For cognition, monkeys were initially trained on a simple visual discrimination problem, followed by reversal learning using the Cambridge Neuropsychological Test Automated Battery (CANTAB). They also performed the Hill-and-Valley task as a measure of fine motor skills. To assess emotional reactivity, each marmoset was separated from their colony for 7 hours. Behavioral assessments, which involved recording the occurrence of approximately 25 behaviors, occurred a total of 6 times: immediately before separation, 3 times during separation, immediately after separation, and 24-hr later. No sex difference was found for simple discrimination, but males tended to perform better than females on the reversal learning task. No sex difference was observed in motor skills. During separation from the colony, females were more reactive than males, as indicated by more agitated locomotion, and vocalizations. Together, these findings expand upon previous studies and demonstrate sex differences in reversal learning and emotional reactivity in gonadally-intact middle-aged marmosets. As the study progresses, we should be able to determine the neural correlates of these sex differences and how they may change with aging. Supported by NIH grant AG046266
Background: Despite the length of time HIV has been wreaking havoc on its victims, improvements in the prevention and treatment of HIV are needed. Anti-retroviral therapy can be effective but is expensive and not entirely accessible for people infected in third world countries. Several promising broadly neutralizing antibodies have been isolated from infected individuals; we propose that generating antigen specific human monoclonal antibodies using humanized mice further represents a promising approach to engineer prophylactic antibodies to reduce spread and infection of HIV.
Methods: Immunodeficient mice were engrafted with fetal liver and thymus (BLT) prior to infection with different HIV isolates. HIV infection of the mice was monitored by viral load and antibody response followed by ELISA using gp120, gp41 or gp120/CD4 complex as antigens. Approximately 8-12 weeks post infection, spleens were harvested and splenocytes fused with human fusion partner HMMA 2.5 to isolate antibody-expressing hybridomas. Lead clones were scaled and purified for testing in functional assays such as TZM-bl neutralization assays as well as ADCVI to determine neutralizing and cytotoxic ability of the antibodies. Antibody sequences were also determined for analysis.
Results: A robust, specific antibody response, of both IgG and IgA isotypes, was generated in response to HIV infection. Over 60 hybridomas were created that were not only immunoreactive with env antigens, but also had neutralization activity. Moreover, variable family usage was not limited and somatic mutation was clearly evident.
Conclusions: These findings suggest that humanized BLT mice are a novel source for well-characterized, stable human monoclonal antibodies to HIV.
Purpose: ErbB2 signaling appears to be increased and may enhance AR activity in a subset of CRPC, but agents targeting ErbB2 have not been effective. This study was undertaken to assess ErbB2 activity in abiraterone-resistant prostate cancer (PCa), and determine whether it may contribute to androgen receptor (AR) signaling in these tumors.
Experimental Design: AR activity and ErbB2 signaling were examined in the radical prostatectomy specimens from a neoadjuvant clinical trial of leuprolide plus abiraterone, and in the specimens from abiraterone-resistant CRPC xenograft models. The effect of ErbB2 signaling on AR activity was determined in two CRPC cell lines. Moreover, the effect of combination treatment with abiraterone and an ErbB2 inhibitor was assessed in a CRPC xenograft model.
Results: We found that ErbB2 signaling was elevated in residual tumor following abiraterone treatment in a subset of patients, and was associated with higher nuclear AR expression. In xenograft models, we similarly demonstrated that ErbB2 signaling was increased and associated with AR reactivation in abiraterone-resistant tumors, while ERBB2 message level was not changed. Mechanistically, we show that ErbB2 signaling and subsequent activation of the PI3K/AKT signaling stabilizes AR protein. Inhibitors targeting ErbB2/PI3K/AKT pathway disrupt AR transcriptional activity. Furthermore, concomitantly treating CRPC xenograft with abiraterone and an ErbB2 inhibitor, lapatinib, blocked AR reactivation and suppressed tumor progression.
Conclusions: ErbB2 signaling is elevated in a subset of abiraterone-resistant prostate cancer patients and stabilizes AR protein. Combination therapy with abiraterone and ErbB2 antagonists may be effective for treating the subset of CRPC with elevated ErbB2 activity.
Characterization of neutrophils and macrophages from ex vivo cultured murine bone marrow for morphologic maturation and functional responses by imaging flow cytometry
Neutrophils and macrophages differentiate from common myeloid progenitors in the bone marrow, where they undergo unique nuclear morphologic changes as they mature into fully functional phagocytes. These changes include condensation of chromatin, the most pronounced exhibited by mature neutrophils. Both myeloid cells acquire multiple functions critical to their ability to kill pathogens, including phagocytosis, the production of proteolytic enzymes and reactive oxygen species (ROS), and in the case of neutrophils, release of nuclear material known as nuclear extracellular traps (NETs). Studies on these functions often rely on the use of cells acquired from mature mouse tissues, but these tend to produce limited numbers of cells. Strategies to analyze the morphologic features and functional responses of these cells include the use of conventional brightfield or fluorescence microscopy to examine changes in nuclear structure, internalization of fluorescein-labeled bacterial or yeast particles, or release of nuclear material. Flow cytometry also is often used, especially for identifying changes in the expression of lineage-specific cell surface markers and ROS production. However, each of these techniques presents certain limitations. Here we describe methods to generate abundant populations highly enriched for neutrophils or macrophages from previously frozen, ex vivo cultured mouse bone marrow. We then apply state-of-the-art imaging flow cytometry, which combines the resolution of microscopy with the speed of flow cytometry, to analyze each lineage for changes in nuclear structure and expression of key cell surface markers. Different gating and masking strategies are applied to characterize phagocytosis of pH-dependent fluorescein-labeled E. coli, ROS production, and NET release by neutrophils. We also demonstrate that neutrophils engulfing E. coli bioparticles produce NETs in a process we term PhagoNETosis. Together these assays reveal the power of imaging flow cytometry for simultaneously assessing the maturation features and functional responses of these critical mediators of innate immunity.
Transferrin Conjugated Polymeric Nanomedicine for Targeting Pancreatic Cancer using Paclitaxel and Gemcitabine
Pancreatic cancer (PanCa) has a dismal prognosis with five-year survival rates under 5%. PanCa is usally diagnosed at very late stages and even if diagnosed early, surgery is rarely an option. These factors contribute towards the bleak statistics for PanCa Chemo and radiation treatments having deleterious side-effects. There is therefore a clinical, unmet need for novel, targeted treatments with low morbidity in PanCa. Gemzar® (gemcitabine-HCl) is an FDA (Food and Drug Administration) approved chemotherapeutic drug that has been used to treat PanCa. However, intrinsic and acquired chemoresistance to gemcitabine contribute to the poor prognosis of PanCa. A combination of Abraxane® (albumin-stabilized paclitaxel nano-formulation) with gemcitabine has shown survival benefits and has now become the first line treatment for PanCa. Desmoplasia is a fundamental characteristic of PanCa that contributes significantly to its chemoresistance, making drug delivery to PanCa cells difficult. Nanomedicines combining multiple drugs can be designed to overcome this hurdle. This project aims at developing a targeted nanomedicine by using a combination of gemcitabine and paclitaxel encapsulated in polymeric nanoparticles for the treatment of PanCa. Oil/ water emulsion technique was employed for the preparation of poly (lactic-co-glycolic acid) (PLGA) nanoparticles encapsulating gemcitabine and paclitaxel. Synthesis protocols yielded drug-loaded PLGA nanoparticles with an average diameter less than 200 nm, with encapsulation efficiencies ranging from 40-70%. In vitro tests for cell viability studies using the MTT assay demonstrated lower cell viability in AsPC-1 cells when treated with these nano-formulations as compared to their free-drug counterparts. Current studies include conjugating drug-loaded PLGA-polyethylene glycol-Maleimide nanoparticles with transferrin peptide for targeted therapy, which is expected to prove more efficacious when tested for cell viability in vitro than its non-targeted formulations that have been obtained. These results therefore certify this nanotherapeutic approach as a potential therapy for PanCa.
A Novel Population Of Natural Killer Cells Pplays A Critical Role in the Depletion of Splenic B2 B Cells During Experimental Africian Trypanosomiasis
Mice infected with Trypanosoma brucei, the causative agent of human sleeping sickness and a contributor to nagana in cattle, rapidly lose the capacity to mount VSG-specific antibody responses, and die with uncontrolled parasitemia. We have shown (Bockstal et al., 2011, PLOS Pathogens) that the loss of humoral immune competence in the infected mice results from depletion of developing and mature splenic B cells. We now report that T. brucei-induced splenic B cell depletion is dependent upon the presence of the pore forming molecule perforin which is present in the cytotoxic granules of cytotoxic T lymphocytes, natural killer T cells and natural killer cells, occurs in the absence of T cells (and natural killer T cells), i.e., in T cell receptor (αβγδ)-/- mice, but does not occur in intact mice that are depleted of natural killer (NK) cells by treatment with monoclonal antibody specific for the NK1.1 differentiation antigen. In the intact mice, B cells are deleted after remission of the first T. brucei parasitemic wave. At this time natural killer cells are expressing the cytotoxic granule marker CD107a, indicating that they have degranulated, executing their effector function. Moreover, in vitro assays show that B cells from T. brucei infected mice are killed by natural killer cells from uninfected C57BL/6 mice but not efficiently killed by CD107a positive natural killer cells isolated from infected mice, which may be functionally exhausted.
The Billiar lab is interested in the interplay between mechanical tension and programmed cell death (namely, apoptosis) in cells growing on micro-contact printed aggregates. The Billiar lab uses a bioinspired hydrogel to develop an in vitro model for mechanosensitive signaling in mammalian cells. The micro-contact printed cell aggregates experience a loss of tensional homeostasis at the center of the aggregates, which results in selective cell death at the center, but not periphery of the aggregates, followed by calcification, similar to excised diseased aortic valves. However, the subcellular mechanisms responsible for transducing the mechanical cues from the loss of tensional homeostasis to pro-apoptotic signaling have yet to be elucidated; the Billiar lab is interested in finding this link.
Mechanotransduction is the functional link between mechanical cues and the consequent subcellular biochemical response.1 Cells sense and respond to their physical surroundings via cell-cell junctions, cell-matrix adhesions, and intracellular actin networks.1 For example, in epithelial cells, restriction of cell growth to spatially patterned circular arrays leads to (1) increased proliferation and (2) higher tractional stresses at the periphery than at the center.2-3 Proliferation at the periphery of these circular cell aggregates is YAP-dependent, with nuclear localization of YAP at the periphery.4 Transcriptional co-activator YAP is (1) a nuclear relay of mechanical signals,5 (2) the main transcriptional effector of the Hippo pathway,6 and (3) involved in both proliferation (via TEAD promoter) and apoptosis (via p73 promoter).7 Cell competition is an apoptosis-dependent cell communication phenomenon based on cell fitness comparisons, and which creates “loser” cells that die via apoptosis and “winner” cells that survive.8-9 For example, co-culture of TEAD-activity-manipulated fibroblasts with WT induces cell competition, in which cells with higher TEAD activity “won,” and cells with lower TEAD activity “lost” (underwent apoptosis).10
Hypothesis: Culture of fibroblasts in geometrically constrained, circular cell aggregates induces cell competition via the formation of “winner” and “loser” cell populations due to differences in tensional homeostasis experienced at the periphery vs. center of the aggregates.
Background: Stroke and myocardial infarction have been reported to occur after the development of herpes zoster (shingles), a common and preventable disease.
Purpose: To evaluate literature describing the association between herpes zoster and its subtypes with the occurrence of cardiovascular events.
Data Sources: PubMed, SCOPUS (Embase), OAIster, Google Scholar (searched in January 2016)
Study Selection: Studies published up to January 2016 examining the association between herpes zoster or subtype of herpes zoster with the occurrence of cardiovascular events, including stroke, transient ischemic attack, or an acute coronary event, were selected. Case reports, case studies, and studies of non-general adult populations were excluded.
Data Extraction: Data from studies meeting criteria were abstracted on a standardized form, and evaluated following modified set of standard guidelines.
Data Synthesis: Nine published articles, with study populations ranging from 2,632 to 4,620,980 patients, met our pre-defined eligibility criteria. Eight studies found at least one positive association between herpes zoster type unspecified and subsequent stroke, transient ischemic attack, or an acute coronary event. Five studies found positive associations between herpes zoster ophthalmicus and stroke or myocardial infarction. Subgroup analyses from three studies were inconsistent regarding the association of cardiovascular events with receipt of antiviral therapy for herpes zoster.
Limitations: Excludes non-English publications and non-published evidence.
Conclusions: A small number of studies showed greater risks of stroke, transient ischemic attack, and acute cardiac events following the development of herpes zoster and herpes zoster ophthalmicus. Further prospective studies should develop strategies to reduce the risk of cardiovascular disease among patients with herpes zoster.
The impact of changing guidelines on prostate cancer screening in a population-based setting, 2000-2014
Introduction: This study evaluates the potential impact of the publication of conflicting prostate cancer (PCa) screening trial results in 2009 and changes to the US Preventive Services Task Force (USPSTF) guidelines to recommend against screening in 2012 on temporal trends in PSA testing at two participating sites in the NCI-funded Cancer Research Network.
Methods: Study participants were men aged 40-80 without a history of PCa who sought care at Fallon Health (Worcester, MA) or Henry Ford Health System (Detroit, MI) between 2000-2014. We used health claims and electronic health record data to identify men who underwent PSA testing per calendar year. We also examined trends in PSA testing among high-risk men (African-American, family history of PCa). Testing rates were compared between 2000-2008, 2009-2012, and 2013-2014.
Results: From a population of 279,350 eligible men, 133,038 (48%) had at least one PSA test during the study period. Mean age at PSA test was 57 years, which increased over time at both sites. Overall, PSA testing rates rose between 2000-2008 (27-32% of eligible men per year), but declined between 2009-2012 (25% of eligible men). Testing rates declined further in 2013-2014 (23% of eligible men). We observed similar rates of decline in testing for men aged 55-69 and those aged ≥70. High-risk men were less likely to be screened across all time periods, although data was limited.
Conclusions: This analysis of two population-based electronic health datasets provides evidence of a recent decrease in PSA testing, following an increase in the early 2000s. Although we are unable to determine causality, it is plausible that results of recent screening trials and/or changes to the USPSTF guidelines have impacted PSA testing practices over the past 14 years.
For many parasitic diseases, high-throughput phenotypic screening is an important tool in finding new drugs. Some of the most important parasitic diseases are caused by nematodes. However, these parasitic nematodes are not typically amenable to high throughput screening. Due to the ease of its maintenance and suitability for high throughput assay, the nematode Caenorhabditis elegans is instead used. To address whether C. elegans is a good model for nematode drug discovery, we compared the drug susceptibility of C. elegans relative to the human hookworm nematode parasite Ancylostoma ceylanicum at several developmental stages using a library of FDA approved drugs. I will present results of these studies that point to how well C. elegans efficacy correlates with hookworm efficacy and how early larval stages (easier to get) correlated with adult stages (more representative of what stage is targeted in human therapy). In addition, we are working on moderate-high throughput system for screening adult parasites. Murine Holigmosomoides polygyrus is a good model for human parasitic nematodes. Using Union Biometrica, Copas, worm sorter we were able to sort H. polygyrus into 384 well format. Here I will discuss the capabilities of this system as well as how we are building de novo, in collaboration with the Albrecht laboratory at WPI, an imaging and image analysis platform for screening adult stages of this parasite against large drug libraries.
Through comparative oncogenomic studies and functional analyses, we have identified the bone morphogenetic protein (BMP) factor GDF6 as a new melanoma oncogene. The secreted, carboxy-terminal portion of GDF6 is the active form that binds to cell-surface receptors to initiate BMP signaling. Targeted antibodies directed against secreted proteins are a proven therapeutic modality in several diseases.
To develop therapeutic antibodies against the active form of GDF6, we generated a panel of monoclonal antibodies. Due to the high similarity of human and mouse GDF6 proteins, the C-terminal GDF6 protein was expressed as bacterial recombinant protein with fusion tags to enhance immunogenicity. The Expresso Screening System (Lucigen) was used to select fusion tags, and MBP and SlyD were chosen for optimal protein solubility and purification recovery. Ten CD1 mice were immunized with GDF6-MBP fusion protein and robust immune responses were observed in all animals after 5 immunizations. Animals were sacrificed for hybridoma fusion, and hybridoma clones were screened by ELISA using GDF6-SlyD fusion protein to select clones with specific binding activity to GDF6. Over 70 monoclonal antibodies were identified with strong reactivity to GDF6, and a subset has been shown to recognize the endogenous, secreted form of GDF6 via western blot. These antibodies will be screened for their activity to block GDF6 binding to melanoma cells and ability to inhibit downstream signaling using both in vitro assays and in vivo xenograft models.
HIV vaccine efforts tend to focus on the induction of IgG neutralizing antibodies. In part, this may stem from the observations that most HIV infected individuals fail to produce significant mucosal IgA. However, this is unlike most other infections and in turn it can be argued that mucosal IgA with appropriate function and specificity may contribute significantly to the prevention of HIV transmission. To explore this, we previously isotype switched F425A1g8, an anti-HIV CD4i human monoclonal antibody that binds to epitopes exposed upon CD4 binding (CD4i) The VH and VL chains were amplified from the IgG hybridoma and inserted into IgA1 or IgA2 and IgKappa vectors respectively. Stable cells lines were produced and antibody was collected and purified. Initial results showed that the IgA1 variant neutralized a number of HIV-1 isolates better than its parental form IgG1. We believe the increased neutralization of HIV is mainly due to the structural differences between IgG1 and IgA1. We hypothesize that the extended hinge of IgA1 may increase segmental flexibility and change the interaction of antibody with CD4i epitopes of the HIV, resulting in greater avidity. To look at this further, we have generated monomeric and dimeric IgA1 and IgA2 variants of three different CD4i antibodies: F425A1g8, 17b and E51. All antibody variants will be tested for immumoreactivity, HIV neutralization, prevention of transmission and ADCVI activity. Consistent with our previous results, there are significant differences in functional activity of the other CD4i antibodies with IgA1 more effective than the IgA2 variants. Additional activities will contribute to the hypothesis that the extended hinge region of the IgA1 antibody increases the antibodies ability to access the CD4i epitopes upon HIV-1 binding to CD4. These studies should impact on the design of active and passive immunotherapy and the prevention of HIV transmission.
Predicting Early Failure in Total Knee Arthroplasty: A Critical Review of Oxinium Femoral Components
Introduction: Retrospectively, it has been shown that significant patient-reported pain 6 months following total knee arthroplasty (TKA) is associated with a 7 times greater revision rate at 5 years. Our goal is to use the FORCE-TJR registry to prospectively evaluate if postoperative pain and function scores can predict increased revision rate 5 years following TKA. Our preliminary analyses have focused on one implant reported by Australia to have a significantly high 5-year revision rate: Oxinium femoral components. Materials and Methods: FORCE-TJR matched implant catalog numbers to the international implant library to define TKA patients who received oxinium femoral components and all other implants. We defined 12-month KOOS pain and function (SF PCS) for patients with the study implant and all others (n=9187). Age, BMI, sex, pre-TKA pain, function, low back pain severity, and Charlson comorbidity index were compared for patients with moderate pain (KOOS pain<75) vs. minimal pain (KOOS pain>75) at 12 months postoperatively. Results: We observed that 27% of oxinium patients reported moderate pain vs. 21% of patients receiving all implants at 12 months postoperatively. Compared to patients with minimal pain, moderate pain patients had greater pre-op pain (KOOS=37 vs. 50; p< 0.0002), poorer pre-op function (PCS=30 vs. 33; p<0.04), and more moderate to severe low back pain (52% vs. 24%; p<0.027). In addition, high 12-month pain patients had poorer 12-month function (PCS=37 vs. 45; p<0.0000). Conclusion: These preliminary results indicate that moderate pain at 12-months post operatively is associated with poorer functional gain following TKA. Surgeons should recognize and potentially intervene on this group if improvement in their ultimate functional gain is desired. By continuing to follow this group of oxinium patients we will be able to determine if early pain and decreased function following TKA is associated with an increased revision rate.
Abstract This clinical trial in autism spectrum disorder (ASD) tests a nontoxic approach to therapy of ASD.
Background: Direct treatment of underlying mechanisms in ASD is limited. Cellular dysfunction in ASD may involve a number of related metabolic pathways. A clinical clue may be found in the “fever effect” in ASD, in which febrile illness dramatically but temporarily ameliorates disordered behavior. Fever stimulates heat shock proteins (HSP) and cellular stress responses that may ultimately lead to improved synaptic function and increased long-range connectivity. The expression of gene transcription by NFE2L2 (Nrf2), which is reduced in ASD, may also increase during fever. Sulforaphane (SF), an isothiocyanate obtained from 3-day-old broccoli sprouts, induces HSP and Nrf2 as well as “cell-protective” responses. SF has several possible modes of action that may benefit ASD through common cellular mechanisms underlying heterogeneous phenotypes. SF crosses the blood brain barrier and is bioavailable orally.
Preliminary data: In a randomized, double-blind, placebo-controlled pilot trial in 44 male adolescents and adults (13-30 years), results showed SF was well tolerated without significant side effects. On average, participants on SF (particularly those with a history of fever effect) showed significantly more improvements in ASD symptoms than placebo participants. Significant improvements for SF participants included social interaction, aberrant/abnormal behavior, repetitive/stereotypical behavior, and verbal communication.
Current study: Our randomized, double-blind, placebo-controlled phase-2 clinical trial at UMass has three aims: To determine: (1) if orally administered SF has measurable effects in children (ages 3-12 years) with ASD; (2) if treatment with sulforaphane is safe and well tolerated; (3) To elucidate cellular biomarkers that support the mechanisms of action of SF in ASD. We hypothesize that SF will have positive effects, and that these effects will be more marked and lasting in the developing – compared to the mature – brain.
Bioavailability of the Antimalarial Drug Artemisinin Delivered Orally as Dried Leaves of Artemisia annua: the Role of Solubility and Protein.
Malaria treatment using orally consumed dried leaves of the artemisinin producing GRAS plant Artemisia annua has recently shown promise. Previously we showed, oral consumption of A. annua dried leaves (DLA) yielded >40 times more artemisinin in the blood of mice than treatment with pure artemisinin. Using the Caco-2 cell culture model of the human intestinal epithelium, we also showed that compared to pure artemisinin, digested DLA doubled the permeability (Papp). Here, using simulated human digestion, we show that artemisinin solubility is about seven times higher in digestates of DLA than in digestates of pure artemisinin, likely contributing to its enhanced bioavailability. Digestion with pure artemisinin combined with levels of essential oils comparable to that in DLA increased the solubility of artemisinin 2.5 times indicating essential oils play a role in increasing artemisinin solubility. Interestingly, increasing the starting concentration of artemisinin in Caco-2 transport studies did not alter Papp. Considering malaria affects mostly young children and about 60% of the population experiences DLA as unpleasant tasting, we also tested several protein rich foods as potential flavor-masking agents for their effects on bioavailability. We showed that while taste was masked, peanuts and a peanut-based paste used to treat malnutrition, PlumpyNut, reduced artemisinin and flavonoid levels in simulated digestates, respectively, likely decreasing their bioavailability. Experiments to further investigate the role of several compounds such as camphor, a principle component of the essential oil fraction, and flavonoids on artemisinin solubility and bioavailability are ongoing. The results of these experiments are helping to explain the increased bioavailability afforded by DLA seen in mice.
Community-based Participatory Research to Promote Healthy Diet and Nutrition and Prevent and Control Obesity among African Americans: A Literature Review
The literature on community-based participatory research (CBPR) approaches for promoting healthy diet, nutrition, and preventing and controlling obesity in African American communities was systematically reviewed.
CBPR studies of diet, nutrition, and weight management among African Americans were identified from 1989 through October 31, 2015 using PubMed and CINAHL databases and MeSH term and keyword searches.
A total of 16 CBPR studies on healthy diet, nutrition, and weight management among African Americans were identified; outcome evaluation results were available for all but two. Of the remaining 14 studies, 11 focused on adults, 1 on children, and 2 on both children and adults. Eight studies employed CBPR methods to address diet, nutrition, and weight management in church settings. Four had a cluster randomized controlled design. Others had a pre-post test, quasi-experimental, or uncontrolled design. Only one study addressed four levels of the socioecological model; none addressed all five levels of the model. The identified studies indicate that CBPR approaches can be effective for promoting healthy diet, nutrition, and weight management among African American adults but there is a need for additional studies with rigorous study designs that overcome methodologic limitations of many existing studies. There is only limited evidence for the effectiveness of CBPR approaches for promoting healthy eating and weight control among African American children and adolescents
To address health disparities, additional CBPR studies are needed to promote healthy diet, nutrition, and weight management in African American communities. Of particular interest are multilevel CBPR studies that include interventions aimed at multiple levels of the socioecological model.