Characterization of Outer Membrane Vesicles Produced by Vibrio vulnificus.

Vibrio vulnificus (V. vulnificus) is a halophilic gram-negative bacterium that inhabits coastal warm water and induce severe diseases such as primary septicemia. To investigate the mechanisms of rapid bacterial translocation on intestinal infection, we focused on outer membrane vesicles (OMVs), which are extracellular vesicles produced by Gram-negative bacteria and deliver virulence factors. However, there are very few studies on the pathogenicity or contents of V. vulnificus OMVs (Vv-OMVs). In this study, we investigated the effects of Vv-OMVs on host cells. Epithelial cells INT407 were stimulated with purified OMVs and morphological alterations and levels of lactate dehydrogenase (LDH) release were observed. In cells treated with OMVs, cell detachment without LDH release was observed, which exhibited different characteristics from cytotoxic cell detachment observed in V. vulnificus infection. Interestingly, OMVs from a Vibrio Vulnificus Hemolysin (VVH) and Multifunctional-autoprocessing repeats-in -toxin (MARTX) double-deletion mutant strain also caused cell detachment without LDH release. Our results suggested that the proteolytic function of a serine protease contained in Vv-OMVs may contribute to pathogenicity of V. vulnificus by assisting bacterial translocation. This study reveals a new pathogenic mechanism during V. vulnificus infections. J. Med. Invest. 71 : 102-112, February, 2024.

GTN057, a komaroviquinone derivative, induced myeloma cells' death in vivo and inhibited c-MET tyrosine kinase.

OBJECTIVE: Despite the development of newly developed drugs, most multiple myeloma (MM) patients with high-risk cytogenetic abnormalities such as t(4;14) or del17p relapse at anin early stage of their clinical course. We previously reported that a natural product,komaroviquinone (KQN), isolated from the perennial semi-shrub Dracocephalum komarovi, i.e., komaroviquinone (KQN) and its derivative GTN024 induced the apoptosis of MM cells by producing reactive oxygen species (ROS), but both exhibited significant hematological toxicity. Aim of this study is to clarify anti-tumor activity, safety and pharmacokinetics of GTN057, an optimization compound of KQN in vivo. METHODS: ICR/SCID xenograft model of KMS11, a t(4;14) translocation-positive MM cell line, was used for in vivo study. Mice pharmacokinetics of GTN057 and the degradation products were analyzed by LC-MS/MS. RESULTS: Herein, our in vitro experiments revealed that GTN057 is much less toxic to normal hematopoietic cells, induced the apoptosis of both MM cell lines andpatient samples, including those with high-risk cytogenetic changes. A xenograft model of a high-risk MM cell line demonstrated that GTN057 significantly delayed the tumor growth with no apparent hematological or systemic toxicities in vivo. The pathological examination of GTN057-treated tumors in vivoshowed revealed apoptosis of MM cells and anti-angiogenesis. In addition to the production of ROS, GTN057 inhibited the downstream signaling of c-MET, a receptor tyrosine kinase a receptor forand hepatocyte growth factor (HGF) receptor. Thus, GTN057 is less toxic and is able tomay be a candidate drug for treating MM patients, via multifunctional mechanisms. We have also extensively studied the pharmacologyical analysis of GTN057. The metabolites of GTN057, (e.g.,such as GTN054), may also have anti-tumorantitumor activity. CONCLUSION: Natural products or and their derivatives can could be good sources of antineoplastic drugs even for high-risk cancer.

Negative E-cadherin expression on bone marrow myeloma cell membranes is associated with extramedullary disease.

Background: The loss of E-cadherin expression and the induction of N-cadherin are known as hallmarks of the epithelial-to-mesenchymal transition, an essential initial step in the process of metastasis in solid tumors. Although several studies have reported expressions of these cadherins in patients with multiple myeloma (MM), their clinical significance is unknown as MM cells are non-epithelial. Methods: In this study, we examined the expression of E- and N-cadherins by immunohistochemistry using bone marrow (BM) biopsy specimens from 31 newly diagnosed MM patients and in subsequent biopsy specimens from six of these. Results: Negative E-cadherin expression on BM myeloma cell membranes was significantly associated with the presence of soft-tissue masses arising from bone lesions and breaking through the cortical bone, referred to as extramedullary disease (EMD). Conclusions: Given the aggressive nature of EMD, our study suggests that screening for E-cadherin using BM immunohistochemistry is one measure that could predict the development of EMD in patients with MM.

[PAX5-positive plasma cell leukemia presenting as lymphocytosis].

An 82-year-old Japanese male patient was initially diagnosed with lymphocytosis. His complete blood count revealed a white blood cell count of 30.9×109/l with 81% abnormal lymphocytes. The abnormal lymphocytes included monoclonal clones of CD38+ and CD138+cytoplasmic κ+ and IgG-κ M-protein, which led to the final diagnosis of plasma cell leukemia (PCL). Bortezomib and dexamethasone therapy was initiated, but the patient succumbed to the disease on the 8th day of hospitalization. A cytogenetic examination revealed a t (9;14)(p13;q32) translocation and the Western blotting confirmed high PAX5 expression. Similar to our present case, PCL cases with "lymphocytosis" have been widely reported, which some speculating the involvement of PAX5 overexpression in the pathogenesis. Such cases, including ours, may be classified as a unique group of disorders (PCL presenting as "lymphocytosis"), which requires accurate differential diagnosis and subsequent urgent multidisciplinary intensive treatment.

Reversible modification of mitochondrial ADP/ATP translocases by paired Legionella effector proteins.

Adenosine diphosphate (ADP) ribosylation is a reversible posttranslational modification involved in the regulation of numerous cellular processes. Prototype ADP ribosyltransferases (ARTs) from many pathogenic bacteria are known to function as toxins, while other bacterial ARTs have just recently emerged. Recent studies have shown that bacteria also possess enzymes that function as poly-ADP ribose (ADPr) glycohydrolases (PARGs), which reverse poly-ADP ribosylation. However, how bacteria manipulate host target proteins by coordinated reactions of ARTs and ADPr hydrolases (ARHs) remains elusive. The intracellular bacterial pathogen Legionella pneumophila, the causative agent of Legionnaires' disease, transports a large array of effector proteins via the Dot/Icm type IV secretion system to host cells. The effector proteins, which mostly function as enzymes, modulate host cellular processes for the bacteria's benefit. In this study, we identified a pair of L. pneumophila effector proteins, Lpg0080 and Lpg0081, which function as an ART and an ARH, respectively. The two proteins were shown to coordinately modulate mitochondrial ADP/adenosine triphosphate (ATP) translocases (ANTs) by their enzymatic activities to conjugate ADPr to, and remove it from, a key arginine residue. The crystal structures of Lpg0081 and the Lpg0081:ADPr complex indicated that Lpg0081 is a macroD-type ARH with a noncanonical macrodomain, whose folding topology is strikingly distinct from that of the canonical macrodomain that is ubiquitously found in eukaryotic PARGs and ARHs. Our results illustrate that L. pneumophila has acquired an effector pair that coordinately manipulate mitochondrial activity via reversible chemical modification of ANTs.

Rare evolution of CSF3R-mutated chronic neutrophilic leukemia to t(4;12)(q12;p13) acute myeloid leukemia with SETBP1 mutation.

Chronic neutrophilic leukemia (CNL) is a rare myeloproliferative disease accompanied by mutations in CSF3R. Here, we present a patient with CNL who developed to acute myeloid leukemia (AML) at the same time that a t(4;12)(q12;p13) translocation appeared. The uniqueness of this cytogenetic abnormality led us to delineate the molecular aberrations relevant for clonal evolution. While the CSF3R mutation was present throughout the course of the disease, the SETBP1 mutation was newly acquired at the AML transformation. The present case suggests that careful monitoring of t(4;12)(q12;p13) and SETBP1 is crucial to predict AML evolution in CNL patients.

Philadelphia Chromosome-positive Acute Lymphoblastic Leukemia Incidentally Detected by Fluorodeoxyglucose-positron Emission Tomography/Computed Tomography at a Health Checkup.

We herein report a case of Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph-ALL) that was incidentally detected by fluorodeoxyglucose-positron emission tomography (18F-FDG PET)/computed tomography (CT) at a health checkup. At that time, the findings of a physical examination and blood tests were all normal, except for the diffuse bone marrow uptake (maximum standardized uptake value: 6.3). One month later, when the blood counts remained in the normal ranges, a bone marrow examination confirmed the diagnosis of Ph-ALL. Although a diffuse bone marrow uptake of 18F-FDG is observed in some benign conditions, physicians should also consider the possibility of hematological malignancies, including acute leukemia, even when that is the only abnormal finding.

A novel homozygous variant of the thrombomodulin gene causes a hereditary bleeding disorder.

We report a 19-year-old Vietnamese woman who experienced several life-threatening bleeding events, including ovarian hemorrhage. Blood analysis revealed a decreased fibrinogen level with markedly elevated fibrinogen/fibrin degradation products and D-dimer levels. Despite hemostatic surgery and administration of several medications, such as nafamostat mesylate, tranexamic acid, and unfractionated heparin, the coagulation abnormalities were not corrected, and the patient experienced repeated hemorrhagic events. We found that administration of recombinant human thrombomodulin (rhTM) remarkably improved the patient's pathophysiology. Screening and sequencing of the TM gene (THBD) revealed a previously unreported homozygous variation: c.793T>A (p.Cys265Ser). Notably, the Cys265 residue forms 1 of 3 disulfide bonds in the epidermal growth factor (EGF)-like domain 1 of TM. Transient expression experiments using COS-1 cells demonstrated markedly reduced expression of TM-Cys265Ser on the plasma membrane relative to wild-type TM. The TM-Cys265Ser mutant was intracellularly degraded, probably because of EGF-like domain 1 misfolding. The reduced expression of TM on the endothelial cell membrane may be responsible for the disseminated intravascular-coagulation-like symptoms observed in the patient. In summary, we identified a novel TM variant, c.793T>A (p.Cys265Ser). Patients homozygous for this variant may present with severe bleeding events; rhTM should be considered a possible treatment option for these patients.