Coincidental outbreak of methicillin-resistant Staphylococcus aureus in a hematopoietic stem cell transplantation unit

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most common nosocomial pathogens among hospital-acquired infections, and immunocompromised patients are highly susceptive to infection. The molecular typing of isolated strains is a common method for tracing an outbreak of MRSA, but experience with this approach is still limited in the hematopoietic stem cell transplantation (HSCT) ward. METHODS: We experienced 6 cases of MRSA infection/colonization in our 26-bed HSCT ward during a 4-week period. This unusual outbreak strongly suggested that the same MRSA strain was involved despite strict isolation and aseptic patient care. Clarification of the transmission pattern was critical, and we applied pulsed-field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP) assays for evaluation. RESULTS AND CONCLUSION: In four of the six cases, the pattern of bands examined by PFGE and AFLP analyses supported the idea that direct person-to-person transmission was very unlikely and the outbreak was coincidental. This experience highlights the clinical value of molecular typing methods for the clinical epidemiological assessment of MRSA outbreak.     

Development and characterization of a new monoclonal antibody against SARS-CoV-2 NSP12 (RdRp)

SARS-CoV-2 NSP12, the viral RNA-dependent RNA polymerase (RdRp), is required for viral replication and is a therapeutic target to treat COVID-19. To facilitate research on SARS-CoV-2 NSP12 protein, we developed a rat monoclonal antibody (CM12.1) against the NSP12 N-terminus that can facilitate functional studies. Immunoblotting and immunofluorescence assay (IFA) confirmed the specific detection of NSP12 protein by this antibody for cells overexpressing the protein. Although NSP12 is generated from the ORF1ab polyprotein, IFA of human autopsy COVID-19 lung samples revealed NSP12 expression in only a small fraction of lung cells including goblet, club-like, vascular endothelial cells, and a range of immune cells, despite wide-spread tissue expression of spike protein antigen. Similar studies using in vitro infection also generated scant protein detection in cells with established virus replication. These results suggest that NSP12 may have diminished steady-state expression or extensive posttranslation modifications that limit antibody reactivity during SARS-CoV-2 replication.     

MFG-E8 Regulates Angiogenesis in Cutaneous Wound Healing

Our research group recently demonstrated that pericytes are major sources of the secreted glycoprotein and integrin ligand lactadherin (MFG-E8) in B16 melanoma tumors, and that MFG-E8 promotes angiogenesis via enhanced PDGF–PDGFRβ signaling mediated by integrin–growth factor receptor crosstalk. However, sources of MFG-E8 and its possible roles in skin physiology are not well characterized. The objective of this study was to characterize the involvement of MFG-E8 in skin wound healing. In the dermis of normal murine and human skin, accumulations of MFG-E8 were found around CD31⁺ blood vessels, and MFG-E8 colocalized with PDGFRβ⁺, αSMA⁺, and NG2⁺ pericytes. MFG-E8 protein and mRNA levels were elevated in the dermis during full-thickness wound healing in mice. MFG-E8 was diffusely present in granulation tissue and was localized around blood vessels. Wound healing was delayed in MFG-E8 knockout mice, compared with the wild type, and myofibroblast and vessel numbers in wound areas were significantly reduced in knockout mice. Inhibition of MFG-E8 production with siRNA attenuated the formation of capillary-like structures in vitro. Expression of MFG-E8 in fibrous human granulation tissue with scant blood vessels was less than that in granulation tissue with many blood vessels. These findings suggest that MFG-E8 promotes cutaneous wound healing by enhancing angiogenesis.Wound healing is a dynamic process involving angiogenesis, production of soluble mediators and extracellular matrix, and migration of various types of cells, including keratinocytes, fibroblasts, macrophages, and leukocytes. Dysregulation of this interactive process may result in delayed wound healing, as is seen in chronic skin ulcers or scarring. Wound healing has three temporally overlapping phases: inflammation, tissue formation, and remodeling.^1,2 The inflammation phase occurs immediately after wounding. It is characterized by hypoxia with fibrin clot formation, as well as recruitment of neutrophils and platelets. Tissue formation occurs 2 to 10 days later and is characterized by epithelialization, formation of granulation tissue and new blood vessels, and accumulation of macrophages and fibroblasts. Activated macrophages release growth factors, such as vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF), and initiate angiogenesis. PDGF receptor β (PDGFRβ) signaling is essential for angiogenesis and for recruitment, proliferation, and normal function of fibroblasts and pericytes during the tissue-formation phase.³ Blockade of VEGF receptor signaling and PDGFRβ signaling inhibits angiogenesis and results in delayed wound healing,^3,4 indicating that angiogenesis is critical for normal wound healing.The secreted glycoprotein lactadherin was initially identified as a component of milk fat globules and is here referred to as milk fat globule-EGF factor 8 (MFG-E8); other names in the literature include secreted protein containing epidermal growth factor (EGF) repeats and discoidin/F5/8 domains 1, or SED1. MFG-E8 comprises two N-terminal EGF-like domains, and two C-terminal discoidin-like domains (C1 and C2) share homology with blood coagulation factors V and VIII.^5–9 One EGF-like domain (E2) contains an RGD consensus integrin-binding motif, and MFG-E8 binds to integrin αvβ3/5.^7–11 The C-terminal domains of MFG-E8 can bind to negatively charged and oxidized phospholipids,^12,13 facilitating opsonization of apoptotic cells for uptake by phagocytes.^10,14 This process has been reported to contribute to autoimmunity, mastitis, sepsis, atherosclerosis, and Alzheimer disease.^15–19 Interactions of MFG-E8 with CD51 (integrin αv) have also been implicated in regulation of angiogenesis and mammary gland branching,^11,20 and interactions mediated via the C1 domain are thought to be important for sperm–egg binding and collagen turnover.^21,22Our research group has previously demonstrated that MFG-E8 enhances angiogenesis in tumors and in oxygen-induced retinopathy in mice.²³ We determined that pericytes and/or pericyte precursors are important sources of MFG-E8 in vivo, that MFG-E8 enhances angiogenesis via actions on pericytes as well as endothelial cells (ECs), and that MFG-E8 can be effectively targeted with therapeutic benefit.²³ In murine melanomas and in retinas of mice with oxygen-induced retinopathy, MFG-E8 colocalized with pericytes rather than with ECs, and pericytes purified from tumors contained large amounts of MFG-E8 mRNA. Tumor- and retinopathy-associated angiogenesis was diminished in MFG-E8 knockout (KO) mice, and pericyte coverage of neovessels was also reduced. Inhibition of MFG-E8 production by pericyte/pericyte precursor-like 10T1/2 cells using siRNAs, or inhibition of MFG-E8 action with some anti–MFG-E8 antibodies, attenuated PDGF-BB–induced 10T1/2 cell migration, but did not affect proliferation or differentiation.²³ We have also determined that MFG-E8 produced by 10T1/2 cells associated with integrin αv and PDGFRβ on cell surfaces after PDGF-BB treatment, altered the distribution of PDGFRβ within cells and delayed PDGF-BB–stimulated degradation of PDGFRβ, thereby enhancing PDGFRβ signaling mediated by integrin–growth factor receptor crosstalk.²⁴In a study of MFG-E8, epithelial tissues, and wound healing, Bu et al²⁵ found that MFG-E8 promotes the migration of intestinal epithelial cells via a PKCε-dependent mechanism engaged by binding of MFG-E8 to phosphatidylserine. Their findings indicate that MFG-E8 is involved in the maintenance of intestinal epithelial homeostasis and the promotion of mucosal healing. The possible role of MFG-E8 in cutaneous wound healing has not been studied previously. In the present study, we analyzed skin wound healing using MFG-E8 wild-type (WT) and KO mice. We demonstrate that MFG-E8 production was increased and that MFG-E8 accumulated in granulation tissue during wound healing, and that wound healing in MFG-E8 KO mice was delayed. We relate delayed wound healing to diminished angiogenesis and myofibroblast infiltration in wounds in MFG-E8 KO mice.     

Estrogen receptor alpha polymorphism as a genetic marker for bone loss, vertebral fractures and susceptibility to estrogen

OBJECTIVE: The purpose of the present study was to investigate the possible roles of PvuII and XbaI polymorphisms of the estrogen receptor alpha (ER(alpha)) in bone mineral density (BMD), vertebral fracture, bone loss rate after menopause and response to hormone replacement therapy (HRT). METHODS: All 286 women were grouped according to the genotypes of PvuII or XbaI polymorphisms of the ER(alpha) gene. We compared the BMD Z-score, incidence of vertebral fracture, changes in Z-score after menopause and response of BMD to HRT among the genotypes. RESULTS: Subjects with the PPxx genotype had significantly (P<0.05) lower Z-scores than did subjects with the other genotypes. A negative correlation was observed between the length of time after menopause and the decrease of the Z-score only in women with the pp genotype, suggesting faster bone loss in this group. In the analysis of the ER(alpha) polymorphism with regard to the effect of HRT on BMD, there appears to be a significantly greater increase of BMD (P<0.01 and 0.05) in women with the pp genotype than in those with the Pp or PP genotype. CONCLUSIONS: PvuII and XbaI polymorphisms of the ER(alpha) gene were associated with BMD in postmenopausal Japanese women. Also, the polymorphisms may be useful genetic markers for predicting vertebral fracture in relatively young postmenopausal women. The PvuII polymorphism may be associated with susceptibility to changes in estrogen level.     

GABA-induced intracellular Ca2+ elevation in the embryonic chick brainstem slice

We examined the intracellular Ca2+ ([Ca2+]i) elevation evoked by GABA in an 8-day embryonic chick brainstem slice using a Ca imaging technique with Ca green-1 AM. When small quantities of GABA were pressure-ejected on the surface of the slice, the [Ca2+]i elevation was clearly detected. The GABA-induced [Ca2+]i elevation was eliminated in a Ca2+-free solution, whereas the previously reported GABA-induced light-scattering change was independent of extracellular Ca2+. Although, micro-application of glycine or glutamate also induced [Ca2+]i elevation, these changes were smaller than that by GABA. These results suggest that the GABA-induced [Ca2+]i elevation is due to Ca2+ entry resulting from membrane depolarization and may play an important role in the development of the central nervous system (CNS).     

Detailed glomerular ultrastructure in Japanese type 2 diabetic patients by the quick-freezing and deep-etching method

Mesangial expansion and glomerular basement membrane (GBM) thickening did not correlate with urinary albumin excretion (UAE) in type 2 diabetic patients in our previous studies; therefore, it was necessary to elucidate more detailed ultrastructural changes in the early stages of diabetic nephropathy (DN) in type 2 diabetic patients. The quick-freezing and deep-etching (QF–DE) method allows us to examine three-dimensional ultrastructures of human renal glomeruli in vivo at high resolution. The QF–DE method was applied to six type 2 diabetic patients without definable renal diseases other than DN. Four patients were normoalbuminuric (NA) and the other two were microalbuminuria (MA). Three control specimens were the normal parts from nephrectomies due to renal cell carcinomas. Electron microscopic morphometric analyses provided quantitative glomerular structural changes. Replica membranes were prepared by the QF–DE method, and diameters of mesh structures at the GBM and mesangial matrix (MM) were measured on electron micrographs as previously described. By the QF–DE method, both the GBM middle layer and MM were composed of polygonal meshwork structures. The mesh pores of the GBM and MM were more enlarged and irregular in shape in NA diabetic patients than those of the controls, and these ultrastructural changes became more obvious in MA patients. The mesh diameters of the GBM and MM in the diabetic patients were also larger than those of the controls. Such a mesh diameter of the GBM was well correlated with the amount of UAE, while the mesh diameter of MM showed a slight correlation with UAE. Although there were small number of subjects in the present study, the detailed ultrastructural changes in NA and MA type 2 diabetic patients, which had not been disclosed by conventional electron microscopy, were revealed by the QF–DE method. Increased mesh diameters of GBM might be related with the increase of UAE.