Prolonged antibiotic treatment for infected low flow vascular malformations.

BACKGROUND: Infection in low flow malformations is difficult to diagnose and treat. Initial presentation can be followed by cycles of recurrent infection lasting several years. The optimal duration of antibiotic therapy to prevent recurrence of infection has not been established. METHODS: All cases of infection in low flow malformations at the Royal Children's Hospital over a ten-year period were reviewed. Clinical markers of infection and duration of initial antibiotic treatment were correlated with the development of recurrent episodes of infection. RESULTS: Twenty-one patients met criteria for inclusion. Nineteen were diagnosed as lymphatic malformations and two as venous malformations. The majority of patients (13 or 62%) received a prolonged course of six weeks or more of antibiotics. Eleven (52%) patients went on to have recurrent infections, but these were significantly less likely to be in those treated with a long course of antibiotics (Fisher's exact test, p=0.026). In only 12 of 21 cases could a bacterium be grown. Elevated CRP was the most consistent abnormal laboratory finding in infection. CONCLUSIONS: Longer courses of antibiotics reduce the risk of recurrent infection in low-flow vascular malformations. We recommend an antibiotic course of three months or more at the initial presentation of infection in a low flow malformation. Elevated CRP is the most sensitive test for diagnosis of infection in low-flow malformations. TYPE OF STUDY: Treatment study. LEVEL OF EVIDENCE: III.

Consensus statement for the treatment of infantile haemangiomas with propranolol.

Although most infantile haemangiomas do not require treatment due to a natural history of spontaneous involution, some require early intervention. The Australasian Vascular Anomalies Network and the Australasian Paediatric Dermatology Network have developed a consensus statement for the treatment of infantile haemangiomas with oral propranolol. Infants with haemangiomas that are life threatening, at risk of ulceration, or at risk of causing a significant functional impairment, psychological impact or physical deformity should be treated early with oral propranolol. Oral propranolol is safe and effective and in most healthy infants oral propranolol can be started in an outpatient setting.

Wound management of ulcerated haemangioma of infancy - an audit.

Haemangioma of infancy, a benign tumour of blood vessels, is the most common tumour of infancy. Ulceration, the most common complication, presents a unique wound care challenge. A retrospective audit of medical records of children with haemangioma of infancy who presented to the Royal Children's Hospital, Melbourne, Australia, between January 2000 and December 2014 was undertaken with an aim to examine wound management of ulcerated haemangioma of infancy. In total, 535 hospital medical records were identified as suitable, of which 352 were randomly selected and audited, of which 84 patients had ulcerated haemangioma of infancy, and 62 were subject to wound management. Of these, 35 were successfully managed by wound dressings, 9 were not fully healed at the time of last review, and 18 were referred for surgical excision. Patients attended an average of five outpatient visits, and the average time from presentation to documented healing was 105 days. There were a total of 225 episodes of wound dressing, for which there was a documented follow-up appointment at which healing could be assessed. Although a wide range of dressings were used, there was no clear pattern of benefit of one dressing over another. Wounds were less likely to be healed after the use of a silver-impregnated dressing. Pain was poorly documented. Clinical assessment of whether wounds were infected was of no help in planning treatment. There is considerable variability in the management of this difficult wound group, and further prospective studies are required.

Blue Rubber Bleb Nevus (BRBN) Syndrome Is Caused by Somatic TEK (TIE2) Mutations.

Blue rubber bleb nevus syndrome (Bean syndrome) is a rare, severe disorder of unknown cause, characterized by numerous cutaneous and internal venous malformations; gastrointestinal lesions are pathognomonic. We discovered somatic mutations in TEK, the gene encoding TIE2, in 15 of 17 individuals with blue rubber bleb nevus syndrome. Somatic mutations were also identified in five of six individuals with sporadically occurring multifocal venous malformations. In contrast to common unifocal venous malformation, which is most often caused by the somatic L914F TIE2 mutation, multifocal forms are predominantly caused by double (cis) mutations, that is, two somatic mutations on the same allele of the gene. Mutations are identical in all lesions from a given individual. T1105N-T1106P is recurrent in blue rubber bleb nevus, whereas Y897C-R915C is recurrent in sporadically occurring multifocal venous malformation: both cause ligand-independent activation of TIE2, and increase survival, invasion, and colony formation when expressed in human umbilical vein endothelial cells.

GAPTrap: A Simple Expression System for Pluripotent Stem Cells and Their Derivatives.

The ability to reliably express fluorescent reporters or other genes of interest is important for using human pluripotent stem cells (hPSCs) as a platform for investigating cell fates and gene function. We describe a simple expression system, designated GAPTrap (GT), in which reporter genes, including GFP, mCherry, mTagBFP2, luc2, Gluc, and lacZ are inserted into the GAPDH locus in hPSCs. Independent clones harboring variations of the GT vectors expressed remarkably consistent levels of the reporter gene. Differentiation experiments showed that reporter expression was reliably maintained in hematopoietic cells, cardiac mesoderm, definitive endoderm, and ventral midbrain dopaminergic neurons. Similarly, analysis of teratomas derived from GT-lacZ hPSCs showed that ß-galactosidase expression was maintained in a spectrum of cell types representing derivatives of the three germ layers. Thus, the GAPTrap vectors represent a robust and straightforward tagging system that enables indelible labeling of PSCs and their differentiated derivatives.

Applying Advanced Imaging Techniques to a Murine Model of Orthotopic Osteosarcoma.

INTRODUCTION: Reliable animal models are required to evaluate novel treatments for osteosarcoma. In this study, the aim was to implement advanced imaging techniques in a murine model of orthotopic osteosarcoma to improve disease modeling and the assessment of primary and metastatic disease. MATERIALS AND METHODS: Intra-tibial injection of luciferase-tagged OPGR80 murine osteosarcoma cells was performed in Balb/c nude mice. Treatment agent [pigment epithelium-derived factor (PEDF)] was delivered to the peritoneal cavity. Primary tumors and metastases were evaluated by in vivo bioluminescent assays, micro-computed tomography, [(18)F]-Fluoride-PET and [(18)F]-FDG-PET. RESULTS: [(18)F]-Fluoride-PET was more sensitive than [(18)F]-FDG-PET for detecting early disease. Both [(18)F]-Fluoride-PET and [(18)F]-FDG-PET showed progressive disease in the model, with fourfold and twofold increases in standardized uptake value (p < 0.05) by the study endpoint, respectively. In vivo bioluminescent assay showed that systemically delivered PEDF inhibited growth of primary osteosarcoma. DISCUSSION: Application of [(18)F]-Fluoride-PET and [(18)F]-FDG-PET to an established murine model of orthotopic osteosarcoma has improved the assessment of disease. The use of targeted imaging should prove beneficial for the evaluation of new approaches to osteosarcoma therapy.

Isolation of human lymphatic endothelial cells by multi-parameter fluorescence-activated cell sorting.

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34(Low)CD31(Pos)VEGFR-3(Pos)PODOPLANIN(Pos) LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

Infantile haemangiomas that failed treatment with propranolol: clinical and histopathological features.

AIM: To describe the clinical and histopathological characteristics of infantile haemangiomas that failed treatment with oral propranolol . DESIGN: This study is a case series from the vascular birthmarks clinic at Royal Children's Hospital, Melbourne. PATIENTS: The patients for this study were infants who commenced treatment with oral propranolol before 6 months of age and who were treated for at least 4 months without a satisfactory result. For histology and immunohistochemistry, tissue from the four non-responding patients who subsequently underwent surgical excision was matched with four historical controls. OUTCOME MEASURES: Based on medical record review and photographic assessments, infants were defined as having failed treatment with oral propranolol if the infantile haemangioma either continued to grow or showed 20% improvement or less. Tissue sections were examined for tissue structure, mast cells, sympathetic innervations and beta-2 adrenergic receptor expression, and the number of mast cells and beta-2 adrenergic positive cells. RESULTS: From a group of 135 infants who met the inclusion criteria, 14 infants failed propranolol treatment. Eleven of these infants had focal facial haemangiomas. No difference was seen in tissue morphology, tissue innervations, beta-2 adrenergic receptor expression, cell number or mast cell distribution, and number between non-responding and control haemangiomas. CONCLUSION: We report a treatment failure rate of 10%, which is higher than previously reported. Focal facial lesions failed to respond twice as frequently as other types of haemangioma. No histopathological reason was identified to indicate why some haemangiomas failed to respond.

Isolation of human lymphatic malformation endothelial cells, their in vitro characterization and in vivo survival in a mouse xenograft model.

Human lymphatic vascular malformations (LMs), also known as cystic hygromas or lymphangioma, consist of multiple lymphatic endothelial cell-lined lymph-containing cysts. No animal model of this disease exists. To develop a mouse xenograft model of human LM, CD34(Neg)CD31(Pos) LM lymphatic endothelial cells (LM-LEC) were isolated from surgical specimens and compared to foreskin CD34(Neg)CD31(Pos) lymphatic endothelial cells (LECs). Cells were implanted into a mouse tissue engineering model for 1, 2 and 4 weeks. In vitro LM-LECs showed increased proliferation and survival under starvation conditions (P < 0.0005 at 48 h, two-way ANOVA), increased migration (P < 0.001, two-way ANOVA) and formed fewer (P = 0.029, independent samples t test), shorter tubes (P = 0.029, independent samples t test) than foreskin LECs. In vivo LM-LECs implanted into a Matrigel™-containing mouse chamber model assembled to develop vessels with dilated cystic lumens lined with flat endothelium, morphology similar to that of clinical LMs. Human foreskin LECs failed to survive implantation. In LM-LEC implanted chambers the percent volume of podoplanin(Pos) vessels was 1.18 ± 2.24 % at 1 week, 6.34 ± 2.68 % at 2 weeks and increasing to 7.67 ± 3.60 % at 4 weeks. In conclusion, the significantly increased proliferation, migration, resistance to apoptosis and decreased tubulogenesis of LM-LECs observed in vitro is likely to account for their survival and assembly into stable LM-like structures when implanted into a mouse vascularised chamber model. This in vivo xenograft model will provide the basis of future studies of LM biology and testing of potential pharmacological interventions for patients with lymphatic malformations.

Extracellular matrix of secondary lymphoid organs impacts on B-cell fate and survival.

We describe a unique extracellular matrix (ECM) niche in the spleen, the marginal zone (MZ), characterized by the basement membrane glycoproteins, laminin α5 and agrin, that promotes formation of a specialized population of MZ B lymphocytes that respond rapidly to blood-borne antigens. Mice with reduced laminin α5 expression show reduced MZ B cells and increased numbers of newly formed (NF) transitional B cells that migrate from the bone marrow, without changes in other immune or stromal cell compartments. Transient integrin α6ß1-mediated interaction of NF B cells with laminin α5 in the MZ supports the MZ B-cell population, their long-term survival, and antibody response. Data suggest that the unique 3D structure and biochemical composition of the ECM of lymphoid organs impacts on immune cell fate.

Hypoxic conditioning enhances the angiogenic paracrine activity of human adipose-derived stem cells.

Human adipose-derived stem cells (ASCs) secrete cytokines and growth factors that can be harnessed in a paracrine fashion for promotion of angiogenesis, cell survival, and activation of endogenous stem cells. We recently showed that hypoxia is a powerful stimulus for an angiogenic activity from ASCs in vitro and here we investigate the biological significance of this paracrine activity in an in vivo angiogenesis model. A single in vitro exposure of ASCs to severe hypoxia (<0.1% O2) significantly increased both the transcriptional and translational level of the vascular endothelial growth factor-A (VEGF-A) and angiogenin (ANG). The angiogenicity of the ASC-conditioned medium (ASC(CM)) was assessed by implanting ASC(CM)-treated polyvinyl alcohol sponges subcutaneously for 2 weeks in mice. The morphometric analysis of anti-CD31-immunolabeled sponge sections demonstrated an increased angiogenesis with hypoxic ASC(CM) treatment compared to normoxic control ASC(CM) treatment (percentage vascular volume; 6.0%±0.5% in the hypoxic ASC(CM) vs. 4.1%±0.7% in the normoxic ASC(CM), P<0.05). Reduction of VEGF-A and ANG levels in the ASC(CM) with respective neutralizing antibodies before sponge implantation showed a significantly diminished angiogenic response (3.5%±0.5% in anti-VEGF-A treated, 3.2%±0.7% in anti-ANG treated, and 3.5%±0.6% in anti-VEGF-A/ANG treated). Further, both the normoxic and hypoxic ASC(CM) were able to sustain in vivo lymphangiogenesis in sponges. Collectively, the model demonstrated that the increased paracrine production of the VEGF-A and ANG in hypoxic-conditioned ASCs in vitro translated to an in vivo effect with a favorable biological significance. These results further illustrate the potential for utilization of an in vitro optimized ASC(CM) for in vivo angiogenesis-related applications as an effective cell-free technology.

Comparative analysis of paracrine factor expression in human adult mesenchymal stem cells derived from bone marrow, adipose, and dermal tissue.

Human adult mesenchymal stem cells (MSCs) support the engineering of functional tissue constructs by secreting angiogenic and cytoprotective factors, which act in a paracrine fashion to influence cell survival and vascularization. MSCs have been isolated from many different tissue sources, but little is known about how paracrine factor secretion varies between different MSC populations. We evaluated paracrine factor expression patterns in MSCs isolated from adipose tissue (ASCs), bone marrow (BMSCs), and dermal tissues [dermal sheath cells (DSCs) and dermal papilla cells (DPCs)]. Specifically, mRNA expression analysis identified insulin-like growth factor-1 (IGF-1), vascular endothelial growth factor-D (VEGF-D), and interleukin-8 (IL-8) to be expressed at higher levels in ASCs compared with other MSC populations whereas VEGF-A, angiogenin, basic fibroblast growth factor (bFGF), and nerve growth factor (NGF) were expressed at comparable levels among the MSC populations examined. Analysis of conditioned media (CM) protein confirmed the comparable level of angiogenin and VEGF-A secretion in all MSC populations and showed that DSCs and DPCs produced significantly higher concentrations of leptin. Functional assays examining in vitro angiogenic paracrine activity showed that incubation of endothelial cells in ASC(CM) resulted in increased tubulogenic efficiency compared with that observed in DPC(CM). Using neutralizing antibodies we concluded that VEGF-A and VEGF-D were 2 of the major growth factors secreted by ASCs that supported endothelial tubulogenesis. The variation in paracrine factors of different MSC populations contributes to different levels of angiogenic activity and ASCs maybe preferred over other MSC populations for augmenting therapeutic approaches dependent upon angiogenesis.

Strategies in cardiac tissue engineering.

In heart failure, post-myocardial infarction and some congenital cardiac anomalies, organ transplantation is the only effective cure. Shortage of organ donors and complications of orthotopic heart transplant remain major challenges to the modern field of transplantation. Tissue engineering using cell-based strategies presents itself as a new way of generating functional myocardium. Engineering functional myocardium de novo requires an abundant source of cells that can form cardiomyocytes. These cells may be used with biocompatible scaffold materials to generate a contractile myocardium. Lastly, to sustain the high metabolism of the construct, a functional vasculature needs to be developed with the forming cardiac tissue. This review provides an update on the progress of stem cell research in the context of cardiac tissue development, types of biomaterials used in cardiac tissue engineering (CTE) and currently employed strategies for vascularization in CTE. In addition, a brief overview of strategies utilized in CTE is provided.

Disparate companions: tissue engineering meets cancer research.

Recreating an environment that supports and promotes fundamental homeostatic mechanisms is a significant challenge in tissue engineering. Optimizing cell survival, proliferation, differentiation, apoptosis and angiogenesis, and providing suitable stromal support and signalling cues are keys to successfully generating clinically useful tissues. Interestingly, those components are often subverted in the cancer setting, where aberrant angiogenesis, cellular proliferation, cell signalling and resistance to apoptosis drive malignant growth. In contrast to tissue engineering, identifying and inhibiting those pathways is a major challenge in cancer research. The recent discovery of adult tissue-specific stem cells has had a major impact on both tissue engineering and cancer research. The unique properties of these cells and their role in tissue and organ repair and regeneration hold great potential for engineering tissue-specific constructs. The emerging body of evidence implicating stem cells and progenitor cells as the source of oncogenic transformation prompts caution when using these cells for tissue-engineering purposes. While tissue engineering and cancer research may be considered as opposed fields of research with regard to their proclaimed goals, the compelling overlap in fundamental pathways underlying these processes suggests that cross-disciplinary research will benefit both fields. In this review article, tissue engineering and cancer research are brought together and explored with regard to discoveries that may be of mutual benefit.

An endogenously deposited fibrin scaffold determines construct size in the surgically created arteriovenous loop chamber model of tissue engineering.

BACKGROUND: An arteriovenous loop (AVL) enclosed in a polycarbonate chamber in vivo, produces a fibrin exudate which acts as a provisional matrix for the development of a tissue engineered microcirculatory network. OBJECTIVES: By administering enoxaparin sodium - an inhibitor of fibrin polymerization, the significance of fibrin scaffold formation on AVL construct size (including the AVL, fibrin scaffold, and new tissue growth into the fibrin), growth, and vascularization were assessed and compared to controls. METHODS: In Sprague Dawley rats, an AVL was created on femoral vessels and inserted into a polycarbonate chamber in the groin in 3 control groups (Series I) and 3 experimental groups (Series II). Two hours before surgery and 6 hours post-surgery, saline (Series I) or enoxaparin sodium (0.6 mg/kg, Series II) was administered intra-peritoneally. Thereafter, the rats were injected daily with saline (Series I) or enoxaparin sodium (1.5 mg/kg, Series II) until construct retrieval at 3, 10, or 21 days. The retrieved constructs underwent weight and volume measurements, and morphologic/morphometric analysis of new tissue components. RESULTS: Enoxaparin sodium treatment resulted in the development of smaller AVL constructs at 3, 10, and 21 days. Construct weight and volume were significantly reduced at 10 days (control weight 0.337 +/- 0.016 g [Mean +/- SEM] vs treated 0.228 +/- 0.048, [P < .001]: control volume 0.317 +/- 0.015 mL vs treated 0.184 +/- 0.039 mL [P < .01]) and 21 days (control weight 0.306 +/- 0.053 g vs treated 0.198 +/- 0.043 g [P < .01]: control volume 0.285 +/- 0.047 mL vs treated 0.148 +/- 0.041 mL, [P < .01]). Angiogenesis was delayed in the enoxaparin sodium-treated constructs with the absolute vascular volume significantly decreased at 10 days (control vascular volume 0.029 +/- 0.03 mL vs treated 0.012 +/- 0.002 mL [P < .05]). CONCLUSION: In this in vivo tissue engineering model, endogenous, extra-vascularly deposited fibrin volume determines construct size and vascular growth in the first 3 weeks and is, therefore, critical to full construct development.

Bisphosphonate-associated jawbone osteonecrosis: a correlation between imaging techniques and histopathology.

OBJECTIVES: Recently, jawbone osteonecrosis has been reported as a potential adverse effect of bisphosphonates administration. This paper considers and highlights histopathologic and radiologic features of this condition. STUDY DESIGN: Eleven patients, owing to unresponsiveness to conservative treatment and uncontrollable pain, underwent surgical resection of diseased jawbone after extensive hyperbaric oxygen therapy. A thorough clinical, laboratory, and imaging study was performed. Surgical specimens underwent histopathologic and immunohistochemical evaluation. RESULTS: Computerized tomography (CT) scans showed increased bone density, periosteal reaction, and bone sequestration in advanced stages. With magnetic resonance imaging (MRI), exposed areas showed a low signal in T1- and T2-weighted and inversion recovery images, which suggests low water content and is histopathologically correlated with paucity in cells and vessels (osteonecrotic pattern). Unexposed diseased bone was characterized by T1 hypointensity and T2 and IR hyperintensity, which suggests high water content and inflammation, associated with hypercellularity, osteogenesis, and hypervascularity (osteomyelitic pattern). CONCLUSIONS: Diseased bone extends beyond the limits of the bone exposed in the oral cavity. Histopathologic examination correlated well with CT and MRI, which are the choice for the evaluation of bisphosphonate-associated jawbone osteonecrosis.

The extracellular matrix of the spleen as a potential organizer of immune cell compartments.

Until recently little information was available on the molecular details of the extracellular matrix (ECM) of secondary lymphoid tissues. There is now growing evidence that these ECMs are unique structures, combining characteristics of basement membranes and interstitial or fibrillar matrices, resulting in scaffolds that are strong and highly flexible and, in certain secondary lymphoid compartments, also forming conduit networks for rapid fluid transport. This review will address the structural characteristics of the ECM of the murine spleen and its potential role as an organizer of immune cell compartments, with reference to the lymph node where relevant.

Time course analysis of hypoxia, granulation tissue and blood vessel growth, and remodeling in healing rat cutaneous incisional primary intention wounds.

Hypoxia and the development and remodeling of blood vessels and connective tissue in granulation tissue that forms in a wound gap following full-thickness skin incision in the rat were examined as a function of time. A 1.5 cm-long incisional wound was created in rat groin skin and the opposed edges sutured together. Wounds were harvested between 3 days and 16 weeks and hypoxia, percent vascular volume, cell proliferation and apoptosis, alpha-smooth muscle actin, vascular endothelial growth factor-A, vascular endothelial growth factor receptor-2, and transforming growth factor-beta1 expression in granulation tissue were then assessed. Hypoxia was evident between 3 and 7 days while maximal cell proliferation at 3 days (123.6+/-22.2 cells/mm2, p<0.001 when compared with normal skin) preceded the peak percent vascular volume that occurred at 7 days (15.83+/-1.10%, p<0.001 when compared with normal skin). The peak in cell apoptosis occurred at 3 weeks (12.1+/-1.3 cells/mm2, p<0.001 when compared with normal skin). Intense alpha-smooth muscle actin labeling in myofibroblasts was evident at 7 and 10 days. Vascular endothelial growth factor receptor-2 and vascular endothelial growth factor-A were detectable until 2 and 3 weeks, respectively, while transforming growth factor-beta1 protein was detectable in endothelial cells and myofibroblasts until 3-4 weeks and in the extracellular matrix for 16 weeks. Incisional wound granulation tissue largely developed within 3-7 days in the presence of hypoxia. Remodeling, marked by a decline in the percent vascular volume and increased cellular apoptosis, occurred largely in the absence of detectable hypoxia. The expression of vascular endothelial growth factor-A, vascular endothelial growth factor receptor-2, and transforming growth factor-beta1 is evident prior, during, and after the peak of vascular volume reflecting multiple roles for these factors during wound healing.