Pharmacological Chaperoning in Fabry Disease

Fabry Disease is an X-­‐linked lysosomal storage disorder characterized by a variety of symptoms including hypohydrosis, seizures, cardiac abnormalities, skin lesions, and chronic pain. These symptoms stem from a lack of functional endogenous α-­‐ Galactosidase A (α-­GAL), which leads to an accrual of its natural substrate. The severity of the disease symptoms can be directly correlated with the amount of residual enzyme activity. It has been shown that an imino sugar, 1-deoxygalactonojirimycin (DGJ), can increase enzymatic activity and clear excess substrate. This pH-­‐dependent chaperoning phenomenon is believed to arise from the presence of aspartic acid 170 in the active site. This key residue may become protonated at lower pH, preventing a buried salt bridge from being formed. We mutated this residue to an alanine, abolishing activity, and making traditional assays impractical. We have measured the KD of chaperone for this modified active site through crystallography. Previous crystallographic studies on this enzyme have also shown a preliminary second binding site on the surface of α-­Galactosidase that prefers the β-­Galactose anomer. When β-­Galactose binds it buries a greater surface area than when α‐Galactose binds to the active site. Binding of this site by a small molecule should stabilize the native state of the enzyme, but would be sterically occluded from inhibiting active site. We have probed this second site by soaking crystals of α‐Galactosidase with a small library of compounds

Time is Money: The True Cost of Helicopter EMS (HEMS)

Background: Multiple studies have been published that attempt to examine relevant outcomes of utilizing helicopter transport over traditional ground-based emergency medical services (EMS). Unfortunately, these studies suffer from poor methodology and are confounded by substantial differences in training and expertise of the personnel involved in the medical transfer. The EMS system in Central Massachusetts is unique in that when a physician from an outside hospital calls for an HEMS transport, the pilot determines if weather conditions permit to fly without having any further knowledge about the patient. If flying is deemed unsafe, the helicopter personnel will retrieve the patient by traditional ground ambulance. This allowed us to compare these two transfer modalities while eliminating the confounder of crew expertise. We hypothesize that the overall mortality of these inter-facility transfers is comparable, regardless of mode of transport and diagnoses, if accompanied by highly-trained and experienced personnel. Purpose: This study aimed to delineate the advantages of helicopter transport (HEMS) over ground transport (GEMS) of critically ill patients undergoing inter-facility transfers. Methods: We performed a retrospective chart review of 2,129 patients that were transferred from an outside hospital to our tertiary care center in Central Massachusetts by a Helicopter EMS (HEMS) crew between January of 2010 and April of 2017. These patients were either transferred by ground ambulance or helicopter ambulance. Transfers occurred by ground ambulance in limited cases where HEMS was grounded due to poor weather. In these instances, the Helicopter crew, consisting of a paramedic and flight nurse, staffed a ground ambulance to complete the transfer. Furthermore, we identified 655 patients that were transferred directly from the scene of the emergency, but these patients were excluded due to inability to standardize for transport distance. After categorizing 1,323 patients that were transferred by HEMS and 151 patients that were transferred by GEMS, patients were matched to their respective All Patients Refined Diagnosis Related Group (APR-DRG) and In-Hospital Mortality data. Scalar and categorical outcomes were analyzed T-Test or Fishers exact test respectively. Sub-group analyses were completed by categorizing patients by diagnosis or APR-DRG severity level. Results: The use of Helicopter EMS significantly reduced the time to definitive care over Ground EMS by at least twenty-one minutes, but no-more than one hour and seventeen minutes depending on the location of the requesting hospital. We found no statistical difference in the age or gender of the population transferred by either HEMS or GEMS. However, the population transferred by GEMS had significantly increased APR-DRG Severity and Risk of Mortality scores over the population transferred by HEMS. Despite this finding, there was no difference in in-hospital mortality between the two transfer modalities. Subgroup analysis did not show any significant difference in mortality based on diagnosis or APR- DRG classification. Conclusions: Despite the significant reduction in the time to definitive care through the utilization of HEMS, there was no statistically significant survival benefit conferred to critically ill patients undergoing inter-facility transfers. This suggests that the transport methodology may be of secondary importance to the level of training of the providers who are delivering the care en route

Systemic inhibition of myeloid dendritic cells by circulating HLA class I molecules in HIV-1 infection

<p>Abstract</p> <p>Background</p> <p>HIV-1 infection is associated with profound dysfunction of myeloid dendritic cells, for reasons that remain ill-defined. Soluble HLA class I molecules can have important inhibitory effects on T cells and NK cells, but may also contribute to reduced functional properties of professional antigen-presenting cells. Here, we investigated the expression of soluble HLA class I isoforms during HIV-1 infection and assessed their functional impact on antigen-presenting characteristics of dendritic cells.</p> <p>Results</p> <p>Soluble HLA class I molecules were highly upregulated in progressive HIV-1 infection as determined by quantitative Western blots. This was associated with strong increases of intracellular expression of HLA class I isoforms in dendritic cells and monocytes. Using mixed lymphocyte reactions, we found that soluble HLA class I molecules effectively inhibited the antigen-presenting properties of dendritic cells, however, there was no significant influence of HLA class I molecules on the cytokine-secretion properties of these cells. The immunomodulatory effects of soluble HLA class I molecules were mediated by interactions with inhibitory myelomonocytic MHC class I receptors from the Leukocyte Immunoglobulin Like Receptor (LILR) family.</p> <p>Conclusions</p> <p>During progressive HIV-1 infection, soluble HLA class I molecules can contribute to systemic immune dysfunction by inhibiting the antigen-presenting properties of myeloid dendritic cells through interactions with inhibitory myelomonocytic HLA class I receptors.</p

LILRB2 Interaction with HLA Class I Correlates with Control of HIV-1 Infection.

Natural progression of HIV-1 infection depends on genetic variation in the human major histocompatibility complex (MHC) class I locus, and the CD8+ T cell response is thought to be a primary mechanism of this effect. However, polymorphism within the MHC may also alter innate immune activity against human immunodeficiency virus type 1 (HIV-1) by changing interactions of human leukocyte antigen (HLA) class I molecules with leukocyte immunoglobulin-like receptors (LILR), a group of immunoregulatory receptors mainly expressed on myelomonocytic cells including dendritic cells (DCs). We used previously characterized HLA allotype-specific binding capacities of LILRB1 and LILRB2 as well as data from a large cohort of HIV-1-infected individuals (N = 5126) to test whether LILR-HLA class I interactions influence viral load in HIV-1 infection. Our analyses in persons of European descent, the largest ethnic group examined, show that the effect of HLA-B alleles on HIV-1 control correlates with the binding strength between corresponding HLA-B allotypes and LILRB2 (p = 10-2). Moreover, overall binding strength of LILRB2 to classical HLA class I allotypes, defined by the HLA-A/B/C genotypes in each patient, positively associates with viral replication in the absence of therapy in patients of both European (p = 10-11-10-9) and African (p = 10-5-10-3) descent. This effect appears to be driven by variations in LILRB2 binding affinities to HLA-B and is independent of individual class I allelic effects that are not related to the LILRB2 function. Correspondingly, in vitro experiments suggest that strong LILRB2-HLA binding negatively affects antigen-presenting properties of DCs. Thus, we propose an impact of LILRB2 on HIV-1 disease outcomes through altered regulation of DCs by LILRB2-HLA engagement

Light Forge: A Microfluidic DNA Melting-based Tuberculosis Test

BACKGROUND: There is a well-documented lack of rapid, low-cost tuberculosis (TB) drug resistance diagnostics in low-income settings across the globe. It is these areas that are plagued with a disproportionately high disease burden and in greatest need of these diagnostics. METHODS: In this study, we compared the performance of Light Forge, a microfluidic high-resolution melting analysis (HRMA) prototype for rapid low-cost detection of TB drug resistance with a commercial HRMA device, a predictive nearest-neighbor thermodynamic model, DNA sequencing, and phenotypic drug susceptibility testing (DST). The initial development and assessment of the Light Forge assay was performed with 7 phenotypically drug resistant strains of Mycobacterium tuberculosis (M.tb) that had their rpoB gene subsequently sequenced to confirm resistance to Rifampin. These isolates of M.tb were then compared against a drug-susceptible standard, H37Rv. Seven strains of M.tb were isolated from clinical specimens and individually analyzed to characterize the unique melting profile of each strain. RESULTS: Light Forge was able to detect drug-resistance linked mutations with 100% concordance to the sequencing, phenotypic DST and the nearest neighbor thermodynamic model. Researchers were then blinded to the resistance profile of the seven M.tb strains. In this experiment, Light Forge correctly classified 7 out of 9 strains as either drug resistant or drug susceptible. CONCLUSIONS: Light Forge represents a promising prototype for a fast, low-cost diagnostic alternative for detection of drug resistant strains of TB in resource constrained settings

Haboob Dust Storms and Motor Vehicle Collision-related Trauma in Phoenix, Arizona

Background: The Sonoran Desert region, encompassing most of southern Arizona, has an extreme climate that is famous for dust storms known as haboobs. These storms lead to decreased visibility and potentially hazardous driving conditions. In this study we evaluate the relationship between haboob events and emergency department (ED) visits due to motor vehicle collisions (MVCs) in Phoenix, Arizona.Methods: This study is a retrospective analysis of MVC-related trauma presentations to Phoenix, AZ, hospitals before and following haboob dust storms. These events were identified from 2009–2017 primarily using Phoenix International Airport weather data. De-identified trauma data were obtained from the Arizona Department of Health Services (ADHS) Arizona State Trauma Registry (ASTR) from seven trauma centers within a 10-mile radius of the airport. We compared MVC-related trauma using six- and 24-hour windows before and following the onset of haboob events.Results: There were 31,133 MVC-related trauma encounters included from 2009–2017 and 111 haboob events meeting meteorological criteria during that period. There was a 17% decrease in MVC-related ED encounters in the six hours following haboob onset compared to before onset (235 vs 283, P = 0.04), with proportionally more injuries among males (P &lt; 0.001) and higher mortality (P = 0.02). There was no difference in frequency of presentations (P = 0.82), demographics, or outcomes among the 24-hour pre-and post-haboob groups.Conclusion: Haboob dust storms in Phoenix, Arizona, are associated with a decrease in MVC-related injuries during the six-hour period following storm onset, likely indicating the success of public safety messaging efforts. Males made up a higher proportion of those injured during the storms, suggesting a target for future interventions. Future public-targeted weather-safety initiatives should be accompanied more closely by monitoring and evaluation efforts to assess for effectiveness

Haboob Dust Storms and Motor Vehicle Collision-related Trauma in Phoenix, Arizona

Background: The Sonoran Desert region, encompassing most of southern Arizona, has an extreme climate that is famous for dust storms known as haboobs. These storms lead to decreased visibility and potentially hazardous driving conditions. In this study we evaluate the relationship between haboob events and emergency department (ED) visits due to motor vehicle collisions (MVCs) in Phoenix, Arizona. Methods: This study is a retrospective analysis of MVC-related trauma presentations to Phoenix, AZ, hospitals before and following haboob dust storms. These events were identified from 2009–2017 primarily using Phoenix International Airport weather data. De-identified trauma data were obtained from the Arizona Department of Health Services (ADHS) Arizona State Trauma Registry (ASTR) from seven trauma centers within a 10-mile radius of the airport. We compared MVC-related trauma using six- and 24-hour windows before and following the onset of haboob events. Results: There were 31,133 MVC-related trauma encounters included from 2009–2017 and 111 haboob events meeting meteorological criteria during that period. There was a 17% decrease in MVC-related ED encounters in the six hours following haboob onset compared to before onset (235 vs 283, P = 0.04), with proportionally more injuries among males ( P < 0.001) and higher mortality ( P = 0.02). There was no difference in frequency of presentations ( P = 0.82), demographics, or outcomes among the 24-hour pre-and post-haboob groups. Conclusion: Haboob dust storms in Phoenix, Arizona, are associated with a decrease in MVC-related injuries during the six-hour period following storm onset, likely indicating the success of public safety messaging efforts. Males made up a higher proportion of those injured during the storms, suggesting a target for future interventions. Future public-targeted weather-safety initiatives should be accompanied more closely by monitoring and evaluation efforts to assess for effectiveness

Spearman correlation between LILRB2 binding strength and mVL in HIV-1-infected individuals.

<p>Spearman correlation between LILRB2 binding strength and mVL in HIV-1-infected individuals.</p

Effect of LILRB2-HLA binding strength and individual class I alleles on viral control (controllers vs. non-controllers) in white patients.

<p>Logistic regression model with stepwise selection included all <i>HLA</i> class I alleles with phenotypic frequencies of >2% and one of the A, B, C or ABC binding scores at a time. The results are shown for the p<0.05 cut-off. The C binding score did not stay in the model. ORs for binding scores reflect a change of 0.1 units.</p>1<p>stayed in the model with the p<0.01 cut-off but not with the p<0.001 cut-off.</p>2<p>stayed in the model with the p<0.01 and p<0.001 cut-offs.</p