Activation of Hepatic Stellate Cells Requires Dissociation of E-Cadherin-Containing Adherens Junctions with Hepatocytes.

Hepatic stellate cells (HSCs) are resident mesenchymal cells in the space of Disse interposed between liver sinusoidal endothelial cells and hepatocytes. Thorn-like microprojections, or spines, project out from the cell surface of HSCs, crossing the space of Disse, to establish adherens junctions with neighboring hepatocytes. Although HSC activation is initiated largely from stimulation by adjacent cells, isolated HSCs also activate spontaneously in primary culture on plastic. Therefore, other unknown HSC-initiating factors apart from paracrine stimuli may promote activation. The dissociation of adherens junctions between HSCs and hepatocytes as an activating signal for HSCs was explored, establishing epithelial cadherin (E-cadherin) as an adhesion molecule linking hepatocytes and HSCs. In vivo, following carbon tetrachloride-induced liver injury, HSCs lost their spines and dissociated from adherens junctions in the early stages of injury, and were subsequently activated along with an increase in YAP/TAZ expression. After abrogation of liver injury, HSCs reconstructed their spines and adherens junctions. In vitro, reconstitution of E-cadherin-containing adherens junctions by forced E-cadherin expression quiesced HSCs and suppressed TAZ expression. Additionally, increase of TAZ expression leading to the activation of HSCs by autocrine stimulation of transforming growth factor-ß, was revealed as a mechanism of spontaneous activation. Thus, we have uncovered a critical event required for HSC activation through enhanced TAZ-mediated mechanotransduction after the loss of adherens junctions between HSCs and hepatocytes.

Catastrophic antiphospholipid syndrome with Epstein-Barr virus-associated hemophagocytosis: A clinicopathological conference.

Catastrophic antiphospholipid syndrome (CAPS) is a severe variant of antiphospholipid syndrome associated with multiorgan thrombosis in a short term. We present the case of a 14-year-old immunocompetent girl who developed renal, intestinal, and pulmonary infarction; thrombocytopenia; and hemolytic anemia within 1 week. She was diagnosed with thrombotic microangiopathy. Hence, plasma exchange and corticosteroid therapy were initiated, which improved thrombocytopenia. However, the patient's platelet count decreased. Her general condition gradually worsened with eventual death. An autopsy revealed multiple infarctions in the kidneys bilaterally, jejunum, ileum, and pulmonary parenchyma. Microthrombi were not detected. Massive hemophagocytosis was observed in the splenic pulp, lymph nodes, and bone marrow. Several Epstein-Barr viruses (EBVs)-encoded small ribonucleic acid (RNA)-positive lymphocytes were also found in the bone marrow. The presence of antibodies to both viral capsid antigen-immunoglobulin G and EBV nuclear antigen indicated past infection. Antiphospholipid antibody was positive after her death. The patient was finally diagnosed with CAPS and EBV-associated hemophagocytosis, possibly due to EBV reactivation. Establishing a clinical diagnosis of CAPS was relatively difficult because two different causes of thrombocytopenia, CAPS and hemophagocytosis, led to a difficulty in understanding this case's pathogenesis.

[Acquired hemophilia A with reappearance of inhibitor 10 years after the completion of immunosuppressive therapy].

Acquired hemophilia A (AHA) is a disease that causes severe bleeding with the appearance of an inhibitor (INH) against blood coagulation factor VIII (FVIII). The prevalence of this condition is low; it occurs in only one in one to four million people per year; however, the number of diagnosed cases has increased in recent years owing to the greater awareness of the disease. It is noteworthy that this is a hemorrhagic disease that suddenly develops in the elderly. AHA treatment is divided into hemostatic treatment for bleeding and immunosuppressive therapy (IST) for removing FVIII-INH. As long as FVIII-INH remains, there is a risk of fatal bleeding; therefore, it is desirable to start IST soon after diagnosis. However, the use of immunosuppressive drugs for the elderly is often challenging due to concerns about adverse events, such as infectious diseases that have a considerable impact on prognosis. Ten years after the end of IST, we managed the case of a patient with AHA who had a relapse of FVIII-INH at the age of 84 years. In this case, relapse was detected early when there was no bleeding symptom, and remission was rapidly achieved with a small amount of IST without any adverse effects. There are few reports on AHA relapse; we believe that the present report will contribute meaningfully to the literature on this subject and would be useful when considering the long-term management of AHA.

Factor VIII-Mimetic Function of Humanized Bispecific Antibody in Hemophilia A.

BACKGROUND: In patients with severe hemophilia A, standard treatment is regular prophylactic and episodic intravenous infusions of factor VIII. However, these treatments are burdensome, especially for children, and may lead to the formation of anti-factor VIII alloantibodies (factor VIII inhibitors). Emicizumab (ACE910), a humanized bispecific antibody mimicking the cofactor function of factor VIII, was developed to abate these problems. METHODS: We enrolled 18 Japanese patients with severe hemophilia A (with or without factor VIII inhibitors) in an open-label, nonrandomized, interindividual dose-escalation study of emicizumab. The patients received subcutaneous emicizumab weekly for 12 weeks at a dose of 0.3, 1.0, or 3.0 mg per kilogram of body weight (cohorts 1, 2, and 3, respectively). The end points were safety and pharmacokinetic and pharmacodynamic profiles. An additional, exploratory end point was the annualized bleeding rate, calculated as 365.25 times the number of bleeding episodes, divided by the number of days in the treatment period as compared with the 6 months before enrollment. RESULTS: Emicizumab was associated with neither serious adverse events nor clinically relevant coagulation abnormalities. Plasma concentrations of emicizumab increased in a dose-dependent manner. Activated partial-thromboplastin times remained short throughout the study. The median annualized bleeding rates in cohorts 1, 2, and 3 decreased from 32.5 to 4.4, 18.3 to 0.0, and 15.2 to 0.0, respectively. There was no bleeding in 8 of 11 patients with factor VIII inhibitors (73%) and in 5 of 7 patients without factor VIII inhibitors (71%). Episodic use of clotting factors to control bleeding was reduced. Antibodies to emicizumab did not develop. CONCLUSIONS: Once-weekly subcutaneous administration of emicizumab markedly decreased the bleeding rate in patients who had hemophilia A with or without factor VIII inhibitors. (Funded by Chugai Pharmaceutical; JapicCTI number, 121934.).

[A Pediatric Case of Acquired Hemophilia A: The Usefulness of the Activated Partial Thromboplastin Time (APTT) Cross-Mixing Test for Early Diagnosis].

Acquired hemophilia A (AHA), a bleeding disorder caused by autoantibodies against FVIII, has the potential for life-threatening bleeding. The annual onset rate is said to be one in 4 million people, but diagnosis examples increase in adults because a disorder concept penetrated. AHA is quite rare in children, with an incidence rate of 0.045 per 1 million, but early detection is crucial because serious bleeding can happen, as in adults. We report a pediatric case who received an early diagnosis of AHA by an activated partial thromboplastin time (APTT) cross-mixing test. The 12-year-old girl had neither a past history nor a family history of bleeding episodes. She presented with intramuscular bleeding and epistaxis without trauma or medication. At diagnosis, her blood test showed prolonged APTT. Other hemostatic tests, such as the platelet count, prothrombin time and fibrinogen concentration, were within the normal range. We administered an APTT cross-mixing test that detected an inhibitor pattern and inhibitory antibodies against factors VIII. As a result, we administered prednisolone and the inhibitor disappeared after 1.5 months. In conclusion, AHA is a bleeding disorder which should be considered even in children due to the potential for life-threatening bleeding. Furthermore, the APTT cross-mixing test is useful for screening coagulation factor deficiencies and inhibitors.

The clinical presentation and genotype of protein C deficiency with double mutations of the protein C gene.

BACKGROUND: Severe protein C (PC) deficiency is a rare heritable thrombophilia leading to thromboembolic events during the neonatal period. It remains unclear how individuals with complete PC gene (PROC) defects develop or escape neonatal stroke or purpura fulminans (PF). PROCEDURE: We studied the onset of disease and the genotype of 22 PC-deficient patients with double mutations in PROC based on our cohort (n = 12) and the previous reports (n = 10) in Japan. RESULTS: Twenty-two patients in 20 unrelated families had 4 homozygous and 18 compound heterozygous mutations. Sixteen newborns presented with PF (n = 11, 69%), intracranial thromboembolism and hemorrhage (n = 13, 81%), or both (n = 8, 50%), with most showing a plasma PC activity of <10%. Six others first developed overt thromboembolism when they were over 15 years of age, showing a median PC activity of 31% (range: 19-52%). Fifteen of the 22 patients (68%) had the five major mutations (G423VfsX82, V339M, R211W, M406I, and F181V) or two others (E68K and K193del) that have been reported in Japan. Three of the six late-onset cases, but none of the 16 neonatal cases, had the K193del mutation, which has been reported to be the most common variant of Chinese thrombophilia. A novel mutation of A309V was determined in a family of two patients with late onset. CONCLUSIONS: The genotype of double-PROC mutants might show less diversity than heterozygous mutants in terms of the timing of the onset of thrombophilia (newborn onset or late onset).

Efficient expansion of mouse primary tenocytes using a novel collagen gel culture method.

Development of regenerative therapies for damaged tendons remains a great challenge, largely because of lack of information regarding the mechanisms responsible for differentiation of tenocytes. Mouse tenocytes have not been fully characterized owing to the absence of efficient and reproducible methods for their in vitro expansion without losing phenotypic features. The objective of the study was to establish an improved and reliable method for stable primary culture of mouse tenocytes by using collagen gel. Achilles and tail tendon tissues were harvested and embedded in collagen gel. After 10 days of continuous culture, the gel was digested and cells were passaged on tissue culture-treated plastic dishes. Mouse tenocytes cultured in collagen gel exhibited significantly shorter doubling time and higher numbers of proliferation when maintained on the plastic dishes compared with those cultured without using gel. Transmission electron microscopic analyses showed that cultured tenocytes retained some morphological features of tenocytes in tendon tissues, such as cell-cell junctional complex formation, well-developed rough endoplasmic reticulum, and mitochondria in their cytoplasm. mRNA expression of tenocyte markers (tenomodulin, type I collagen, periostin, and scleraxis) was higher in cells cultured in collagen gel than in those cultured in the absence of gel. Our results show that tenocytes cultured using the collagen gel method express typical lineage markers and exhibit improved growth characteristics, thus providing a stable platform for studying molecular mechanisms that control their differentiation.

Osteoclastogenesis is influenced by modulation of gap junctional communication with antiarrhythmic peptides.

Osteoclasts are formed by the fusion of mononuclear precursor cells of the monocyte-macrophage lineage. Among several putative mechanisms, gap-junctional intercellular communication (GJC) has been proposed to have a role in osteoclast fusion and bone resorption. We examined the role of GJC in osteoclastogenesis and in vitro bone resorption with mouse bone marrow hematopoietic stem cells and RAW 264.7 cells. Blocking of gap junctions with 18-α-glycyrrhetinic acid (18GA) led to inhibition of osteoclastogenesis and in vitro bone resorption. Similarly, the GJC inhibitor GAP27 inhibited osteoclast formation. GJC modulation with the antiarrhythmic peptides (AAPs) led to increased amounts of multinuclear RAW 264.7 osteoclasts as well as increased number of nuclei per multinuclear cell. In the culture of bone marrow hematopoietic stem cells in the presence of bone marrow stromal cells AAP reduced the number of osteoclasts, and coculture of MC3T3-E1 preosteoblasts with RAW 264.7 macrophages prevented the action of AAPs to promote osteoclastogenesis. The present data indicate that AAPs modulate the fusion of the pure culture of cells of the monocyte-macrophage lineage. However, the fusion is influenced by GJC in cells of the osteoblast lineage.

A comparative electron microscopic study of bone repair after internal fracture, osteotomy, and perforation of rat tibia.

BACKGROUND AND OBJECTIVE. Although previous studies have provided new information on bone repair, there are still gaps in knowledge about resorptive and formative processes during bone repair at the electron microscopic level. The aim of this study was to compare bone repair after the internal fracture, osteotomy, and bicortical perforation of the tibia by means of electron microscopy. MATERIAL AND METHODS. An electron microscopic study of bone repair after the internal fracture, osteotomy, and bicortical perforation of the tibia was performed on 72 male Wistar rats. Rats undergoing osteotomy and perforation were further subdivided into the control and immobilization subgroups. Bone repair was observed during the first posttraumatic weeks. RESULTS. Although bone repair in general had similar bone healing stages in all the groups, the repair process depended on the mode and degree of injury thus being different in the experimental groups. After the internal fracture, indirect ossification was observed; after osteotomy, primary periosteal, secondary endosteal ossification was noted; and after perforation, primary endosteal, secondary periosteal ossification was documented. Immobilization had an inhibitory effect on bone repair. CONCLUSIONS. The results of the present study gave new information at the electron microscopic level about intracellular changes and intercellular matrix synthesis during different types of posttraumatic bone repair and confirmed our previous reports on similar posttraumatic bone repair in histomorphometric and immunohistochemical studies.

Spectrum of MHC class II variability in Darwin's finches and their close relatives.

The study describes >400 major histocompatibility complex (MHC) class II B exon 2 and 114 intron 2 sequences of 36 passerine bird species, 13 of which belong to the group of Darwin's finches (DFs) and the remaining 23 to close or more distant relatives of DFs in Central and South America. The data set is analyzed by a combination of judiciously selected statistical methods. The analysis reveals that reliable information concerning MHC organization, including the assignment of sequences to loci, and evolution, as well as the process of species divergence, can be obtained in the absence of genomic sequence data, if the analysis is taken several steps beyond the standard phylogenetic tree construction approach. The main findings of the present study are these: The MHC class II B region of the passerine birds is as elaborate in its organization, divergence, and genetic diversity as the MHC of the eutherian mammals, specifically the primates. Hence, the reported simplicity of the fowl MHC is an oddity. With the help of appropriate markers, the divergence of the MHC genes can be traced deep in the phylogeny of the bird taxa. Transspecies polymorphism is rampant at many of the bird MHC loci. In this respect, the DFs behave as if they were a single, genetically undifferentiated population. There is thus far no indication of alleles that could be considered species, genus, or even DF group specific. The implication of these findings is that DFs are in the midst of adaptive radiations, in which morphological differentiation into species is running ahead of genetic differentiation in genetic systems such as the MHC or the mitochondrial DNA. The radiations are so young that there has not been enough time to sort out polymorphisms at most of the loci among the morphologically differentiating species. These findings parallel those on Lake Victoria haplochromine fishes. Several of the DF MHC allelic lineages can be traced back to the MHC genes of the species Tiaris obscura, which we identified previously as the closest extant relative of DFs in continental America.

Organization of Mhc class II A and B genes in the tilapiine fish Oreochromis.

Perch-like fishes of the family Cichlidae are models for the study of speciation. An important tool in these studies is the major histocompatibility complex (Mhc) and its organization. The present study takes the first step toward the elucidation of the Mhc class II gene organization in the tilapiine fish Oreochromis niloticus (Orni). Using class II A- and class II B-specific probes, Mhc-bearing clones were identified and isolated from a bacterial artificial chromosome (BAC) library. The analysis of these clones by a combination of molecular, genetic-mapping, and phylogenetic methods led to the identification of nine class II A and 15 class II B loci. Genes at these loci constitute two families, which we designate as class IIa and class IIb families. Each of the families contains A and B loci. Some genes in both families are expressed and functional. The two families differ in their chromosomal location (they are unlinked) and their mode of evolution. The class IIa family genes are conserved across different teleost taxonomical orders, whereas the class IIb family genes are apparently products of multiple, more recent, rounds of gene duplications. The rounds established at least five monophyletic groups of genes. The founding unit of each monophyletic group might have been a pair of class II A and B loci.

Successful treatment of a patient with tumor necrosis factor receptor-associated periodic syndrome using a half-dose of etanercept.

TNF receptor-associated periodic syndrome (TRAPS) is caused by mutations of TNFRSF1A gene and characterized by recurrent febrile episodes of prolonged duration and initial good response to steroids. Etanercept, a TNF blocker, has been used as a putative molecular-targeted agent for TRAPS, with some patients showing limited efficacy. Here, we report a patient with TRAPS who recovered from steroid dependency by etanercept and kept remission with a reduced dose of etanercept. The pathophysiology of TRAPS still remains to be elucidated and several hypotheses have been proposed. In the most recent hypothesis, the concerted action of wild-type and mutant TNF receptors plays an important role in provoking enhanced inflammation in TRAPS. The excellent response to etanercept in our patient suggested that there is heterogeneity in TRAPS patients in terms of the contribution of normal TNF signaling to autoinflammation.

Hemophilic pseudotumor of the maxillary sinus in an inhibitor-positive patient with hemophilia A receiving emicizumab: a case report.

A 14-year-old inhibitor-positive male patient with severe hemophilia A and severe psychomotor disability was admitted due to left buccal swelling and impaired downward movement of the left eye. He had been on noninvasive positive-pressure ventilation (NPPV) through a nasal mask for upper airway obstruction. The patient began to have repeated epistaxis 16 months after initiation of treatment with 6 mg/kg Q4W of emicizumab, and was thus administered a bypassing agent. Left buccal swelling and impaired downward movement of the left eye appeared during the subsequent month. Imaging examination revealed a mass in the left maxillary sinus and bone destruction. Endoscopic tumor resection and tracheostomy were performed using recombinant activated factor VII. NPPV was discontinued thereafter. Pathological examination revealed that the mass was a hemophilic pseudotumor (HP). After discharge the emicizumab-regimen dose was changed to 3 mg/kg, Q2W to increase serum emicizumab levels. No recurrent HP or bleeding requiring treatment was observed. Pressure applied to the damaged nasal mucosa by NPPV was suspected as the main cause of HP development. If a mass is observed in a patient with hemophilia, HP should be considered as a possible diagnosis even if the patient is receiving emicizumab.

Cathepsin L in bone marrow-derived cells is required for retinal and choroidal neovascularization.

Many vision-threatening diseases are characterized by intraocular neovascularization, (e.g., proliferative diabetic retinopathy and age-related macular degeneration). Although a new therapy with anti-VEGF antibodies is being used to treat these intraocular neovascular disorders, the visual recovery is limited, mainly because of the remnants of fibrovascular tissues. The ideal goal of the treatment is to prevent the invasion of new vessels into the avascular tissue through a matrix barrier. The purpose of this study was to determine the role played by cathepsin L, a matrix degrading enzyme, on intraocular angiogenesis. Used established animal models of retinal and choroidal neovascularization, we demonstrated that an inhibition of cathepsin L by specific inhibitors resulted in a significant decrease of intraocular neovascularization. A similar decrease of neovascularization was found in cathepsin L-deficient mice. Transplantation of bone marrow from cathepsin L-deficient mice into wild-type mice significantly reduced the degree of intraocular neovascularization. In addition, immunocytochemical analyses demonstrated that VE cadherin-positive endothelial progenitor cells, but not CD43-positive or Iba-1-positive cells, were the major cells contributing to the production of cathepsin L. These data indicate that cathepsin L expressed in endothelial progenitor cells plays a critical role in intraocular angiogenesis and suggest a potential therapeutic approach of targeting cathepsin L for neovascular ocular diseases.

Amyloid-beta up-regulates complement factor B in retinal pigment epithelial cells through cytokines released from recruited macrophages/microglia: Another mechanism of complement activation in age-related macular degeneration.

One of the earliest signs of age-related macular degeneration (AMD) is the formation of drusen which are extracellular deposits beneath the retinal pigmented epithelium (RPE). To investigate the relationship between drusen and AMD, we focused on amyloid beta (Abeta), a major component of drusen and also of senile plaques in the brain of Alzheimer's patients. We previously reported that Abeta was accumulated in drusen-like structure in senescent neprilysin gene-disrupted mice. The purpose of this study was to investigate the influence of Abeta on factor B, the main activator of the complement alternative pathway. The results showed that Abeta did not directly modulate factor B expression in RPE cells, but increased the production of monocyte chemoattractant protein-1 (MCP-1). Abeta also increased the production of IL-1beta and TNF-alpha in macrophages/microglia, and exposure of RPE cells to IL-1beta and TNF-alpha significantly up-regulated factor B. Co-cultures of RPE cells and macrophages/microglia in the presence of Abeta significantly increased the expression of factor B in RPE. These findings indicate that cytokines produced by macrophages/microglia that were recruited by MCP-1 produced in RPE cells stimulated by Abeta up-regulate factor B in RPE cells. Thus, a combined mechanism exists for Abeta-induced for the activation of the complement alternative pathway in the subretinal space; cytokine-induced up-regulation of activator factor B and dysfunction of the inhibitor factor I by direct binding to Abeta as suggested in our earlier study.

Effects of basic fibroblast growth factor on the development of the stem cell properties of human dental pulp cells.

We isolated adherent fibroblastic cells after collagenase and dispase treatment of human dental pulp. When human dental pulp cells (hDPCs) were cultured in the presence of basic fibroblast growth factor (bFGF), the ratio of hDPCs in the S-phase was significantly higher in comparison with incubation without bFGF. The ratio of hDPCs expressing STRO-1 as a marker of stem cell populations increased approximately eightfold in the presence of bFGF as opposed to that in the absence of bFGF. We demonstrated the characterization and distinctiveness of the hDPCs and showed that, when cultured with the medium containing serum and bFGF, they were highly proliferative and capable of differentiating in vitro into osteoblasts, chondrocytes, and adipocytes. Furthermore, the in vitro differentiation was confirmed at both the protein and gene expression levels. Transplantation of hDPCs -- expanded ex vivo in the presence of bFGF into immunocompromised mice -- revealed the formation of bone, cartilage, and adipose tissue. The donor hDPC-derived cells were labeled in the bone tissues located near the PLGA in the subcutaneous tissues of recipient mice using a human-specific Alu probe. When cultured with a serum-free medium containing bFGF, the hDPCs strongly expressed STRO-1 immunoreactive products and sustained self-renewal, and thus were almost identical in differentiation potential and proliferation activity to hDPCs cultured with the medium containing serum and bFGF. The present results suggest that the hDPCs cultured in the presence of bFGF irrespective of the presence or absence of the bovine serum are rich in mesenchymal stem cells or progenitor cells and useful for cell-based therapies to treat dental diseases.

Effect of transfusion on the venous blood lactate level in very low-birthweight infants.

BACKGROUND: In recent years the blood lactate level can be easily and quickly measured with a small amount of blood, and the availability of an arterial blood lactate level has been reported as an indicator of oxygen deficit in adults. To determine whether venous blood lactate level can serve as such a marker for determining the indications for transfusion, blood lactate and hemoglobin level were monitored before and after transfusion. METHODS: The study subjects consisted of 12 very low-birthweight infants admitted to the neonatal intensive care unit and who had transfusion between June 2005 and June 2007. The data on the blood lactate and hemoglobin were collected retrospectively by referring to the clinical records. RESULTS: A total of 18 transfusions was performed. There was no significant relationship between venous blood lactate and hemoglobin concentration before transfusion. The subjects were classified into two groups according to the lactate level before transfusion: > or =3.3 mmol/L and <3.3 mmol/L. In the high-lactate group the lactate decreased significantly after transfusion (P < 0.01) and it continued to decrease thereafter. In the low-lactate group, however, the lactate remained unchanged. CONCLUSIONS: Venous blood lactate measurements may offer some additional information regarding the optimal time for performing a transfusion. To the authors' knowledge this is the first report to study the changes in lactate levels using venous blood sampling in red blood cell transfusion in very low-birthweight infants.

Elastic fiber system evaluated in the digestive organ of rats.

According to our previous reports, the intraperiodontal elastic fiber system comprises oxytalan fibers, whereas all types of elastic system fibers are present in the gingiva. Much remains to be elucidated regarding the topographic development of the elastic fiber system that constitutes the walls of the digestive organs. This study aimed to examine the topographic development of the elastic fiber system in the periodontal tissue, oral cavity and digestive tract of rats at light- and electron microscopic levels. At embryonic day 20, in situ hybridization revealed the mRNA expression of tropoelastin in the putative gingival lamina propria but not in the dental follicle. At the postnatal stage, the masticatory mucous membrane of the gingiva and hard palate comprised three different types of elastic system fibers (oxytalan, elaunin and elastic fibers). Conversely, the elastic fiber system comprised elaunin and elastic fibers in other oral mucosae and the lining mucosae of digestive tract organs (the esophagus, stomach and small intestine). The findings of our study suggest that the elastic fiber system is mainly related to tissue resistance in the periodontal ligament and tissue elasticity in the oral mucosae without masticatory mucosae and the overlying mucosa of digestive tracts and both functions in the gingiva and hard palate, respectively. The appearance of elaunin fibers in the periodontium of rats aged 14 weeks suggests the expression of tropoelastin induced by mechanical stressors such as mastication. The intraperiodontal difference in the distribution of elaunin fibers suggests heterogeneity among fibroblasts constituting the periodontium.