Activation dependent clustering of the erbB2 receptor tyrosine kinase detected by scanning near-field optical microscopy

ErbB2 (HER2, Neu), a member of the epidermal growth factor (EGF) receptor tyrosine kinase family, is often overexpressed in breast cancer and other malignancies, ErbB2 homodimerizes but also presents as a common auxiliary subunit of the EGF and heregulin receptors (erbB1 or EGFR; and erbB3-4, respectively), with which it heteroassociates, ErbB2 is generally regarded as an orphan (ligand-less) receptor with a very potent kinase domain activated either via its associated partners or constitutively as a consequence of discrete mutations. It follows that the extent and regulation of its cell surface interactions are of central importance. We have studied the large-scale association pattern of erbB2 in quiescent and activated cells labeled with fluorescent anti-erbB2 monoclonal antibodies using scanning near-field optical microscopy (SNOM), ErbB2 was found to be concentrated in irregular membrane patches with a mean diameter of approx. 0.5 mu m in nonactivated SKBR3 and MDA453 human breast tumor cells. The average number of erbB2 proteins in a single cluster on nonactivated SKBR3 cells was about 10(3). Activation of SKBR3 cells with EGF, heregulin as well as a partially agonistic anti-erbB2 monoclonal antibody led to an increase in the mean cluster diameter to 0.6-0.9 mu m, irrespective of the ligand, The EGF-induced increase in the erbB2 cluster size was inhibited by the EGFR-specific tyrosine kinase inhibitor PD153035, The average size of erbB2 clusters on the erbB2-transfected line of CHO cells (CB2) was similar to that of activated SKBR3 cells, a finding correlated with the increased base-line tyrosine phosphorylation of erbB2 in cells expressing only erbB2, We conclude that an increase in cluster size may constitute a general phenomenon in the activation of erbB2

Grading of recommendations, assessment, development and evaluations concept 7: issues and insights linking guideline recommendations to trustworthy essential medicine lists

Objectives: Guidelines and essential medicine lists (EMLs) bear similarities and differences in the process that lead to decisions. Access to essential medicines is central to achieve universal health coverage. The World Health Organization (WHO) EML has guided prioritization of essential medicines globally for nearly 50 years, and national EMLs (NEMLs) exist in over 130 countries. Guideline and EML decisions, at WHO or national levels, are not always coordinated and aligned. We sought to explore challenges, and potential solutions, for decision-making to support trustworthy medicine selection for EMLs from a Grading of Recommendations, Assessment, Development and Evaluations (GRADE) Working Group perspective. We primarily focus on the WHO EML; however, our findings may be applicable to NEML decisions as well. Study Design and Setting: We identified key challenges in connecting the EML to health guidelines by involving a broad group of stakeholders and assessing case studies including real applications to the WHO EML, South Africa NEML, and a multiple sclerosis guideline connected to a WHO EML application for multiple sclerosis treatments. To address challenges, we utilized the results of a survey and feedback from the stakeholders, and iteratively met as a project group. We drafted a conceptual framework of challenges and potential solutions. We presented a summary of the results for feedback to all attendees of the GRADE Working Group meetings in November 2022 (approximately 120 people) and in May 2023 (approximately 100 people) before finalizing the framework. Results: We prioritized issues and insights/solutions that addressed the connections between the EML and health guidelines. Our suggested solutions include early planning alignment of guideline groups and EMLs, considering shared participation to strengthen linkage, further clarity on price/cost considerations, and using explicit shared criteria to make guideline and EML decisions. We also provide recommendations to strengthen the connection between WHO EML and NEMLs including through contextualization methods. Conclusion: This GRADE concept article, jointly developed by key stakeholders from the guidelines and EMLs field, identified key conceptual issues and potential solutions to support the continued advancement of trustworthy EMLs. Adopting structured decision criteria that can be linked to guideline recommendations bears the potential to advance health equity and gaps in availability of essential medicines within and between countries

Primary alterations during the development of hidradenitis suppurativa

BACKGROUND: Hidradenitis suppurativa (HS) is a chronic, inflammatory disease of the apocrine gland‐rich (AGR) skin region. The initial steps of disease development are not fully understood, despite intense investigations into immune alterations in lesional HS skin. OBJECTIVES: We aimed to systematically investigate the inflammatory molecules involved in three stages of HS pathogenesis, including healthy AGR, non‐lesional HS and lesional HS skin, with the parallel application of multiple mRNA and protein‐based methods. METHODS: Immune cell counts (T cells, dendritic cells, macrophages), Th1/Th17‐related molecules (IL‐12B, TBX21, IFNG, TNFA, IL‐17, IL10, IL‐23A, TGFB1, RORC, CCL20), keratinocyte‐related sensors (TLR2,4), mediators (S100A7, S100A8, S100A9, DEFB4B, LCN2, CAMP, CCL2) and pro‐inflammatory molecules (IL1B, IL6, TNFA, IL‐23A) were investigated in the three groups by RNASeq, RT‐qPCR, immunohistochemistry and immunofluorescence. RESULTS: Epidermal changes were already detectable in non‐lesional HS skin; the epidermal occurrence of antimicrobial peptides (AMPs), IL‐1β, TNF‐α and IL‐23 was highly upregulated compared with healthy AGR skin. In lesional HS epidermis, TNF‐α and IL‐1β expression remained at high levels while AMPs and IL‐23 increased even more compared with non‐lesional skin. In the dermis of non‐lesional HS skin, signs of inflammation were barely detectable (vs. AGR), while in the lesional dermis, the number of inflammatory cells and Th1/Th17‐related mediators were significantly elevated. CONCLUSIONS: Our findings that non‐lesional HS epidermal keratinocytes produce not only AMPs and IL‐1β but also high levels of TNF‐α and IL‐23 confirm the driver role of keratinocytes in HS pathogenesis and highlight the possible role of keratinocytes in the transformation of non‐inflammatory Th17 cells (of healthy AGR skin) into inflammatory cells (of HS) via the production of these mediators. The fact that epidermal TNF‐α and IL‐23 appear also in non‐lesional HS seems to prove these cytokines as excellent therapeutic targets

Induction of fibroblast senescence generates a non-fibrogenic myofibroblast phenotype that differentially impacts on cancer prognosis

Cancer-associated fibroblasts (CAF) remain a poorly characterized, heterogeneous cell population. Here we characterized two previously described tumor-promoting CAF sub-types, smooth muscle actin (SMA)-positive myofibroblasts and senescent fibroblasts, identifying a novel link between the two. Analysis of CAF cultured ex vivo, showed that senescent CAF are predominantly SMA-positive; this was confirmed by immunochemistry in head & neck (HNSCC) and esophageal (EAC) cancers. In vitro, we found that fibroblasts induced to senesce develop molecular, ultrastructural and contractile features typical of myofibroblasts and this is dependent on canonical TGF-? signaling. Similar to TGF-?1-generated myofibroblasts, these cells secrete soluble factors that promote tumor cell motility. However, RNA-sequencing revealed significant transcriptomic differences between the two SMA-positive CAF groups, particularly in genes associated with extracellular matrix (ECM) deposition and organization, which differentially promote tumor cell invasion. Notably, second harmonic generation imaging and bioinformatic analysis of SMA-positive human HNSCC and EAC showed that collagen fiber organization correlates with poor prognosis, indicating that heterogeneity within the SMA-positive CAF population differentially impacts on survival. These results show that non-fibrogenic, SMA-positive myofibroblasts can be directly generated through induction of fibroblast senescence and suggest that senescence and myofibroblast differentiation are closely linked processes

Novel experimental setup for megahertz X-ray diffraction in a diamond anvil cell at the High Energy Density (HED) instrument of the European X-ray Free-Electron Laser (EuXFEL)

The high-precision X-ray diffraction setup for work with diamond anvil cells (DACs) in interaction chamber 2 (IC2) of the High Energy Density instrument of the European X-ray Free-Electron Laser is described. This includes beamline optics, sample positioning and detector systems located in the multipurpose vacuum chamber. Concepts for pump-probe X-ray diffraction experiments in the DAC are described and their implementation demonstrated during the First User Community Assisted Commissioning experiment. X-ray heating and diffraction of Bi under pressure, obtained using 20 fs X-ray pulses at 17.8 keV and 2.2 MHz repetition, is illustrated through splitting of diffraction peaks, and interpreted employing finite element modeling of the sample chamber in the DAC