Research Spotlight

Posted August 15th 2019

The migalastat GLP-HEK assay is the gold standard for determining amenability in patients with Fabry disease.

Raphael Schiffmann M.D.

Raphael Schiffmann M.D.

Schiffmann, R., D. G. Bichet, E. Benjamin, X. Wu and R. Giugliani (2019). “The migalastat GLP-HEK assay is the gold standard for determining amenability in patients with Fabry disease.” Mol Genet Metab Rep 20: 1-2 100494.

Full text of this article.

The pharmacological chaperone migalastat is indicated for the treatment of Fabry disease in patients with an amenable GLA variant. Amenability is determined by an in vitro, good laboratory practice (GLP)-validated assay using HEK293 cells (GLP-HEK assay) performed at a single, highly experienced, GLP-certified laboratory using rigorous standards and extensive analytical validation to limit inter-assay variability. The recent report by Oommen et al. entitled “Inter-assay variability influences migalastat amenability assessments among Fabry disease variants” showed, despite technical differences between a non-GLP-validated assay and the GLP-HEK assay, 53 out of the 59 GLA variants tested in the non-GLP assay matched the GLP-HEK amenability classification. Considering the non-GLP assay was done without identical procedures and validated quality standards as in the GLP-HEK assay, differences in results are expected. We noted at least two deviations from the GLP-HEK assay that likely account for the discrepancies reported for 6 variants. First, the GLP-HEK assay uses qPCR to directly measure the amount of transfected plasmid DNA for transfection efficiency control. The method employed by Oommen et al., an indirect measurement of co-transfected, secreted embryonic alkaline phosphatase (SEAP), may be inaccurate because overexpression of mutant α-galactosidase A (α-Gal A) can affect trafficking and secretion of SEAP. Second, Oommen et al. used the relative activity (% of wild type) instead of absolute activity (nmol/mg/h) to calculate the fold-increase in α-Gal A activity in response to migalastat, causing values for 4 variants to narrowly miss the amenability criteria . . . In conclusion, the concern over assay variability seems unfounded, since amenability to migalastat is determined in a single GLP-certified laboratory. We believe physicians can have a high level of confidence in the approved GLP-HEK assay, which identifies GLA variants with the potential to respond to migalastat. Of course, individual response will need to be assessed clinically. (Excerpts from text, p. 1.)


Posted August 15th 2019

New Developments in Hemodynamic Monitoring.

Michael A.E. Ramsay M.D.

Michael A.E. Ramsay M.D.

Scheeren, T. W. L. and M. A. E. Ramsay (2019). “New Developments in Hemodynamic Monitoring.” J Cardiothorac Vasc Anesth 33 Suppl 1: S67-s72.

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Hemodynamic monitoring is an essential part of the perioperative management of the cardiovascular patient. It helps to detect hemodynamic alterations, diagnose their underlying causes, and optimize oxygen delivery to the tissues. Furthermore, hemodynamic monitoring is necessary to evaluate the adequacy of therapeutic interventions such as volume expansion or vasoactive medications. Recent developments include the move from static to dynamic variables to assess conditions such as cardiac preload and fluid responsiveness and the transition to less-invasive or even noninvasive monitoring techniques, at least in the perioperative setting. This review describes the available techniques that currently are being used in the care of the cardiovascular patient and discusses their strengths and limitations. Even though the thermodilution method remains the gold standard for measuring cardiac output (CO), the use of the pulmonary artery catheter has declined over the last decades, even in the setting of cardiovascular anesthesia. The transpulmonary thermodilution method, in addition to accurately measuring CO, provides the user with some additional helpful variables, of which extravascular lung water is probably the most interesting. Less-invasive monitoring techniques use, for example, pulse contour analysis to originate flow-derived variables such as stroke volume and CO from the arterial pressure signal, or they may measure the velocity-time integral in the descending aorta to estimate the stroke volume, using, for example, the esophageal Doppler. Completely noninvasive methods such as the volume clamp method use finger cuffs to reconstruct the arterial pressure waveform, from which stroke volume and CO are calculated. All of these less-invasive CO monitoring devices have percentage errors around 40% compared with reference methods (thermodilution), meaning that the values are not interchangeable.


Posted August 15th 2019

Differential expression and release of exosomal miRNAs by human islets under inflammatory and hypoxic stress.

Bashoo Naziruddin Ph.D.

Bashoo Naziruddin Ph.D.

Saravanan, P. B., S. Vasu, G. Yoshimatsu, C. M. Darden, X. Wang, J. Gu, M. C. Lawrence and B. Naziruddin (2019). “Differential expression and release of exosomal miRNAs by human islets under inflammatory and hypoxic stress.” Diabetologia Aug 1. [Epub ahead of print].

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AIMS/HYPOTHESIS: Pancreatic islets produce non-coding microRNAs (miRNAs) that regulate islet cell function and survival. Our earlier investigations revealed that human islets undergo significant damage due to various types of stresses following transplantation and release miRNAs. Here, we sought to identify and validate exosomal miRNAs (exo-miRNAs) produced by human islets under conditions of cellular stress, preceding loss of cell function and death. We also aimed to identify islet stress signalling pathways targeted by exo-miRNAs to elucidate potential regulatory roles in islet cell stress. METHODS: Human islets were subjected to proinflammatory cytokine and hypoxic cell stress and miRNA from exosomes was isolated for RNA sequencing and analysis. Stress-induced exo-miRNAs were evaluated for kinetics of expression and release by intact islets for up to 48 h exposure to cytokines and hypoxia. A subset of stress-induced exo-miRNAs were assessed for recovery and detection as biomarkers of islet cell stress in a diabetic nude mouse xenotransplant model and in patients undergoing total pancreatectomy with islet auto-transplantation (TPIAT). Genes and signalling pathways targeted by stress-induced exo-miRNAs were identified by Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and direct interactions of miRNAs with downstream signalling targets were validated in human islet cells using the miRNA Tests for Read Analysis and Prediction (MirTrap) system. RESULTS: Global exo-miRNA sequencing revealed that 879 miRNA species were released from human islets and 190 islet exo-miRNAs were differentially expressed in response to proinflammatory cytokines, hypoxia or both. Release of exo-miRNAs hsa-miR-29b-3p and hsa-miR-216a-5p was detected within 6 h of exposure to cytokines and hypoxia. The remaining subset of stress-induced exo-miRNAs, including hsa-miR-148a-3p and islet cell damage marker hsa-miR-375, showed delayed release at 24-48 h, correlating with apoptosis and cell death. Stress and damage exo-miRNAs were significantly elevated in the circulation in human-to-mouse xenotransplant models and in human transplant recipients. Elevated blood exo-miRNAs negatively correlated with post-transplant islet function based on comparisons of stress and damage exo-miRNA indices with Secretory Unit of Islet Transplant Objects (SUITO) indices. KEGG analysis and further validation of exo-miRNA targets by MirTrap analysis revealed significant enrichment of islet mRNAs involved in phosphoinositide 3-kinase/Akt and mitogen-activated protein kinase signalling pathways. CONCLUSIONS/INTERPRETATION: The study identifies exo-miRNAs differentially expressed and released by islets in response to damage and stress. These exo-miRNAs could serve as potential biomarkers for assessing islet damage and predicting outcomes in islet transplantation. Notably, exo-miRNAs 29b-3p and 216a-5p could be detected in islets prior to damage-released miRNAs and indicators of cellular apoptosis and death. Thus, these stress-induced exo-miRNAs may have potential diagnostic value for detecting early islet stress prior to progressive loss of islet cell mass and function. Further investigations are warranted to investigate the utility of these exo-miRNAs as early indicators of islet cell stress during prediabetic conditions.


Posted August 15th 2019

Libman-Sacks Endocarditis Involving a Bioprosthesis in the Aortic Valve Position in Systemic Lupus Erythematosus.

William C. Roberts M.D.

William C. Roberts M.D.

Roberts, W. C., A. Y. Lee, S. R. Lander, C. S. Roberts and B. L. Hamman (2019). “Libman-Sacks Endocarditis Involving a Bioprosthesis in the Aortic Valve Position in Systemic Lupus Erythematosus.” Am J Cardiol 124(2): 316-318.

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Described herein is a 39-year-old man with systemic lupus erythematosus not receiving corticosteroid therapy who developed Libman-Sacks endocarditis causing stenosis of a bioprosthesis in the aortic valve position.


Posted August 15th 2019

Does time from diagnosis to treatment of high- or very-high-risk prostate cancer affect outcome?

Ashley E. Ross, M.D.

Ashley E. Ross, M.D.

Reichard, C. A., Y. A. Nyame, D. Sundi, J. Tosoian, L. Wilkins, R. Alam, M. F. Achim, X. Wang, A. J. Stephenson, E. A. Klein, A. E. Ross, J. W. Davis and B. F. Chapin (2019). “Does time from diagnosis to treatment of high- or very-high-risk prostate cancer affect outcome?” BJU Int 124(2): 282-289.

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OBJECTIVE: To determine whether time from diagnosis to treatment impacted outcomes in a multicentre cohort of high- and very-high-risk (VHR) patients with prostate cancer undergoing radical prostatectomy (RP). PATIENTS AND METHODS: In all, 1392 patients from three tertiary centres who underwent RP for either high-risk or VHR disease, from 2005 to 2015, were identified. The cohort was divided into tertiles based on time from diagnostic biopsy to RP. Cumulative incidence of biochemical recurrence (BCR), metastasis, and prostate cancer-specific mortality (PCSM) were calculated for each tertile. The Kaplan-Meier method was used to evaluate for differences in all-cause mortality (ACM) amongst tertiles. Competing risks regression models, as well as Cox proportional hazards regression models, were fitted to assess the association between time-to-event outcomes and patient characteristics. RESULTS: The median (interquartile range [IQR]) time from biopsy to RP was 68 (50-94) days. The median (IQR) follow-up was 31 (12.1-55.7) months. The cumulative incidence of BCR (P = 0.14), metastasis (P = 0.15), and PCSM (P = 0.69) did not differ amongst time-to-treatment tertiles of VHR patients. Also, Kaplan-Meier estimates of ACM (P = 0.53) did not differ amongst time-to-treatment tertiles. Similarly, BCR, metastasis, PCSM, and ACM did not significantly differ amongst time-to-treatment tertiles in multivariable modelling. CONCLUSION: In this pooled meta-dataset of patients with high-risk or VHR prostate cancer, time from diagnosis to RP did not appear to significantly contribute to differences in clinical outcomes. This finding supports the safety of enrollment of such patients into neoadjuvant clinical trials.