The Versican G1 Fragment and Serum-Derived Hyaluronan-Associated Proteins Interact and Form a Complex in Granulation Tissue of Pressure Ulcers.

The hyaluronan (HA)-rich extracellular matrix plays dynamic roles during tissue remodeling. Versican and serum-derived HA-associated protein (SHAP), corresponding to the heavy chains of inter-α-trypsin inhibitor, are major HA-binding molecules in remodeling processes, such as wound healing. Versican G1-domain fragment (VG1F) is generated by proteolysis and is present in either remodeling tissues or the mature dermis. However, the macrocomplex formation of VG1F has not been clarified. Therefore, we examined the VG1F-containing macrocomplex in pressure ulcers characterized by chronic refractory wounds. VG1F colocalized with SHAP-HA in specific regions of the granulation tissue but not with fibrillin-1. A unique VG1F-SHAP-HA complex was isolated from granulation tissues using gel filtration chromatography and subsequent cesium chloride-gradient ultracentrifugation under dissociating conditions. Consistent with this molecular composition, recombinant versican G1, but not versican G3, interacted with the two heavy chains of inter-α-trypsin inhibitor. The addition of recombinant VG1 in fibroblast cultures enhanced VG1F-SHAP-HA complex deposition in the pericellular extracellular matrix. Comparison with other VG1F-containing macrocomplexes, including dermal VG1F aggregates, versican-bound microfibrils, and intact versican, highlighted the tissue-specific organization of HA-rich extracellular matrix formation containing versican and SHAP. The VG1F-SHAP-HA complex was specifically detected in the edematous granulation tissues of human pressure ulcers and in inflamed stages in a mouse model of moist would healing, suggesting that the complex provides an HA-rich matrix suitable for inflammatory reactions.

Polypoid granulation tissue in pressure ulcers: Significance of describing individual ulcers.

Granulation tissue formation is required for the healing of deep pressure ulcers. The wound healing process is often delayed at the stage of granulation tissue formation. The pathogenesis of pressure ulcers showing granulation tissue may vary; however, no terminology has been defined to describe existing ulcers. Thus, we previously defined terminology for granulation tissue to describe individual ulcers. Based on these terms, we retrospectively evaluated the findings of deep pressure ulcers. In particular, we focused on polypoid granular tissue, a unique morphological feature. Polypoid granulation tissues were frequently observed in pressure ulcers over the sacrum compared with those over the foot. Chronological observation of a few cases indicated that external forces from specific directions during the healing process caused the development of polypoid granulation tissue. In addition, most pressure ulcers showing polypoid granulation tissue exhibited a trench-like appearance in individual wounds. Based on these observations, polypoid granulation tissue may generate from specific external forces, which lead to wound deformity. Morphological findings in an individual wound may be useful to predict the mechanical factors on existing pressure ulcers.

Evaluation of Drug-Induced Photosensitivity Using the Japanese Adverse Drug Event Report (JADER) Database.

Drug-induced photosensitivity (DIP) refers to the development of cutaneous disorders caused by the combined effects of different medications and light. The aim of this study was to obtain new information on drug risk comparisons and on DIP onset profiles, including seasonal variations, for clinically used prescription drugs. We analyzed reports of DIP recorded in the Japanese Adverse Drug Event Report (JADER) database using a reporting odds ratio (ROR). We also used Weibull proportional-hazards models for each drug to examine the patterns of DIP. The JADER database contains 430587 reports recorded from April 2004 to November 2016. The ROR values (95% confidence interval [CI]) of losartan/hydrochlorothiazide (HCTZ), valsartan/HCTZ, and ketoprofen were 214.5 (162.1-283.9), 104.7 (66.3-165.5), and 117.9 (76.6-181.5), respectively. For time-to-onset analysis, the median durations (interquartile range) for DIP caused by losartan/HCTZ, valsartan/HCTZ, and ketoprofen were 56 (41-78), 49 (38-88), and 8 (2-14) days, respectively. The lower limit of the 95% CI for the Weibull shape parameter ß value for losartan/HCTZ was greater than 1. More than half of the reports of DIP onset following the administration of ketoprofen were recorded within 10 d of treatment initiation. The seasonal variation of photosensitivity reactions was shown to follow an annual sinusoidal pattern with a peak in April and May. Based on the results, losartan/HCTZ, valsartan/HCTZ, and ketoprofen should be used carefully in clinical practice to avoid DIP.

Orally active prostacyclin analogue beraprost sodium in patients with chronic kidney disease: a randomized, double-blind, placebo-controlled, phase II dose finding trial.

BACKGROUND: Evidence increasingly points to the importance of chronic hypoxia in the tubulointerstitium as a final common pathway to end-stage renal disease (ESRD). Beraprost sodium (BPS) is an orally active prostacyclin (PGI2) analogue demonstrating prevention of the progression of chronic kidney disease (CKD) in various animal models by maintaining renal blood flow and attenuating renal ischemic condition. METHODS: This multicenter, randomized, double-blind, placebo-controlled, phase II trial was designed to determine the recommended dose of the sustained-release form of BPS (TRK-100STP 120 µg/day or 240 µg/day) in Japanese patients with CKD. TRK-100STP was administered to a total of 112 patients. The primary efficacy endpoint was the difference in the slope of the regression line of reciprocal of serum creatinine (1/SCr) over time, obtained by the least-squares method. RESULTS: Regarding the primary endpoint, statistical superiority of TRK-100STP 240 µg over placebo was not confirmed and so a recommended dose was not determined. Compared to placebo, however, the slope of regression line of 1/SCr, elevation of SCr and serum cystatin C during the treatment period revealed greater improvement at 120 µg, at both doses, and at 240 µg, respectively. In terms of safety, both TRK-100STP treatment groups were well tolerated. CONCLUSIONS: Although the study failed to meet the primary endpoint, results indicate that TRK-100STP may potentially prevent the decline in renal function of CKD patients independent of blood pressure or urinary protein levels. TRIAL REGISTRATION: NCT02480751. June 21, 2015.

A multinational phase IIb/III trial of beraprost sodium, an orally active prostacyclin analogue, in patients with primary glomerular disease or nephrosclerosis (CASSIOPEIR trial), rationale and study design.

BACKGROUND: Chronic kidney disease (CKD) is public health concern even in Asian countries. TRK-100STP, a sustained release tablet of an orally-active prostacyclin analogue, beraprost sodium, is suggested to suppress worsening of some parameters of renal filtration function, containing in slope of 1/serum creatinine (1/SCr) vs. time in a phase II clinical trial. METHODS/DESIGN: We describe the design of the phase IIb/III trial of TRK-100STP, CASSIOPEIR (CRF Asian Study with Oral PGI2 derivative for Evaluating Improvement of Renal function) conducted in approximately 160 centers in China, Hong Kong, Japan, Malaysia, Republic of Korea, Taiwan, and Thailand. A total of 750 patients (n = 250 per group) with primary glomerular disease or nephrosclerosis were planned to be enrolled. Patients were randomized into one of three treatment groups in a double-bind, placebo-controlled manner: TRK-100STP 60 µg b.i.d.; TRK-100STP 120 µg b.i.d.; or placebo. The treatment period is planned to last 2 to 4 years. The primary efficacy endpoint is the renal composite endpoint including doubling of SCr and ESRD (dialysis induction, renal transplantation, or increase in SCr to ≥ 6.0 mg/dL). DISCUSSION: This trial targeting CKD patients is designed to (a) demonstrate the superiority of TRK-100STP over placebo using renal composite endpoints, (b) determine the recommended clinical dose, and (c) assess the safety of TRK-100STP in this population and setting. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01090037.

In vitro analysis of the functional effects of an NLRP3 G809S variant with the co-existence of MEFV haplotype variants in atypical autoinflammatory syndrome.

PURPOSE: Hereditary periodic fever syndromes have been considered monogenic diseases. However, some recent reports have described patients with co-existence of recurrent fever responsible genes. This study assessed whether a rare variant, found in Japanese children showing atypical autoinflammatory syndrome, located in the leucine-rich repeat domain of Nod-like receptor family, pyrin domain containing 3 (NLRP3) with co-existence of Mediterranean fever (MEFV) haplotype variants may contribute to a proinflammatory phenotype using a systematic approach. METHODS: Cytokine production in serum or from peripheral blood monocytes was measured by ELISA. DNA sequence analysis of genes including NLRP3, MEFV, mevalonate kinase (MVK), and tumor necrosis factor receptor superfamily, member 1A (TNFRSF1A) were performed on patient samples. In vitro functional assays determined the effects of the NLRP3 variants and pyrin using NF-κB activation and speck formation assays. RESULTS: A heterozygous genetic variant of NLRP3, G809S, was found in samples from both patients. Additionally the previously reported heterozygous MEFV variants (P369S-R408Q or E148Q-P369S-R408Q) were also detected in both patients. Serum IL-1ra and sTNFR1 levels increased in the attack phase of the disease in both patients. The production levels of IL-1ß from monocytes isolated from both cases were elevated following LPS and IFN-γ stimulation. The NLRP3 G809S variant demonstrated no increase of NF-κB activity following monosodium urate stimulation, whereas it significantly increased speck formation by interacting with apoptosis-associated speck-like protein with caspase recruitment domain. CONCLUSIONS: The phenotype of atypical autoinflammatory disease in patients could be modified by a synergistic effect with two other variants of autoinflammatory-associated genes.

Possible role of a septin, SEPT1, in spreading in squamous cell carcinoma DJM-1 cells.

We performed biochemical, histochemical and cell biological characterization of septins by focusing on SEPT1 in human skin tissues and a squamous cell carcinoma (SCC) cell line DJM-1. In immunoblotting, SEPT1, together with other septins, was detected in normal human epidermis, SCC and DJM-1. In immunohistochemical analyses, SEPT1 was detected diffusely in the cytoplasm of human epidermal cells and eccrine gland epithelial cells, and the protein level was increased in some skin tumors. In DJM-1 cells, SEPT1 together with other members of SEPT2-subgroup, SEPT4 and SEPT5, was enriched in lamellipodia and the localization was dependent on the cortical actin structure. SEPT1 distribution at lamellipodia was also observed in melanoma B16 cells. SEPT9, SEPT11 and SEPT14, in contrast, were localized along with microtubules in DJM-1 cells. In immunoprecipitation assays, SEPT1 and SEPT5 were found to form a complex in DJM-1 cells, whereas SEPT9, SEPT11 and SEPT14 formed a distinct complex with other septins including SEPT7, SEPT8 and SEPT10, in which SEPT5 was not included. When SEPT1 was silenced in DJM-1 cells, cell spreading was inhibited. These results suggest that SEPT1 may participate in cell-cell and/or cell-substrate interaction in DJM-1 and exert its function in a coordinated manner with other septins.

Sphingosine kinase 1 plays a role in the upregulation of CD44 expression through extracellular signal-regulated kinase signaling in human colon cancer cells.

Our previous study has shown that the activity and expression of sphingosine kinase (SPHK) regulated the sensitivity of human colon cancer cells to the chemotherapeutic oxaliplatin (L-OHP). In addition, the cancer stem cell marker CD44 increases cell resistance to anticancer drugs. Here, we use colon cancer cell lines to examine the relationship between SPHK1 activity and CD44 expression.CD44 expression was measured by western blotting and quantitative PCR in two human colon cancer cell lines: L-OHP-resistant RKO and L-OHP-sensitive HCT116. The regulation of CD44 by SPHK1 was examined by either blocking or overexpressing SPHK1 and by using an L-OHP-resistant HCT116 clone (HCT116-R).The levels of SPHK1, CD44, phosphorylated-Akt, and phosphorylated-extracellular signal-regulated kinase (ERK) were much higher in the RKO cells than in the HCT116 cells. The treatment of RKO cells with the SPHK inhibitor or SPHK1 silencing by RNA interference suppressed CD44 protein expression. SPHK1 and CD44 levels were much higher in HCT116-R cells compared with the parental HCT116 cells. Transfection of HCT116 cells with SPHK1 cDNA enhanced the expression of both CD44 and phosphorylated-ERK. The increase in the CD44 protein level was abolished by the inhibition of ERK phosphorylation. Treatment of RKO cells with the sphingosine-1-phosphate (S1P)2 receptor antagonist suppressed ERK phosphorylation and the expression of CD44 mRNA and protein. Exogenous stimulation with S1P increased ERK phosphorylation and CD44 protein expression in HCT116 cells, but treatment with an MEK inhibitor and S1P2 receptor antagonist blocked this effect.These findings indicate that SPHK1 and its product, S1P, contribute toward the regulation of CD44 protein expression through the ERK signaling pathway through S1P2 in human colon cancer cells.

Location-dependent depth and undermining formation of pressure ulcers.

AIM OF THE STUDY: We examined the location-specific properties of pressure ulcers, focusing on depth and undermining formation, which are often unfavorable factors for ulcer healing. METHODS: We conducted a retrospective observational study of 2 independent databases on pressure ulcers. Databases from a 200-bed hospital (database A) and a 300-bed hospital (database B) were collected during different time periods. Relationships between ulcer location, ulcer depth, and undermining formation were analyzed. All pressure ulcers were accurately diagnosed and classified according to their locations. RESULTS: A total of 282 pressure ulcers in 189 patients from database A and 232 pressure ulcers in 154 patients from database B were analyzed. It was found that pressure ulcers primarily developed over the sacrum. Ratio of stages III and IV pressure ulcers was high in pressure ulcers of the foot, ankle, and crus on the lower leg. Among the deep pressure ulcers, undermining formation was frequently observed on the greater trochanter, ilium, and sacrum. In contrast, pressure ulcers of the foot, ankle, and crus did not exhibit undermining formation. CONCLUSION: Our results revealed marked differences in pressure ulcer properties depending on their location. Factors affecting depth and undermining of pressure ulcers appear to be related to anatomical and physical properties of the bone and subcutaneous tissue.

Cell biological characterization of a multidomain adaptor protein, ArgBP2, in epithelial NMuMG cells, and identification of a novel short isoform.

ArgBP2 is a member of the SoHo (sorbin-homology) family of adaptor proteins believed to play roles in cell adhesion, cytoskeletal organization, and signaling. We show here a novel splicing isoform of ArgBP2, i.e., ArgBP2™, composed of only three SH3 (src-homology 3) domains and structurally similar to vinexinß. We then characterized the biochemical and cell biological properties of ArgBP2 to compare these with vinexin. Similar to vinexin, ArgBP2 was enriched at focal adhesions in REF52 fibroblast cells and induced anchorage-dependent extracellular signal-regulated kinase activation in NIH3T3 fibroblast cells. In epithelial NMuMG cells, immunofluorescence analyses revealed localization of ArgBP2 at tight junctions (TJs), whereas vinexin was distributed in cytoplasm as well as cell-cell boundaries. During TJ formation, recruitment of ZO-1 to TJs was followed by ArgBP2. Based on mutation analyses, a second SH3 domain was found to be important for ArgBP2 localization to the cell-cell contact sites. These data suggest some role of ArgBP2 in NMuMG cells at TJs that may be distinct from the function of vinexin.

Alternatively activated macrophages are associated with the α2AP production that occurs with the development of dermal fibrosis : The role of alternatively activated macrophages on the development of fibrosis.

BACKGROUND: Fibrotic diseases are characterized by tissue overgrowth, hardening, and/or scarring because of the excessive production, deposition, and contraction of the extracellular matrix (ECM). However, the detailed mechanisms underlying these disorders remain unclear. It was recently reported that α2-antiplasmin (α2AP) is elevated in fibrotic tissue and that it is associated with the development of fibrosis. In the present study, we examined the mechanism underlying the production of α2AP on the development of fibrosis. METHODS: To clarify the mechanism underlying the production of α2AP on the development of fibrosis, we focused on high-mobility group box 1 (HMGB1), which is associated with the development of fibrosis. The mouse model of bleomycin-induced fibrosis was used to evaluate the production of α2AP on the development of fibrosis. RESULTS: We found that HMGB1 induced the production of α2AP through receptor for advanced glycation end products (RAGE) in fibroblasts. Next, we showed that macrophage reduction by a macrophage-depleting agent, clodronate, attenuated the progression of fibrosis and the production of α2AP and HMGB1 in the bleomycin-induced mice. We also showed that IL-4-stimulated alternatively activated macrophages induced the production of HMGB1, that IL-4-stimulated alternatively activated macrophage conditioned media (CM) induced pro-fibrotic changes and α2AP production, and that the inhibition of HMGB1 and RAGE attenuated these effects in fibroblasts. Furthermore, the blockade of IL-4 signaling by IL-4Rα neutralizing antibodies attenuated the progression of fibrosis and the production of α2AP and HMGB1 in the bleomycin-induced mice. CONCLUSION: These findings suggest that alternatively activated macrophage-derived HMGB1 induced the production of α2AP through RAGE and that these effects are associated with the development of fibrosis. Our findings may provide a clinical strategy for managing fibrotic disorders.

Effects of Sustained-Release Beraprost in Patients With Primary Glomerular Disease or Nephrosclerosis: CASSIOPEIR Study Results.

TRK-100STP, a sustained-release preparation of the orally active prostacyclin analogue beraprost sodium, targets renal hypoxia. This study aimed to show the superiority of TRK-100STP over placebos in patients with chronic kidney disease (with either primary glomerular disease or nephrosclerosis) to determine the recommended dose. CASSIOPEIR (Chronic Renal Failure Asian Study with Oral PGI2 Derivative for Evaluating Improvement of Renal Function) was a randomized, double-blind, placebo-controlled study conducted at 160 sites in seven Asia-Pacific countries and regions. Eligible patients (n = 892) were randomized to TRK-100STP 120, 240 µg, or placebo for a treatment period of up to 4 years. The primary efficacy endpoint was time to first occurrence of a renal composite: doubling of serum creatinine or occurrence of end-stage renal disease. No significant differences were observed in composite endpoints between TRK-100STP and placebo (P = 0.5674). Hazard ratios (95% CI) in the TRK-100STP 120 and 240 µg vs. placebo groups were 0.98 (0.78, 1.22) and 0.91 (0.72, 1.14), respectively. The overall incidence of adverse events and adverse drug reactions was comparable between treatment arms.

Wound, pressure ulcer and burn guidelines - 6: Guidelines for the management of burns, second edition.

"Wound, pressure ulcer and burn guidelines - 6: Guidelines for the management of burns, second edition" is revised from the first edition which was published in the Japanese Journal of Dermatology in 2016. The guidelines were drafted by the Wound, Pressure Ulcer and Burn Guidelines Drafting Committee delegated by the Japanese Dermatological Association, and intend to facilitate physicians' clinical decisions in preventing, diagnosing and treating burn injury. All sections are updated by collecting documents published since the publication of the first edition. Especially, the recommendation levels of dressing materials newly covered by the Japanese national health insurance are mentioned. In addition, the clinical questions (CQ) regarding the initial treatment of electrical (CQ15) and chemical burns (CQ16), and also the use of escharotomy (CQ22), are newly created.

Wound, pressure ulcer and burn guidelines - 1: Guidelines for wounds in general, second edition.

The Japanese Dermatological Association prepared the clinical guidelines for the "Wound, pressure ulcer and burn guidelines", second edition, focusing on treatments. Among them, "Guidelines for wounds in general" is intended to provide the knowledge necessary to heal wounds, without focusing on particular disorders. It informs the basic principles of wound treatment, before explanations are provided in individual chapters of the guidelines. We updated all sections by collecting references published since the publication of the first edition. In particular, we included new wound dressings and topical medications. Additionally, we added "Question 6: How should wound-related pain be considered, and what should be done to control it?" as a new section addressing wound pain, which was not included in the first edition.

Wound, pressure ulcer and burn guidelines - 4: Guidelines for the management of connective tissue disease/vasculitis-associated skin ulcers.

The Japanese Dermatological Association prepared guidelines focused on the treatment of skin ulcers associated with connective tissue disease/vasculitis practical in clinical settings of dermatological care. Skin ulcers associated with connective tissue diseases or vasculitis occur on the background of a wide variety of diseases including, typically, systemic sclerosis but also systemic lupus erythematosus (SLE), dermatomyositis, rheumatoid arthritis (RA), various vasculitides and antiphospholipid antibody syndrome (APS). Therefore, in preparing the present guidelines, we considered diagnostic/therapeutic approaches appropriate for each of these disorders to be necessary and developed algorithms and clinical questions for systemic sclerosis, SLE, dermatomyositis, RA, vasculitis and APS.

IFN-γ Reduces Epidermal Barrier Function by Affecting Fatty Acid Composition of Ceramide in a Mouse Atopic Dermatitis Model.

IFN-γ is detected in chronic lesions of atopic dermatitis (AD); however, its specific role remains to be elucidated. An impaired stratum corneum barrier function is a hallmark of AD, and it is associated with a reduction in ceramides with long-chain fatty acids (FAs) in the stratum corneum. FA elongases, ELOVL1 and ELOVL4, are essential for the synthesis of these ceramides, together with ceramide synthase 3 (CerS3). We have previously shown that IFN-γ, but not other cytokines, induced the downregulation of these enzymes in cultured keratinocytes. Our aim was to investigate the in vivo role of IFN-γ in the lesional skin of AD by analyzing mouse dermatitis models. The local mRNA expression of IFN-γ increased in mite fecal antigen-induced AD-like dermatitis in NC/Nga mice but not in imiquimod-induced psoriasis-like dermatitis in BALB/c mice. The mRNA expression of ELOVL1 and ELOVL4 significantly decreased in AD-like dermatitis, whereas ELOVL1 increased in psoriasis-like dermatitis. The expression of CerS3 increased slightly in AD-like dermatitis, but it increased by 4.6-fold in psoriasis-like dermatitis. Consistently, the relative amount of ceramides with long-chain FAs decreased in AD-like dermatitis but not in psoriasis-like dermatitis. These results suggest that IFN-γ in the lesional skin may reduce ceramides with long-chain FAs by decreasing the expression of ELOVL. Thus, IFN-γ may contribute to the chronicity of AD by impairing barrier function.