Comprehensive targeted next-generation sequencing in patients with slow-flow vascular malformations.

Recent studies have shown that the PI3K signaling pathway plays an important role in the pathogenesis of slow-flow vascular malformations (SFVMs). Analysis of genetic mutations has advanced our understanding of the mechanisms involved in SFVM pathogenesis and may identify new therapeutic targets. We screened for somatic variants in a cohort of patients with SFVMs using targeted next-generation sequencing. Targeted next-generation sequencing of 29 candidate genes associated with vascular anomalies or with the PI3K signaling pathway was performed on affected tissues from patients with SFVMs. Fifty-nine patients with SFVMs (venous malformations n = 21, lymphatic malformations n = 27, lymphatic venous malformations n = 1, and Klippel-Trenaunay syndrome n = 10) were included in the study. TEK and PIK3CA were the most commonly mutated genes in the study. We detected eight TEK pathogenic variants in 10 samples (16.9%) and three PIK3CA pathogenic variants in 28 samples (47.5%). In total, 37 of 59 patients (62.7%) with SFVMs harbored pathogenic variants in these three genes involved in the PI3K signaling pathway. Inhibitors of this pathway may prove useful as molecular targeted therapies for SFVMs.

CXCR3high CD8+ T cells with naïve phenotype and high capacity for IFN-γ production are generated during homeostatic T-cell proliferation.

Naïve phenotype (NP) T cells spontaneously initiate homeostatic proliferation (HP) as T-cell output is reduced because of physiologic thymic involution with age. However, the effects of sustained HP on overall immune function are poorly understood. We demonstrated that the NP CD8+ T cell population in adult thymectomized mice showing accelerated HP has an increased capacity for TCR-mediated interferon-γ and tumor necrosis factor α production, which is attributed to an increase in CXCR3+ cells in the NP CD8+ T cell population. The CXCR3+ NP CD8+ T cells developed during persistent HP with a slow cell division rate, but rarely during robust antigen-driven proliferation with a fast cell division rate. In ontogeny, the proportions of CXCR3+ cells in the NP CD8+ T cell population showed a biphasic profile, which was high at the newborn and aged stages. Upon transfer, CXCR3+ NP CD8+ T cells, but not CXCR3- NP CD8+ T cells, potently enhanced Th17-mediated inflammatory tissue reactions in vivo. Furthermore, CXCR3high NP CD8+ T cells with similar features were also detected at variable levels in healthy human blood. These results suggest that CXCR3+ NP CD8+ T cells generated during physiological HP significantly impact overall immunity at the immunologically vulnerable neonatal and aged stages.

Physiologic Thymic Involution Underlies Age-Dependent Accumulation of Senescence-Associated CD4+ T Cells.

Immune aging may underlie various aging-related disorders, including diminished resistance to infection, chronic inflammatory disorders, and autoimmunity. PD-1+ and CD153+ CD44high CD4+ T cells with features of cellular senescence, termed senescence-associated T (SA-T) cells, increasingly accumulate with age and may play a role in the immune aging phenotype. In this article, we demonstrate that, compared with young mice, the aged mouse environment is highly permissive for spontaneous proliferation of transferred naive CD4+ T cells, and it drives their transition to PD-1+ and CD153+ CD44high CD4+ T cells after extensive cell divisions. CD4+ T cells with essentially the same features as SA-T cells in aged mice are also generated from naive CD4+ T cells after extensive cell divisions under severe T-lymphopenic conditions by gamma irradiation or in developmental T cell defect, often in association with spontaneous germinal centers, as seen in aged mice. The increase in SA-T cells is significantly enhanced after thymectomy at the young adult stage, along with accelerated T cell homeostatic proliferation, whereas embryonic thymus implantation in the late adult stage markedly restricts the homeostatic proliferation of naive CD4+ T cells in the host and delays the increase in SA-T cells. Our results suggest that reduced T cell output due to physiologic thymic involution underlies the age-dependent accumulation of SA-T cells as a result of increasing homeostatic proliferation of naive CD4+ T cells. SA-T cells may provide a suitable biomarker of immune aging, as well as a potential target for controlling aging-related disorders.

Serum IgA level, monocyte count, and international prognostic index are independently associated with overall survival in patients with HTLV-I-negative nodal peripheral T cell lymphoma.

Peripheral T cell lymphomas (PTCL) account for 10-15 % of non-Hodgkin's lymphomas and are associated with poor prognosis. Although many prognostic factors for PTCL have been proposed, the heterogeneity of PTCL seems to be an obstacle in the establishment of clinically useful prognostic system, such as the International Prognostic Index (IPI) in diffuse large B cell lymphoma. PTCL with nodal manifestation include the HTLV-I-negative histologic subtypes of PTCL not otherwise specified (PTCL-NOS), angioimmunoblastic T cell lymphoma (AITL), and anaplastic large cell lymphoma (ALCL). As PTCL-NOS encompasses a group of similar tumors and mostly shares their clinical pictures, we retrospectively analyzed clinical data from 77 patients diagnosed with ALCL, AITL, and PTCL-NOS at Kobe City Medical Center General Hospital from May 1994 to February 2012 to identify the prognostic factor for nodal PTCL. The median age of patients was 64 years, ranging from 23 to 83 years. With a median follow-up of 50 months, 5-year overall survival (OS) was 43 %. Multivariate analysis identified high-risk IPI (hazard ratio (HR), 4.04; P = 0.015), absolute monocyte count > 0.8 × 10(9)/L (HR, 3.44; P = 0.001), and serum concentration of IgA > 410 mg/dL (HR, 2.31; P = 0.013) as poor prognostic factors for OS. Thus, we have identified novel prognostic factors of monocyte count and serum IgA level for nodal PTCL. Although conventional prognostic models mainly reflect both tumor characteristics and host factors, the present model indicates the importance of host immune response as the unfavorable prognosis.

The Prospective Natural History Study of Patients with Intractable Venous Malformation and Klippel-Trenaunay Syndrome to Guide Designing a Proof-of-Concept Clinical Trial for Novel Therapeutic Intervention.

Background: The natural history of venous malformation (VM) and Klippel-Trenaunay Syndrome (KTS) has not been quantitatively studied. To obtain benchmarks to guide designing clinical trials to assess safety and efficacy of novel drug candidates, the clinical course of the patients was followed for 6 months. Methods and Results: This is a multicenter prospective observational study evaluating the change rate in lesion volume from baseline with magnetic resonance images, as the primary endpoint. In addition, disease severities, performance status (PS), pain visual analog scale (VAS) score, quality of life (QoL), infections, and coagulation markers were also evaluated. Thirty-four patients (VM = 17, KTS = 17, 1-53 of age; median 15.9 years) with measurable lesion volume were analyzed. There was no statistically significant difference in the lesion volume between baseline and day 180, and the mean change rate (standard deviation) was 1.06 (0.28). There were no baseline characteristics that affected the change in lesion volume over 6 months. However, there were patients who showed more than 20% volume change and it was suggested that the lesion volume was largely impacted by local infection. There were no statistically significant changes in pain VAS score, severity, PS, QoL score, D-dimer, and platelet count over 6 months within all patients analyzed. Conclusion: The results showed the representative natural course of VM and KTS for a 6-month period with objective change of lesion volume and other factors, suggesting that it is scientifically reasonable to conduct a Phase 2 proof-of-concept study without a placebo arm, using the results of this study as the control. Clinical Trial Registration: NCT04285723, NCT04589650.

Algorithm for reconstruction of composite cranial defects using the fascial component of free anterolateral thigh flaps.

BACKGROUND: In case of composite cranial defect including the dura mater, the cranial bone, and the scalp, the fascial component of the anterolateral thigh flap can be used for dural reconstruction. However, the advantages and applications of the fascial component depending on the type of defect have not been thoroughly discussed. We made the algorithm for reconstruction of composite cranial defects using the fascial component of free anterolateral thigh flaps. PATIENTS AND METHODS: Six cases of composite cranial defects were reconstructed using free anterolateral thigh flaps with the fascial component. The type of method used was classified into 3 types. Type 1 involves separating the fascia from the flap completely and using it as a nonvascularized component. In type 2, the fascia is not separated from the flap and is instead used as a vascularized component. Type 3 involves separating the vascularized adipofascial component from the skin paddle and using it as a chimeric pattern flap. The algorithm for determining the type of fascial component is applied depending on the condition of the defect. RESULTS: All flaps were transferred successfully in every case. In 4 cases, the type 1 method was used. The type 2 and 3 methods were used in 1 case each. Cranial bone reconstruction was performed in 3 cases. There were no major complications after the procedures. CONCLUSIONS: The fascial component is useful for dural reconstruction. The type of fascial component used is selected depending on the condition of the defect.

Dermatopontin interacts with fibronectin, promotes fibronectin fibril formation, and enhances cell adhesion.

We report that dermatopontin (DP), an abundant dermal extracellular matrix protein, is found in the fibrin clot and in the wound fluid, which comprise the provisional matrix at the initial stage of wound healing. DP was also found in the serum but at a lower concentration than that in wound fluid. DP co-localized with both fibrin and fibronectin on fibrin fibers and interacted with both proteins. Both normal fibroblast and HT1080 cell adhesion to the fibrin-fibronectin matrix were dose-dependently enhanced by DP, and the adhesion was mediated by α5ß1 integrin. The cytoskeleton was more organized in the cells that adhered to the fibrin-fibronectin-DP complex. When incubated with DP, fibronectin formed an insoluble complex of fibronectin fibrils as visualized by electron microscopy. The interacting sites of fibronectin with DP were the first, thirteenth, and fourteenth type III repeats (III(1), III(13), and III(14)), with III(13) and III(14) assumed to be the major sites. The interaction between III(2-3) and III(12-14) was inhibited by DP, whereas the interaction between I(1-5) and III(12-14) was specifically and strongly enhanced by DP. Because the interaction between III(2-3) and III(12-14) is involved in forming a globular conformation of fibronectin, and that between I(1-5) and III(12-14) is required for forming fibronectin fibrils, DP promotes fibronectin fibril formation probably by changing the fibronectin conformation. These results suggest that DP has an accelerating role in fibroblast cell adhesion to the provisional matrix in the initial stage of wound healing.

Asynchronous osteoradionecrosis of the mandible treated with sequential fibula osteoseptocutaneous flaps: a report of two cases.

Osteroradionecrosis of the mandible is one of the most serious complications of radiotherapy in head and neck cancer. Once osteoradionecrosis of the mandible has been established, conservative therapy is often useless. Two cases of asynchronous bilateral osteoradionecrosis of the mandible are presented. In both cases, successful reconstruction was performed with 2 free fibula osteoseptocutaneous flap transfers from both legs. We believe that adequate debridement, with removal of dead or hypovascular bone and surrounding soft tissue, followed by vascularized composite bone grafts, is the key for a successful outcome. In our cases, the oral function after 2 reconstructive surgeries with the fibula osteoseptocutaneous flap was acceptable. Furthermore, the patient could walk without difficulty.

[Fatal hepatic failure due to AL amyloidosis in a patient with multiple myeloma].

Although about 10 to 15% of patients with multiple myeloma (MM) develop AL amyloidosis, liver-restricted fatal amyloidosis is rare. We encountered such an MM patient. A 73-year-old female without a history of carpal tunnel syndrome was diagnosed with IgG-κ MM (Stage I by Durie & Salmon) in January, 2005. Because MM was exacerbated to Stage III in May, 2007, VAD (vincristine, adriamycin, dexamethasone) chemotherapy was performed with minor response, despite 3 courses of this regimen. Three courses of salvage chemotherapy (cyclophosphamide+melphalan; CM) were then performed with near partial response. In March, 2008, just before the 4th cycle of CM chemotherapy, she was slightly icteric with elevated biliary tract enzymes; therefore, treatment was switched to oral cyclophosphamide and prednisolone. At this time, she did not have macroglossia, skin eruption, gastrointestinal dysfunction, or bleeding. Echocardiography was also non-specific. One month later, she developed a marked bleeding tendency and leg edema. Laboratory tests showed a severe deterioration in liver function. In the middle of May, 2008, she progressed to hepatic coma and died of intracranial hemorrhage several days later. Autopsy showed that the liver was almost substituted by AL amyloid substance.

Allogeneic stem cell transplantation for trisomy 8-positive myelodysplastic syndrome or myelodysplastic/myeloproliferative disease with refractory Behçet's disease: Case report and the review of literature.

We had two cases of trisomy 8-positive myelodysplastic syndrome (MDS) with incomplete Behçet's disease (BD) in which the remissions of both diseases were maintained by allogeneic stem cell transplantation (allo-SCT). Among MDS with BD patients, sometimes it is difficult to control the symptoms of BD with standard therapies such as corticosteroids and tumor necrosis factor (TNF) inhibitors. Although there should be careful consideration regarding indications for transplantation, our two cases, in which refractory BD was completely controlled by allo-SCT, suggest that allo-SCT can be one of the treatment options for higher-risk MDS with BD patients.

Conservative surgical treatment for early-stage vulvar malignant melanoma.

PURPOSE: To investigate the efficacy of wide local excision as a surgical treatment for early-stage vulvar melanomas. METHODS: Wide local excision with or without lymph node dissection was performed in three patients with stage I vulvar melanoma (American Joint Committee on Cancer classification, 1992). RESULTS: All three patients were successfully treated by wide local excision. There was no evidence of recurrence at long-term follow-up in any of the patients. CONCLUSIONS: Wide local excision with adequate tumor-free margins should be considered the treatment of choice for early-stage vulvar melanomas.

Sural perforator flap: assessment of the posterior calf region as donor site for a free fasciocutaneous flap.

Three kinds of free fasciocutaneous flap from the posterior calf region have been described in the literature: the medial sural perforator flap, the lateral sural perforator flap, and the traditional posterior calf fasciocutaneous flap that is supplied by superficial cutaneous vessels. Moreover, it has been reported that superficial cutaneous vessels are of a suitable size for microanastomosis when deep musculocutaneous perforators are absent or relatively tiny. To establish a safe technique for free fasciocutaneous flap elevation from the posterior calf region, we examined the number and location of the musculocutaneous perforators and the size of superficial cutaneous vessels at their origin from the popliteal artery in six formalinized cadavers. We found that all legs had at least one perforator either from the medial sural artery or the lateral sural artery. By contrast, we failed to find superficial cutaneous vessels of suitable size for microanastomosis in three legs, and there was no significant inverse relationship between the diameter of the superficial cutaneous artery and the number of musculocutaneous perforators. Our results suggest that the medial sural perforator flap and the lateral sural perforator flap might be the surgeon's first and second choice, respectively. The traditional posterior calf fasciocutaneous flap should be the third choice because our study suggests that its availability is doubtful. Another site is recommended, when preoperative Doppler study suggests that the existence of musculocutaneous perforator is in doubt. Two clinical cases, with a medial sural perforator flap and a lateral sural perforator flap, respectively, are presented.

Unusual regioselective mercuration of metalloporphyrins and its potential applications.

The reaction of Hg(CF3CO2)2 with metalloporphyrins produces mercurated porphyrins regioselectively, the reaction, surprisingly occurring at the most hindered betaB-position; this behavior is in marked contrast to the usual electrophilic substitution reactions of porphyrins, whose reactions produce meso-substituted porphyrins; the obtained mercurated porphyrins are active to transition metal-catalyzed coupling reactions, such as the Mizoroki-Heck reaction.

Artepillin C, a Typical Brazilian Propolis-Derived Component, Induces Brown-Like Adipocyte Formation in C3H10T1/2 Cells, Primary Inguinal White Adipose Tissue-Derived Adipocytes, and Mice.

Induction of brown-like adipocytes (beige/brite cells) in white adipose tissue (WAT) suggests a new approach for preventing and treating obesity via induction of thermogenesis associated with uncoupling protein 1 (UCP1). However, whether diet-derived factors can directly induce browning of white adipocytes has not been well established. In addition, the underlying mechanism of induction of brown-like adipocytes by diet-derived factors has been unclear. Here, we demonstrate that artepillin C (ArtC), which is a typical Brazilian propolis-derived component, significantly induces brown-like adipocytes in murine C3H10T1/2 cells and primary inguinal WAT (iWAT)-derived adipocytes. This significant induction is due to activation of peroxisome proliferator-activated receptor γ and stabilization of PRD1-BF-1-RIZ1 homologous domain-containing protein-16 (PRDM16). Furthermore, the oral administration of ArtC (10 mg/kg) for 4 weeks significantly induced brown-like adipocytes accompanied by significant expression of UCP1 and PRDM16 proteins in iWAT of mice, and was independent of the ß3-adrenergic signaling pathway via the sympathetic nervous system. These findings may provide insight into browning of white adipocytes including the molecular mechanism mediated by dietary factors and demonstrate that ArtC has a novel biological function with regard to increasing energy expenditure by browning of white adipocytes.

A case of Werner syndrome with three primary lesions of malignant melanoma.

Three primary lesions of malignant melanoma developed in a 44-year-old Japanese woman with Werner syndrome. One lesion was on the right large pudental lip and the others in distinct locations on her left sole. After the wide local excision of these tumors, the wound of the large pudental lip was sutured, and the defects on the sole were covered with skin grafts. After one course of chemotherapy consisting of dacarbazine, nimustine, vincristine sulfate and local injection of Interferon beta were performed, severe myelosupression occurred and continued for two months. Defective production of WRN protein was confirmed by Western blotting, although the three representative mutations in Japanese patients, mutations 1, 4 and 6, which include over 90% of the Japanese patients, were not detected. We also reviewed 26 cases of malignant melanoma associated with Werner syndrome (WS), including ours.

HIV-related NK/T-cell lymphoma in the brain relapsed during intensive chemotherapy but regressed after chemotherapy discontinuation: the importance of maintaining cellular immunity.

This study reports a case of human immunodeficiency virus (HIV)-related natural killer/T-cell lymphoma with an unexpected clinical course. The lymphoma cells were positive for Epstein-Barr virus and the primary nodal lesions regressed after chemotherapy and combined antiretroviral therapy (c-ART); however, brain metastasis progressed along with a reduction in the CD8+ T-cell count. Chemotherapy was discontinued and the patient was treated with c-ART alone, resulting in regression of the brain lesions and recovery of the CD8+ T-cell count. This case highlights the importance of maintaining anti-tumor immunity in patients with HIV-related lymphoma.

Dermatopontin regulates fibrin formation and its biological activity.

Dermatopontin (DP) is a small extracellular matrix component in the dermis. Fibrin is a major component of a provisional matrix that is formed just after wounding. Previously, we found that DP was present in the provisional matrix, and it interacted with fibrin. Here, we examined the role of DP on fibrin function. DP interacted with both the fibrin monomer and fibrils, and was incorporated into the fibrils during fibrin formation. A DP sequence, PHGQVVVAVRS, was identified as a fibrin-binding site, and a globular D domain of fibrin was the binding site for DP. DP accelerated fibrin fibril formation into structurally modified fibrils. Fibrin fibrils formed in the presence of DP enhanced both endothelial cell attachment and cell spreading. The attached cells developed a more organized cytoskeleton when compared with those that attached to fibrin fibrils only. The main receptor for cell adhesion was identified as αvß3 integrin, and a cooperating receptor was a ß1-containing integrin species, probably α5ß1 integrin. These results indicate that DP can modify certain biological functions of fibrin, and thus a another function of this extracellular matrix protein was revealed. In addition, the fibrin-DP complex might become useful for developing an improved artificial matrix for improving wound healing.

Functional peptide of dermatopontin produces fibrinogen fibrils and modifies its biological activity.

BACKGROUND: Dermatopontin (DP), a small extracellular matrix protein, interacts with both fibrinogen and fibrin. DP accelerates fibrin fibril formation and enhances cell adhesion to fibrin fibrils but DP does not influence fibrinogen fibril formation. We have previously demonstrated that DP-4 (PHGQVVVAVRS) is a functional dermatopontin peptide (Wu et al., 2014). OBJECTIVE: Identification of biological functions of DP-4. METHODS: Protein-protein interactions were examined by solid-phase assay. The kinetics of fibrinogen/fibrin polymer formation was monitored by turbidity change, SDS-PAGE, and electron microscopy. A cell adhesion assay was performed using human umbilical vein endothelial cells. RESULTS: Although DP promoted fibrin formation, the DP-4 peptide promoted fibrinogen polymerization but did not apparently affect fibrin formation. The polymerized fibrinogen formed straight solid fibrils comparable to the normally formed fibrin fibrils. A minimum functional sequence of the DP-4 peptide was determined to be VVVAVRS. An αC domain in fibrinogen was involved in the fibril formation. Fibrinogen fibrils made by DP-4 enhanced endothelial cell adhesion and spreading in a dose-dependent manner. This cell adhesion was inhibited by heparin and by anti-αvß3 and ß1 integrin antibodies. CONCLUSION: DP-4 did not reproduce the full functional biological activities of DP with fibrin but DP-4 did promote fibrinogen fibril formation. The fibrinogen fibrils produced by DP-4 are useful as a novel synthetic biomaterial for therapeutic applications.