The viral vector vaccine VSV-GP boosts immune response upon repeated applications

Background: Vesicular stomatitis virus (VSV) is a potent candidate vaccine vector for various viral diseases (e.g. HIV, HCV, RSV). The biggest limitation of VSV, however, is its neurotoxicity, which limits application in humans. The second drawback is that VSV induces neutralizing antibodies rapidly and is thus ineffective as a vaccine vector upon repeated applications. Our group has recently shown that VSV pseudotyped with the glycoprotein (GP) of the lymphocytic choriomeningitis virus (LCMV), VSV-GP, is not neurotoxic. The aim of this project was to evaluate the potential of VSV-GP as a vaccine vector. Methods: For this purpose, we used Ovalbumin (OVA) as a model antigen and analyzed immunogenicity of GP-pseudotyped and wildtype VSV containing OVA (VSV-GP-OVA and VSV-OVA) in vitro and in vivo in mouse models. Results: We showed that both vectors infected murine bone marrow-derived dendritic cells (bmDCs) in vitro. These bmDCs were able to activate OVA specific CD8+ and CD4+ T cells. Immunization experiments in mice revealed that both VSV-OVA and VSV-GP-OVA induced functional OVA-specific cytotoxic T cells (CTLs) after a single immunization. In addition, with both viruses, mice generated antibodies against OVA. However, boosting with the same virus was only possible for the GP-pseudotyped virus but not for wild type VSV. The efficacy of repeated immunization with VSV-OVA was most likely limited by high levels of neutralizing antibodies, which we detected after the first immunization. In contrast, no neutralizing antibodies against VSV-GP were induced even after boosting. Conclusion: Taken together, we showed that the non-neurotoxic VSV-GP is able to induce specific T cell and B cell responses against the model antigen OVA to the same level as the wild type VSV vector. However, in contrast to wild type VSV, VSV-GP-OVA boosted the immune response upon repeated applications. Thus, VSV-GP is a promising novel vaccine vector

Characterization of DLK1(PREF1)+/CD34+ cells in vascular stroma of human white adipose tissue

AbstractSorting of native (unpermeabilized) SVF-cells from human subcutaneous (s)WAT for cell surface staining (cs) of DLK1 and CD34 identified three main populations: ~10% stained cs-DLK1+/cs-CD34−, ~20% cs-DLK1+/cs-CD34+dim and ~45% cs-DLK1−/cs-CD34+. FACS analysis after permeabilization showed that all these cells stained positive for intracellular DLK1, while CD34 was undetectable in cs-DLK1+/cs-CD34− cells. Permeabilized cs-DLK1−/cs-CD34+ cells were positive for the pericyte marker α-SMA and the mesenchymal markers CD90 and CD105, albeit CD105 staining was dim (cs-DLK1−/cs-CD34+/CD90+/CD105+dim/α-SMA+/CD45−/CD31−). Only these cells showed proliferative and adipogenic capacity. Cs-DLK1+/cs-CD34− and cs-DLK1+/cs-CD34+dim cells were also α-SMA+ but expressed CD31, had a mixed hematopoietic and mesenchymal phenotype, and could neither proliferate nor differentiate into adipocytes. Histological analysis of sWAT detected DLK1+/CD34+ and DLK1+/CD90+ cells mainly in the outer ring of vessel-associated stroma and at capillaries. DLK1+/α-SMA+ cells were localized in the CD34− perivascular ring and in adventitial vascular stroma. All these DLK1+ cells possess a spindle-shaped morphology with extremely long processes. DLK1+/CD34+ cells were also detected in vessel endothelium. Additionally, we show that sWAT contains significantly more DLK1+ cells than visceral (v)WAT. We conclude that sWAT has more DKL1+ cells than vWAT and contains different DLK1/CD34 populations, and only cs-DLK1−/cs-CD34+/CD90+/CD105+dim/α-SMA+/CD45−/CD31− cells in the adventitial vascular stroma exhibit proliferative and adipogenic capacity

Quiescence, Stemness and Adipogenic Differentiation Capacity in Human DLK1−/CD34+/CD24+ Adipose Stem/Progenitor Cells

We explore the status of quiescence, stemness and adipogenic differentiation capacity in adipose stem/progenitor cells (ASCs) ex vivo, immediately after isolation from human subcutaneous white adipose tissue, by sorting the stromal vascular fraction into cell-surface DLK1+/CD34−, DLK1+/CD34dim and DLK1−/CD34+ cells. We demonstrate that DLK1−/CD34+ cells, the only population exhibiting proliferative and adipogenic capacity, express ex vivo the bonafide quiescence markers p21Cip1, p27Kip1 and p57Kip2 but neither proliferation markers nor the senescence marker p16Ink4a. The pluripotency markers NANOG, SOX2 and OCT4 are barely detectable in ex vivo ASCs while the somatic stemness factors, c-MYC and KLF4 and the early adipogenic factor C/EBPβ are highly expressed. Further sorting of ASCs into DLK1−/CD34+/CD24− and DLK1−/CD34+/CD24+ fractions shows that KLF4 and c-MYC are higher expressed in DLK1−/CD34+/CD24+ cells correlating with higher colony formation capacity and considerably lower adipogenic activity. Proliferation capacity is similar in both populations. Next, we show that ASCs routinely isolated by plastic-adherence are DLK1−/CD34+/CD24+. Intriguingly, CD24 knock-down in these cells reduces proliferation and adipogenesis. In conclusion, DLK1−/CD34+ ASCs in human sWAT exist in a quiescent state, express high levels of somatic stemness factors and the early adipogenic transcription factor C/EBPβ but senescence and pluripotency markers are barely detectable. Moreover, our data indicate that CD24 is necessary for adequate ASC proliferation and adipogenesis and that stemness is higher and adipogenic capacity lower in DLK1−/CD34+/CD24+ relative to DLK1−/CD34+/CD24− subpopulations

Complement as an Endogenous Adjuvant for Dendritic Cell-Mediated Induction of Retrovirus-Specific CTLs

Previous studies have demonstrated the involvement of complement (C) in induction of efficient CTL responses against different viral infections, but the exact role of complement in this process has not been determined. We now show that C opsonization of retroviral particles enhances the ability of dendritic cells (DCs) to induce CTL responses both in vitro and in vivo. DCs exposed to C-opsonized HIV in vitro were able to stimulate CTLs to elicit antiviral activity significantly better than non-opsonized HIV. Furthermore, experiments using the Friend virus (FV) mouse model illustrated that the enhancing role of complement on DC-mediated CTL induction also occurred in vivo. Our results indicate that complement serves as natural adjuvant for DC-induced expansion and differentiation of specific CTLs against retroviruses

The Impact of Human Lipoaspirate and Adipose Tissue-Derived Stem Cells Contact Culture on Breast Cancer Cells: Implications in Breast Reconstruction

Background: Autologous fat transfer in the form of lipoaspirates for the reconstruction of the breast after breast cancer surgery is a commonly used procedure in plastic surgery. However, concerns regarding the oncologic risk of nutrient-rich fat tissue are widely debated. Previous studies have primarily focused on studying the interaction between adipose-derived stem cells (ASCs) and breast cancer cells. Methods: In this study, we performed a comprehensive analysis of the paracrine- and contact-based interactions between lipoaspirates, ASCs and breast cancer cell lines. An inverted flask culture method was used to study the contact-based interaction between lipoaspirates and breast cancer cells, while GFP-expressing breast cancer cell lines were generated to study the cell–cell contact interaction with ASCs. Three different human breast cancer cell lines, MCF-7, MDA-MB-231 and BT-474, were studied. We analyzed the impact of these interactions on the proliferation, cell cycle and epithelial-to-mesenchymal (EMT) transition of the breast cancer cells. Results: Our results revealed that both lipoaspirates and ASCs do not increase the proliferation rate of the breast cancer cells either through paracrine- or contact-dependent interactions. We observed that lipoaspirates selectively inhibit the proliferation of MCF-7 cells in contact co-culture, driven by the retinoblastoma (Rb) protein activity mediating cell cycle arrest. Additionally, ASCs inhibited MDA-MB-231 breast cancer cell proliferation in cell–cell contact-dependent interactions. Quantitative real-time PCR revealed no significant increase in the EMT-related genes in breast cancer cells upon co-culture with ASCs. Conclusion: In conclusion, this study provides evidence of the non-oncogenic character of lipoaspirates and supports the safety of clinical fat grafting in breast reconstruction after oncological surgical procedures. In vivo studies in appropriate animal models and long-term post-operative clinical data from patients are essential to reach the final safety recommendations

Application of Adipose-Tissue Derived Products for Burn Wound Healing

Burn injuries are a significant global health concern, leading to high morbidity and mortality. Deep burn injuries often result in delayed healing and scar formation, necessitating effective treatment options. Regenerative medicine, particularly cell therapy using adipose-derived stem cells (ASCs), has emerged as a promising approach to improving burn wound healing and reducing scarring. Both in vitro and preclinical studies have demonstrated the efficacy of ASCs and the stromal vascular fraction (SVF) in addressing burn wounds. The application of ASCs for burn healing has been studied in various forms, including autologous or allogeneic cells delivered in suspension or within scaffolds in animal burn models. Additionally, ASC-derived non-cellular components, such as conditioned media or exosomes have shown promise. Injection of ASCs and SVF at burn sites have been demonstrated to enhance wound healing by reducing inflammation and promoting angiogenesis, epithelialization, and granulation tissue formation through their paracrine secretome. This review discusses the applications of adipose tissue derivatives in burn injury treatment, encompassing ASC transplantation, as well as the utilization of non-cellular components utilization for therapeutic benefits. The application of ASCs in burn healing in the future will require addressing donor variability, safety, and efficacy for successful clinical application

Metformin Improves Stemness of Human Adipose-Derived Stem Cells by Downmodulation of Mechanistic Target of Rapamycin (mTOR) and Extracellular Signal-Regulated Kinase (ERK) Signaling

Adipose tissue plays an important role in regulating metabolic homeostasis by storing excess fat and protecting other organs from lipotoxicity. Aging is associated with central fat redistribution, culminating in a decrease in insulin-sensitive subcutaneous and an increase in insulin-resistant visceral adipose depots. Adipose-derived stem cells (ASCs) play an important role in the regeneration of adipose tissue. Aged ASCs show decreased stemness and regenerative potential due to the accumulation of oxidative stress and mitochondrial dysfunction-related cell damage. Metformin is a well-established anti-diabetic drug that has shown anti-aging effects in different organisms and animal models. In this study, we analyzed the effect of metformin treatment on the stemness of human ASCs in cell culture and whole adipose tissue culture models. Our results demonstrate that metformin improves the stemness of ASCs, reducing their rate of proliferation and adipocyte differentiation. Investigating the possible underlying mechanism, we observed a decrease in the mTOR and ERK activity in metformin-treated ASCs. In addition, we observed an increase in autophagy activity upon metformin treatment. We conclude that metformin treatment improves ASCs stemness by reducing mTOR and ERK signaling and enhancing autophagy. Future in vivo evaluations in animal models and humans will pave the way for the clinical adaptation of this well-established drug for reviving the stemness of aged stem cells

Protective Effect of Butanolic Fraction of Delphinium brunonianum on Fructose-Mediated Metabolic Alterations in Rats

The present study was conducted with an intent to evaluate the protective effect of butanolic fraction of Delphinium brunonianum on fructose mediated metabolic abnormalities in rats. Rats in all groups except control group were fed on 10% fructose for 6 weeks; however, rats in the treated group also received butanolic fraction for the last 3 weeks, along with the fructose. Moreover, phytoconstituents present in butanolic fraction were analyzed using LC-MS. All doses of butanolic fraction profoundly reduce the fructose-induced blood pressure, sympathetic over-activity, and weight gain. Furthermore, butanolic fraction prominently reduces the glucose intolerance and hyperinsulinemia in fructose-fed rats. On treatment with butanolic fraction, oxidative enzymes and the functionality of the aorta was also restored. Phytochemical analysis revealed the presence of several active constituents including bergenin, scopolin, rutinoside, kaempferol, coumaric acid, apigenin, and gingerol. In conclusion, butanolic fraction of Delphinium brunonianum has the potential to prevent and recover the fructose-induced metabolic perturbations

Targeting viral antigens to CD11c on dendritic cells induces retrovirus-specific T cell responses.

Dendritic cells (DC) represent the most potent antigen presenting cells and induce efficient cytotoxic T lymphocyte (CTL) responses against viral infections. Targeting antigens (Ag) to receptors on DCs is a promising strategy to enhance antitumor and antiviral immune responses induced by DCs. Here, we investigated the potential of CD11c-specific single-chain fragments (scFv) fused to an immunodominant peptide of Friend retrovirus for induction of virus-specific T cell responses by DCs. In vitro CD11c-specific scFv selectively targeted viral antigens to DCs and thereby significantly improved the activation of virus-specific T cells. In vaccination experiments DCs loaded with viral Ag targeted to CD11c provided improved rejection of FV-derived tumors and efficiently primed virus-specific CTL responses after virus challenge. Since the induction of strong virus-specific T cell responses is critical in viral infections, CD11c targeted protein vaccines might provide means to enhance the cellular immune response to prophylactic or therapeutic levels