VEGF microsphere technology to enhance vascularization in fat grafting.

Vascularization is crucial for implantation of engineered tissues in reconstructive surgery. Polypeptides encapsulated in microspheres can be efficiently transported to their site of action and released in a sustained dosage. We evaluated the effect of delivering vascular endothelial growth factor (VEGF)-encapsulated microspheres in a lipoaspirate scaffold on vascularization and tissue survival. The VEGF-loaded (n=6) and empty (n=6) poly(lactic-co-glycolic acid) microspheres in human lipoaspirate and the human lipoaspirate alone (n=6) were injected subcutaneously into the flanks of athymic nude mice. Three mice from each group were killed, and grafts were explanted at weeks 3 and 6. Increases in mass and volume of VEGF samples, as well as decreases in empty and lipoaspirate-only samples, were observed at 3 and 6 weeks, reaching statistical significance at 6 weeks. Hematoxylin and eosin and CD31+ imaging demonstrated significantly greater vascularization in VEGF samples than in both the empty and lipoaspirate-only groups at both 3 and 6 weeks.

Co-immunization with an optimized plasmid-encoded immune stimulatory interleukin, high-mobility group box 1 protein, results in enhanced interferon-gamma secretion by antigen-specific CD8 T cells.

DNA vaccination is a novel immunization strategy that has great potential for the development of vaccines and immune therapeutics. This strategy has been highly effective in mice, but is less immunogenic in non-human primates and in humans. Enhancing DNA vaccine potency remains a challenge. It is likely that antigen-presenting cells (APCs), and especially dendritic cells (DCs), play a significant role in the presentation of the vaccine antigen to the immune system. A new study reports the synergistic recruitment, expansion and activation of DCs in vivo by high-mobility group box 1 (HMGB1) protein. Such combinational strategies for delivering vaccine in a single, simple platform will hypothetically bolster the cellular immunity in vivo. Here, we combined plasmid encoding human immunodeficiency virus-1 (HIV-1) Gag and Env with an HMGB1 plasmid as a DNA adjuvant in BALB/c mice (by intramuscular immunization via electroporation), and humoral and cellular responses were measured. Co-administration of this potent immunostimulatory adjuvant strongly enhanced the cellular interferon-gamma (IFN-gamma) and humoral immune response compared with that obtained in mice immunized with vaccine only. Our results show that co-immunization with HMGB1 can have a strong adjuvant activity, driving strong cellular and humoral immunity that may be an effective immunological adjuvant in DNA vaccination against HIV-1.

Human immunodeficiency virus type 1 Nef induces programmed death 1 expression through a p38 mitogen-activated protein kinase-dependent mechanism.

Chronic viral infection is characterized by the functional impairment of virus-specific T-cell responses. Recent evidence has suggested that the inhibitory receptor programmed death 1 (PD-1) is specifically upregulated on antigen-specific T cells during various chronic viral infections. Indeed, it has been reported that human immunodeficiency virus (HIV)-specific T cells express elevated levels of PD-1 and that this expression correlates with the viral load and inversely with CD4(+) T-cell counts. More importantly, antibody blockade of the PD-1/PD-L1 pathway was sufficient to both increase and stimulate virus-specific T-cell proliferation and cytokine production. However, the mechanisms that mediate HIV-induced PD-1 upregulation are not known. Here, we provide evidence that the HIV type 1 (HIV-1) accessory protein Nef can transcriptionally induce the expression of PD-1 during infection in vitro. Nef-induced PD-1 upregulation requires its proline-rich motif and the activation of the downstream kinase p38. Further, inhibition of Nef activity by p38 MAPK inhibitor effectively blocked PD-1 upregulation, suggesting that p38 MAPK activation is an important initiating event in Nef-mediated PD-1 expression in HIV-1-infected cells. These data demonstrate an important signaling event of Nef in HIV-1 pathogenesis.

HIV-1 viral genes and mitochondrial apoptosis.

The mitochondrion is an organelle that regulates various cellular functions including the production of energy and programmed cell death. Aberrant mitochondrial function is often concomitant with various cytopathies and medical disorders. The mitochondrial membrane plays a key role in the induction of cellular apoptosis, and its destabilization, as triggered by both intracellular and extracellular stimuli, results in the release of proapoptotic factors into the cytosol. Not surprisingly, proteins from the human immunodeficiency virus type 1 (HIV) have been implicated in exploiting this organelle to promote the targeted depletion of key immune cells, which assists in viral evasion of the immune system and contributes to the characteristic global immunodeficiency observed during progression of disease. Here we review the mechanisms by which HIV affects the mitochondrion, and suggest that various viral-associated genes may directly regulate apoptotic cell death.

Risk factors for pannus formation in the post-bariatric surgery population.

BACKGROUND: Previous studies describe a relationship between pannus mass and panniculectomy-related complication rates. Patient management may be improved by elucidating the key factors influencing pannus formation. METHODS: A retrospective review was conducted of 135 patients who had undergone laparoscopic Roux-en-Y gastric bypass from 1996 to 2010 and subsequent panniculectomy. Outcome measures included age, sex, body mass index, time of surgery, resected pannus mass, comorbidities, and panniculectomy-related complications. Nonparametric continuous and nominal variables were assessed using Spearman rank-correlation and Mann-Whitney U tests, respectively. RESULTS: One hundred thirty-five patients (123 women and 12 men; mean age, 44.7 years) were included in analysis. All patients had body contouring surgery more than 1 year after bariatric surgery (median time interval, 2.1 years). Median body mass index at the time of bypass, 1 year after bypass, and at the time of body contouring surgery was 48.7, 30.0, and 29.4 kg/m, respectively. Median pannus mass was 2.2 kg. Larger pannus mass was associated with greater age at gastric bypass surgery (p = 0.034), higher pre-gastric bypass body mass index (p = 0.031), higher prepanniculectomy body mass index (p < 0.001), and longer time interval between gastric bypass and panniculectomy (p = 0.046). Female patients requiring blood transfusions had a significantly larger pannus mass than those who did not (p = 0.048). CONCLUSION: Performing bariatric surgery on patients at a younger age or having patients reduce body mass index as much as possible before bariatric surgery may be useful for minimizing symptomatic pannus formation and in turn may decrease rates of panniculectomy-related complications. CLINICAL QUESTION/LEVEL OF EVIDENCE: Risk, III.

Prevalence of endogenous CD34+ adipose stem cells predicts human fat graft retention in a xenograft model.

BACKGROUND: Fat grafting is a promising technique for soft-tissue augmentation, although graft retention is highly unpredictable and factors that affect graft survival have not been well defined. Because of their capacity for differentiation and growth factor release, adipose-derived stem cells may have a key role in graft healing. The authors' objective was to determine whether biological properties of adipose-derived stem cells present within human fat would correlate with in vivo outcomes of graft volume retention. METHODS: Lipoaspirate from eight human subjects was processed using a standardized centrifugation technique and then injected subcutaneously into the flanks of 6-week-old athymic nude mice. Graft masses and volumes were measured, and histologic evaluation, including CD31+ staining for vessels, was performed 8 weeks after transplantation. Stromal vascular fraction isolated at the time of harvest from each subject was analyzed for surface markers by multiparameter flow cytometry, and also assessed for proliferation, differentiation capacity, and normoxic/hypoxic vascular endothelial growth factor secretion. RESULTS: Wide variation in percentage of CD34+ progenitors within the stromal vascular fraction was noted among subjects and averaged 21.3 ± 15 percent (mean ± SD). Proliferation rates and adipogenic potential among stromal vascular fraction cells demonstrated moderate interpatient variability. In mouse xenograft studies, retention volumes ranged from approximately 36 to 68 percent after 8 weeks, with an overall average of 52 ± 11 percent. A strong correlation (r = 0.78, slope = 0.76, p < 0.05) existed between stromal vascular fraction percentage of CD34+ progenitors and high graft retention. CONCLUSION: Inherent biological differences in adipose tissue exist between patients. In particular, concentration of CD34+ progenitor cells within the stromal vascular fraction may be one of the factors used to predict human fat graft retention.

Immunogenicity of novel consensus-based DNA vaccines against Chikungunya virus.

Chikungunya virus (CHIKV) is an emerging arbovirus and is an important human pathogen. Infection of humans by CHIKV can cause a syndrome characterized by fever, headache, rash, nausea, vomiting, myalgia, arthralgia and occasionally neurological manifestations such as acute limb weakness. It is also associated with a fatal haemorrhagic condition. CHIKV is geographically distributed from Africa through Southeast Asia and South America, and its transmission to humans is mainly through the Aedes aegypti species mosquitoes. The frequency of recent epidemics in the Indian Ocean and La Reunion islands suggests that a new vector perhaps is carrying the virus, as A. aegypti are not found there. In fact, a relative the Asian tiger mosquito, Aedes albopictus, may be the culprit which has raised concerns in the world health community regarding the potential for a CHIK virus pandemic. Accordingly steps should be taken to develop methods for the control of CHIKV. Unfortunately, currently there is no specific treatment for Chikungunya virus and there is no vaccine currently available. Here we present data of a novel consensus-based approach to vaccine design for CHIKV, employing a DNA vaccine strategy. The vaccine cassette was designed based on CHIKV capsid- and envelope-specific consensus sequences with several modifications, including codon optimization, RNA optimization, the addition of a Kozak sequence, and a substituted immunoglobulin E leader sequence. The expression of capsid, envelope E1 and E1 was evaluated using T7-coupled transcription/translation and immunoblot analysis. A recently developed, adaptive constant-current electroporation technique was used to immunize C57BL/6 mice with an intramuscular injection of plasmid coding for the CHIK-Capsid, E1 and E2. Analysis of cellular immune responses, including epitope mapping, demonstrates that electroporation of these constructs induces both potent and broad cellular immunity. In addition, antibody ELISAs demonstrate that these synthetic immunogens are capable of inducing high titer antibodies capable of recognizing native antigen. Taken together, these data support further study of the use of consensus CHIK antigens in a potential vaccine cocktail.