Molecular engineering of a minimal E-cadherin inhibitor protein derived from Clostridium botulinum hemagglutinin.

Hemagglutinin (HA), a nontoxic component of the botulinum neurotoxin (BoNT) complex, binds to E-cadherin and inhibits E-cadherin-mediated cell-cell adhesion. HA is a 470 kDa protein complex comprising six HA1, three HA2, and three HA3 subcomponents. Thus, to prepare recombinant full-length HA in vitro, it is necessary to reconstitute the macromolecular complex from purified HA subcomponents, which involves multiple purification steps. In this study, we developed NanoHA, a minimal E-cadherin inhibitor protein derived from Clostridium botulinum HA with a simple purification strategy needed for production. NanoHA, containing HA2 and a truncated mutant of HA3 (amino acids 380-626; termed as HA3mini), is a 47 kDa single polypeptide (one-tenth the molecular weight of full-length HA, 470 kDa) engineered with three types of modifications: (i) a short linker sequence between the C terminus of HA2 and N terminus of HA3; (ii) a chimeric complex composed of HA2 derived from the serotype C BoNT complex and HA3mini from the serotype B BoNT complex; and (iii) three amino acid substitutions from hydrophobic to hydrophilic residues on the protein surface. We demonstrated that NanoHA inhibits E-cadherin-mediated cell-cell adhesion of epithelial cells (e.g., Caco-2 and Madin-Darby canine kidney cells) and disrupts their epithelial barrier. Finally, unlike full-length HA, NanoHA can be transported from the basolateral side to adherens junctions via passive diffusion. Overall, these results indicate that the rational design of NanoHA provides a minimal E-cadherin inhibitor with a wide variety of applications as a lead molecule and for further molecular engineering.

Increased Myosin light chain 9 expression during Kawasaki disease vasculitis.

Introduction: Kawasaki disease (KD) is an acute systemic vasculitis that predominantly afflicts children. KD development is known to be associated with an aberrant immune response and abnormal platelet activation, however its etiology is still largely unknown. Myosin light chain 9 (Myl9) is known to regulate cellular contractility of both non-muscle and smooth muscle cells, and can be released from platelets, whereas any relations of Myl9 expression to KD vasculitis have not been examined. Methods: Plasma Myl9 concentrations in KD patients and children with febrile illness were measured and associated with KD clinical course and prognosis. Myl9 release from platelets in KD patients was also evaluated in vitro. Myl9 expression was determined in coronary arteries from Lactobacillus casei cell wall extract (LCWE)-injected mice that develop experimental KD vasculitis, as well as in cardiac tissues obtained at autopsy from KD patients. Results and discussion: Plasma Myl9 levels were significantly higher in KD patients during the acute phase compared with healthy controls or patients with other febrile illnesses, declined following IVIG therapy in IVIG-responders but not in non-responders. In vitro, platelets from KD patients released Myl9 independently of thrombin stimulation. In the LCWE-injected mice, Myl9 was detected in cardiac tissue at an early stage before inflammatory cell infiltration was observed. In tissues obtained at autopsy from KD patients, the highest Myl9 expression was observed in thrombi during the acute phase and in the intima and adventitia of coronary arteries during the chronic phase. Thus, our studies show that Myl9 expression is significantly increased during KD vasculitis and that Myl9 levels may be a useful biomarker to estimate inflammation and IVIG responsiveness to KD.

The pronounced lung lesions developing in LATY136F knock-in mice mimic human IgG4-related lung disease.

RATIONALE: Immunoglobulin (Ig) G4-related disease (IgG4-RD) is a novel clinical disease entity characterized by an elevated serum IgG4 concentration and tumefaction or tissue infiltration by IgG4-positive plasma cells. Pathological changes are most frequently seen in the pancreas, lacrimal glands, and salivary glands, but pathological changes in the lung also exist. Linker for activation of T cell (LAT)Y136F knock-in mice show Th2-dominant immunoreactions with elevated serum IgG1 levels, corresponding to human IgG4. We have reported that LATY136F knock-in mice display several characteristic features of IgG4-RD and concluded that they constitute an appropriate model of human IgG4-RD in salivary glands, pancreas, and kidney lesions. OBJECTIVES: The aim of this study is to evaluate whether lung lesions in LATY136F knock-in mice can be a model of IgG4-related lung disease. METHODS: Lung tissue samples from LATY136F knock-in mice (LAT) and wild-type mice (WT) were immunostained for IgG1 and obtained for pathological evaluation, and cell fractions and cytokine levels in broncho-alveolar lavage fluid (BALF) were analyzed. RESULTS: In the LAT group, IgG1-positive inflammatory cells increased starting at 4 weeks of age and peaked at 10 weeks of age. The total cell count and percentage of lymphocytes increased significantly in BALF in the LAT group compared to the WT group. In BALF, Th2-dominant cytokines and transforming growth factor-ß were also increased. In the LAT group, marked inflammation around broncho-vascular bundles peaked at 10 weeks of age. After 10 weeks, fibrosis around broncho-vascular bundles and bronchiectasis were observed in LATY136F knock-in mice but not WT mice. CONCLUSIONS: LATY136F knock-in mice constitute an appropriate model of lung lesions in IgG4-RD.

Pre-administration of low-dose methamphetamine enhances movement and neural activity after high-dose methamphetamine administration in the striatum.

Methamphetamine (METH) is a powerful stimulant drug of abuse, with potent addictive and neurotoxic properties. In this study, the effects of low-dose METH administration prior to high-dose METH administration on movement and neural activity in rats were examined. Rats were administered low-dose (1 mg/kg/day) METH or saline for 5 consecutive days (m5 and s5, respectively), followed by high-dose (10 mg/kg) METH on day 6 (m5M and s5M, respectively). An accelerometer was used to evaluate the frequency of movement when rats were placed in a cage for 30 min. The expression of c-fos, a neuronal activity marker, in the striatum was analyzed using immunohistochemistry. Striatal protein expression of neuronal markers, including vesicular glutamate transporter 2 (VGLUT2), glutamate decarboxylase 67 (GAD67), tyrosine hydroxylase (TH), tryptophan hydroxylase 2 (TPH2), and the glial marker, glial fibrillary acidic protein (GFAP), was analyzed by western blot. Accelerometer counts and the numbers of c-fos-positive cells in the striatum were significantly higher in the m5M than in the s5, m5, and s5M groups. The expression levels of VGLUT2 and GAD67, but not those of TH, TPH2, or GFAP, were significantly higher in the m5M than in the s5M group. These results suggest that pre-administration of low-dose METH prior to high-dose METH administration in rats may alter excitatory and inhibitory neurons in the striatum, thereby affecting movement and neural activity in rats.

LatY136F knock-in mouse model for human IgG4-related disease.

BACKGROUND: The adaptor protein Linker for activation of T cell (LAT) is a key signaling hub used by the T cell antigen receptor. Mutant mice expressing loss-of-function mutations affecting LAT and including a mutation in which tyrosine 136 is replaced by a phenylalanine (LatY136F) develop lymphoproliferative disorder involving T helper type 2 effector cells capable of triggering a massive polyclonal B cell activation that leads to hypergammaglobulinemia G1 and E and to non-resolving inflammation and autoimmunity. The purpose of this study was to evaluate whether the phenotypes of LatY136F knock-in mice resemble the immunohistopathological features of immunoglobulin G4-related disease (IgG4-RD). METHODS: LatY136F knock-in mice were sacrificed at 4-20 weeks of age, and pancreas, kidney, salivary gland and lung were obtained. All organs were stained with hematoxylin-eosin and with Azan for estimation of collagen in fibrosis, and the severity scores of inflammation and fibrosis were evaluated. Immunostainings were performed to analyze the types of infiltrating cells. In addition, the effects of corticosteroid treatment on the development of tissue lesions and serum levels of IgG1 were assessed. RESULTS: Tissue lesions characterized by inflammatory mononuclear cell infiltration and fibrosis were detected in pancreas, kidney, and salivary gland starting from 6 weeks of age. Immunostainings showed pronounced infiltration of plasma cells, CD4-positive T cells, and macrophages. Infiltrating plasma cells predominantly expressed IgG1. The extent of inflammation in pancreas and salivary glands was markedly reduced by corticosteroid treatment. CONCLUSIONS: LatY136F knock-in mice displayed increased production of Th2-type IgG1 (a homologue of human IgG4) and developed multiple organ tissue lesions reminiscent of those seen in patients with IgG4-RD. Moreover, the development of these tissue lesions was highly sensitive to corticosteroid treatment like in IgG4-RD. For these reasons we consider the LatY136F knock-in mouse strain to represent a promising model for human IgG4-RD.

Activated tumor cell integrin αvß3 cooperates with platelets to promote extravasation and metastasis from the blood stream.

Metastasis is the main cause of death in cancer patients, and understanding mechanisms that control tumor cell dissemination may lead to improved therapy. Tumor cell adhesion receptors contribute to cancer spreading. We noted earlier that tumor cells can expressing the adhesion receptor integrin αvß3 in distinct states of activation, and found that cells which metastasize from the blood stream express it in a constitutively high affinity form. Here, we analyzed steps of the metastatic cascade in vivo and asked, when and how the affinity state of integrin αvß3 confers a critical advantage to cancer spreading. Following tumor cells by real time PCR, non-invasive bioluminescence imaging, intravital microscopy and histology allowed us to identify tumor cell extravasation from the blood stream as a rate-limiting step supported by high affinity αvß3. Successful transendothelial migration depended on cooperation between tumor cells and platelets involving the high affinity tumor cell integrin and release of platelet granules. Thus, this study identifies the high affinity conformer of integrin αvß3 and its interaction with platelets as critical for early steps during hematogenous metastasis and target for prevention of metastatic disease.

Tracheal injury added to cervical bone destruction due to the impact of hitting the water surface: four immersed adult bodies.

In the forensic examinations of cases of falling, two properties of the water surface, namely its nature as a hard, flat object and as a soft and ungraspable substance must be appreciated. Namely, at the moment of impact, the water surface exerts a greater resistance against relatively broad areas like the head, face and trunk than against the extremities that have a small area. Therefore, total resistance against the whole body would promote flexure. We experienced 72 autopsy cases of immersed bodies during a 4-year period. The cause of death for 64 of these with or without cervical vertebra fracture was drowning. In these cases, the various heights of the falls could often be estimated at the scene. A characteristic pattern of cervical injury with involvement of hyoid bone and thyroid cartilage in addition to cervical vertebra fracture plus rare involvement of the trachea was identified. When a fall from a relatively low height is broken by the water surface, to a certain degree physical findings that differ from those seen in falls to the ground from extreme heights are left mediated by different underlying mechanisms.

Fatal and non-fatal cases of lime sulfide exposure and pathogenetic mechanisms underlying pancreatic injury: Case reports with an animal experiment.

Lime sulfide poisoning by the oral route is rarely encountered in the practice of forensic science, whereas hydrogen sulfide poisoning is seen frequently. We report here two cases of fatal lime sulfide poisoning with several related cases and in addition induced histological damage with acute inflammation in animal models under at similar concentrations. We also evaluated sulfide and thiosulfate concentrations and speculated as to the cause of pancreatic damage in these cases.

Impact of adrenomedullin on dextran sulfate sodium-induced inflammatory colitis in mice: insights from in vitro and in vivo experimental studies.

BACKGROUND: Although adrenomedullin (AM) is known to ameliorate inflammatory processes, few data exist regarding the effect of AM on inflammatory colitis. Therefore, we examined the effect of AM on inflammatory response in vitro and in vivo colitis model. METHODS: In mice experimental colitis induced by 3% dextran sulfate sodium (DSS) in drinking water for 7 days, AM with 225-900 µg/kg in 0.5 ml of saline or saline alone were given intraperitoneally once a day. In the in vitro experiment, we determined the cytokine response in THP-1 cell activated by lipopolysaccharide with or without AM of 10 nM. Additionally, we performed wound healing assay in Caco-2 cell interfered by DSS with or without AM of 100 nM. RESULTS: In the colitis model, AM significantly reduced the disease activity index, histological score, and local production of inflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6 in accordance with reduction of serum amyloid A levels. Secretion of TNF-α in lipopolysaccharide-stimulated THP-1 cells was significantly reduced in the presence of AM. The distance of wound healing interfered by 0.25% DSS was significantly improved in the presence of AM of 100 nM. CONCLUSIONS: These results demonstrate that AM could ameliorate DSS-induced experimental colitis possibly through suppression of systemic and local production of cytokines such as TNF-α, associated with acceleration of ulcer reepithelialization and colon tissue regeneration.

Sudden death of a young male with previously undiagnosed autosomal dominant polycystic kidney disease (ADPKD).

Autosomal dominant polycystic kidney disease (ADPKD) is one of the best known genetic diseases. However, in only very rare cases does it present as an abnormal death followed by clarification of its genetic background. We experienced a case in which ADPKD first became evident from the results of forensic autopsy, on the basis of which all potentially affected family members were offered genetic and other medical examinations. In this way, forensic medicine was able not only to determine the cause of death but to contribute to preventive medicine as well.

Impact of anti-apoptotic and anti-oxidative effects of bone marrow mesenchymal stem cells with transient overexpression of heme oxygenase-1 on myocardial ischemia.

Although mesenchymal stem cells (MSCs) have therapeutic potential for tissue injury, intolerance and poor cell viability limit their reparative capability. Therefore, we examined the impact of bone marrow-derived MSCs, in which heme oxygenase-1 (HO-1) was transiently overexpressed, on the repair of an ischemic myocardial injury. When MSCs and HO-1-overexpressed MSCs (MSC(HO-1)) were exposed to serum deprivation/hypoxia or H(2)O(2)-induced oxidative stress, MSC(HO-1) exhibited increased resistance to cell apoptosis compared with MSCs (17 +/- 1 vs. 30 +/- 2%, P < 0.05) and were markedly resistant to cell death (2 +/- 1 vs. 32 +/- 2%, P < 0.05). Under these conditions, vascular endothelial growth factor (VEGF) production was 2.1-fold greater in MSC(HO-1) than in MSCs. Pretreatment of MSCs and MSC(HO-1) with phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B (Akt) pathway inhibitors such as LY-294002 (50 muM) or wortmannin (100 nM) significantly decreased VEGF production. In a rat infarction model with MSCs or MSC(HO-1) (5 x 10(6) +/- 0.1 x 10(6) cells/rat) transplantation, the number of TdT-mediated dUTP nick end-labeling-positive cells was significantly lower in the MSC(HO-1) group than in the MSC group (12.1 +/- 1.0 cells/field vs. 26.5 +/- 2.6, P < 0.05) on the 4th day after cell transplantation. On the 28th day, increased capillary density associated with decreased infarction size was observed in the MSC(HO-1) group (1,415 +/- 47/mm(2) with 21.6 +/- 2.3%) compared with those in the MSCs group (1,215 +/- 43/mm(2) with 28.2 +/- 2.3%, P < 0.05), although infarction size relative to area at risk was not different in each group at 24 h after transplantation. These results demonstrate that MSC(HO-1) exhibit markedly enhanced anti-apoptotic and anti-oxidative capabilities compared with MSCs, thus contributing to improved repair of ischemic myocardial injury through cell survival and VEGF production associated with the PI 3-kinase/Akt pathway.

Secretion of pleiotrophin stimulates breast cancer progression through remodeling of the tumor microenvironment.

Pleiotrophin (PTN, Ptn) is an 18-kDa secretory cytokine expressed in many breast cancers; however, the significance of Ptn expression in breast cancer has not been established. We have now tested three models to determine the role of inappropriate expression of Ptn in breast cancer. Mouse mammary tumor virus (MMTV) promoter-driven Ptn expressed in MMTV-polyoma virus middle T antigen (PyMT)-Ptn mouse breast cancers was first shown to induce rapid growth of morphologically identified foci of "scirrhous" carcinoma and to extensively remodel the microenvironment, including increased tumor angiogenesis and striking increases in mouse protocollagens Ialpha2, IValpha5, and XIalpha1, and elastin. Ectopic Ptn expression in MCF-7 (human breast cancer)-Ptn cell xenografts also was shown to markedly increase MCF-7-Ptn cell xenograft growth in nude mice; furthermore, it induced extensive remodeling of the microenvironment and tumor angiogenesis. In a coculture model of equal numbers of NIH 3T3 stromal fibroblasts and MCF-7-Ptn cells, PTN secreted from MCF-7-Ptn cells was then shown to induce a more malignant MCF-7-Ptn breast cancer cell phenotype and extensive remodeling of the MCF-7-Ptn/NIH 3T3 cell microenvironment; it up-regulated expression of markers of aggressive breast cancers, including PKCdelta and matrix metalloproteinase-9 in both MCF-7-Ptn and NIH 3T3 cells. The morphological phenotypes of MCF-7-Ptn cell xenografts and MCF-7-Ptn cell/NIH 3T3 cell cocultures closely resembled breast cancers in MMTV-PyMT-Ptn mice. Inappropriate expression of Ptn thus promotes breast cancer progression in mice; the data suggest that secretion of PTN through stimulation of the stromal cell microenvironment alone may be sufficient to account for significant features of breast cancer progression.

Platelet glycoprotein Ib alpha supports experimental lung metastasis.

The platelet paradigm in hemostasis and thrombosis involves an initiation step that depends on platelet membrane receptors binding to ligands on a damaged or inflamed vascular surface. Once bound to the surface, platelets provide a unique microenvironment supporting the accumulation of more platelets and the elaboration of a fibrin-rich network produced by coagulation factors. The platelet-specific receptor glycoprotein (GP) Ib-IX, is critical in this process and initiates the formation of a platelet-rich thrombus by tethering the platelet to a thrombogenic surface. A role for platelets beyond the hemostasis/thrombosis paradigm is emerging with significant platelet contributions in both tumorigenesis and inflammation. We have established congenic (N10) mouse colonies (C57BL/6J) with dysfunctional GP Ib-IX receptors in our laboratory that allow us an opportunity to examine the relevance of platelet GP Ib-IX in syngeneic mouse models of experimental metastasis. Our results demonstrate platelet GP Ib-IX contributes to experimental metastasis because a functional absence of GP Ib-IX correlates with a 15-fold reduction in the number of lung metastatic foci using B16F10.1 melanoma cells. The results demonstrate that the extracellular domain of the alpha-subunit of GP Ib is the structurally relevant component of the GP Ib-IX complex contributing to metastasis. Our results support the hypothesis that platelet GP Ib-IX functions that support normal hemostasis or pathologic thrombosis also contribute to tumor malignancy.

Vascular tissue fragility assessed by a new double stain method.

Although matrix metalloproteinases (MMPs) are known to be involved in the development of atherosclerosis and the instability of atheromatous plaques, much remains to be learned about their roles at the tissue level. To help clarify this area, we established a new double staining method using film in situ zymography and immunohistochemistry. Using this technique, a comprehensive analysis of the gelatinolytic activity in human vessel tissue demonstrated that gelatinolytic activity is enhanced in the shoulder region and fibrous cap at superficial areas of the atheromatous plaque in the presence of thrombolysis. Enzyme assay clarified high activity in the superficial area (7.50 +/- 5.04 U/mg weight; P < 0.001). Gelatin zymography also indicated that addition of the antiplatelet agent, trapidil, alters the amount of secretion of MMP-2 and MMP-9 and their activation ratio. This novel approach to detect the activity of gelatinases in resected tissues may aid in the selection of optimal treatment of individual patients.

Intraperitoneal fluid accumulation induced by Clostridium perfringens alpha-toxin (phospholipase C).

We report that the intraperitoneal injection of Clostridium perfringens alpha-toxin into mice induces ascites. This phenomenon was monitored by measuring fluid volume and analyzing hematologic data. The mouse toxicity test provides a simple and useful model for examining C. perfringens alpha-toxin-induced vascular permeability.