Current Concepts in the Management of Primary Lymphedema.

Primary lymphedema is a heterogeneous group of conditions encompassing all lymphatic anomalies that result in lymphatic swelling. Primary lymphedema can be difficult to diagnose, and diagnosis is often delayed. As opposed to secondary lymphedema, primary lymphedema has an unpredictable disease course, often progressing more slowly. Primary lymphedema can be associated with various genetic syndromes or can be idiopathic. Diagnosis is often clinical, although imaging can be a helpful adjunct. The literature on treating primary lymphedema is limited, and treatment algorithms are largely based on practice patterns for secondary lymphedema. The mainstay of treatment focuses on complete decongestive therapy, including manual lymphatic drainage and compression therapy. For those who fail conservative treatment, surgical treatment can be an option. Microsurgical techniques have shown promise in primary lymphedema, with both lymphovenous bypass and vascularized lymph node transfers demonstrating improved clinical outcomes in a few studies.

Noninvasive Monitoring of Allograft Rejection Using a Novel Epidermal Sampling Technique.

Despite promising short- and long-term results to date in vascularized composite allotransplantation (VCA), acute rejection remains the most common major complication in recipients. Currently, diagnosis of acute rejection relies on clinical inspection correlated with histopathological analysis. However, disagreement exists regarding the value of full-thickness skin and mucosal biopsies and histopathology remains semiquantitative, subject to sampling bias, and prone to intra- and inter-observer variabilities. Additionally, biopsies may cause infection, scarring, and/or potentially incite rejection through immune activation after injury. Noninvasive methods to diagnose rejection represent a critical unmet need for the emerging field of VCA. Here, we propose a novel technique utilizing skin stripping of the epidermis and subsequent molecular analysis to detect known markers of acute rejection. Using a small animal VCA model, we sought to validate our epidermal sampling technique as a noninvasive diagnostic test for acute rejection.

Dysregulation of Nrf2/Keap1 Redox Pathway in Diabetes Affects Multipotency of Stromal Cells.

The molecular and cellular level reaches of the metabolic dysregulations that characterize diabetes are yet to be fully discovered. As mechanisms underlying management of reactive oxygen species (ROS) gain interest as crucial factors in cell integrity, questions arise about the role of redox cues in the regulation and maintenance of bone marrow-derived multipotent stromal cells (BMSCs) that contribute to wound healing, particularly in diabetes. Through comparison of BMSCs from wild-type and diabetic mice, with a known redox and metabolic disorder, we found that the cytoprotective nuclear factor erythroid-related factor 2 (Nrf2)/kelch-like erythroid cell-derived protein 1 (Keap1) pathway is dysregulated and functionally insufficient in diabetic BMSCs (dBMSCs). Nrf2 is basally active, but in chronic ROS, we found irregular inhibition of Nrf2 by Keap1, altered metabolism, and limited BMSC multipotency. Forced upregulation of Nrf2-directed transcription, through knockdown of Keap1, restores redox homeostasis. Normalized Nrf2/Keap1 signaling restores multipotent cell properties in dBMSCs through Sox2 expression. These restored BMSCs can resume their role in regenerative tissue repair and promote healing of diabetic wounds. Knowledge of diabetes and hyperglycemia-induced deficits in BMSC regulation, and strategies to reverse them, offers translational promise. Our study establishes Nrf2/Keap1 as a cytoprotective pathway, as well as a metabolic rheostat, that affects cell maintenance and differentiation switches in BMSCs.

Microenvironmental cues enhance mesenchymal stem cell-mediated immunomodulation and regulatory T-cell expansion.

Mesenchymal stem cells (MSCs) are known to both have powerful immunosuppressive properties and promote allograft tolerance. Determining the environmental oxygen tension and inflammatory conditions under which MSCs are optimally primed for this immunosuppressive function is essential to their utilization in promoting graft tolerance. Of particular interest is the mechanisms governing the interaction between MSCs and regulatory T cells (Tregs), which is relatively unknown. We performed our experiments utilizing rat bone marrow derived MSCs. We observed that priming MSCs in hypoxia promotes maintenance of stem-like characteristics, with greater expression of typical MSC cell-surface markers, increased proliferation, and maintenance of differentiation potential. Addition of autologous MSCs to CD4+/allogeneic endothelial cell (EC) co-culture increases regulatory T cell (Treg) proliferation, which is further enhanced when MSCs are primed in hypoxia. Furthermore, MSC-mediated Treg expansion does not require direct cell-cell contact. The expression of indolamine 2,3-dioxygenase, a mediator of MSC immunomodulation, increases when MSCs are primed in hypoxia, and inhibition of IDO significantly decreases the expansion of Tregs. Priming with inflammatory cytokines IFNγ and TNFα increases also expression of markers associated with MSC immunomodulatory function, but decreases MSC proliferation. The expression of IDO also increases when MSCs are primed with inflammatory cytokines. However, there is no increase in Treg expansion when MSCs are primed with IFNγ, suggesting an alternate mechanism for inflammatory-stimulated MSC immunomodulation. Overall, these results suggest that MSCs primed in hypoxia or inflammatory conditions are optimally primed for immunosuppressive function. These results provide a clearer picture of how to enhance MSC immunomodulation for clinical use.

Ex vivo allotransplantation engineering: Delivery of mesenchymal stem cells prolongs rejection-free allograft survival.

Current pharmacologic regimens in transplantation prevent allograft rejection through systemic recipient immunosuppression but are associated with severe morbidity and mortality. The ultimate goal of transplantation is the prevention of allograft rejection while maintaining recipient immunocompetence. We hypothesized that allografts could be engineered ex vivo (after allotransplant procurement but before transplantation) by using mesenchymal stem cell-based therapy to generate localized immunomodulation without affecting systemic recipient immunocompetence. To this end, we evaluated the therapeutic efficacy of bone marrow-derived mesenchymal stem cells in vitro and activated them toward an immunomodulatory fate by priming in inflammatory or hypoxic microenvironments. Using an established rat hindlimb model for allotransplantation, we were able to significantly prolong rejection-free allograft survival with a single perioperative ex vivo infusion of bone marrow-derived mesenchymal stem cells through the allograft vasculature, in the absence of long-term pharmacologic immunosuppression. Critically, transplanted rats rejected a second, nonengineered skin graft from the same donor species to the contralateral limb at a later date, demonstrating that recipient systemic immunocompetence remained intact. This study represents a novel approach in transplant immunology and highlights the significant therapeutic opportunity of the ex vivo period in transplant engineering.

Estimating pressure gradients by auscultation: How technology (echocardiography) can help improve clinical skills.

AIM: To extend our previously-published experience in estimating pressure gradients (PG) via physical examination in a large patient cohort. METHODS: From January 1, 1997 through December 31, 2009, an attending pediatric cardiologist compared clinical examination (EXAM) with Doppler-echo (ECHO), in 1193 patients with pulmonic stenosis (PS, including tetralogy of Fallot), aortic stenosis (AS), and ventricular septal defect (VSD). EXAM PG estimates were based primarily on a murmur's pitch, grade, and length. ECHO peak instantaneous PG was derived from the modified Bernoulli equation. Patients were 0-38.4 years old (median 4.8). RESULTS: For all patients, EXAM correlated highly with ECHO: ECHO = 0.99 (EXAM) + 3.2 mmHg; r = +0.89; P < 0.0001. Agreement was excellent (mean difference = -2.9 ± 16.1 mmHg). In 78% of all patients, agreement between EXAM and ECHO was within 15 mmHg and within 5 mmHg in 45%. Clinical estimates of PS PG were more accurate than of AS and VSD. A palpable precordial thrill and increasing loudness of the murmur predicted higher gradients (P < 0.0001). Weight did not influence accuracy. A learning curve was evident, such that the most recent quartile of patients showed ECHO = 1.01 (EXAM) + 1.9, r = +0.92, P < 0.0001; during this time, the attending pediatric cardiologist had been > 10 years in practice. CONCLUSION: Clinical examination can accurately estimate PG in PS, AS, or VSD. Continual correlation of clinical findings with echocardiography can lead to highly accurate diagnostic skills.