Standardization of extracellular vesicle measurements by flow cytometry through vesicle diameter approximation

Essentials

• Platelet extracellular vesicles (EVs) concentrations measured by flow cytometers are incomparable.
• A model is applied to convert ambiguous scatter units to EV diameter in nanometer.
• Most included flow cytometers lack the sensitivity to detect EVs of 600 nm and smaller.
• The model outperforms polystyrene beads for comparability of platelet EV concentrations.

Summary

Background

Detection of extracellular vesicles (EVs) by flow cytometry has poor interlaboratory comparability, owing to differences in flow cytometer (FCM) sensitivity. Previous workshops distributed polystyrene beads to set a scatter‐based diameter gate in order to improve the comparability of EV concentration measurements. However, polystyrene beads provide limited insights into the diameter of detected EVs.

Objectives

To evaluate gates based on the estimated diameter of EVs instead of beads.

Methods

A calibration bead mixture and platelet EV samples were distributed to 33 participants. Beads and a light scattering model were used to set EV diameter gates in order to measure the concentration of CD61–phycoerythrin‐positive platelet EVs.

Results

Of the 46 evaluated FCMs, 21 FCMs detected the 600–1200‐nm EV diameter gate. The 1200–3000‐nm EV diameter gate was detected by 31 FCMs, with a measured EV concentration interlaboratory variability of 81% as compared with 139% with the bead diameter gate. Part of the variation in both approaches is caused by precipitation in some of the provided platelet EV samples. Flow rate calibration proved essential because systems configured to 60 μL min^?1 differed six‐fold in measured flow rates between instruments. Conclusions EV diameter gates improve the interlaboratory variability as compared with previous approaches. Of the evaluated FCMs, 24% could not detect 400‐nm polystyrene beads, and such instruments have limited utility for EV research. Finally, considerable differences were observed in sensitivity between optically similar instruments, indicating that maintenance and training affect the sensitivity.

     

Clinical,Biochemical, Tumoural and Mutation Profile of VHL- and MEN2A-Associated Pheochromocytoma: A Comparative Study

World Journal of Surgery - To compare clinical, biochemical, tumoural and mutational characteristics of Von Hippel Lindau Syndrome (VHL)-associated pheochromocytoma (PCC) to multiple endocrine...     

Immunotherapy of Fungal Infections

Fungal organisms are ubiquitous in the environment. Pathogenic fungi, although relatively few in the whole gamut of microbial pathogens, are able to cause disease with varying degrees of severity in individuals with normal or impaired immunity. The disease state is an outcome of the fungal pathogen’s interactions with the host immunity, and therefore, it stands to reason that deep/invasive fungal diseases be amenable to immunotherapy. Therefore, antifungal immunotherapy continues to be attractive as an adjunct to the currently available antifungal chemotherapy options for a number of reasons, including the fact that existing antifungal drugs, albeit largely effective, are not without limitations, and that morbidity and mortality associated with invasive mycoses are still unacceptably high. For several decades, intense basic research efforts have been directed at development of fungal immunotherapies. Nevertheless, this approach suffers from a severe bench-bedside disconnect owing to several reasons: the chemical and biological peculiarities of the fungal antigens, the complexities of host-pathogen interactions, an under-appreciation of the fungal disease landscape, the requirement of considerable financial investment to bring these therapies to clinical use, as well as practical problems associated with immunizations. In this general, non-exhaustive review, we summarize the features of ongoing research efforts directed towards devising safe and effective immunotherapeutic options for mycotic diseases, encompassing work on antifungal vaccines, adoptive cell transfers, cytokines, antimicrobial peptides (AMPs), monoclonal antibodies (mAbs), and other agents.     

Multimeric composition of endothelial cell-derived von Willebrand factor

The multimeric composition of human endothelial cell (EC)-derived von Willebrand factor (vWF) was studied using SDS-agarose gel electrophoresis and autoradiography. Two multimers were found in lysates prepared from confluent cultures of human umbilical vein endothelial cells. The smaller multimer had a molecular weight (mol wt) of approximately 950 Kd, while the second was larger than those seen in plasma. When electrophoresis was performed using the discontinuous buffer system of Ruggeri and Zimmerman, the small multimer consisted of a single band migrating with the slowest-moving component of the corresponding plasma triplet. The large EC-vWF multimer was detected in culture media conditioned with EC monolayers for ten minutes. It remained the only multimer in media conditioned for up to three days. Calcium ionophore A23187 increased the amount of the large vWF multimer released into the culture media, but did not change its multimeric composition. The small multimer was never detected in the EC- conditioned media. These findings suggest that (1) a large, fully polymerized multimer of vWF is released from the ECs, while the small multimer probably represents a major intermediate component in the process of multimerization, and (2) plasma vWF multimers are probably generated from the large endothelial vWF after it is released into the circulation.     

Occurrence of hepatopulmonary syndrome in Budd-Chiari syndrome and the role of venous decompression

BACKGROUND & AIMS: Hepatopulmonary syndrome (HPS) has been predominantly detected in cirrhotic patients and rarely in patients with noncirrhotic portal hypertension. The aim of this study was to determine the occurrence of HPS in patients with Budd-Chiari syndrome (only anecdotal reports available) and evaluate the role of venous decompression in its reversal. METHODS: Twenty-nine consecutive cases of Budd-Chiari syndrome without primary cardiopulmonary disease were investigated by air contrast echocardiography and arterial blood gas analysis. Venous decompression (e.g., by balloon cavoplasty) was attempted when feasible. RESULTS: Eight cases (27.6%) of HPS and 9 cases (31.0%) with positive contrast echocardiography but unimpaired oxygenation were detected. Duration of disease was longer (P = 0.026) among those with positive contrast echocardiography. Cavoplasty reversed 4 of 5 cases of HPS and 2 of 2 cases with positive contrast echocardiography alone. Venous decompression by drainage of amebic liver abscess (which was compressing hepatic venous outflow) also reversed 1 case of HPS. HPS was relieved by venous decompression in 5 of 6 cases. CONCLUSIONS: HPS developed in a substantial fraction of our patients with Budd-Chiari syndrome, with positive contrast echocardiography occurring mainly in the benign, slowly progressing variety. Venous decompression showed promise in reversing such cases.     

Evaluation of Aerosol and Water Contamination and Control of Cross Infection in Dental Clinics

Background

The oral cavity is an unique environment which provides an ideal medium for bacterial growth. As a result of repeated exposure to the microorganisms present in blood and saliva, the dental health professionals and the patients are at a higher risk for developing many infectious diseases. A pilot study was carried out in the Dept of Dental Surgery, Armed Forces Medical College to assess the risk of cross infection in dental clinics.

Methods

Samples were collected from different dental surgeries of the Dept of Dental Surgery, Armed Forces Medical College and sent for microbiological culture and identification to the Dept of Microbiology, Armed Forces Medical College. The sampling was carried out in two stages, before and after implementing a set protocol.

Result

All dental unit waterlines were coated with a well established biofilm made up of filamentous and bacillus-like microorganisms in first stage of study. There was marked reduction in the number of colonies from the samples collected during second stage. Same findings were observed in the samples of aerosol produced by ultrasonic scalers.

Conclusion

The present study concluded that the new set protocol followed is significantly effective in reducing the microbial load in the water tubing, container and aerosol production. It is an effective measure for reducing the chances of cross infection in the dental surgeryKey Words: Aerosol, Biofilm, Cross infection, Colony forming units     

Urinary albumin concentration and long-term cardiovascular risk in acute coronary syndrome patients: a PROVE IT-TIMI 22 substudy

Albuminuria has been shown to be associated with mortality and cardiovascular events, independent of traditional cardiovascular risk factors. This suggests that albuminuria may not just represent glomerular damage, but may be a marker of more diffuse endothelial dysfunction. We investigated the relationship between urinary albumin levels after an acute coronary syndrome and cardiovascular outcomes in statin treated subjects after acute coronary syndromes (ACS). Furthermore we assessed the effect of intensive statin treatment on albuminuria among patients in the PROVE IT-TIMI 22 trial, in which patients who had been hospitalized with ACS were randomized to pravastatin 40 mg (standard therapy) or atorvastatin 80 mg daily (intensive therapy). In univariate analyses, increasing urine albumin concentration was associated with increased risk of myocardial infarction, stroke, heart failure, and composite of death, myocardial infarction and stroke at 2 years. However, in a multivariable model containing traditional cardiovascular risk factors, albuminuria was not an independent predictor of the primary PROVE IT endpoint of death, myocardial infarction, unstable angina, revascularization and stroke, and was only an independent predictor of all-cause mortality at urinary albumin concentration >300 mcg/ml. There was no significant change in urinary albumin concentration from enrolment to end of study in either the standard or intensive statin therapy groups, and no significant difference between treatment groups. Our results suggest that after an acute coronary syndrome in statin treated patients, microalbuminuria may reflect traditional cardiovascular risk factor burden and offer little prognostic information independent of those factors.     

Chromone linked nitrone derivative induces the expression of iNOS2 and Th1 cytokines but reduces the Th2 response in experimental visceral leishmaniasis

In our previous work we have shown that the novel synthetic chromone derivative could effectively inhibit the Leishmania donovani replication in vitro and in vivo with less cytotoxicity on murine splenocytes. The aim of the present study is to explore the possible mechanism of anti-leishmanial effect of C-(6-methyl-4-oxo-4H-1-benzopyran-3-yl)-N-(p-tolyl) nitrone (designated as NP1) in vitro and in vivo in experimental visceral leishmaniasis caused by L. donovani. The cytotoxic effect of this derivative was studied in murine peritoneal macrophages by MTT method. NP1 at a dose of 17.06 μM showed 50% inhibition on L. donovani promastigotes but found less cytotoxic to the RAW 264.7 cells. Even the higher concentration of IC50 (up to four fold) did not exert much cytotoxic effect on RAW 264.7. Interestingly, NP1 at lower concentration (8.53 μM) could inhibit 50% of intracellular amastigotes in murine peritoneal macrophages. L. donovani is known to exert its pathogenic effects mainly by the suppression of NO generation and subversion of the cellular inflammatory responses in the macrophages. NP1 was found to induce a potent host-protective immune response by enhancing NO generation and iNOS2 expression at mRNA level and by up-regulating proinflammatory cytokines such as IL-12 and IFN-γ and limiting the expression of IL-10 in vivo. The NO dependent killing was further confirmed in iNOS^?/? mice compared to wild type. In agreement with the fact, induced synthesis of IL-12 and IFN-γ and associated down-regulation of IL-10 by the treatment of NP1 clearly indicated the possibility of novel strategy of drug development against Leishmania infection.