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A man in his 60s developed a pancreatic pseudocyst postoperatively after an open graft replacement for a ruptured abdominal aortic aneurysm. Endoscopic drainage was performed; however, this led to an aortic graft infection due to macroscopic communication with the perigraft cavity. Percutaneous drainage was performed to manage the pancreatic fistula and graft infection simultaneously. Although the pancreatic pseudocyst diminished, the aortic graft infection persisted. Subsequently, partial aortic graft replacement with greater omental flap coverage was performed. He was discharged with oral antibiotics, with no recurrence of infections at 10 months., (@ 2024 The Editorial Committee of Annals of Vascular Diseases.)

Small intestinal mononuclear cells that express CX3CR1 (CX3CR1 + cells) regulate immune responses 1-5 . CX3CR1 + cells take up luminal antigens by protruding their dendrites into the lumen 1-4,6 . However, it remains unclear how dendrite protrusion by CX3CR1 + cells is induced in the intestine. Here we show in mice that the bacterial metabolites pyruvic acid and lactic acid induce dendrite protrusion via GPR31 in CX3CR1 + cells. Mice that lack GPR31, which was highly and selectively expressed in intestinal CX3CR1 + cells, showed defective dendrite protrusions of CX3CR1 + cells in the small intestine. A methanol-soluble fraction of the small intestinal contents of specific-pathogen-free mice, but not germ-free mice, induced dendrite extension of intestinal CX3CR1 + cells in vitro. We purified a GPR31-activating fraction, and identified lactic acid. Both lactic acid and pyruvic acid induced dendrite extension of CX3CR1 + cells of wild-type mice, but not of Gpr31b -/- mice. Oral administration of lactate and pyruvate enhanced dendrite protrusion of CX3CR1 + cells in the small intestine of wild-type mice, but not in that of Gpr31b -/- mice. Furthermore, wild-type mice treated with lactate or pyruvate showed an enhanced immune response and high resistance to intestinal Salmonella infection. These findings demonstrate that lactate and pyruvate, which are produced in the intestinal lumen in a bacteria-dependent manner, contribute to enhanced immune responses by inducing GPR31-mediated dendrite protrusion of intestinal CX3CR1 + cells.

We have examined the role of Fam60a , a gene highly expressed in embryonic stem cells, in mouse development. Fam60a interacts with components of the Sin3a-Hdac transcriptional corepressor complex, and most Fam60a -/- embryos manifest hypoplasia of visceral organs and die in utero. Fam60a is recruited to the promoter regions of a subset of genes, with the expression of these genes being either up- or down-regulated in Fam60a -/- embryos. The DNA methylation level of the Fam60a target gene Adhfe1 is maintained at embryonic day (E) 7.5 but markedly reduced at E9.5 in Fam60a -/- embryos, suggesting that DNA demethylation is enhanced in the mutant. Examination of genome-wide DNA methylation identified several differentially methylated regions, which were preferentially hypomethylated, in Fam60a -/- embryos. Our data suggest that Fam60a is required for proper embryogenesis, at least in part as a result of its regulation of DNA methylation at specific gene promoters., Competing Interests: RN, ON, TM, NS, TI, KY, YS, CM, MK, HS, SK, HH No competing interests declared, (© 2018, Nabeshima et al.)

Lrrc6 encodes a cytoplasmic protein that is expressed specifically in cells with motile cilia including the node, trachea and testes of the mice. A mutation of Lrrc6 has been identified in human patients with primary ciliary dyskinesia (PCD). Mutant mice lacking Lrrc6 show typical PCD defects such as hydrocephalus and laterality defects. We found that in the absence of Lrrc6, the morphology of motile cilia remained normal, but their motility was completely lost. The 9 + 2 arrangement of microtubules remained normal in Lrrc6(-/-) mice, but the outer dynein arms (ODAs), the structures essential for the ciliary beating, were absent from the cilia. In the absence of Lrrc6, ODA proteins such as DNAH5, DNAH9 and IC2, which are assembled in the cytoplasm and transported to the ciliary axoneme, remained in the cytoplasm and were not transported to the ciliary axoneme. The IC2-IC1 interaction, which is the first step of ODA assembly, was normal in Lrrc6(-/-) mice testes. Our results suggest that ODA proteins may be transported from the cytoplasm to the cilia by an Lrrc6-dependent mechanism., (© 2016 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

Axonemal dynein complexes are preassembled in the cytoplasm before their transport to cilia, but the mechanism of this process remains unclear. We now show that mice lacking Pih1d3, a PIH1 domain-containing protein, develop normally but manifest male sterility. Pih1d3(-/-) sperm were immotile and fragile, with the axoneme of the flagellum lacking outer dynein arms (ODAs) and inner dynein arms (IDAs) and showing a disturbed 9+2 microtubule organization. Pih1d3 was expressed specifically in spermatogenic cells, with the mRNA being most abundant in pachytene spermatocytes. Pih1d3 localized to the cytoplasm of spermatogenic cells but was not detected in spermatids or mature sperm. The levels of ODA and IDA proteins were reduced in the mutant testis and sperm, and Pih1d3 was found to interact with an intermediate chain of ODA as well as with Hsp70 and Hsp90. Our results suggest that Pih1d3 contributes to cytoplasmic preassembly of dynein complexes in spermatogenic cells by stabilizing and promoting complex formation by ODA and IDA proteins.

Vesnarinone is a synthetic quinolinone derivative used in the treatment of cardiac failure and cancer. It is also known to cause agranulocytosis as a side effect, which restricts its use, although the mechanism underlying agranulocytosis is not well understood. Here, we show that vesnarinone binds to valosin-containing protein (VCP), which interacts with polyubiquitinated proteins and is essential for the degradation of IκBα to activate nuclear factor (NF)κB. We show that vesnarinone impairs the degradation of IκBα, and that the impairment of the degradation of IκBα is the result of the inhibition of the interaction between VCP and the 26S proteasome by vesnarinone. These results suggest that vesnarinone suppresses NFκB activation by inhibiting the VCP-dependent degradation of polyubiquitinated IκBα, resulting in the suppression of tumor necrosis factor-α mRNA expression.

Accumulating evidence suggests that PD-1, an immuno-inhibitory receptor expressed on activated T cells, regulates peripheral T cell tolerance. In particular, PD-1 is involved in the induction and/or maintenance of T cells' intrinsic unresponsiveness to previously encountered Ags, although the mechanism is yet to be determined. We used a simple experimental model to dissect the mechanism for anergy establishment, in which 2C TCR transgenic rag2(-/-) PD-1(+/+) mice were anergized by a single injection of a cognate peptide. Interestingly, 2C rag2(-/-) PD-1(-/-) mice were totally resistant to anergy induction by the same treatment; thus, PD-1 was responsible for anergy induction. Furthermore, PD-1 expression was induced within 24 h of the initial Ag exposure. The establishment of anergy was associated with a marked down-regulation of IL-2 from the CD8(+) T cells. In fact, IL-2 blockade resulted in anergy even in 2C rag2(-/-)PD-1(-/-) T cells. Furthermore, the complementation of the IL-2 signal in 2C rag2(-/-) PD-1(+/+) mice reversed the anergy induction. We propose that CD8(+) T cell anergy is induced by a reduction of cell-autonomous IL-2 synthesis, which is caused by the quick expression of PD-1 in response to Ag stimulation and the subsequent stimulation of this receptor by its ligands on surrounding cells.

We have previously reported that human promyelocytic leukemia HL-60 cells can be induced to differentiate into mature granulocytes when HL-60 co-cultivated with human bone marrow stromal LP101 cells. In the present study, we investigated which factors produced by LP101 cells induce HL-60 cells to differentiate into mature granulocytes. The expression of the cell surface antigen CD11b on HL-60 cells was increased after a 72-h culture with the conditioned medium (CM) obtained from LP101 cells. LP101 cells were observed to produce various cytokines, including TNF-alpha, GM-CSF and IL-6. The neutralizing antibodies against these cytokines partially suppressed the CM-induced differentiation of HL-60 cells. Recombinant TNF-alpha induced the differentiation of HL-60 cells, and GM-CSF and IL-6 additionally enhanced the effect of TNF-alpha. When the CM was divided into a low molecular weight (LMW) fraction and a high molecular weight (HMW) fraction by ultrafiltration, the LMW fraction synergistically enhanced the differentiation inducible activity of TNF-alpha. These results demonstrate that LP101 cells induce the differentiation of HL-60 cells by producing various cytokines including TNF-alpha, IL-6, and GM-CSF, and that unknown low molecular weight factors also participate.

It has been reported that vesnarinone, a new inotropic agent used in the treatment of cardiac failure, causes agranulocytosis as a side effect. To study the mechanisms by which this complication occurs, vesnarinone was introduced into a coculture system of HL60 and bone marrow (BM) stromal cells, in which HL60 cells were able to differentiate into mature granulocytes with no inducible exogenous factors added to the culture. When HL60 cells were cocultured with the human BM-derived stromal cell line LP101, HL60 cells were induced to differentiate into mature granulocytes, and expression of the mature granulocyte-macrophage surface antigen, CD11b was increased. Conditioned medium (CM) obtained from LP101 cells also showed the capacity to induce the maturation of HL60 cells, in a dose- and time-dependent manner. The differentiation of HL60 cells induced by CM was also determined by morphological analysis, expression of myeloperoxidase, and a nitroblue tetrazolium (NBT) reduction test. When HL60 cells were cocultured with LP101 in the presence of vesnarinone, the CD11b expression was greatly suppressed. CM obtained from vesnarinone-treated LP101 (ves-CM) lost the capacity to induce the differentiation of HL60 cells, at a concentration of 1 microg/mL of vesnarinone. Vesnarinone itself did not affect the proliferation of HL60 cells. Furthermore, the addition of vesnarinone or ves-CM to HL60 cultures incubated with CM did not alter the induction of CD11b expression, suggesting that vesnarinone has no effect on HL60 cells, but that it inhibits stromal cells from producing soluble factor(s) required for the differentiation of HL60 cells to mature granulocytes. All these findings indicate that vesnarinone causes the hematopoietic disorder agranulocytosis, via impairment of stromal function.

Vesnarinone, an oral therapeutic agent for cardiac failure, causes agranulocytosis as a side effect. To elucidate the mechanism of occurrence of the agranulocytosis, we examined the effect of vesnarinone on granulopoiesis using an in vitro human long-term bone marrow culture system. Addition of vesnarinone to the culture decreased the total number of hematopoietic cells, mainly composed of mature granulocytes and macrophages, but increased the number of granulocyte-macrophage progenitor cells (CFU-GM) and CD33-CD34+ cells as compared with an untreated control. Differentiation of CFU-GM was induced by removing the agent from the culture medium, indicating that the effect of vesnarinone was reversible. The agent did not directly affect CFU-GM in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). Furthermore, treatment of stromal cells with vesnarinone repressed the production of G, GM, M-CSF, suggesting that the agent may cause a hematopoietic disorder, agranulocytosis, through the impairment of stromal cell function.

We have previously reported the establishment of a variety of human bone marrow stromal cell lines using a recombinant SV40-adenovirus vector. Using this vector, we obtained more clonal stromal cells. Here, we have characterized these cells and analyzed their capacity to support the proliferation and differentiation of hematopoietic cells. The stromal cells were cocultured with nonadherent human bone marrow cells used as hematopoietic cells. The total numbers of hematopoietic cells and CFU-GM in culture were counted every week. Two of the six stromal cell lines, AA101 and HAS303, supported the proliferation and differentiation of hematopoietic cells and CFU-GM for more than 9 weeks. Further, granulocytes, macrophages, and megakaryocytes were detected when cocultured with these cells. When hematopoietic cells were cocultured but separated from the two stromal cell lines by a 0.45-microns millipore membrane to prevent their attachment, almost all CFU-GM disappeared within 7 weeks. The supportive stromal cells produced GM-CSF and IL-6. However, other cell lines producing these humoral factors did not support hematopoietic cell proliferation for such a long time. These findings suggest that these established human bone marrow stromal cell lines will be useful, in that analysis of their supportive function in hematopoietic cell proliferation and differentiation through cell-to-cell interaction will shed some light on this area.