Immune-bone interplay in the structural damage in rheumatoid arthritis.

The immune and bone systems maintain homeostasis by interacting closely with each other. Rheumatoid arthritis is a pathological consequence of their interplay, as activated T cell immune responses result in osteoclast-mediated bone erosion. An imbalance between forkhead box protein 3 (Foxp3)+ regulatory T (Treg ) cells and T helper type 17 (Th17) cells is often linked with autoimmune diseases, including arthritis. Th17 cells contribute to the bone destruction in arthritis by up-regulating receptor activator of nuclear factor kappa-Β ligand (RANKL) on synovial fibroblasts as well as inducing local inflammation. Studies on the origin of Th17 cells in inflammation have shed light on the pathogenic conversion of Foxp3+ T cells. Th17 cells converted from Foxp3+ T cells (exFoxp3 Th17 cells) comprise the most potent osteoclastogenic T cell subset in inflammatory bone loss. It has been suggested that osteoclastogenic T cells may have developed originally to stop local infection in periodontitis by inducing tooth loss. In addition, Th17 cells also contribute to the pathogenesis of arthritis by modulating antibody function. Antibodies and immune complexes have attracted considerable attention for their direct role in osteoclastogenesis, and a specific T cell subset in joints was shown to be involved in B cell antibody production. Here we summarize the recent advances in our understanding of the immune-bone interplay in the context of the bone destruction in arthritis.

Spectrum of clinical and genetic features of patients with inherited platelet disorder with suspected predisposition to hematological malignancies: a nationwide survey in Japan.

BACKGROUND: Inherited thrombocytopenia (IT) contains several forms of familial thrombocytopenia and some of them have propensity to hematological malignancies. The etiological and genetic features of this heterogeneous syndrome have not yet been elucidated. PATIENTS AND METHODS: We conducted a nationwide survey to collect clinical information and samples from patients with familial thrombocytopenia and/or hematological malignancies in order to obtain a comprehensive understanding of IT. RESULTS: Among the 43 pedigrees with clinical samples, RUNX1 mutations were identified in 8 pedigrees (18.6%). While MYH9 and ANKRD26 mutations were identified in 2 and 1 pedigrees, respectively, no gene mutations were detected in the remaining 32 pedigrees from a panel of previously reported pathogenetic mutations. Clinical data were comparable between FPD/AML and non-FPD/AML probands. CONCLUSIONS: Our study clarified that it is unexpectedly difficult to diagnose FPD/AML based on clinical information alone, and thus, genetic testing is strongly recommended. Our survey also identified some pedigrees with a strong family history of myelodysplastic syndromes of unknown origin. Additionally, there were 14 pedigrees in which three or more members were affected by immune thrombocytopenia (ITP), and a computer-aided simulation suggested that such a distribution almost never happens by coincidence, which implicates a genetic predisposition to ITP.

IL-28B (IFN-λ3) and IFN-α synergistically inhibit HCV replication.

Genetic variation in the IL-28B (interleukin-28B; interferon lambda 3) region has been associated with sustained virological response (SVR) rates in patients with chronic hepatitis C treated with peginterferon-α and ribavirin. However, the mechanisms by which polymorphisms in the IL-28B gene region affect host antiviral responses are not well understood. Using the HCV 1b and 2a replicon system, we compared the effects of IFN-λs and IFN-α on HCV RNA replication. The anti-HCV effect of IFN-λ3 and IFN-α in combination was also assessed. Changes in gene expression induced by IFN-λ3 and IFN-α were compared using cDNA microarray analysis. IFN-λs at concentrations of 1 ng/mL or more exhibited concentration- and time-dependent HCV inhibition. In combination, IFN-λ3 and IFN-α had a synergistic anti-HCV effect; however, no synergistic enhancement was observed for interferon-stimulated response element (ISRE) activity or upregulation of interferon-stimulated genes (ISGs). With respect to the time course of ISG upregulation, the peak of IFN-λ3-induced gene expression occurred later and lasted longer than that induced by IFN-α. In addition, although the genes upregulated by IFN-α and IFN-λ3 were similar to microarray analysis, interferon-stimulated gene expression appeared early and was prolonged by combined administration of these two IFNs. In conclusion, IFN-α and IFN-λ3 in combination showed synergistic anti-HCV activity in vitro. Differences in time-dependent upregulation of these genes might contribute to the synergistic antiviral activity.

A new species of cockroach, Periplaneta gajajimana sp. nov., collected in Gajajima, Kagoshima Prefecture, Japan.

We described a new species of cockroach, Periplaneta gajajimana sp. nov., which was collected in Gajajima, Kagoshima-gun Toshimamura, Kagoshima Prefecture, Japan, on November 2012. The new species is characterized by its reddish brown to blackish brown body, smooth surface pronotum, well developed compound eyes, dark brown head apex, dark reddish brown front face and small white ocelli connected to the antennal sockets. In male, the tegmen tip reach the abdomen end or are slightly shorter, while in the female, it does not reach the abdominal end and exposes the abdomen beyond the 7th abdominal plate. We confirmed the validity of this new species by breeding the specimens in our laboratory to demonstrate that the features of the progeny were maintained for several generations. For comparison and easy identification of this new species, the key to species identification of the genus Periplaneta that had been reported in Japan to date are also presented.

Discovery of cockroach specimens of the genus Squamoptera (Ectobiidae: Pseudophyllodromiinae) from Okinawa, Japan, showing wing polymorphism.

Cockroach specimens of the genus, Squamoptera were collected from the Iriomote island of Okinawa prefecture, Japan. The morphological features of the specimens were characterized as having a white band on the dorsal surface of its thorax, its tegmen reduced into a tiny scale-like structure and the hindwing was absent. Ocelli was also absent and the small compound eyes not extending to apex of the head nor to the frontal face but extend further lower than the base of the antennae. When the specimens were reared in the laboratory, besides the short wing form, the long wing form began to appear in the rearing colony. In our reproductive biological study, we observed that hatching of the ootheca from the short wing female takes about 30 days, with an average of 6.6 nymphs being hatched from one ootheca. The male to female ratio of the offspring was 36:30. However, the frequency appearance of the offspring from the ootheca of the short wing female was 98.5% short wing and 1.5% long wing form. Our specimens occasionally show body polymorphism in the form of individuals having long wings instead of the usual short one. The long wing form does not show the white band on the dorsal surface of its thorax.

A truncated erythropoietin receptor that fails to prevent programmed cell death of erythroid cells.

A form of the human erythropoietin receptor (EPOR) was identified in which the cytoplasmic region is truncated by alternative splicing. The truncated form of the receptor (EPOR-T) is the most prevalent form of EPOR in early-stage erythroid progenitor cells, but the full-length EPOR (EPOR-F) becomes the most prevalent form in late-stage progenitors. EPOR-T can transduce a mitogenic signal. However, cells transfected with EPOR-T are more prone to programmed cell death than those expressing EPOR-F. EPOR-F may transduce a signal to prevent programmed cell death that is independent of the mitogenic signal, and alternative splicing of the EPOR gene may have an important role in erythropoiesis.

Appropriate hinge position for prevention of unstable lateral hinge fracture in open wedge high tibial osteotomy.

AIMS: Open wedge high tibial osteotomy (OWHTO) for medial-compartment osteoarthritis of the knee can be complicated by intra-operative lateral hinge fracture (LHF). We aimed to establish the relationship between hinge position and fracture types, and suggest an appropriate hinge position to reduce the risk of this complication. PATIENTS AND METHODS: Consecutive patients undergoing OWHTO were evaluated on coronal multiplanar reconstruction CT images. Hinge positions were divided into five zones in our new classification, by their relationship to the proximal tibiofibular joint (PTFJ). Fractures were classified into types I, II, and III according to the Takeuchi classification. RESULTS: Among 111 patients undergoing OWHTOs, 22 sustained lateral hinge fractures. Of the 89 patients without fractures, 70 had hinges in the zone within the PTFJ and lateral to the medial margin of the PTFJ (zone WL), just above the PTFJ. Among the five zones, the relative risk of unstable fracture was significantly lower in zone WL (relative risk 0.24, confidence interval 0.17 to 0.34). CONCLUSION: Zone WL appears to offer the safest position for the placement of the osteotomy hinge when trying to avoid a fracture at the osteotomy site. Cite this article: Bone Joint J 2017;99B10:1313-18.

Live imaging of extracellular signal-regulated kinase and protein kinase A activities during thrombus formation in mice expressing biosensors based on Förster resonance energy transfer.

Essentials Spatiotemporal regulation of protein kinases during thrombus formation remains elusive in vivo. Activities of protein kinases were live imaged in mouse platelets at laser-ablated arterioles. Protein kinase A was activated in the dislodging platelets at the downstream side of the thrombus. Extracellular signal-regulated kinase was activated at the core of contracting platelet aggregates. SUMMARY: Background The dynamic features of thrombus formation have been visualized by conventional video widefield microscopy or confocal microscopy in live mice. However, owing to technical limitations, the precise spatiotemporal regulation of intracellular signaling molecule activities, which have been extensively studied in vitro, remains elusive in vivo. Objectives To visualize, by the use of two-photon excitation microscopy of transgenic mice expressing Förster resonance energy transfer (FRET) biosensors for extracellular signal-regulated kinase (ERK) and protein kinase A (PKA), ERK and PKA activities during thrombus formation in laser-injured subcutaneous arterioles. Results When a core of densely packed platelets had developed, ERK activity was increased from the basal region close to the injured arterioles. PKA was activated at the downstream side of an unstable shell overlaying the core of platelets. Intravenous administration of a MEK inhibitor, PD0325901, suppressed platelet tethering and dislodged platelet aggregates, indicating that ERK activity is indispensable for both initiation and maintenance of the thrombus. A cAMP analog, dbcAMP, inhibited platelet tethering but failed to dislodge the preformed platelet aggregates, suggesting that PKA can antagonize thrombus formation only in the early phase. Conclusion In vivo imaging of transgenic mice expressing FRET biosensors will open a new opportunity to visualize the spatiotemporal changes in signaling molecule activities not only during thrombus formation but also in other hematologic disorders.

MSC/ECM Cellular Complexes Induce Periodontal Tissue Regeneration.

Transplantation of mesenchymal stem cells (MSCs), which possess self-renewing properties and multipotency, into a periodontal defect is thought to be a useful option for periodontal tissue regeneration. However, developing more reliable and predictable implantation techniques is still needed. Recently, we generated clumps of an MSC/extracellular matrix (ECM) complex (C-MSC), which consisted of cells and self-produced ECM. C-MSCs can regulate their cellular functions in vitro and can be grafted into a defect site, without any artificial scaffold, to induce bone regeneration. Accordingly, this study aimed to evaluate the effect of C-MSC transplantation on periodontal tissue regeneration in beagle dogs. Seven beagle dogs were employed to generate a premolar class III furcation defect model. MSCs isolated from dog ilium were seeded at a density of 7.0 × 104 cells/well into 24-well plates and cultured in growth medium supplemented with 50 µg/mL ascorbic acid for 4 d. To obtain C-MSCs, confluent cells were scratched using a micropipette tip and were then torn off as a cellular sheet. The sheet was rolled up to make round clumps of cells. C-MSCs were maintained in growth medium or osteoinductive medium (OIM) for 5 or 10 d. The biological properties of C-MSCs were evaluated in vitro, and their periodontal tissue regenerative activity was tested by using a dog class III furcation defect model. Immunofluorescence analysis revealed that type I collagen fabricated the form of C-MSCs. OIM markedly elevated calcium deposition in C-MSCs at day 10, suggesting its osteogenic differentiation capacity. Both C-MSCs and C-MSCs cultured with OIM transplantation without an artificial scaffold into the dog furcation defect induced periodontal tissue regeneration successfully compared with no graft, whereas osteogenic-differentiated C-MSCs led to rapid alveolar bone regeneration. These findings suggested that the use of C-MSCs refined by self-produced ECM may represent a novel predictable periodontal tissue regenerative therapy.

Induced megakaryocytic maturation of the human leukemia cell line UT-7 results in down-modulation of erythropoietin receptor gene expression.

Erythropoietin (Epo) affects not only erythrocyte production but, in vitro, also promotes megakaryocyte maturation. However, the mechanism of action of Epo on megakaryocytic development remains to be determined. Recently, we reported the establishment of a human Epo-dependent megakaryoblastic leukemic cell line UT-7. Exposure of UT-7 to the tumor promoter, phorbol myristate acetate (PMA), resulted in the appearance of mature megakaryocytic properties, including the expression of platelet factor 4 and beta-thromboglobulin. With exposure to PMA, however, UT-7 cells lost their responsiveness to Epo and Scatchard analysis showed an 85% decrease in the number of Epo receptors after 24 h. While the number of binding sites declined, the affinity of Epo binding was unchanged. Associated with the decline in the number of Epo receptors was a profound decrease (> 95%) in the level of Epo receptor (Epo-R) mRNA. To determine the level of regulation of the Epo-R gene, its rate of transcription was measured by nuclear run-off assay in untreated cells and in cells exposed to PMA for 6, 12, and 24 h. The rate of transcription was nearly identical at all time points in control and PMA-treated cells. Stability of Epo-R mRNA also was measured in the presence of actinomycin D, an inhibitor of transcription. The half-life of Epo-R mRNA in untreated and PMA-treated cells was 90 and 30 min, respectively. These results indicate that the down-modulation of the expression of the Epo-R gene is mainly caused by increased instability of mature mRNA of Epo-R. Posttranscriptional regulation may be an important mechanism in the regulation of hematopoietic growth factor receptor genes and one of the mechanisms by which lineage restriction is achieved.

Survival of highly proliferative colony forming cells after treatment of bone marrow cells with 4-hydroperoxycyclophosphamide.

We investigated the in vitro effects of 4-hydroperoxycyclophosphamide (4-HC) on human hemopoietic stem cells. Marrow cells were exposed to 4-HC and then assayed for mixed (CFU-GEMM), erythroid (BFU-E), megakaryocyte (CFU-M), and granulocyte-macrophage (CFU-GM) colony forming cells. We found that highly proliferative colony forming cells, especially CFU-GEMM and BFU-E, were relatively spared by 4-HC treatment. One third of the surviving progenitors formed large colonies, some of which contained more than 50,000 cells. By sequential examination of the formation of these large colonies, we found immature colonies consisting of blasts at the early stage of culture. The morphology of these "blast cell colonies" in situ was arbitrarily classified into four types. Among them were the blast cell colonies consisting of the individual cells that were dispersed and had a few granules within the cytoplasm (type A); these cells finally formed very large colonies on day 22 of culture. Approximately 70% of the single cells derived from type A blast cell colonies produced secondary colonies consisting of erythroblasts, macrophages, eosinophils, and/or basophils. These results show that the blast cells in type A colonies have a highly proliferative capacity. The availability of a highly enriched population of primitive hemopoietic progenitors will provide us with a unique opportunity to study the interaction between a single stem cell and purified hemopoietic factors.

Establishment and characterization of a human leukemic cell line with megakaryocytic features: dependency on granulocyte-macrophage colony-stimulating factor, interleukin 3, or erythropoietin for growth and survival.

A new human leukemia cell line with megakaryocytic features, designated UT-7, was established from the bone marrow of a patient with acute megakaryoblastic leukemia. Surface marker analysis revealed that the majority of the cells reacted with monoclonal antibodies against platelet glycoprotein Ib (CD42b), glycoprotein IIb/IIIa (CD41a), MY 7 (CD13), MY 9 (CD33), and glycophorin A antigens. Cytogenetic analysis showed a human male near-tetraploid karyotype with a modal chromosome number of 92-96. Flow cytometry-derived DNA histograms demonstrated that the majority of the cells spontaneously contained 4 N DNA ploidy levels. Ultrastructural study showed that platelet peroxidase activity was weakly positive but myeloperoxidase activity was negative. Ferritin and theta-granule, which have been used as ultrastructural markers for the erythroid lineage, could not be detected. In response to phorbol myristate acetate, platelet factor 4 and beta-thromboglobulin, which were specifically synthesized in the process of megakaryocyte maturation, dramatically increased in UT-7 cells. This was accompanied by an increase in cell size, ploidy level, platelet peroxidase activity, and the surface density of glycoprotein IIb/IIIa antigen. These findings suggest that UT-7 is a new leukemic cell line with megakaryocytic features and with the potential to differentiate into cells with more mature megakaryocytic properties in response to phorbol myristate acetate. This cell line showed strict dependency on interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor, or erythropoietin. The maximal effective doses of IL-3, granulocyte-macrophage colony-stimulating factor, and erythropoietin for proliferation in liquid culture were 10 units/ml, 1 ng/ml, and 1 unit/ml, respectively. These concentrations were comparable to the doses that maximally stimulate the clonal growth of normal hemopoietic cells. IL-6 could stimulate the proliferation of UT-7 cells but not maintain the line in long-term culture. UT-7 cells may be a useful model for (a) the analysis of gene regulation of megakaryocytic maturation-associated proteins expressed in the process of megakaryocytic differentiation and (b) the study of signal transduction of hemopoietic factors associated with megakaryocytopoiesis.

Characterization of stage progression in chronic myeloid leukemia by DNA microarray with purified hematopoietic stem cells.

Chronic myeloid leukemia (CML) is characterized by the clonal expansion of hematopoietic stem cells (HSCs). Without effective treatment, individuals in the indolent, chronic phase (CP) of CML undergo blast crisis (BC), the prognosis for which is poor. It is therefore important to clarify the mechanism underlying stage progression in CML. DNA microarray is a versatile tool for such a purpose. However, simple comparison of bone marrow mononuclear cells from individuals at different disease stages is likely to result in the identification of pseudo-positive genes whose change in expression only reflects the different proportions of leukemic blasts in bone marrow. We have therefore compared with DNA microarray the expression profiles of 3456 genes in the purified HSC-like fractions that had been isolated from 13 CML patients and healthy volunteers. Interestingly, expression of the gene for PIASy, a potential inhibitor of STAT (signal transducer and activator of transcription) proteins, was down-regulated in association with stage progression in CML. Furthermore, forced expression of PIASy has induced apoptosis in a CML cell line. These data suggest that microarray analysis with background-matched samples is an efficient approach to identify molecular events underlying the stage progression in CML.

Purification and immunohistochemical localization of rat liver glutathione peroxidase.

Glutathione peroxidase was purified from the rat liver to give a single protein band in polyacrylamide gel electrophoresis. Rabbits were immunized with this purified enzyme, and a highly specific anti-glutathione peroxidase antiserum was obtained. Using this antibody, an immunohistochemical technique (the indirect method of peroxidase-labeled antibody) was applied to study the localization of the enzyme in the liver cells. On immunohistochemical observation, glutathione peroxidase was localized exclusively in the cytoplasm of hepatocytes, and a stronger 'immuno-staining' was exhibited in the peripheries of the hepatic lobules than in the central zone.

Arachidonate 5-lipoxygenase of porcine pancreas: its localization in acinar cells.

Arachidonate 5-lipoxygenase has been found so far in various types of leukocyte. When a homogenate of porcine pancreas was incubated with arachidonic acid, 5-hydroxy-6,8,11,14-eicosatetraenoic acid was predominantly produced concomitant with small amounts of compounds derived from leukotriene A4. After differential centrifugation of the homogenate, the 5-lipoxygenase activity was found predominantly in the 1000 x g pellet and 105,000 x g supernatant. When porcine pancreas was investigated immunohistochemically with anti-5-lipoxygenase antibody, Langerhans islets were unstained, and infiltration of 5-lipoxygenase-positive leukocytes was hardly observed. In contrast, acinar cells were positively stained. Immunoelectron microscopy demonstrated the localization of the enzyme along the nuclear membranes of the acinar cells.

Cloning and characterization of Xenopus laevis xSox7 cDNA.

A family of SRY-related genes has been termed SOX. We have isolated and sequenced a cDNA encoding a novel Sox protein from Xenopus laevis ovary. This cDNA contains an open reading frame (ORF) coding for 362 amino acids, which encompasses an HMG box and exhibits a strong (90%) identity to that of mouse Sox7; the cDNA was named xSox7 in this study. Northern analysis revealed that the xSox7 mRNA was 2.0 kb in length. Various adult frog tissues were tested by reverse transcription/polymerase chain reaction for xSox7 mRNA, and the results showed that xSox7 is expressed in a wide range of tissues. Furthermore, electrophoretic mobility shif assay indicated that recombinant xSox7 is capable of binding to AACAAT sequence.

Cloning and expression of Xenopus laevis xSox12 cDNA.

A family of SRY-related genes has been termed SOX. We have isolated and sequenced a cDNA encoding xSox12 from Xenopus laevis ovary. The cDNA contained an open reading frame (ORF) coding for 470 amino acids encompassing an HMG box characteristic of the SOX family, a leucine zipper motif and glutamine-rich segments. The size of the xSox12 mRNA was determined to be 3.0 knt by Northern analysis. The ovary was the most prominent in the expression of the Sox mRNA among the various tissues of adult frog as far as examined.

Mechanism of superoxide anion generation in the toxic red tide phytoplankton Chattonella marina: possible involvement of NAD(P)H oxidase.

Red tide phytoplankton Chattonella marina is known to produce reactive oxygen species (ROS), such as superoxide anion (O(2)(-)), hydrogen peroxide (H(2)O(2)) and hydroxyl radical (&z.rad;OH), under normal physiological conditions. Although several lines of evidence suggest that ROS are involved in the mortality of fish exposed to C. marina, the mechanism of ROS generation in C. marina remains to be clarified. In this study, we found that the cell-free supernatant prepared from C. marina cells showed NAD(P)H-dependent O(2)(-) generation, and this response was inhibited by diphenyleneiodonium, an inhibitor of mammalian NADPH oxidase. When the cell-free supernatant of C. marina was analyzed by immunoblotting using antibody raised against the human neutrophil cytochrome b558 large subunit (gp91phox), a main band of approximately 110 kDa was detected. The cell surface localization of the epitope recognized with this antibody was also demonstrated in C. marina by indirect immunofluorescence. Furthermore, Southern blot analysis performed on genomic DNA of C. marina with a probe covering the C-terminal region of gp91phox suggested the presence of a single-copy gene coding for gp91phox homologous protein in C. marina. These results provide evidence for the involvement of an enzymatic system analogous to the neutrophil NADPH oxidase as a source of O(2)(-) production in C. marina.

Phorbol ester treatment of two megakaryoblastic cell lines, CMK and UT-7, induces specific protein composition changes with non-coincident time-courses.

The expressions of platelet-specific glycoprotein(GP)s Ib, IIb and IIIa were analyzed in 2 megakaryoblastic cell lines: CMK and UT-7. Phorbol-12 myristate 13-acetate (PMA) treatment of CMK induced expressions of GPs IIb and IIIa that peaked on the 4th day post-treatment, while treated UT-7 cells showed maximal levels of these GPs during the 6-8th days. Antibody staining to detect the formation of GPIb alpha after PMA induction showed the presence of the intact GP in UT-7 and the degraded form in CMK. In UT-7, synthesis of GPIIb mRNA increased on days 4-6 after PMA treatment, in parallel with the increase of GPIIb. PMA increased the cytoskeletal protein (actin and myosin) contents in both lines, but in contrast to the two GPs, the increase in these proteins started immediately after PMA addition to the cells. Cell surface proteins of CMK and UT-7 cells were rapidly modified after PMA induction. Especially notable were the degradations of 93-kDa and 140-kDa proteins that occurred on days 1-2 after PMA treatment. These studies demonstrate that the expression of platelet-specific proteins shows a different time dependency than the increment of cytoskeletal proteins, indicating that the syntheses of these two classes of proteins are most likely induced through different mechanisms.

PLP-I: a novel prolactin-like gene in rodents.

In this report, we describe molecular cloning and characterization of cDNAs encoding a novel rat prolactin-like protein. The rat cDNAs were isolated from the decidua and the gene was named PLP-I. cDNAs for the mouse equivalent were also cloned by the cross-hybridization technique. Pregnancy-specific expression of the rat PLP-I gene was observed in the rat placenta by Northern analysis. Location of signal peptide cleavage sites in rat and mouse pre-PLP-I proteins was predicted using a theoretical method. A molecular phylogenetic tree for the growth hormone-prolactin superfamily including the novel member, PLP-I, constructed using the neighbor-joining method, places rat/mouse PLP-I closest to rat/mouse placental lactogen I and II.