The Effects of Negative Pressure Therapy on Hair Growth of Mouse Models.

Negative pressure therapy (NPT) has been shown to facilitate wound healing and promote hair growth in a porcine model. However, there is a paucity of research on the impact of negative pressure on hair growth in murine models. Despite the ability of nude mice to develop hair follicles, the hair they produce is often flawed towing to genetically induced keratin disorders, rendering them a pertinent animal model for assessing hair regeneration. Therefore, this study aims to investigate the effects of negative pressure on hair follicle growth in a nude mouse model. To achieve this, a customized external tissue expansion device was developed to apply negative pressure to the dorsum of nude mice. The mice were subjected to several treatment courses consisting of 15 and 30 min of continuous negative pressure at 10 mmHg, which were repeated 5 and 10 times every other day until sacrifice. Dorsal skin samples were subsequently extracted from the suction and nonsuction areas. The sections were stained with various antibodies to assess the expression of SOX-9, LHX-2, Keratin-15, ß-catenin, CD31, and vascular endothelial growth factor-A, and a TUNEL assay was used to analyze cell apoptosis. The results showed that the number of hair follicles and angiogenesis were significantly higher in the suction area than in the nonsuction area in all groups. Moreover, mice that received NPT for 15 min for 10 times had a higher hair follicle density than the other three groups. Immunofluorescence staining for LHX-2 and Keratin 15 further validated the results of these findings. In conclusion, this study demonstrated that negative pressure effectively promotes hair follicle growth and angiogenesis in nude mice through SOX-9- and LHX-2-mediated follicular regeneration and ß-catenin-mediated hair follicle morphogenesis.

Vascularized lymph node transplantation successfully reverses lymphedema and maintains immunity in a rat lymphedema model.

Vascularized lymph node transplantation (VLNT) has shown inspiring results for the treatment of lymphedema. Nevertheless, it remains unclear how VLNT restores lymphatic drainage and whether or not immunity recovers after surgery. Hindlimb lymphedema model was created using rats with extensive groin and popliteal lymph node removable following with radiotherapy, and the lymphedema was confirmed using indocyanine green (ICG) lymphangiography and micro-computer tomography for volume measurement. VLNT was performed 1 month later. Volume measurement, ICG lymphangiography, histology, and immune reaction were done 1 month after surgery. VLNT successfully reduced the volume of the lymphedema hindlimb, restored lymphatic drainage function with proven lymphatic channel, and reduced lymphedema-related inflammation and fibrosis. It promotes lymphangiogenesis shown from ICG lymphangiography, histology, and enhanced lymphangiogenesis gene expression. Dendritic cell trafficking via the regenerated lymphatic channels was successfully restored, and maintained systemic immune response was proved using dinitrofluorobenzene sensitization and challenge. VLNT effectively reduces lymphedema and promotes lymphatic regeneration in the capillary lymphatic but not the collecting lymphatic vessels. Along with the re-established lymphatic system was the restoration of immune function locally and systemically. This correlated to clinical experience regarding the reduction of swelling and infection episodes after VLNT in lymphedema patients.

Enhancing lymphangiogenesis and lymphatic drainage to vascularized lymph nodes with nanofibrillar collagen scaffolds.

BACKGROUND: This study investigated the effect of nanofibrillar collagen scaffold (BioBridge) implantation from the affected limb to the unaffected contralateral femoral vein or lymph node in a rat model. METHODS: Hind limb lymphedema in Lewis rats was created with lymphadenectomy and inguinal circumcision without radiation. The volumetric difference (greater than 5%) using computed tomography and indocyanine green fluorescence evaluated the progress of lymphedema at 4 weeks. The lymphedema rats have separated into Group I: Controls; Group II: implanted BioBridge to the contralateral femoral vein; and Group III: implanted BioBridge to the contralateral inguinal lymph node. RESULTS: A total of 14 of 30 (46.7%) rats developed hind limb lymphedema with a mean volume difference of 5.83 ± 0.99% and showed diffuse dermal backflow at 4 weeks postlymphadenectomy. Four weeks postimplantation of BioBridge, the mean volumetric difference was 5.62 ± 2.11%, 4.97 ± 0.59%, and -2.47 ± 2.37% in Group I, II, and III, respectively (p < 0.05). The dermal backflow on the affected limb increased in Groups I and II but decreased in Group III. CONCLUSIONS: Implantation of BioBridge from the affected limb to the contralateral inguinal lymph node significantly reduced the hind limb lymphedema at 4 weeks.

Edaravone use in acute intracerebral hemorrhage: A systematic review and meta-analysis of randomized controlled trials.

Background: Edaravone alleviates neurological deficits among patients with intracerebral hemorrhage; however, its effects on mortality and long-term functional outcomes remain unknown. Objective: To assess clinical outcomes associated with edaravone initiated within 7 days of symptoms onset in intracerebral hemorrhage. Methods: We systematically searched PubMed, Embase, Cochrane Library, CiNii, China National Knowledge Infrastructure, Chinese VIP information, Wanfang Data, and SinoMed for relevant randomized controlled trials from their inception to 1 May 2021 and conducted a comprehensive systematic review and meta-analysis (PROSPERO registration number: CRD42019147801). All-cause mortality and long-term functional outcomes were taken as the primary outcomes. Results: A total of 38 randomized controlled trials including 3,454 participants with acute intracerebral hemorrhage were included. The selected articles were of poor quality. Meta-analysis revealed that edaravone could not reduce all-cause mortality [relative risk (RR) = 0.51; 95% confidence interval (CI) (0.11-2.32); p = 0.38]. No studies reported on long-term functional outcomes in those trials. In addition, edaravone alleviated neurological deficits [mean difference (MD) = -5.44; 95% CI (-6.44 to -4.44); p<0.00001], improved the activities of daily living [MD = 8.44; 95% CI (7.65-9.23); p<0.00001], reduced the hematoma volume [MD = -4.71; 95% CI (-5.86 to -3.56); p<0.00001], and increased treatment response [RR = 1.26; 95% CI (1.22-1.31); p<0.00001]. In terms of safety outcome, there was no significant difference between the edaravone group and the control groups [RR = 1.67; 95% CI (0.92 to 3.06); p = 0.09]. Conclusion: Till date, edaravone does not associate with mortality reduction when initiated within 7 days of intracerebral hemorrhage onset. The effect of edaravone on long-term functional outcomes remains unknown due to lack of data. Although edaravone alleviated neurological deficits, improved activities of daily living, and reduced hematoma volume, we cautiously interpreted the results owing to the overall poor quality and high heterogeneity of the included trials. Presently, the results are insufficient to support edaravone as a routine treatment option for acute intracerebral hemorrhage.

Long-term outcomes of arterial ischemia or venous occlusion on vascularized groin lymph nodes in a rat model.

BACKGROUND: This study investigated the long-term effects of arterial ischemia and venous occlusion on lymph node drainage function in a rat model. METHODS: Bilateral groin lymph node flaps of 18 Lewis rats were dissected. The pedicle artery was clamped for 4, 5, and 6 h (A4, A5, and A6 groups), and the vein for 3, 4, and 5 h (V3, V4, and V5 groups) in six flaps. At 4 weeks, the evaluations included gross morphomics, indocyanine green (ICG) lymphography, histological section, immunofluorescence of terminal deoxynucleotidyl transferase assay, and heme oxygenase-1 (HO-1) stain. RESULTS: The lymph node flaps developed shrinkage and partial necrosis in A5, A6, V4, and V5 groups. Hemorrhage in the lymph node cortex and medulla was observed histologically in A5, A6, and V5 groups. ICG lymphography showed loss of lymphatic drainage function in 2 of 6 flaps in A6 and V5 groups. Cell death was shown partly in cortical follicles in A5 and V4 groups and completely in A6 and V5 groups. The HO-1 expression was statistically increased in A5 and V5 groups, respectively (p < 0.05). CONCLUSIONS: The critical arterial ischemia and venous occlusion time were 4 h at 4 weeks of follow-up.

Impacts of arterial ischemia or venous occlusion on vascularized groin lymph nodes in a rat model.

BACKGROUND: Reported ischemia time of vascularized lymph nodes was 5 hours. This study investigated the effects of arterial ischemia and venous occlusion on vascularized lymph node function in rats. METHODS: Bilateral pedicled groin lymph node flaps were raised in 27 Lewis rats. Femoral artery and vein were separated and clamped for 1, 3, 4, or 5 hour(s). Lymph node flap perfusion and drainage were assessed by laser Doppler flowmetry and indocyanine green lymphography. Histologic changes were assessed using hematoxylin and eosin stain, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL), and glutathione assays. RESULTS: Perfusion units of 2.84 ± 1.41, 2.46 ± 0.64, 2.42 ± 0.37, and 2.01 ± 0.90 were measured in arterial ischemia groups, and 1.71 ± 0.45, 2.20 ± 0.98, 1.49 ± 0.35, and 0.81 ± 0.20 in venous occlusion groups after 1, 3, 4, and 5 hours of clamping, respectively. Lymphatic drainage showed mean latency periods of 5.33 ± 0.88, 9.00 ± 3.21, 10.00 ± 2.08, and 24.50 ± 11.50 seconds in arterial clamping groups, and 25.00 ± 3.61, 26.00 ± 3.06, 23.33 ± 4.41, and 152.00 ± 0 seconds in venous clamping groups, respectively. Severe medullary and cortical congestion and hemorrhage on histology and cell damage by glutathione levels and TUNEL assay were found after 4 hours of venous clamping. CONCLUSIONS: Arterial ischemia and venous occlusion impact the function and viability of vascularized lymph node flaps differently. The critical venous occlusion time was 4 hours.

Critical Ischemia Time, Perfusion, and Drainage Function of Vascularized Lymph Nodes.

BACKGROUND: Vascularized lymph node transfer is a promising surgical treatment for lymphedema. This study investigated the effect of ischemia on the lymphatic drainage efficiency of vascularized lymph node flaps and the critical ischemia time of lymph nodes. METHODS: Twenty-four lymph nodes containing groin flaps in 12 Sprague-Dawley rats were dissected. Clamping of the vascular pedicle was performed for 0, 1, 3, 5, 6, or 7 hours; then, each was allowed to reperfuse by means of the vascular pedicle for 1 hour. Perfusion and ischemic changes were assessed using indocyanine green lymphography; laser Doppler flowmetry; and histologic studies with associated lymphatic vessel endothelial hyaluronan receptor-1, CD68, 4',6-diamidino-2-phenylindole, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling, and glutathione assay stains. RESULTS: The mean latency period of the groin lymph node flaps was 247 ± 67, 83 ± 15, 72 ± 42, 30 ± 18, and 245 ± 85 seconds in the 0-, 1-, 3-, 5-, and 6-hour groups, respectively. Perfusion detected by laser Doppler was 85.2 ± 14.5, 87.2 ± 36.7, 129.8 ± 33.7, 140.4 ± 148.5, 156.1 ± 91.4, and 41.2 ± 34.8 perfusion units at ischemia times of 0, 1, 3, 5, 6, and 7 hours, respectively. Cell damage measured by glutathione was 46.8 ± 10.2, 67.7 ± 14.2, 62.8 ± 15.4, 126.6 ± 5.9, 259.0 ± 70.3, and 109.1 ± 27.5 at ischemia times of 0, 1, 3, 5, 6, and 7 hours, respectively. Histologically, as ischemia time increased, hemorrhage and congestion became more severe. CONCLUSIONS: The critical ischemia time of vascularized lymph nodes is 5 hours in the rodent animal model, verified by indocyanine green lymphatic fluid uptake, laser Doppler perfusion, and histologic assessments. Interestingly, lymphatic drainage and perfusion of vascularized lymph nodes were improved with an increased ischemia time before the critical 5 hours was reached.

Periosteal Osteogenic Capacity Depends on Tissue Source.

Periosteal osteogenic capacity can be exploited to enhance bone formation in the fields of tissue engineering and regenerative medicine. Despite this importance, there have been no studies examining the composition, structure, and osteogenic capacity of periostea from different bone sources. In this study, structure and osteogenic factor content were compared among periostea from rib, calvarial, femoral, and tibial bones, in which the native bones of these four regions were harvested and subjected to histological analysis. The osteogenic capacity of grafted periosteum was evaluated using an in vivo vascularized pedicle model of bone tissue engineering. Poly(ethylene glycol)-poly(l-lactic acid) (PEG-PLLA) copolymer hydrogels were seeded with bone marrow mesenchymal stem cells and implanted with grafted periosteum harvested from either calvarial or tibial bone, which were representative of thin and thick native periostea, respectively. The cambium layer thickness of periostea from the femoral and tibial bones (36.9% ± 2.5% and 36.8% ± 2.6%) was greater than that from the calvarial and rib bones (26.8% ± 2.4% and 25.5% ± 1.9%). The osteocalcin and alkaline phosphatase levels were comparatively higher in the femoral and tibial periostea than those in periostea harvested from the calvarial and rib bones. The construct implanted with grafted tibial periosteum resulted in greater neo-bone regeneration and higher osteocalcin and alkaline phosphatase expression. This study is the first investigation of the osteogenic capacity of periostea from diverse sources. The results can be used to guide clinical strategies that exploit periostea for tissue engineering and clinical applications.

Synthesis and evaluation of dual crosslinked alginate microbeads.

Alginate hydrogels have been investigated for a broad variety of medical applications. The ability to assemble hydrogels at neutral pH and mild temperatures makes alginate a popular choice for the encapsulation and delivery of cells and proteins. Alginate has been studied extensively for the delivery of islets as a treatment for type 1 diabetes. However, poor stability of the encapsulation systems after implantation remains a challenge. In this paper, alginate was modified with 2-aminoethyl methacrylate hydrochloride (AEMA) to introduce groups that can be photoactivated to generate covalent bonds. This enabled formation of dual crosslinked structure upon exposure to ultraviolet light following initial ionic crosslinking into bead structures. The degree of methacrylation was varied and in vitro stability, long term swelling, and cell viability examined. At low levels of the methacrylation, the beads could be formed by first ionic crosslinks followed by exposure to ultraviolet light to generate covalent bonds. The methacrylated alginate resulted in more stable beads and cells were viable following encapsulation. Alginate microbeads, ionic (unmodified) and dual crosslinked, were implanted into a rat omentum pouch model. Implantation was performed with a local injection of 100 µl of 50 µg/ml of Lipopolysaccharide (LPS) to stimulate a robust inflammatory challenge in vivo. Implants were retrieved at 1 and 3 weeks for analysis. The unmodified alginate microbeads had all failed by week 1, whereas the dual-crosslinked alginate microbeads remained stable up through 3 weeks. The modified alginate microbeads may provide a more stable alternative to current alginate-based systems for cell encapsulation. STATEMENT OF SIGNIFICANCE: Alginate, a naturally occurring polysaccharide, has been used for cell encapsulation to prevent graft rejection of cell transplants for people with type I diabetes. Although some success has been observed in clinical trials, the lack of reproducibility and failure to reach insulin dependence for longer periods of time indicates the need for improvements in the procedure. A major requirement for the long-term function of alginate encapsulated cells is the mechanical stability of microcapsules. Insufficient mechanical integrity of the capsules can lead to immunological reactions in the recipients. In this work, alginate was modified to allow photoactivatable groups in order to allow formation of covalent crosslinks in addition to ionic crosslinking. The dual crosslinking design prevents capsule breakdown following implantation in vivo.

Promoting chondrocyte cell clustering through tuning of a poly(ethylene glycol)-poly(peptide) thermosensitive hydrogel with distinctive microarchitecture.

Hydrogels are considered to be attractive cell-matrix for chondrocytes due to their similarity in properties to the natural cartilage. However, the formation of chondrocyte cell clusters in hydrogels has been mostly limited to naturally-derived or relatively fast degrading materials. In this study, a series of diblock copolymer poly(ethylene glycol)-poly(alanine) (mPEG-PA) was synthesized and investigated as injectable biomimic hydrogels for the culturing of chondrocytes. Depending on the poly(alanine) chain length, afforded hydrogels exhibited variable mechanical property and microarchitecture due to difference in secondary structure arrangement. After 21days of culture, cell clusters were observed in all hydrogels with longer PA chains and these hydrogels supported more homogenous and established clustering as well as significantly higher glycosaminoglycan and collagen deposition. Interestingly, scanning electron microscopy revealed a distinct micron range fibrillar-like microarchitecture that may be responsible for maintaining chondrocyte phenotype and matrix production. In addition, micrographs revealed the presence of collagen fibrils and an extensive extracellular matrix network. Therefore, it is reasonable to conclude that mPEG-PA hydrogels possess the desirable properties for chondrocyte cluster formation and serve as potential candidate in cartilage tissue engineering.

Proposed pathway and mechanism of vascularized lymph node flaps.

OBJECTIVE: To investigate the pump mechanism and pathway of lymph transit in vascularized lymph node flaps. BACKGROUND: Microsurgical treatment of lymphedema with vascularized lymph node transfer can improve signs and symptoms of disease, but the pathways and mechanisms of these flaps warrant further exploration. METHODS: (Animal model) 72 flaps were raised in 18 rats: 36 groin flaps contained lymph nodes (LN), 36 deep inferior epigastric artery perforator flaps did not (non-LN). Indocyanine green (ICG) was added into normal saline (NS), 1%, 3%, 5%, 7% and 10% albumin. Three rats were assigned to each group. LN and non-LN flaps were submerged in solution and surveyed for venous fluorescence. In the 7% albumin and NS groups, volumetric change of solution was measured. (Human model) A similar experiment was performed in humans using five submental LN flaps. RESULTS: (Animal model) Fluorescence was detected in the venous pedicle of LN flaps submerged in 5%, 7% and 10% albumin, and half of flaps submerged in 3% albumin. Fluorescence was not detected in LN node flaps submerged in ICG-containing NS or 1% albumin solution. Fluorescence was not detected in non-LN flaps. There was greater volume reduction with LN flaps than non-LN flaps (p<0.001). (Human model) Fluorescence was detected in the venous pedicle of all flaps immersed in lymph. CONCLUSIONS: ICG fluorescence was detected in the venous pedicle of rat and human LN flaps submerged in lymph or albumin when the concentration was greater than 3%. Based on these results, a pathway for lymphatic uptake is presented.

Quantity of lymph nodes correlates with improvement in lymphatic drainage in treatment of hind limb lymphedema with lymph node flap transfer in rats.

PURPOSE: This study was conducted to investigate the correlation between the number of vascularized lymph nodes (LN) transferred and resolution of hind limb lymphedema in a rat model. METHODS: Unilateral hind limb lymphedema was created in 18 male Sprague-Dawley rats following inguinal and popliteal LN resection and radiation. A para-aortic LN flap based on the celiac artery was subsequently transferred to the affected groin. The three study groups consisted of Group A (no LN transfer), Group B (transfer of a single vascularized LN), and Group C (transfer of three vascularized LNs). Volumetric analysis of bilateral hind limbs was performed using micro-CT imaging at 1, 2, and 3 months postoperatively. Lymphatic drainage was assessed with Tc(99) lymphoscintigraphy preoperatively and at 3 months postoperatively. RESULTS: A statistically significant volume reduction was seen in Groups B and C compared to Group A at all time points. Volume reduction of Group A vs.Group B at 1 month (8.6% ± 2.0% vs. 2.7% ± 2.6%, P < 0.05), 2 months (9.3% ± 2.2% vs. -4.3% ± 2.7%, P < 0.05), and 3 months (7.6% ± 3.3% vs. -8.9% ± 5.2%, P < 0.05). Volume reduction of Group A vs. Group C at 1 month (8.6% ± 2.0% vs. -6.6% ± 3.1%, P < 0.05), 2 months (9.3% ± 2.2% vs. -10.2% ± 4.6%, P < 0.05), and 3 months (7.6% ± 3.3% vs. -9.1% ± 3.1%, P < 0.05). Of note, comparison of Groups B and C demonstrated greater volume reduction in Group C at 1 (P < 0.02) and 2 (P = 0.07) months postoperatively. CONCLUSIONS: LN flap transfer is an effective procedure for the treatment of lymphedema. The number of vascularized LNs transferred correlates positively with the degree of volume reduction.

Developing a Lower Limb Lymphedema Animal Model with Combined Lymphadenectomy and Low-dose Radiation.

BACKGROUND: This study was aimed to establish a consistent lower limb lymphedema animal model for further investigation of the mechanism and treatment of lymphedema. METHODS: Lymphedema in the lower extremity was created by removing unilateral inguinal lymph nodes followed by 20, 30, and 40 Gy (groups IA, IB, and IC, respectively) radiation or by removing both inguinal lymph nodes and popliteal lymph nodes followed by 20 Gy (group II) radiation in Sprague-Dawley rats (350-400 g). Tc(99) lymphoscintigraphy was used to monitor lymphatic flow patterns. Volume differentiation was assessed by microcomputed tomography and defined as the percentage change of the lesioned limb compared to the healthy limb. RESULTS: At 4 weeks postoperatively, 0% in group IA (n = 3), 37.5% in group IB (n = 16), and 50% in group IC (n = 26) developed lymphedema in the lower limb with total mortality and morbidity rate of 0%, 56.3%, and 50%, respectively. As a result of the high morbidity and mortality rates, 20 Gy was selected, and the success rate for development of lymphedema in the lower limb in group II was 81.5% (n = 27). The mean volume differentiation of the lymphedematous limb compared to the health limb was 7.76% ± 1.94% in group II, which was statistically significant compared to group I (P < 0.01). CONCLUSIONS: Removal of both inguinal and popliteal lymph nodes followed by radiation of 20 Gy can successfully develop lymphedema in the lower limb with minimal morbidity in 4 months.

The mechanism of vascularized lymph node transfer for lymphedema: natural lymphaticovenous drainage.

BACKGROUND: Vascularized lymph node flap transfer for the treatment of upper and lower limb lymphedema has had promising results. This study was performed to investigate the mechanism of lymph drainage of a vascularized lymph node flap both experimentally and clinically. METHODS: In the experimental study, 18 Sprague-Dawley rats were used to create 36 flaps, either a groin lymph node flap or an abdominal cutaneous flap that did not contain lymph nodes. Indocyanine green dye was injected into the edge of 12 lymph node flaps, directly into a lymph node of 12 lymph node flaps, and into the edge of 12 cutaneous flaps. In the clinical study, an identical study design was used, with 24 vascularized lymph node flaps and 12 cutaneous flaps not containing lymph nodes. RESULTS: Experimentally, fluorescence was detected in the pedicle vein after a mean latency period of 153 ± 129 seconds when the edge of the lymph node flap was injected and 12.8 ± 8.1 seconds when the lymph node was directly injected. Fluorescence was not detected in the pedicle vein of the cutaneous flaps (p < 0.01). Clinically, fluorescence was detected in the pedicle vein after a mean latency period of 346 ± 249 seconds when the edge of the lymph node flap was injected and 23.5 ± 27.1 seconds when the lymph node was directly injected. Fluorescence was not detected in the pedicle vein of the cutaneous flaps (p < 0.01). CONCLUSION: The vascularized lymph node flap drains lymph into the pedicle vein, both experimentally and clinically. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

An ectopic approach for engineering a vascularized tracheal substitute.

Tissue engineering can provide alternatives to current methods for tracheal reconstruction. Here we describe an approach for ectopic engineering of vascularized trachea based on the implantation of co-cultured scaffolds surrounded by a muscle flap. Poly(L-lactic-co-glycolic acid) (PLGA) or poly(ε-caprolactone) (PCL) scaffolds were seeded with chondrocytes, bone marrow stem cells and co-cultured both cells respectively (8 groups), wrapped in a pedicled muscle flap, placed as an ectopic culture on the abdominal wall of rabbits (n = 24), and harvested after two and four weeks. Analysis of the biochemical and mechanical properties demonstrated that the PCL scaffold with co-culture cells seeding displayed the optimal chondrogenesis with adequate rigidity to maintain the cylindrical shape and luminal patency. Histological analysis confirmed that cartilage formed in the co-culture groups contained a more homogeneous and higher extracellular matrix content. The luminal surfaces appeared to support adequate epithelialization due to the formation of vascularized capsular tissue. A prefabricated neo-trachea was transferred to the defect as a tracheal replacement and yielded satisfactory results. These encouraging results indicate that our co-culture approach may enable the development of a clinically applicable neo-trachea.

The DNA methylation inhibitor 5-azacytidine increases regulatory T cells and alleviates airway inflammation in ovalbumin-sensitized mice.

BACKGROUND: Asthma is characterized as a chronic inflammatory disorder of the airways associated with an enhanced TH2 response to inhaled allergens. CD4+ T regulatory (Treg) cells are controlled by the master transcription factor FoxP3 and strictly maintain peripheral immunotolerance. Epigenetic regulation of FoxP3 by DNA methyltransferase inhibitors, such as 5-azacytidine (Aza), can generate a steady supply of functional Treg cells. Therefore, we propose that Aza can augment Treg cells in vivo to prevent the pathogenesis of asthma. METHODS: BALB/c mice were sensitized with chicken ovalbumin (OVA) and treated with different doses of Aza. Airway hyperresponsiveness to methacholine, eosinophilia in bronchoalveolar lavage fluid, circulating titers of OVA-specific IgG1 and IgE, and stimulating levels of TH2 cytokines from splenocytes were then determined. Cellular populations were examined by flow cytometry. PC61 antibody, which depletes CD25+ cells, was used to verify the role of CD25+ cells in Aza-induced tolerance. RESULTS: Administration of Aza to OVA-sensitized mice diminished airway hyperreactivity, pulmonary eosinophilia, levels of OVA-specific IgG1 and IgE in serum, and secretion of TH2 cytokines from OVA-stimulated splenocytes in a dose-dependent manner. Percentages of CD25+ and FoxP3+ cells in the CD4+ cell population were notably increased in Aza-treated mice compared to sensitized control mice. Furthermore, the major symptoms of asthma were exacerbated by depleting CD25+ cells in Aza-treated mice. CONCLUSIONS: Aza may have applications as a novel clinical strategy to increase the production of Treg cells in order to modulate the airway inflammation associated with asthma.

Profiling lipid-protein interactions using nonquenched fluorescent liposomal nanovesicles and proteome microarrays.

Fluorescent liposomal nanovesicles (liposomes) are commonly used for lipid research and/or signal enhancement. However, the problem of self-quenching with conventional fluorescent liposomes limits their applications because these liposomes must be lysed to detect the fluorescent signals. Here, we developed a nonquenched fluorescent (NQF)1 liposome by optimizing the proportion of sulforhodamine B (SRB) encapsulant and lissamine rhodamine B-dipalmitoyl phosphatidylethanol (LRB-DPPE) on a liposomal surface for signal amplification. Our study showed that 0.3% of LRB-DPPE with 200 µm of SRB provided the maximal fluorescent signal without the need to lyse the liposomes. We also observed that the NQF liposomes largely eliminated self-quenching effects and produced greatly enhanced signals than SRB-only liposomes by 5.3-fold. To show their application in proteomics research, we constructed NQF liposomes that contained phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) and profiled its protein interactome using a yeast proteome microarray. Our profiling led to the identification of 162 PI(3,5)P2-specific binding proteins (PI(3,5)P2-BPs). We not only recovered many proteins that possessed known PI(3,5)P2-binding domains, but we also found two unknown Pfam domains (Pfam-B_8509 and Pfam-B_10446) that were enriched in our dataset. The validation of many newly discovered PI(3,5)P2-BPs was performed using a bead-based affinity assay. Further bioinformatics analyses revealed that the functional roles of 22 PI(3,5)P2-BPs were similar to those associated with PI(3,5)P2, including vesicle-mediated transport, GTPase, cytoskeleton, and kinase. Among the 162 PI(3,5)P2-BPs, we found a novel motif, HRDIKP[ES]NJLL that showed statistical significance. A docking simulation showed that PI(3,5)P2 interacted primarily with lysine or arginine side chains of the newly identified PI(3,5)P2-binding kinases. Our study showed that this new tool would greatly benefit profiling lipid-protein interactions in high-throughput studies.

Elesclomol induces cancer cell apoptosis through oxidative stress.

Elesclomol (formerly STA-4783) is a novel small molecule undergoing clinical evaluation in a pivotal phase III melanoma trial (SYMMETRY). In a phase II randomized, double-blinded, controlled, multi-center trial in 81 patients with stage IV metastatic melanoma, treatment with elesclomol plus paclitaxel showed a statistically significant doubling of progression-free survival time compared with treatment with paclitaxel alone. Although elesclomol displays significant therapeutic activity in the clinic, the mechanism underlying its anticancer activity has not been defined previously. Here, we show that elesclomol induces apoptosis in cancer cells through the induction of oxidative stress. Treatment of cancer cells in vitro with elesclomol resulted in the rapid generation of reactive oxygen species (ROS) and the induction of a transcriptional gene profile characteristic of an oxidative stress response. Inhibition of oxidative stress by the antioxidant N-acetylcysteine blocked the induction of gene transcription by elesclomol. In addition, N-acetylcysteine blocked drug-induced apoptosis, indicating that ROS generation is the primary mechanism responsible for the proapoptotic activity of elesclomol. Excessive ROS production and elevated levels of oxidative stress are critical biochemical alterations that contribute to cancer cell growth. Thus, the induction of oxidative stress by elesclomol exploits this unique characteristic of cancer cells by increasing ROS levels beyond a threshold that triggers cell death.