Human Germinal Center B Cells Differ from Naïve and Memory B Cells in CD40 Expression and CD40L-Induced Signaling Response.

CD40 expression is required for germinal center (GC) formation and function, but the kinetics and magnitude of signaling following CD40 engagement remain poorly characterized in human B cells undergoing GC reactions. Here, differences in CD40 expression and signaling responses were compared across differentiation stages of mature human tonsillar B cells. A combination of mass cytometry and phospho-specific flow cytometry was used to quantify protein expression and CD40L-induced signaling in primary human naïve, GC, and memory B cells. Protein expression signatures of cell subsets were quantified using viSNE and Marker Enrichment Modeling (MEM). This approach revealed enriched expression of CD40 protein in GC B cells, compared to naïve and memory B cells. Despite this, GC B cells responded to CD40L engagement with lower phosphorylation of NFκB p65 during the first 30 min following CD40L activation. Before CD40L stimulation, GC B cells expressed higher levels of suppressor protein IκBα than naïve and memory B cells. Following CD40 activation, IκBα was rapidly degraded and reached equivalently low levels in naïve, GC, and memory B cells at 30 min following CD40L. Quantifying CD40 signaling responses as a function of bound ligand revealed a correlation between bound CD40L and degree of induced NFκB p65 phosphorylation, whereas comparable IκBα degradation occurred at all measured levels of CD40L binding. These results characterize cell-intrinsic signaling differences that exist in mature human B cells undergoing GC reactions. © 2019 International Society for Advancement of Cytometry.

Raman microspectroscopy and laser-induced breakdown spectroscopy for the analysis of polyethylene microplastics in human soft tissues.

People are exposed to microplastics (MPs) on a large scale in everyday life. However, it is not clear whether MPs can also be distributed and retained in certain tissues. Therefore, the development of analytical methods capable of detecting MPs in specific human organs/tissues is of utmost importance. In this study, the use and combination of spectroscopic techniques, namely Raman microspectroscopy and laser-induced breakdown spectroscopy (LIBS), was tested for the detection of polyethylene (PE) MPs in human tonsils. Preliminary results showed that Raman microspectroscopy was able to detect MPs down to 1 µm in size and LIBS down to 10 µm. In the next step, human tonsils were spiked with PE MPs, and digested. The filtered particles were analyzed using Raman microspectroscopy and LIBS, and complemented by X-ray fluorescence (XRF). The results showed that Raman microspectroscopy could reliably detect PE MPs in spiked human tonsils, while LIBS and XRF served as a reference analytical method to characterize particles that could not be classified by Raman microspectroscopy for their non-organic origin. The results of this study, supported by a current feasibility study conducted on clinical samples, demonstrated the reliability and feasibility of this approach for monitoring MPs in biotic samples.

Histochemical Approach for Simultaneous Detection of Ectonucleotidase and Alkaline Phosphatase Activities in Tissues.

Studies on pathophysiology and the therapeutic potential of extracellular ATP and other purines represent an important and rapidly evolving field. The integral response of the cell is determined by multiple factors, including the release of endogenous ATP, co-expression of different types of nucleotide- and adenosine-selective receptors, as well as the specific makeup of ectoenzymes governing the duration and magnitude of purinergic signaling. Current findings support the presence of an extensive network of purine-converting ectoenzymes that are co-expressed to a variable extent among the mammalian tissues and share similarities in substrate specificity. Here, we describe a histochemical approach for simultaneous detection of ecto-nucleotidase and tissue-nonspecific alkaline phosphatase (TNAP) activities in the same tissue slice. Further employment of this technique for staining human palatine tonsil cryosections revealed selective distribution of the key ectoenzymes within certain tonsillar structures, including germinal centers and connective tissues (ecto-5'-nucleotidase/CD73), as well as interfollicular area (TNAP and NTPDase1/CD39).

Prevalence of Fusobacterium necrophorum in tonsils from patients with chronic tonsillitis.

CONCLUSION: There was a high prevalence of Fusobacterium necrophorum (FN) in patients with chronic tonsillitis in the age group 15-23 years. This indicates that FN might play an important role in the pathogenesis of chronic tonsillitis in this age group, which is also the age group in which chronic or recurrent tonsillitis is most common. OBJECTIVES: The role of FN in patients with acute and chronic tonsillitis is unclear. Thus, this study investigated the occurrence of FN in tonsils of patients with chronic tonsillitis. The aim of the study was to determine the prevalence of FN in patients that underwent tonsillectomy due to chronic tonsillitis. This study also investigated if FN was found at different areas in the tonsils. METHOD: One hundred and twenty-six consecutive patients undergoing tonsillectomy due to chronic tonsillitis were included from the ENT clinics at Sunderby Hospital and Gällivare Hospital, Sweden. Both children and adults were included to encompass various age groups (age =2-57 years). Culture swabs were taken from three different levels of the tonsils - the surface, the crypts, and the inner core of the tonsils. Selective agar plates for detecting FN were used for culture. Culture was also made for detecting ß-hemolytic streptococci, Haemophilus influenzae, and Arcanobacterium. RESULTS: FN was the most common pathogen (19%). The highest prevalence of FN was found in the age group 15-23 years (in 34% of the patients). FN was detected both at the surface and in the core of the tonsils. Furthermore, in the few patients where FN was not detected in all three areas, FN was always detected at the tonsillar surface, in spite of being an anaerobic bacterium. Streptococci group G and C also occurred most frequently (30%) in the same age group as FN (15-23 years), whereas Streptococci group A was more evenly spread among the age groups.