Trehangelins ameliorate inflammation-induced skin senescence by suppressing the epidermal YAP-CCN1 axis.

Trehangelins (THG) are newly identified trehalose compounds derived from broth cultures of an endophytic actinomycete, Polymorphospora rubra. THG are known to suppress Cellular Communication Network factor 1 (CCN1), which regulates collagen homeostasis in the dermis. Although the physical properties of THG suggest a high penetration of the stratum corneum, the effect of THG on the epidermis has not been reported. Here we describe a possible mechanism involved in skin aging focusing on the effect of THG on epidermal CCN1. This study shows that: (1) THG suppress epidermal CCN1 expression by inhibiting the translocation of Yes-Associated Protein (YAP) to nuclei. (2) Epidermal CCN1, localized at the basement membrane, regulates the balance between the growth and differentiation of keratinocytes. (3) Keratinocytes secrete more CCN1 than fibroblasts, which leads to disruption of the basement membrane and extracellular matrix components. (4) The secretion of CCN1 from keratinocytes is increased by ultraviolet B exposure, especially in aged keratinocytes, and deteriorates the elastic fiber structures in the underlying dermis. (5) Topical application of THG ameliorates the structure of the basement membrane in ex vivo human skin explants. Taken together, THG might be a promising treatment for aged skin by suppressing the aberrant YAP-CCN1 axis.

Elastin microfibril interface-located protein 1 and its catabolic enzyme, cathepsin K, regulate the age-related structure of elastic fibers in the skin.

INTRODUCTION: The elastic fiber structure becomes shorter, thicker, and curved with age. Nonetheless, the proteins and catabolic enzymes influencing the maintenance of and change in the three-dimensional (3D) structure of elastic fibers remain unknown. This study aimed to identify the proteins involved in the maintenance and degeneration of elastic fiber structures. METHODS: We performed a combined 3D structural analysis using tissue decolorization technology and mRNA abundance and comprehensive protein expression of tissue-derived cells. The relationship between the proteins was evaluated. RESULTS: Elastin microfibril interface-located protein 1 (EMILIN-1) and cathepsin K (CTSK) were implicated in structural changes in elastic fibers with aging. EMILIN-1 and CTSK levels were highly correlated and changed with age. CTSK was identified as the degrading enzyme of EMILIN-1. CTSK fragmented the otherwise linearly existing dermal elastic fiber structure, with more evident changes in oxytalan fibers. EMILIN-1 expression in fibroblasts was increased by co-culturing with keratinocytes. Furthermore, CTSK expression was increased by UV stress in keratinocytes, resulting in decreased EMILIN-1 expression. CONCLUSION: Using our new assessment strategy, we observed that EMILIN-1 and CTSK are highly linked to changes in the elastic fiber structure with aging. These results indicate that suppressing CTSK expression and increasing EMILIN-1 expression might be an effective approach to prevent elastic fiber morphological changes that lead to wrinkles and sagging. Furthermore, EMILIN-1 in the dermis increases due to interaction with the epidermis, which could provide a new target for the therapeutic care of elastic fibers (including preservation of oxytalan fibers) in epidermis-dermis interaction.

Spinal infarction caused by hypovolemic shock following massive bleeding from stab wounds to the neck.

A 59-year-old female was brought to our emergency department with hypovolemic shock caused by massive bleeding from neck stab wounds inflicted by herself in a committed suicide. The patient complained of comparatively strong pain on her lower back and there was sensory and motor disturbance of bilateral lower limbs, but there was no trauma on the lumber region, the spine, or the vertebrae. After hemostasis, we performed magnetic resonance imaging, which demonstrated high intensity signal in the spinal and longitudinal area from the Th8 to the conus medullaris, and at center of the frontal horn on the upper thoracic spinal cord (owl's eye appearance) on T2 weighted images. This case was diagnosed as spinal infarction caused by low blood pressure as a result of massive bleeding. The basis of diagnosis were as follows: 1) an acute onset; 2) when the ambulance arrived, she was in hypovolemic shock caused by massive hemorrhage; 3) there was no trauma on the lumber region, the spine, or the vertebrae; 4) with CT taken on admission, aortic disease was not detected; and 5) she was not on any antipsychotic drugs which could cause thrombosis. We treated the patient following management protocol of cerebral infarction, but recovery of sensory and motor disorders was minimal.

Free flap transfer to preserve main arterial flow in early reconstruction of open fracture in the lower extremity.

The selection of recipient vessels is crucial when reconstructing traumatized lower extremities using a free flap. When the dorsalis pedis artery and/or posterior tibial artery cannot be palpated, we utilize computed tomography angiography to verify the site of vascular injury prior to performing free flap transfer. For vascular anastomosis, we fundamentally perform end-to-side anastomosis or flow-through anastomosis to preserve the main arterial flow. In addition, in open fracture of the lower extremity, we utilize the anterolateral thigh flap for moderate soft tissue defects and the latissimus dorsi musculocutaneous flap for extensive soft tissue defects. The free flaps used in these two techniques are long and include a large-caliber pedicle, and reconstruction can be performed with either the anterior or posterior tibial artery. The preparation of recipient vessels is easier during the acute phase early after injury, when there is no influence of scarring. A free flap allows flow-through anastomosis and is thus optimal for open fracture of the lower extremity that requires simultaneous reconstruction of main vessel injury and soft tissue defect from the middle to distal thirds of the lower extremity.

Immediate reconstruction after removal of nasal prosthesis using diced cartilage wrapped with temporal fascia.

Various materials are used for nasal augmentations. Silicone is the most prevalent because it is durable and facilitates sculpturing. However, the unfortunate patient who presents with complication of the nasal implants and wants to remove them is vexed with a significant resultant cosmetic defect if the implant is removed. However, the patients who have some troubles after augmentation by the implants tend to hate the use of the prosthesis again. Ideally, immediate reconstruction would be offered to the patient, sparing him/her the deformity left by removal of the implant. We treated 16 patients who had undergone immediate nasal reconstruction after removal of foreign body. We reconstructed nasal deformity by diced cartilage wrapped with temporal fascia. The cartilage harvested from the ear concha was finally diced into 1- to 2-mm cubes. A bag was made from deep or superficial temporal fascia, and diced cartilage cubes were placed in the bag, which was grafted onto the nasal dorsum. This procedure had several advantages including getting natural contouring and enough volume and absence of foreign body reaction. It was also soft to the touch compared with prosthesis. The fascia could support the thin dorsum skin. The nasal augmentation effect of this procedure was comparable with that of prosthesis methods. It had lower risks for infection and exposure and provided more psychologic comfort. The nasal deformities were successfully reconstructed using diced cartilage wrapped with temporal fascia. We believe that this is the good method for the immediate nasal reconstruction after the removal of foreign body.

Effects of neuropeptides and their local administration to cutaneous wounds in sensory-impaired areas.

Wound healing in sensory-impaired areas such as diabetic foot and spinal cord injuries is intractable. Previous studies have shown that delayed wound healing both of wound contraction and epithelialization in denervated rat skin. The aim of this study was to investigate whether the wound healing process was affected by the administration of substance P to skin defects and histological analysis in denervated skin. Full thickness circular skin defects 15 mm in diameter were made symmetrically on the denervated area and the normal innervated area, and substance P and vehicle was administered over a period of 3 days by injecting with a syringe. The rate of wound contraction and epithelialization were measured. The wound surface area in saline injections were larger than the group of substance P injections in denervated area and controls (p < 0.05) on day 3. Wound healing in local administration of substance P to denervated skin defect was equal to in normal animals. It seems that the presence of substance P in the wound area positively affects the early stages of wound healing.

Replaced platelet concentrates containing a new additive solution, M-sol: safety and efficacy for pediatric patients.

BACKGROUND: Allergic transfusion reactions (ATRs), particularly those caused by plasma-rich platelet concentrates (P-PCs), are an important concern in transfusion medicine. Replacing P-PCs with PCs containing M-sol (M-sol-R-PCs) is expected to prevent ATRs. However, this has not yet been verified by sufficient clinical evidence. STUDY DESIGN AND METHODS: A retrospective cohort study was performed between 2008 and 2011. Pediatric patients with hematologic disorders, solid tumors, primary immunodeficiency disorders, or inherited metabolic disorders were transfused with M-sol-R-PCs between 2010 and 2011; the transfusions of P-PCs administered between 2008 and 2011 were compared in terms of frequency and severity of ATRs, corrected count increment (CCI), and occurrence of bleeding. Data were collected for 6 consecutive months on a per-patient basis. RESULTS: Data obtained during 2008 to 2011 showed that of the 78 patients receiving 515 P-PC transfusions, 14 (17.9%) had 17 ATRs (3.3%); 14 and three ATRs were of Grades 1 and 2, respectively. In 2010 to 2011, 49 patients received 620 transfusions of M-sol-R-PCs, and two patients (4.1%) had Grade 1 ATRs (0.3%). Thus, the frequency of ATRs per bag and per patient differed significantly between the two transfusions. No steroid agents were used for the prevention or treatment of ATRs in the M-sol-R-PC group. The CCI (24 hr) for M-sol-R-PCs did not differ from that for P-PCs. CONCLUSION: M-sol-R-PCs were found to be effective in preventing ATRs without loss of transfusion efficiency in children; however, its efficacy should be further evaluated in prospective clinical trials.

A layered ionic crystal of polar Li@C(60) superatoms.

If the physical properties of C(60) fullerene molecules can be controlled in C(60) products already in use in various applications, the potential for industrial development will be significant. Encapsulation of a metal atom in the C(60) fullerene molecule is a promising way to control its physical properties. However, the isolation of C(60)-based metallofullerenes has been difficult due to their insolubility. Here, we report the complete isolation and determination of the molecular and crystal structure of polar cationic Li@C(60) metallofullerene. The physical and chemical properties of Li@C(60) cation are compared with those of pristine C(60). It is found that the lithium cation is located at off-centre positions in the C(60)-I(h) cage interior and that the [Li(+)@C(60)] salt has a unique two-dimensional structure. The present method of purification and crystallization of C(60)-based metallofullerenes provides a new C(60) fullerene material that contains a metal atom.

[Analysis of antibody reactivity for FDP D-dimer fragments by Western blotting].

We evaluated three test kits for fibrin degradation products (FDP) D-dimer. We found that six of 217 plasma sample values obtained by Nanopia test were markedly higher than the values obtained using the other two kits. The regression equation for 211 samples (excluding six) was y=0.64x+3.05 (y: Nanopia, x: LIAS AUTO) and the correlation coefficient was 0.915. Therefore, we classified these samples into three categories, namely correlated(y< 1.0x), incompatible (y= 1.0x-2.9x) and markedly incompatible (y> or =3.0x). Selected samples, eight correlated, four incompatible and four markedly incompatible, were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blotting(WB). WB analysis using anti-fibrinogen antibody showed that both high molecular weight fragments of cross-linked fibrin (HMW-XDP) and DD/E fragments were present in the correlated samples, but there was less HMW-XDP than DD/E in the incompatible samples and mostly DD/E (HMW-XDP was significantly less than DD/E) in the markedly incompatible samples. These data suggest that plasma FDP samples that contain mostly DD/E and little HMW-XDP demonstrated markedly incompatible values using the three D-dimer test kits. These data was reflected by markedly elevated plasmin alpha2-plasmin inhibitor complex values in the incompatible and markedly incompatible samples. Unfortunately, we did not directly demonstrate these phenomena by WB analysis with two anti-D-dimer antibodies used Nanopia or LPIA reagent. In the near future, we expect that standardization of FDP D-dimer assay will be accomplished.

[Analysis of hypofibrinogenemias found on routine coagulation screening tests and identification of heterozygous dysfibrinogenemia or fibrinogen deficiency].

We analyzed the clinical factors resulting in hypofibrinogenemia, which is defined as less than 100mg/dl of plasma fibrinogen values determined by a procedure based on the Thrombin-time method. Within a 12-month period, we assayed 5,746 patients (19,309 plasmas) and found 113 patients (1.97%) with hypofibrinogenemia. We categorized these patients as having decreased synthesis of fibrinogen (less than 3.0g/dl of albumin, 140 IU/l of Cholinesterase, and/or 50% on Hepaplastin Test), increased consumption of fibrinogen (more than 10 microg/ml of FDP D-dimer), known side effect of L-asparaginase administration, or other causes. Details are follows: 1) decreased synthesis: 26 patients, suspected of decreased synthesis (albumin: 3.1-3.4 g/dl): 4 patients, 2) increased consumption: 15 patients, suspected of increased consumption (FDP D-dimer: 5.0-9.9 g/dl): 1 case, 3) decreased synthesis combined with increased consumption: 24 patients, suspected of decreased synthesis and/or suspected of increased consumption: 14 patients, 4) side-effect of L-asparaginase administration: 24 patients, 5) heterozygous dysfibrinogenemia: 1 patient, 6) heterozygous fibrinogen deficiency: 1 patient, suspected of heterozygous fibrinogen deficiency: 1 patient, 7) unidentified: 2 patients with West syndrome treated with a combination of ACTH and valproic acid. Three patients with dysfibrinogenemia or fibrinogen deficiency showed normal or slightly prolonged PT values and normal APTT values. These data and our previous reports suggest that heterozygous patients with dysfibrinogenemia or fibrinogen deficiency do not demonstrate markedly prolonged PT and APTT values, differing from patients with afibrinogenemia.

In vitro expression of beta-thalassaemia gene (IVS1-1G>C) reveals complete inactivation of the normal 5' splice site and alternative aberrant RNA splicing.

We previously reported a case of heterozygous beta-thalassaemia with IVS1-1G > C substitution in the beta-globin gene and a non-detectable level of mutant mRNA in the patient's reticulocytes. The purpose of this study was to determine whether the transcription and RNA splicing and processing of the mutant gene occurred. We analysed the expression of the mRNA encoded by the cloned mutant gene in COS-1 cells by reverse transcription-polymerase chain reaction followed by agarose gel electrophoresis and nucleotide sequencing. The G > C mutation completely inactivated the normal 5' splice site and resulted in the activation of two cryptic 5' splice sites, located 16 and 38 nt upstream of the normal site. The usage of these two cryptic sites accords with the findings of reports on IVS1-1G > A or IVS1-1G > C substitution of exon 1 of the beta-globin gene. Additional experiments that involved transfection of equal amounts of both normal and mutant vectors into COS-1 cells indicated the presence of mutant mRNAs. In conclusion, the beta-thalassaemia gene (IVS1-1G > C) was expressed in transfected cells, but showed aberrant RNA splicing. Further studies will be required to clarify the molecular mechanism that results in severe reduction in the mutant mRNA level in vivo.

[Two cases of classical polyarteritis nodosa associated with MPO-ANCA].

Case 1: An 82 year-old female had a drop-foot, livedo reticularis and paresthesia. MPO-ANCA was positive. Because of developing renal dysfunction, she underwent steroid pulse therapy and dialysis, but died from the complication of congestive heart failure. Autopsy revealed necrotizing arteritis in the interlobular arteries and the arcuate arteries of the kidneys. Crescent formation was not found in the glomeruli. Case 2: A 49-year-old male had a drop foot and numbness. MPO-ANCA was positive. Because of developing renal failure, he underwent dialysis and steroid pulse therapy. MPO-ANCA became negative. Nevertheless, he developed perforation of multiple ulcers in the small intestine and died. Pathohistology revealed arteritis in medium-size arteries at the branch level of the mesenteric arteries. Since the presented two cases showed rapidly and irreversibly aggravated renal function with positive MPO-ANCA, MPA was suspected. However, pathological findings from the autopsy and operation and not the kidney biopsy supported the diagnosis of polyarteritis nodosa (PN). Vasculitis in the arterioles was absent. MPO-ANCA is not specific, and it can be detected in not only MPA, but also PN. The clinical course and pathological findings must be considered to differentiate PN from MPA.

Alcoholic ketoacidosis associated with multiple complications: report of 3 cases.

We report 3 patients with alcoholic ketoacidosis (AKA). All had a history of excessive intake and abrupt termination of alcohol. They showed tachypnea, tachycardia, abdominal tenderness, and epigastralgia. Metabolic acidosis with an increased anion gap, decreased PaCO2 and ketonemia were present. One patient whose ratio of 3-hydroxybutyric acid to acetoacetic acid was 4.0 was associated with diabetic ketoacidosis. All patients were successfully hydrated with electrolyte, glucose and thiamine. Complications such as liver dysfunction, lactic acidosis, acute pancreatitis, Wernicke's encephalopathy, rhabdomyolysis and heart failure were present. Attention should be paid to multiple complications in the treatment of AKA.

Characterization of factor XII Tenri, a rare CRM-negative factor XII deficiency.

Factor XII Tenri (Y34C), a rare cross-reacting material (CRM)-negative factor XII deficiency, was identified in a 71-yr-old Japanese woman with angina pectoris. In the patient's plasma, factor XII activity and antigen levels were only 1.6% and 5.0%, respectively, of those seen in a normal subject. Immunoblot analysis showed that the secreted factor XII Tenri existed not only as a monomer (76 kDa), but also in complexes with apparent molecular weights of approximately 115, 140, 190, 215, and 225 kDa. After reduction with 2-mercaptoethanol, the factor XII Tenri contained in the complexes was completely converted to monomeric form on immunoblot patterns. It appeared that some of the secreted factor XII Tenri formed several types of disulfide-linked complexes, including a factor XII-alpha1-microglobulin complex, through a newly generated Cys residue. The monomeric form of factor XII Tenri, like normal factor XII, was degraded into 2 major fragments with molecular weights of approximately 45 kDa and 30 kDa following mixing with activated partial-thromboplastin-time measuring reagent (cephalin and ellagic acid), whereas the factor XII Tenri that formed the complexes was not. This indicates that the factor XII Tenri present in disulfide-linked complexes with other proteins (and itself) is not converted to active forms, suggesting that attached proteins obstruct or delay the activation of factor XII via an inhibition of its binding to a negatively charged surface in vitro.

[Two autopsy cases of ANCA-associated glomerulonephritis manifested after contrast medium use].

[Case 1]: An 81-year old man was referred to our hospital with dyspnea and bloody sputum. Computed tomography with contrast medium for the evaluation of metastasis of urinary bladder carcinoma had been performed 4 months previously. On admission, his serum creatinine and potassium were 15.3 mg/dl and 6.9 mEq/l, respectively. His chest X ray revealed cardiomegaly, butterfly shadow and interstitial change, indicating congestive heart failure and interstitial pneumonia. His electrocardiogram showed that he was on the brink of cardiac arrest due to hyperkalemia. Mechanical ventilation and hemodialysis were initiated. Myeloperoxidase anti-neutrophil cytoplasmic antibody (MPO-ANCA) was highly positive(321 EU), leading to the diagnosis of MPO-ANCA-associated rapidly progressive glomerulonephritis (RPGN) with interstitial pneumonia. Treatment with pulse methylprednisolone was not effective and he died. Autopsy findings showed crescentic glomerulonephritis, alveolar hemorrhage and interstitial pneumonia with honeycomb formation. [Case 2]: A 73-year old man was referred to our hospital with rapid deterioration of his renal function. He had received a cardiac catheter examination 3 weeks previously. On admission, his serum creatinine was 4.5 mg/dl. His chest X ray showed cardiomegaly and interstitial change. Renal biopsy findings showed crescentic formation in the glomeruli. Moreover, MPO-ANCA was 494 EU, leading to the diagnosis of MPO-ANCA-associated RPGN with interstitial pneumonia. Treatment with pulse methylprednisolone and cyclophosphamide was not effective and he died. Autopsy findings revealed crescentic glomerulonephritis and interstitial pneumonia with honeycomb formation. Here we described two cases of ANCA-associated RPGN complicated by microscopic polyantitis and interstitial pneumonia after the use of contrast medium. The relation between ANCA-associated RPGN and the contrast medium was unclear. However, in the case of rapid deterioration of renal function, MPO-ANCA should be measured even after the use of contrast medium. The complication of lung diseases, especially interstitial pneumonia, should be investigated simultaneously.

Novel beta-thalassemia trait (IVS I-1 G-->C) in a Japanese family.

A rare beta-thalassemia mutation at the splicing junction [namely, G-->C in intervening sequence (IVS) I-1] was found in a Japanese family. The proband and his mother were heterozygous for the mutation. Analysis of mRNA extracted from the reticulocyte-rich fraction obtained from the proband's mother revealed that the mutant beta-globin gene did not produce any detectable, stable mRNA including exon 1 and exon 2, since the polymorphism in exon 1 on her mutant gene was not detected in the RT-PCR products.