A clinical effect of disease-modifying treatment on alloimmunisation in transfused patients with myelodysplastic syndromes:Data from a population-based study

BACKGROUND: Alloimmunisation against blood products is an adverse event, causing time-consuming compatibility testing. Current literature has not yet identified the influence of treatment on the risk of alloimmunisation in patients with myelodysplastic syndromes (MDS). MATERIALS AND METHODS: An observational, population-based study, using the HemoBase registry, was performed including all transfused patients who were diagnosed with MDS between 2005 and 2017 in Friesland, a province in the Netherlands. Information about transfusion dates, types, and treatment regimens was collected from the health records. Blood products were matched for ABO and Rhesus D. The effect of disease-modifying treatment was estimated with incidence rates and a Cox time-dependent analysis. RESULTS: 233 patients were included in this study, with a median follow-up of 13.0 months. Alloimmunisation occurred in 21 patients (9.0%) and predominantly occurred early in follow-up. Three (5%) and 18 (11%) alloimmunisation events occurred in patients with and without disease-modifying treatment, respectively. The hazard ratio for alloimmunisation without treatment compared to during treatment was 2.7 (95% CI: 0.35–20.0), with incidence rates of 7.18 and 2.41 per 100 patient-years, respectively. DISCUSSION: In a non-selected real-world population of MDS patients receiving blood transfusions, the percentage of patients with alloimmunisation was below 10%. The results of this study support the hypothesis that disease-modifying treatment affects the ability of the immune system to mount an antibody response to non-self blood group antigens

Par1b induces asymmetric inheritance of plasma membrane domains via LGN-dependent mitotic spindle orientation in proliferating hepatocytes

The development and maintenance of polarized epithelial tissue requires a tightly controlled orientation of mitotic cell division relative to the apical polarity axis. Hepatocytes display a unique polarized architecture. We demonstrate that mitotic hepatocytes asymmetrically segregate their apical plasma membrane domain to the nascent daughter cells. The non-polarized nascent daughter cell can form a de novo apical domain with its new neighbor. This asymmetric segregation of apical domains is facilitated by a geometrically distinct “apicolateral” subdomain of the lateral surface present in hepatocytes. The polarity protein partitioning-defective 1/microtubule-affinity regulating kinase 2 (Par1b/MARK2) translates this positional landmark to cortical polarity by promoting the apicolateral accumulation of Leu-Gly-Asn repeat-enriched protein (LGN) and the capture of nuclear mitotic apparatus protein (NuMA)–positive astral microtubules to orientate the mitotic spindle. Proliferating hepatocytes thus display an asymmetric inheritance of their apical domains via a mechanism that involves Par1b and LGN, which we postulate serves the unique tissue architecture of the developing liver parenchyma

Myosin Vb and Rab11a regulate phosphorylation of ezrin in enterocytes

Microvilli at the apical surface of enterocytes allow the efficient absorption of nutrients in the intestine. Ezrin activation by its phosphorylation at T567 is important for microvilli development, but how such ezrin phosphorylation is controlled is not well understood. We demonstrate that a subset of kinases that phosphorylate ezrin closely co-distributes with apical recycling endosome marker Rab11a in the subapical domain. Expression of dominant-negative Rab11a mutant or depletion of the Rab11a-binding motor protein myosin Vb prevents the subapical enrichment of Rab11a and these kinases and inhibits ezrin phosphorylation and microvilli development, without affecting the polarized distribution of ezrin itself. We observe a similar loss of the subapical enrichment of Rab11a and the kinases and reduced phosphorylation of ezrin in microvillus inclusion disease, which is associated with MYO5B mutations, intestinal microvilli atrophy and malabsorption. Thus, part of the machinery for ezrin activation depends on recycling endosomes controlled by myosin Vb and Rab11a which, we propose, might act as subapical signaling platforms that enterocytes use to regulate development of microvilli and maintain human intestinal function

The special case of hepatocytes:unique tissue architecture calls for a distinct mode of cell division

Columnar epithelia (e.g., kidney, intestine) and hepatocytes embody the two major organizational phenotypes of non-stratified epithelial cells. Columnar epithelia establish their apical and basal domains at opposing poles and organize in monolayered cysts and tubules, in which their apical surfaces form a single continuous lumen whereas hepatocytes establish their apical domains in the midst of their basolateral domains and organize a highly branched capillary luminal network, the bile canaliculi, in which a single hepatocyte can engage in lumen formation with multiple neighbors. To maintain their distinct tissue architectures, columnar epithelial cells bisect their luminal domains during symmetric cell divisions, while the cleavage furrow in dividing hepatocytes avoids bisecting the bile canalicular domains. We discuss recently discovered molecular mechanisms that underlie the different cell division phenotypes in columnar and hepatocytic model cell lines. The serine/threonine kinase Par1b determines both the epithelial lumen polarity and cell division phenotype via cell adhesion signaling that converges on the small GTPase RhoA

Implications of mitotic spindle orientation during the development of the unique liver architecture.

<p>(1) LGN localizes to the apicolateral plasma membrane area during hepatocyte cell division. (2) The mitotic spindle orients one of its (NuMA-containing) spindle poles towards the LGN-enriched apicolateral plasma membrane. (3) This orientation of the mitotic spindle results in the cleavage furrow not bisecting the apical plasma membrane, resulting in asymmetric segregation of the apical plasma membrane. (4) New apical surfaces are created de novo at the site of abscission. (5) During early liver development, apical pockets are created between hepatocytes. (6) These pockets grow out to bile canalicular/channel-like structures during later phases of liver development. (7) When Par1b is impaired, LGN migrates away from the apicolateral plasma membrane area and is subsequently found on basal or lateral membranes. (8) The mitotic spindle orients its poles towards LGN-enriched cortical areas. (9) The cleavage furrow has an increased chance of bisecting the apical plasma membrane, resulting in symmetric segregation of the apical plasma membrane. (10) Both cells now share the same apical surface (“simple” epithelial polarity). (11) Continued cell division likely results in the generation of “simple” epithelial cyst-like structures.</p

Par1b stimulates apicolateral-directed spindle orientation and asymmetric segregation of the apical domain in MDCK cells.

<p>(A) Schematic overview of the polarity phenotype in control and Par1b-overexpressing MDCK cells. (B) Fixed control and MDCK-Par1b cells were labeled for the apical marker gp135 (left panel, red) and LGN (right panel, yellow). Par1b-overexpressing MDCK cells asymmetrically segregate their apical domain (left panel, red arrowheads; black arrowheads mark the ingressing cleavage furrow) during cell division. LGN localizes to the apicolateral plasma membrane domain in Par1b-overexpressing MDCK cells (right panel, black arrowheads). The dashed line marks the common lateral plasma membrane domain. (C) Histogram analysis of the SA/PA angle shows that dividing MDCK-Par1b cells exhibit a bias towards lower angles (0–30°) in metaphase, as observed for hepatocytes. All figures: red arrowheads mark the apical domain. *<i>p</i><0.05. ***<i>p</i><0.001. Scale bars: 5 µm.</p

Hepatocytes predominantly orient their mitotic spindle axis towards the apicolateral subdomain.

<p>(A) SA axes were quantified as crossing (marked by black arrowheads) the apicolateral membrane (situation 1) or other membranes (situation 2), indicating a bias of the SA axis to cross the apicolateral membrane. (B) Localization of LGN (white outline arrowheads) in polarized HepG2 cells. The apical domain is labeled with ABCB1 and marked by a red arrowhead. The outline diagram (“PM domains”) shows the identity of the cell membranes of the dividing cell (#). Grey, red, orange, and green lines represent the basal, apical, lateral, and apicolateral plasma membrane domains, respectively. (C) Schematic overview of how the orientation of the mitotic spindle (angle between the SA and PA [angle SA/PA]) was measured (see Materials and Methods). (D) Dot plot of the SA/PA angle for dividing HepG2 cells in metaphase. Shown is mean (green bar) and standard error of the mean (SEM) (blue error bars). (E) Histogram analysis reveals a strong bias for HepG2 cells to divide with an SA/PA angle between 0° and 30° during metaphase. *<i>p</i><0.05; **<i>p</i><0.01. BC, bile canaliculus. Scale bars: 5 µm.</p

Rat and mouse hepatocytes predominantly orient their mitotic spindle axis towards the apicolateral subdomain.

<p>(A) Hepatocytes from mouse livers 48 h post-hepatectomy orient their spindle poles (labeled with NuMA) towards the apicolateral subdomain. (B) Quantification of (A) (<i>n</i> = 61). Dividing hepatocytes predominantly orient their SA towards the apicolateral subdomain. (C) Dividing hepatocytes from weaned rat livers orient their spindle poles (marked by NuMA) towards the apicolateral subdomain (marked by DPPIV and ABCC2) in metaphase and telophase. (D) Apicolateral localization of LGN (white outline arrowheads) in dividing rat liver hepatocytes. The apical domain is labeled with ABBC2. Tight junctions are labeled with ZO-1. The outline diagrams (“PM domains”) show the identity of the cell membranes of the dividing cells (#) shown in (D). Grey, red, orange, and green lines represent the basal, apical, lateral, and apicolateral plasma membrane domains, respectively. All figures: filled white arrowheads mark the bile canaliculus or apical domain. Dotted white lines outline the sinusoid (si). Dashed lines indicate the SA. *<i>p</i><0.05 (calculated using a paired two-tailed Student's <i>t</i>-test). Scale bars: 5 µm.</p

Synaptotagmin-like proteins control the formation of a single apical membrane domain in epithelial cells

The formation of epithelial tissues requires both the generation of apical-basal polarity and the coordination of this polarity between neighbouring cells to form a central lumen. During de novo lumen formation, vectorial membrane transport contributes to the formation of a singular apical membrane, resulting in the contribution of each cell to only a single lumen. Here, from a functional screen for genes required for three-dimensional epithelial architecture, we identify key roles for synaptotagmin-like proteins 2-a and 4-a (Slp2-a/4-a) in the generation of a single apical surface per cell. Slp2-a localizes to the luminal membrane in a PtdIns(4,5)P(2)-dependent manner, where it targets Rab27-loaded vesicles to initiate a single lumen. Vesicle tethering and fusion is controlled by Slp4-a, in conjunction with Rab27/Rab3/Rab8 and the SNARE syntaxin-3. Together, Slp2-a/4-a coordinate the spatiotemporal organization of vectorial apical transport to ensure that only a single apical surface, and thus the formation of a single lumen, occurs per cell