In vitro investigations of mast cell (MC) degranulation are essential for studying many diseases, particularly allergy and urticaria. Many MC-degranulation inducers are currently available. However, there is no previous systematic comparative analysis of these available inducers in term of their efficacies to induce MC degranulation. Herein, we performed systematic comparisons of efficacies of five well-known and commonly used MC-degranulation inducers. RBL-2H3 cells were sensitized with 50 ng/ml anti-DNP IgE or biotinylated IgE followed by stimulation with 100 ng/ml DNP-BSA or streptavidin, respectively. For non-IgE-mediated inducers, the cells were treated with 5 µg/ml substance P, compound 48/80, or A23187. At 15-, 30-, 45- and 60-min post-induction, several common MC-degranulation markers (including intracellular [Ca2+], β-hexosaminidase release, tryptase expression by immunofluorescence staining, cellular tryptase level by immunoblotting, secretory tryptase level by immunoblotting, CD63 expression by immunofluorescence staining, and CD63 expression by flow cytometry) were evaluated. The data showed that all these markers significantly increased after activation by all inducers. Among them, A23187 provided the greatest degrees of increases in intracellular [Ca2+] and β-hexosaminidase release at all time-points and upregulation of CD63 at one time-point. These data indicate that all these IgE-mediated (anti-DNP IgE/DNP-BSA and biotinylated IgE/streptavidin) and non-IgE-mediated (substance P, compound 48/80, and A23187) inducers effectively induce MC degranulation, while A23187 seems to be the most effective inducer for MC degranulation.

BACKGROUND: The clinical and prognostic implications of platelet reactivity (PR) testing in a P2Y12-inhibitor naïve population are poorly understood.
OBJECTIVE: This explorative study aims to assess the role of PR and explore factors that may modify elevated mortality risk in patients with altered PR.
METHODS: Platelet ADP-induced CD62P and CD63 expression were measured by flow-cytometry in 1520 patients who were referred for coronary angiography in the Ludwigshafen Risk and Cardiovascular Health Study (LURIC).
RESULTS: High- and Low-platelet reactivity to ADP were strong predictors of cardiovascular and all-cause mortality and risk equivalent to the presence of coronary artery disease. (High platelet reactivity 1.4 [95% CI 1.1-1.9]; Low platelet reactivity: 1.4 [95% CI 1.0-2.0]). Relative weight analysis indicated glucose control (HbA1c), renal function ([eGFR]), inflammation (high-sensitive C-reactive protein [hsCRP]) and antiplatelet therapy by Aspirin as consistent mortality risk modifiers in patients with Low- and High-platelet reactivity. Pre-specified stratification of patients by risk modifiers HbA1c (<7.0%), eGFR (>60 mL/min/1.73 m2) and CRP (<3 mg/L) was associated with a lower mortality risk, however irrespective of platelet reactivity. Aspirin treatment was associated with reduced mortality in patients with high platelet reactivity only (p for interaction: 0.02 for CV-death [<0.01 for all-cause mortality].
CONCLUSIONS: Cardiovascular mortality risk in patients with High- and Low platelet reactivity is equivalent to the presence of coronary artery disease. Targeted glucose control, improved kidney function and lower inflammation are associated with reduced mortality risk, however independent of platelet reactivity. In contrast, only in patients with High-platelet reactivity was Aspirin treatment associated with lower mortality.

Objective: Inflammation has been linked to progression of diabetic foot ulcers (DFU); however, specific predictive markers of nonhealing are scarce. The goal of this study was to identify biochemical and immunological parameters from the blood as predictors of nonhealing in grade 1 and grade 2 DFU. Approach: Individuals with low-grade foot ulcers were enrolled in the study to determine if histopathological, biochemical, and immunological parameters could be used to predict individuals whose ulcers would not heal. Data analysis was performed using traditional univariate analyses as well as univariate and multivariable logistic regression, and STROBE guidelines were used for reporting data. Results: Among the 52 individuals who completed the study, we observe that no single histopathological and biochemical parameter was predictive. Conventional univariate analysis and univariate logistic regression analysis showed that the expression of the cell surface proteins CD63, HLA-DR, and CD11b on monocytes was significantly lower in nonhealed individuals, but with moderate discriminative ability. In comparison, a multivariable logistic regression model identified four of the 31 parameters to be salient predictors with low density lipoprotein (LDL) cholesterol (odds ratio [OR] 18.83, confidence interval [CI] 18.83-342) and cell-surface expression of CD63 on monocytes (OR 0.12, CI 0.12-0.45) showing significance and demonstrating high discrimination ability. Innovation: The approach of using a combination of biochemical and immunological parameters to predict ulcer healing is new. Conclusion: Through this study we conclude that LDL cholesterol and cell-surface expression of CD63 on monocytes strongly correlate with nonhealing in individuals with grade 1 and grade 2 DFU.

No reliable biological marker for the diagnosis of asthma in younger children is currently available. In this study, we analyzed the differences in basophil activation test (BAT) results among children with recurrent wheezing episodes who had different asthma outcomes.
A prospective cohort study was conducted in children aged under 5 years who visited our pediatric respiratory clinic and ward for wheezing. After enrollment, the participants provided samples for a CD63-based BAT performed using an inhalant allergen mixture as a stimulant. Histories of personal allergic diseases and family allergic diseases were evaluated by using a questionnaire. All participants were followed up for 2 years, and their asthma outcomes were evaluated at the end of the follow-up period. The correlation between the BAT results and asthma outcomes was analyzed. Of the 45 originally enrolled children, 38 completed both the follow-up and a BAT. After stimulation with the inhalant mixture, the CD63 expression on basophils and the rate of positive CD63-based BAT results in children diagnosed with asthma were both significantly higher than those in children who were not diagnosed with asthma (p < 0.05 and p < 0.01, respectively). For the prediction of asthma, the positive predictive value and negative predictive value of CD63-based BAT was 71.8 and 69.2%, respectively. The positive likelihood ratio and negative likelihood ratio of CD63-based BAT were 1.70 and 0.3, respectively.
Our pilot study indicates that CD63-based BAT has potential clinical value for predicting asthma outcome in young children with wheezing episodes.

Many cancers commonly metastasize to bone. After entering the bone, cancer cells can interact with surrounding stromal cells, which ultimately influences metastasis progression. Extracellular vesicles, direct cell contact and gap junctions, and cytokines are all mechanisms of intercellular communication that have been observed to occur in the bone microenvironment. These methods of cellular crosstalk can occur between cancer cells and a variety of stromal cells, with each interaction having a different impact on cancer progression. Communication between cancer cells and bone-resident cells has previously been implicated in processes such as cancer cell trafficking and arrest in bone, cancer cell dormancy, cancer cell reactivation, and proliferation. In this chapter we review innovative techniques and model systems that can be used to study bidirectional crosstalk between cancer cells and stromal cells in the bone, with an emphasis specifically on bone-metastatic breast cancer. Investigating how metastatic cancer cells interact with, and are influenced by, the bone microenvironment is crucial to better understanding of the progression of bone metastasis.

BACKGROUND: To evaluate the relationship between the plasmatic CD63 and CAV1 positive exosome levels, in patients with OSCC before and after surgical treatment and to correlate it with their overall survival.
METHODS: A double-blind pilot study over 10 patients OSCC and T4 stage without distant metastases or local bone invasion has been performed. The average follow-up period was 37.64 months (34.3⁻40.84). We obtained 2 plasma tubes of 1 mL each before surgery and 7 days after surgery. Before performing the immunocapture-based analysis, EVs (Extracellular Vesicles) were isolated from the plasma and characterized with western blot analysis.
RESULTS: Mean values of CD63 positive plasmatic exosomes (EXO-CD63) after surgery decreased from 750.88 ± 286.67 to 541.71 ± 244.93 (p = 0.091). On the other hand, CAV-1 positive plasmatic exosomes (EXO-CAV-1) increased after surgery from 507 ± 483.39 to 1120.25 ± 1151.17 (p = 0.237). Patients with EXO-CD63 levels lower than the mean global value before the surgery had a survival of 36.04 months compared with the group with EXO-CD63 higher than the average who only survived 12.49 ± 1.67 months from the diagnosis, p = 0.225. When EXO-CAV-1 levels before surgery was lower than the average (813.94 ± 801.21) overall survival was 24.69 ± 22.23 months in contrast when it was higher that was only 11.64 months, p = 0.157. Patients with lower EXO-CD63 levels after surgery lived an average of 23.84 ± 23.9 months, while those with higher plasmatic levels of EXO-CD63 live 13.35 months, p = 0.808. When EXO-CAV-1 levels after surgery were lower, the average overall survival was 20.344 ± 15.40 months, in contrast when the EXO-CAV-1 levels were higher showing rather an estimate survival expectation of 1.64 months.
CONCLUSIONS: Surgical treatment induced a dramatic reduction of the plasmatic levels of exosomes expressing CD63 as early as 1 week after resection. This first result suggests that the tumour mass is responsible of the high levels of circulating exosomes detected in cancer patients. At the same time point exosome expressing CAV-1 increased, possibly due to the inflammatory reaction immediately after surgery. Lastly, statistical analysis showed that lower levels of plasmatic exosomes both before and after surgery correlated with a better life expectancy of OSCC patients. Hopefully, this approach will prove useful in the clinical follow-up of cancer patients.

Cells release membrane vesicles in their surrounding medium either constitutively or in response to activating signals. Two main types of extracellular vesicles (EVs) are commonly distinguished based on their mechanism of formation, membrane composition and size. According to the current model, EVs shed from the plasma membrane, often called microvesicles, expose phosphatidylserine (PS) and range in size from 100 nm to 1 µm, while EVs originating from endosomal multi-vesicular bodies, called exosomes, contain tetraspanin proteins, including CD63, and range in size from 50 to 100 nm. Heijnen et al. [1] have shown that activated platelets release EVs corresponding to these two types of vesicles, using negative staining electron microscopy (EM) and immuno-gold labeling. Here, we apply cryo-EM and immuno-gold labeling to provide a quantitative analysis of EVs released by platelets activated by thrombin, TRAP and CRP-XL, as well as EVs from serum. We show that EVs activated by these three agonists present a similar size distribution, the majority of them forming a broad peak extending from 50 nm to 1 µm, about 50% of them ranging from 50 to 400 nm. We show also that 60% of the EVs from TRAP or CRP-XL activation expose CD41, a majority of them exposing also PS. To explain the presence of large EVs CD41-negative or PS-negative, several alternative mechanisms of EV formation are proposed. We find also that the majority of EVs in activated platelet samples expose CD63, and distinguish two populations of CD63-positive EVs, namely large EVs with low labeling density and small EVs with high labeling density.

OBJECTIVE: The quantification of basophil activation by flow cytometry is a useful tool for the assessment of immediate-type responses to food allergens and the prediction of clinical tolerance in food allergy patients. The aim of this study is to investigate how the analysis of allergen-induced CD63 up-regulation by flow cytometry can be effective in monitoring the acquisition of clinical tolerance by specific oral desensitization in food allergy. To our knowledge, this is the first study to examine this topic.
METHODS: Three male patients affected by cow\'s milk allergy underwent successful oral desensitization to cow\'s milk. In order to monitor the acquired clinical tolerance that occurred after treatment, we performed laboratory tests for total and specific IgE, specific IgG4 and the Basophil Activation Test (BAT) both at baseline and at the end of the desensitization protocol.
RESULTS: Using a fluorescent enzyme immunoassay, the comparison of specific cow\'s milk antibodies before and after treatment showed a decrease of specific IgE levels, without reaching normal values, and an increase of specific IgG4 levels. A complete suppression of cow\'s milk proteins (α-lactoalbumin, β-lactoglobulin and casein) induced CD63 regulation was observed in all three reported cases.
CONCLUSIONS: Using flow cytometry, food allergen-specific basophil responses could be monitored in order to identify an acquired tolerance induced by desensitization treatment. Although further studies are needed to develop this important new topic, it was interesting to note that the BAT seemed to be more sensitive and characterized by a close correlation with clinical tolerance.