Phenotypic and functional characterization of in vitro cultured human mast cells.

BACKGROUND: Mast cell progenitor cells, derived from CD34+ hematopoietic stem cells, enter the circulation and subsequently mucosal or connective tissues where they mature to mast cells. Upon activation, mast cells increase the expression of activation markers, e.g. CD63, and release histamine amongst other mediators. Traditionally, release of these mediators is quantified using assays measuring their extracellular concentration in the supernatant of stimulated cells. METHODS: Human mast cells (HuMC) were cultured from peripheral blood, phenotypically characterized, passively sensitized with allogenic IgE antibodies and finally stimulated by anti-IgE that crosslinks IgE/FcεRI complexes. Alterations in the number of cells positive for CD63 and release of histamine were quantified simultaneously by flow cytometry. RESULTS: In culture, two distinct CD45+ cell populations were identified: CD117+ CD203c+hi and CD117- CD203c+low cells. Both populations showed positivity for FcεRI, tryptase and chymase, and contained histamine. Activation resulted in a significant increase of cells positive for CD63+ up to 21% (range: 11-39) for CD117+ CD203c+hi cells (P = 0.005), and 27% (18-55) CD63+ for CD117- CD203c+low cells (P = 0.02). Baseline histamine content was higher for CD117+ CD203c+hi cells than for CD117- CD203c+low cells, respectively 994 (695-6815) Molecules of Equivalent Specific Fluorochrome V500 per cell (MESF-V500/cell) and 797 (629-4978) MESF-V500/cell (P = 0.02). After activation, CD117+ CD203c+hi cells showed significant histamine release of 578 (366-1521) MESF-V500/cell, whilst CD117- CD203c+low cells resulted in 310 (217-366) MESF-V500/cell histamine release. CONCLUSION: This study discloses that culturing HuMC from CD34+ progenitors yields 2 phenotypically distinct cell populations that display a greatly similar response upon cross-linking of IgE/FcεRI complexes. © 2016 International Clinical Cytometry Society.

Cannabis sativa allergy: looking through the fog.

IgE-mediated Cannabis (C. sativa, marihuana) allergy seems to be on the rise. Both active and passive exposure to cannabis allergens may trigger a C. sativa sensitization and/or allergy. The clinical presentation of a C. sativa allergy varies from mild to life-threatening reactions and often seems to depend on the route of exposure. In addition, sensitization to cannabis allergens can result in various cross-allergies, mostly for plant foods. This clinical entity, designated as the 'cannabis-fruit/vegetable syndrome', might also imply cross-reactivity with tobacco, natural latex and plant-food-derived alcoholic beverages. Hitherto, these cross-allergies are predominantly reported in Europe and appear mainly to rely upon cross-reactivity between nonspecific lipid transfer proteins or thaumatin-like proteins present in C. sativa and their homologues, ubiquitously distributed throughout plant kingdom. At present, diagnosis of cannabis-related allergies predominantly rests upon a thorough history completed with skin testing using native extracts from crushed buds and leaves. However, quantification of specific IgE antibodies and basophil activation tests can also be helpful to establish correct diagnosis. In the absence of a cure, treatment comprises absolute avoidance measures. Whether avoidance of further use will halt the extension of related cross-allergies remains uncertain.

Flow cytometric analysis of drug-Induced basophil histamine release.

Histamine and its release can be studied by multicolor flow cytometry on a single cell level by an enzyme affinity method (HistaFlow®). However, for the time-being, the clinical and scientific application of the HistaFlow® technique remains limited. This study aims at verifying the reliability of the HistaFlow® as an instrument to quantify IgE-mediated basophil responses to drugs, i.e., rocuronium, which are believed to be less potent basophil activators than large proteinaceous allergens. Ten patients and three exposed control individuals were included in this study. Each subject underwent in vitro basophil activation tests (HistaFlow®) with 0.16 and 1.6 mmol/L rocuronium. Patients showed an activation of basophils ranging from 11 to 86% of CD63 positive basophils and a median histamine release per cell from 68 to 100% after stimulation with an optimal concentration of 1.6 mmol/L rocuronium. For the control individuals no activation was demonstrable. This study confirms that the HistaFlow® technique is a reliable tool to study histamine release by individual cells in response to drugs. Although the HistaFlow® technique will probably not add to the diagnostic management of rocuronium allergy, our findings suggest that the technique could constitute an important asset for future studies on the pathomechanism(s) of immediate drug hypersensitivity reactions. © 2015 International Clinical Cytometry Society.

Transcranial Doppler in smoking relapse prevention strategy.

Nicotine dependence is a progressive, chronic, relapsing disorder. Nicotine is the principal and most potent psychopharmacologically active component of tobacco smoke. Through activation of nicotine receptors in the central nervous system, nicotine can lead to tolerance and dependence. Cessation of smoking is followed by severe pathophysiologic withdrawal and by long-term craving. TCD measurement of cerebral blood flow velocity (BFV) and nicotine dependence degree measured by Fragestrom questionnaire was analyzed in relation to smoking relapse. This study includes 47 participants (25 females and 22 males) included in Breathe Free Plan To Stop Smoking in Non Smoking School in Zagreb. 12 month following the end of treatment participants were divided in three groups: continued abstinence, interrupted abstinence and non abstinence. High nicotine dependence combined with TCD pathological finding significantly discriminated successes and failures, suggesting that smokers with pathological TCD need specific therapeutic approach with more social support, individualized coping skills and cognitive restructuring. Measuring cerebral flow velocity by transcranial Doppler in smokers showed the practical validity in prediction of smoking relapse.

Haemodynamic changes in patients with whiplash injury measured by transcranial Doppler sonography (TCD).

The daily increasing number of cervical whiplash injuries presents ever-greater requirement for vertebrobasilar diagnostics. A cervical spine injury, which is quite frequent injury, may occur during a fall, or industrial, traffic, sport or war injury. Transcranial Doppler (TCD) sonography with Transcan 3-D EME device and 2 MHz probe was used for the assessment of vertebrobasilar circulation in patients with a whiplash injury of the cervical spine, that occurred mostly in car accident. This study includes 47 patients with clinically verified cervical spine trauma with x-ray evidence of no bone lesion. The patients were examined by TCD within a month, and then six months following the accident. The obtained values were compared to normal blood flow velocities and correlated with the severity of clinical picture. During the first month after the injury, statistically significant disturbances in the vertebrobasilar circulation were recorded, such as the increase in mean blood flow velocities in AVL (68%), AVR (62%) and BA (51%) (mostly as spasam). Six months later, normal findings were obtained in about 50% of the vessels, whereas in rest of the patients vasospasam persisted in one, two or all examined blood vessels. TCD of the vertebrobasilar circulation was found to be a very useful method in the diagnostics and follow-up of patients with a whiplash injury.

Intra- and extracranial circulatory changes in cigarette smokers assessed by ultrasound: transcranial Doppler (TCD) and duplex scanning investigation.

The group of 98 "healthy smokers" was analyzed in order to evaluate potential influence of cigarette smoking on intracranial blood flow disturbances and extracranial carotid intimal changes, performing transcranial Doppler (TCD) and Duplex scanning of the carotid arteries. Almost 70% of smokers analyzed in our study had some intracranial circulatory changes, predominantly in the vertebral arteries (35%). Half of smokers analyzed had some pathological intimal changes in the carotid arteries. The most frequent finding was wall thickening (23%) and then calcified (13%) and soft plaques (10%). Dose response relationship between smoking and atherosclerosis is also introduced. More than 30 cigarettes smoked per day can significantly influence plaque development and already 20 cigarettes smoked per day can cause significant intracranial blood flow disturbances. The majority of our investigated smokers were in high risk category for development of cerebrovascular disease and 5% were in a very high risk category. Cessation of cigarette smoking will eliminate it as a risk factor. A low dose-response relation and the development of tolerance produced by exposure to nicotine were also introduced.