Logo: to the web site of Uppsala University

PURPOSE: To evaluate the association between upper-limb (UL) clinical tests and UL accelerometry-derived metrics in children with unilateral Cerebral Palsy (CP).

METHODS: In this cross-sectional study, twenty children with unilateral CP and Manual Ability Classification System level I-III were included. Outcomes of the Assisting Hand Assessment, Box and Block-Test and accelerometry metrics were collected in the clinical setting and in daily life. UL asymmetry index (i.e., the ratio between the well-functioning UL and the affected UL use) was evaluated in different physical activity levels and relative use of UL was evaluated during daily living. Spearman's correlation was used to determine the association between UL clinical tests and accelerometry metrics in a clinical setting and in daily life.

RESULTS: The strongest negative association was between the Assisting Hand Assessment units and accelerometry metrics during the sedentary time in daily life (r_s = -0.64). The asymmetries between ULs were highest during the child's sedentary time (asymmetry index: 45.15) compared to when the child was in light (asymmetry index: 23.97) or higher intensity physical activity (asymmetry index: 13.39). The children used both ULs simultaneously for 44% of the time during daily life.

CONCLUSION: Accelerometry metrics may provide additional objective information to clinical tests by quantifying the amount of UL movements and the amount of asymmetry between the upper limbs in daily life.

Background

Therapies targeting interleukin-5 (IL-5) or its receptor (IL-5Rα) are currently used to treat patients with severe eosinophilic asthma.

Objective

To investigate the impact of anti-IL-5 and anti-IL-5Rα biological therapies on mast cells (MCs) and their progenitors.

Methods

Surface IL-5Rα expression was investigated on MCs and their progenitors in mouse lungs and bone marrow, and in human lungs and blood. Isolated human MC progenitors cultured in the presence or absence of IL-5 were analyzed in vitro. Circulating MC progenitors were quantified in patients with severe asthma, before and after anti-IL-5 (mepolizumab) or anti-IL-5Rα (benralizumab) therapy. Gene expression analysis of MC progenitors was performed before and after anti-IL-5Rα therapy.

Results

Approximately 50% of the human primary lung MCs, and 30% of the human MC progenitors from individuals with allergic asthma expressed IL-5Rα. In patients with mild-to-moderate allergic asthma and mice with acute allergic airway inflammation, the fraction of IL-5Rα ⁺ MC progenitors was elevated. Additionally, IL-5 promoted the proliferation and/or survival of isolated human MC progenitors. Further, patients with severe asthma from two independent cohorts demonstrated a reduction of blood MC progenitors after anti-IL-5 or anti-IL-5Rα treatment. This was associated with improved asthma control, as well as a decline in both blood eosinophils and Th2 cells. Finally, the blood MC progenitors remaining after anti-IL-5Rα (benralizumab) treatment exhibited a downregulated expression of genes involved in growth and proliferation.

Conclusion

This study introduces the possibility that the clinical effects of targeting IL-5/IL-5Rα in severe asthma also may involve reduction of MC populations.

Mast cells localize to mucosal tissues and contribute to innate immune defense against infection. How mast cells sense, differentiate between, and respond to bacterial pathogens remains a topic of ongoing debate. Using the prototype enteropathogen Salmonella Typhimurium (S.Tm) and other related enterobacteria, here we show that mast cells can regulate their cytokine secretion response to distinguish between extracellular and invasive bacterial infection. Tissue-invasive S.Tm and mast cells colocalize in the mouse gut during acute Salmonella infection. Toll-like Receptor 4 (TLR4) sensing of extracellular S.Tm, or pure lipopolysaccharide, causes a modest induction of cytokine transcripts and proteins, including IL-6, IL-13, and TNF. By contrast, type-III-secretion-system-1 (TTSS-1)-dependent S.Tm invasion of both mouse and human mast cells triggers rapid and potent inflammatory gene expression and >100-fold elevated cytokine secretion. The S.Tm TTSS-1 effectors SopB, SopE, and SopE2 here elicit a second activation signal, including Akt phosphorylation downstream of effector translocation, which combines with TLR activation to drive the full-blown mast cell response. Supernatants from S.Tm-infected mast cells boost macrophage survival and maturation from bone-marrow progenitors. Taken together, this study shows that mast cells can differentiate between extracellular and host-cell invasive enterobacteria via a two-step activation mechanism and tune their inflammatory output accordingly.

Quantitative transcriptomics offers a new way to obtain a detailed picture of freshly isolated cells. By direct isolation, the cells are unaffected by in vitro culture, and the isolation at cold temperatures maintains the cells relatively unaltered in phenotype by avoiding activation through receptor cross-linking or plastic adherence. Simultaneous analysis of several cell types provides the opportunity to obtain detailed pictures of transcriptomic differences between them. Here, we present such an analysis focusing on four human blood cell populations and compare those to isolated human skin mast cells. Pure CD19⁺ peripheral blood B cells, CD14⁺ monocytes, and CD4⁺ and CD8⁺ T cells were obtained by fluorescence-activated cell sorting, and KIT+ human connective tissue mast cells (MCs) were purified by MACS sorting from healthy skin. Detailed information concerning expression levels of the different granule proteases, protease inhibitors, Fc receptors, other receptors, transcription factors, cell signaling components, cytoskeletal proteins, and many other protein families relevant to the functions of these cells were obtained and comprehensively discussed. The MC granule proteases were found exclusively in the MC samples, and the T-cell granzymes in the T cells, of which several were present in both CD4⁺ and CD8⁺ T cells. High levels of CD4 were also observed in MCs and monocytes. We found a large variation between the different cell populations in the expression of Fc receptors, as well as for lipid mediators, proteoglycan synthesis enzymes, cytokines, cytokine receptors, and transcription factors. This detailed quantitative comparative analysis of more than 780 proteins of importance for the function of these populations can now serve as a good reference material for research into how these entities shape the role of these cells in immunity and tissue homeostasis.

The extended cleavage specificities of two hematopoietic serine proteases originating from the ray-finned fish, the spotted gar (Lepisosteus oculatus), have been characterized using substrate phage display. The preference for particular amino acids at and surrounding the cleavage site was further validated using a panel of recombinant substrates. For one of the enzymes, the gar granzyme G, a strict preference for the aromatic amino acid Tyr was observed at the cleavable P1 position. Using a set of recombinant substrates showed that the gar granzyme G had a high selectivity for Tyr but a lower activity for cleaving after Phe but not after Trp. Instead, the second enzyme, gar DDN1, showed a high preference for Leu in the P1 position of substrates. This latter enzyme also showed a high preference for Pro in the P2 position and Arg in both P4 and P5 positions. The selectivity for the two Arg residues in positions P4 and P5 suggests a highly specific substrate selectivity of this enzyme. The screening of the gar proteome with the consensus sequences obtained by substrate phage display for these two proteases resulted in a very diverse set of potential targets. Due to this diversity, a clear candidate for a specific immune function of these two enzymes cannot yet be identified. Antisera developed against the recombinant gar enzymes were used to study their tissue distribution. Tissue sections from juvenile fish showed the expression of both proteases in cells in Peyer's patch-like structures in the intestinal region, indicating they may be expressed in T or NK cells. However, due to the lack of antibodies to specific surface markers in the gar, it has not been possible to specify the exact cellular origin. A marked difference in abundance was observed for the two proteases where gar DDN1 was expressed at higher levels than gar granzyme G. However, both appear to be expressed in the same or similar cells, having a lymphocyte-like appearance.

BACKGROUND: Chronic spontaneous urticaria (CSU) is a common, debilitating skin disorder characterized by recurring episodes of raised, itchy and sometimes painful wheals lasting longer than 6 weeks. CSU is mediated by mast cells which are absent from peripheral blood. However, lineage^-CD34^hiCD117^int/hiFcεRI⁺ cells in blood have previously been shown to represent a mast cell precursor.

METHODS: We enumerated FcεRI^-, FcεRI⁺ and FcεRI^hi lineage^-CD34⁺CD117⁺ cells using flow cytometry in blood of patients with CSU (n = 55), including 12 patients receiving omalizumab and 43 not receiving omalizumab (n = 43). Twenty-two control samples were studied. Disease control and patient response to omalizumab was evaluated using the urticaria control test. We performed single-cell RNA sequencing (scRNA-Seq) on lineage^-CD34^hiCD117^hi blood cells from a subset of patients with CSU (n = 8) and healthy controls (n = 4).

RESULTS: CSU patients had more lineage^-CD34⁺CD117⁺FcεRI⁺ blood cells than controls. Lineage^-CD34⁺CD117⁺FcεRI⁺ cells were significantly higher in patients with CSU who had an objective clinical response to omalizumab when compared to patients who had poor disease control 90 days after initiation of omalizumab. scRNA-Seq revealed that lineage^-CD34⁺CD117⁺FcεRI⁺ cells contained both lymphoid and myeloid progenitor lineages, with omalizumab responsive patients having proportionally more myeloid progenitors. The myeloid progenitor lineage contained small numbers of true mast cell precursors along with more immature FcεRI^- and FcεRI⁺ myeloid progenitors.

CONCLUSION: Increased blood CD34⁺CD117⁺FcεRI⁺ cells may reflect enhanced bone marrow egress in the setting of CSU. High expression of these cells strongly predicts better clinical responses to the anti-IgE therapy, omalizumab.

Interleukin (IL)-33 released from airway epithelial cells plays a vital role in shaping type 2 immune responses by binding to the ST2 receptor present in many immune cells, including mast cells (MCs). Intranasal administration of IL-33 in mice induces type 2 lung inflammation, an increase in lung MC progenitors, and transepithelial migration of leukocytes to the bronchoalveolar space. The aim of this study was to determine the contribution of MCs in IL-33-induced lung pathology. Four daily intranasal administrations of IL-33 reduced spirometry-like lung function parameters, induced airway hyperresponsiveness, and increased leukocytes in bronchoalveolar lavage fluid (BAL) in an ST2-dependent manner. MC-deficient (Cpa3^cre/+) mice, which lack MCs, had intact spirometry-like lung function but slightly reduced airway hyperresponsiveness, possibly related to reduced IL-33 or serotonin. Strikingly, Cpa3^cre/+ mice exposed to IL-33 had 50% reduction in BAL T-cells, and CXCL1 and IL-33 were reduced in the lung. Intranasal IL-33 induced CXCR2 expression in T-cells in a MC-independent fashion. Furthermore, IL-33-induced lung MCs were immunopositive for CXCL1 and localized in the epithelium of wild-type mice. These results suggest that MCs are required to sustain intact lung IL-33 and CXCL1 levels in mice with IL-33-induced airway inflammation, thereby facilitating T-cell accumulation in the bronchoalveolar space.

Background: Mast cells (MCs) develop from a rare population of peripheral blood circulating MC progenitors (MCps). Here, we investigated whether the frequency of circulating MCps is altered in asthma patients sensitized to birch pollen during pollen season, compared to out of season.

Methods: Asthma patients were examined during birch pollen season in late April to early June (May), and out of season in November–January. Spirometry measurements, asthma and allergy-related symptoms, asthma control questionnaire (ACQ), and asthma control test (ACT) scores were assessed at both time points. The MCp frequency was determined by flow cytometry in ficoll-separated blood samples from patients with positive birch pollen-specific IgE, and analyzed in relation to basic and disease parameters.

Results: The frequency of MCps per liter of blood was higher in May than in November (p = .004), particularly in women (p = .009). Patients that reported moderate to severe asthma symptoms (<.0001), nose or eye symptoms (p = .02; p = .01), or reduced asthma control (higher ACQ, p = .01) had higher MCp frequency in May than those that did not report this. These associations remained significant after adjusting for sex and BMI. The change in asthma control to a lower ACT score in May correlated with an increase in MCp frequency in May (p = .006, rho = 0.46).

Conclusions: The data suggest that the frequency of MCps increases in symptomatic patients with allergic asthma. Our results unravel a link between asthma symptoms and circulating MCps, and bring new insight into the impact of natural allergen exposure on the expansion of MCs.

Granzymes A and K are two highly homologous serine proteases expressed by mammalian cytotoxic T cells (CTL) and natural killer cells (NK). Granzyme A is the most abundant of the different granzymes (gzms) expressed by these two cell types. Gzms A and K are found in all jawed vertebrates and are the most well conserved of all hematopoietic serine proteases. Their potential functions have been studied extensively for many years, however, without clear conclusions. Gzm A was for many years thought to serve as a key component in the defense against viral infection by the induction of apoptosis in virus-infected cells, similar to gzm B. However, later studies have questioned this role and instead indicated that gzm A may act as a potent inducer of inflammatory cytokines and chemokines. Gzms A and K form clearly separate branches in a phylogenetic tree indicating separate functions. Transcriptional analyses presented here demonstrate the presence of gzm A and K transcripts in both CD4⁺ and CD8⁺ T cells. To enable screening for their primary biological targets we have made a detailed analysis of their extended cleavage specificities. Phage display analysis of the cleavage specificity of the recombinant enzymes showed that both gzms A and K are strict tryptases with high selectivity for Arg over Lys in the P1 position. The major differences in the specificities of these two enzymes are located N-terminally of the cleavage site, where gzm A prefers small amino acids such as Gly in the P3 position and shows a relatively relaxed selectivity in the P2 position. In contrast, gzm K prefers large amino acids such as Phe, Tyr, and Trp in both the P2 and P3 positions and does not tolerate negatively charged residues in the P2 position. This major distinction in extended specificities is likely reflected also in preferred in vivo targets of these two enzymes. This information can now be utilized for high-precision screening of primary targets for gzms A and K in search of their highly conserved but still poorly defined functions in vertebrate immunity.