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Studies investigating the correlates of immune protection against Yersinia infection have established that both humoral and cell mediated immune responses are required for the comprehensive protection. In our previous study, we established that the bivalent fusion protein (rVE) comprising immunologically active regions of Y pestis LcrV (100-270 aa) and YopE (50-213 aa) proteins conferred complete passive and active protection against lethal Y enterocolitica 8081 challenge. In the present study, cohort of BALB/c mice immunized with rVE or its component proteins rV, rE were assessed for cell mediated immune responses and memory immune protection against Y enterocolitica 8081 rVE immunization resulted in extensive proliferation of both CD4 and CD8 T cell subsets; significantly high antibody titer with balanced IgG1: IgG2a/IgG2b isotypes (1:1 ratio) and up regulation of both Th1 (INF-\(\alpha\), IFN-\(\gamma\), IL 2, and IL 12) and Th2 (IL 4) cytokines. On the other hand, rV immunization resulted in Th2 biased IgG response (11:1 ratio) and proliferation of CD4+ T-cell; rE group of mice exhibited considerably lower serum antibody titer with predominant Th1 response (1:3 ratio) and CD8+ T-cell proliferation. Comprehensive protection with superior survival (100%) was observed among rVE immunized mice when compared to the significantly lower survival rates among rE (37.5%) and rV (25%) groups when IP challenged with Y enterocolitica 8081 after 120 days of immunization. Findings in this and our earlier studies define the bivalent fusion protein rVE as a potent candidate vaccine molecule with the capability to concurrently stimulate humoral and cell mediated immune responses and a proof of concept for developing efficient subunit vaccines against Gram negative facultative intracellular bacterial pathogens.
Atherosclerotic lesions that critically narrow the artery can necessitate an angioplasty and stent implantation. Long-term therapeutic effects, however, are limited by excessive arterial remodeling. We here employed a miniaturized nitinol-stent coated with star-shaped polyethylenglycole (star-PEG), and evaluated its bio-functionalization with RGD and CXCL1 for improving in-stent stenosis after implantation into carotid arteries of mice. Nitinol foils or stents (bare metal) were coated with star-PEG, and bio-functionalized with RGD, or RGD/CXCL1. Cell adhesion to star-PEG-coated nitinol foils was unaltered or reduced, whereas bio-functionalization with RGD but foremost RGD/CXCL1 increased adhesion of early angiogenic outgrowth cells (EOCs) and endothelial cells but not smooth muscle cells when compared with bare metal foils. Stimulation of cells with RGD/CXCL1 furthermore increased the proliferation of EOCs. In vivo, bio-functionalization with RGD/CXCL1 significantly reduced neointima formation and thrombus formation, and increased re-endothelialization in apoE\(^{-/-}\) carotid arteries compared with bare-metal nitinol stents, star-PEG-coated stents, and stents bio-functionalized with RGD only. Bio-functionalization of star-PEG-coated nitinol-stents with RGD/CXCL1 reduced in-stent neointima formation. By supporting the adhesion and proliferation of endothelial progenitor cells, RGD/CXCL1 coating of stents may help to accelerate endothelial repair after stent implantation, and thus may harbor the potential to limit the complication of in-stent restenosis in clinical approaches.
In this study, the ability of a multiwalled carbon nanotube functionalized with fluorescein isothiocyanate (MWCNT-FITC) was assessed as a prospective central nervous system-targeting drug delivery system to permeate the blood-brain barrier. The results indicated that the MWCNT-FITC conjugate is able to penetrate microvascular cerebral endothelial monolayers; its concentrations in the Transwell® system were fully equilibrated after 48 hours. Cell viability test, together with phase-contrast and fluorescence microscopies, did not detect any signs of MWCNT-FITC toxicity on the cerebral endothelial cells. These microscopic techniques also revealed presumably the intracellular localization of fluorescent MWCNT-FITCs apart from their massive nonfluorescent accumulation on the cellular surface due to nanotube lipophilic properties. In addition, the 1,000 ps molecular dynamics simulation in vacuo discovered the phenomenon of carbon nanotube aggregation driven by van der Waals forces via MWCN-TFITC rapid dissociation as an intermediate phase.
Background
Oncolytic viruses, including vaccinia virus (VACV), are a promising alternative to classical mono-cancer treatment methods such as surgery, chemo- or radiotherapy. However, combined therapeutic modalities may be more effective than mono-therapies. In this study, we enhanced the effectiveness of oncolytic virotherapy by matrix metalloproteinase (MMP-9)-mediated degradation of proteins of the tumoral extracellular matrix (ECM), leading to increased viral distribution within the tumors.
Methods
For this study, the oncolytic vaccinia virus GLV-1h255, containing the mmp-9 gene, was constructed and used to treat PC-3 tumor-bearing mice, achieving an intra-tumoral over-expression of MMP-9. The intra-tumoral MMP-9 content was quantified by immunohistochemistry in tumor sections. Therapeutic efficacy of GLV-1h255 was evaluated by monitoring tumor growth kinetics and intra-tumoral virus titers. Microenvironmental changes mediated by the intra-tumoral MMP-9 over-expression were investigated by microscopic quantification of the collagen IV content, the blood vessel density (BVD) and the analysis of lymph node metastasis formation.
Results
GLV-1h255-treatment of PC-3 tumors led to a significant over-expression of intra-tumoral MMP-9, accompanied by a marked decrease in collagen IV content in infected tumor areas, when compared to GLV-1h68-infected tumor areas. This led to considerably elevated virus titers in GLV-1h255 infected tumors, and to enhanced tumor regression. The analysis of the BVD, as well as the lumbar and renal lymph node volumes, revealed lower BVD and significantly smaller lymph nodes in both GLV-1h68- and GLV-1h255- injected mice compared to those injected with PBS, indicating that MMP-9 over-expression does not alter the metastasis-reducing effect of oncolytic VACV.
Conclusions
Taken together, these results indicate that a GLV-1h255-mediated intra-tumoral over-expression of MMP-9 leads to a degradation of collagen IV, facilitating intra-tumoral viral dissemination, and resulting in accelerated tumor regression. We propose that approaches which enhance the oncolytic effect by increasing the intra-tumoral viral load, may be an effective way to improve therapeutic outcome.
Ziel dieser Arbeit war es, NF-AT1-Gen-defiziente Mauslinien zu erzeugen und die Folgen dieser genetischen Manipulation in vivo zu untersuchen. Die Untersuchung sollte die durch die Gendefizienz erwarteten Mängel während der Entwicklung (Embryogenese) und, im Besonderen, die Auswirkungen auf das Immunsytem und die Entwicklung und Differenzierung der T-Zellen aufzeigen. Zur Untersuchung der genomischen Organisation des Maus-NF-AT1-Gens wurde eine genomische l-Phagen-DNA-Bibliothek "gescreent" (durchgeführt von Dr. E. Jankevics, Universität von Riga, Lettland), die entsprechenden l-Phagen, die das NF-AT1-Gen enthielten, isoliert und die DNA präpariert. Nach Analyse der klonierten Phagen (Subklonierung und Sequenzierung) wurde eine Restriktionskarte der entsprechenden Bereiche erstellt und der "targeting-vector" erstellt. Der "targeting-vector" wurde durch Elektroporation in embryonale Stammzellen (ES-Zellen) eingebracht und die Integration in das Genom ("Homologe Rekombination") durch Southern Blotting- bzw. PCR-Analyse untersucht. Manipulierte ES-Zellklone wurden in C57Bl/6-Blastozysten injiziert, diese in scheinschwangere Ammentiere transferiert und die Nachkommen nach Geburt anhand der Fellfarben klassifiziert. Nachkommen mit einem hohen Anteil hellen Fells wurden mit C57 Bl/6-Tieren verpaart und die Integration des manipulierten Zellklons in die Keimbahn wurde anhand der wildtypischen Fellfarbe und Genotypisierung nachgewiesen. Bezüglich des manipulierten NF-ATp-Gens heterozygote F1-Tiere wurden miteinander verpaart, um eine homozygote NF-ATp-defiziente Mäuse zu erhalten. Die Deletion des NF-ATp-Proteins wurde durch in Western-Blotting-Experimenten und EMSAs ("electrophoretic mobility shift assays") nachgewiesen. Die NF-ATp-/--Tiere zeigten keine augenscheinlichen Veränderungen während der Entwicklung und, bei jungen Tieren, keine offensichtlichen Veränderungen bei der Entwicklung des Immunsystems. In älteren Tieren (> 6 Wochen) war eine Hyperproliferation der Zellen des Immunsystems zu beobachten, was mit einer Splenomegalie, einer verstärkten Bildung von Keimzentren in lymphatischen Organen, vergrößerten Lymphknoten und einer verlangsamten Involution des Thymus einherging. Weitergehende Untersuchungen der Ursache dieser hyperproliferativen Erkrankung offenbarten eine verminderte klonale Deletion nach Aktivierung. Die Ursachen dieses überraschenden Effekts sind wahrscheinlich vielfältig, da NF-AT-Faktoren an der Regulation der Expression vieler Gene beteiligt sind, u.a. des Apoptose-assoziierten CD95-Liganden. Da sich bezüglich der IL-2-Expression keine Unterschiede zwischen NF-ATp-defizienten Tieren und Kontrollen zeigten, jedoch eine erhöhte IL-2-Konzentration im Medium kultivierter NF-ATp-defizienter T-Zellen beobachtet wurde, wurde die Bindung von NF-ATp an putative NF-AT-Bindungssequenzen des CD25-Promotors, die transkriptionelle Aktivierung des Promotors mittels Luciferase-Assays und die Expression der IL-2R-alpha-Kette (CD25) untersucht. Es konnte gezeigt werden, daß (1.) NF-ATp an zwei Regionen des CD25-Promotors bindet, (2.) der CD25-Promotor durch NF-ATp transkriptionell stark stimuliert wird und (3.) T-Zellen NF-ATp-defizienter Tiere nach Stimulation eine verminderte CD25-Expression zeigen. In NF-ATp-defizienten Tieren war die Expression von CD25 moderat reduziert, was eine Erklärung für den abgeschwächten Phänotyp dieser Tiere - im Vergleich zu IL-2- oder CD25-defizienten Tieren - sein kann. Die hyperproliferativen Erkrankungen dieser verschiedenen Mauslinien weisen auf eine Beteiligung der NF-AT-/IL-2-/IL-2R-Signalwege nicht nur während der T-Zell-Aktivierung hin, sondern auch auf eine Beteiligung an Signalwegen, die zur anschließenden Inaktivierung und Apoptose der T-Lymphozyten nötig sind.
Prenatal stress-induced programming of genome-wide promoter DNA methylation in 5-HTT-deficient mice
(2014)
The serotonin transporter gene (5-HTT/SLC6A4)-linked polymorphic region has been suggested to have a modulatory role in mediating effects of early-life stress exposure on psychopathology rendering carriers of the low-expression short (s)-variant more vulnerable to environmental adversity in later life. The underlying molecular mechanisms of this gene-by-environment interaction are not well understood, but epigenetic regulation including differential DNA methylation has been postulated to have a critical role. Recently, we used a maternal restraint stress paradigm of prenatal stress (PS) in 5-HTT-deficient mice and showed that the effects on behavior and gene expression were particularly marked in the hippocampus of female 5-Htt+/- offspring. Here, we examined to which extent these effects are mediated by differential methylation of DNA. For this purpose, we performed a genome-wide hippocampal DNA methylation screening using methylated-DNA immunoprecipitation (MeDIP) on Affymetrix GeneChip Mouse Promoter 1.0 R arrays. Using hippocampal DNA from the same mice as assessed before enabled us to correlate gene-specific DNA methylation, mRNA expression and behavior. We found that 5-Htt genotype, PS and their interaction differentially affected the DNA methylation signature of numerous genes, a subset of which showed overlap with the expression profiles of the corresponding transcripts. For example, a differentially methylated region in the gene encoding myelin basic protein (Mbp) was associated with its expression in a 5-Htt-, PS- and 5-Htt × PS-dependent manner. Subsequent fine-mapping of this Mbp locus linked the methylation status of two specific CpG sites to Mbp expression and anxiety-related behavior. In conclusion, hippocampal DNA methylation patterns and expression profiles of female prenatally stressed 5-Htt+/- mice suggest that distinct molecular mechanisms, some of which are promoter methylation-dependent, contribute to the behavioral effects of the 5-Htt genotype, PS exposure and their interaction.
Background
While all efforts have been undertaken to propagate the vaccination and develop remedies against SARS-CoV-2, no satisfactory management of this infection is available yet. Moreover, poor availability of any preventive and treatment measures of SARS-CoV-2 in economically disadvantageous communities aggravates the course of the pandemic. Here, we studied a new immunomodulatory phytotherapy (IP), an extract of blackberry, chamomile, garlic, cloves, and elderberry as a potential low-cost solution for these problems given the reported efficacy of herbal medicine during the previous SARS virus outbreak.
Methods
The key feature of SARS-CoV-2 infection, excessive inflammation, was studied in in vitro and in vivo assays under the application of the IP. First, changes in tumor-necrosis factor (TNF) and lnteurleukin-1 beta (IL-1β) concentrations were measured in a culture of human macrophages following the lipopolysaccharide (LPS) challenge and treatment with IP or prednisolone. Second, chronically IP-pre-treated CD-1 mice received an agonist of Toll-like receptors (TLR)-7/8 resiquimod and were examined for lung and spleen expression of pro-inflammatory cytokines and blood formula. Finally, chronically IP-pre-treated mice challenged with LPS injection were studied for “sickness” behavior. Additionally, the IP was analyzed using high-potency-liquid chromatography (HPLC)-high-resolution-mass-spectrometry (HRMS).
Results
LPS-induced in vitro release of TNF and IL-1β was reduced by both treatments. The IP-treated mice displayed blunted over-expression of SAA-2, ACE-2, CXCL1, and CXCL10 and decreased changes in blood formula in response to an injection with resiquimod. The IP-treated mice injected with LPS showed normalized locomotion, anxiety, and exploration behaviors but not abnormal forced swimming. Isoquercitrin, choline, leucine, chlorogenic acid, and other constituents were identified by HPLC-HRMS and likely underlie the IP immunomodulatory effects.
Conclusions
Herbal IP-therapy decreases inflammation and, partly, “sickness behavior,” suggesting its potency to combat SARS-CoV-2 infection first of all via its preventive effects.
Controversy surrounds neutrophil function in cancer because neutrophils were shown to provide both pro-and antitumor functions. We identified a heterogeneous subset of low-density neutrophils (LDNs) that appear transiently in self-resolving inflammation but accumulate continuously with cancer progression. LDNs display impaired neutrophil function and immunosuppressive properties, characteristics that are in stark contrast to those of mature, high-density neutrophils (HDNs). LDNs consist of both immature myeloid-derived suppressor cells (MDSCs) and mature cells that are derived from HDNs in a TGF-beta-dependent mechanism. Our findings identify three distinct populations of circulating neutrophils and challenge the concept that mature neutrophils have limited plasticity. Furthermore, our findings provide a mechanistic explanation to mitigate the controversy surrounding neutrophil function in cancer.
Intestinal glucose absorption is mediated by SGLT1 whereas GLUT2 is considered to provide basolateral exit. Recently, it was proposed that GLUT2 can be recruited into the apical membrane after a high luminal glucose bolus allowing bulk absorption of glucose by facilitated diffusion. Moreover, SGLT1 and GLUT2 are suggested to play an important role in intestinal glucose sensing and incretin secretion. In mice that lack either SGLT1 or GLUT2 we re-assessed the role of these transporters in intestinal glucose uptake after radiotracer glucose gavage and performed Western blot analysis for transporter abundance in apical membrane fractions in a comparative approach. Moreover, we examined the contribution of these transporters to glucose-induced changes in plasma GIP, GLP-1 and insulin levels. In mice lacking SGLT1, tissue retention of tracer glucose was drastically reduced throughout the entire small intestine whereas GLUT2-deficient animals exhibited higher tracer contents in tissue samples than wild type animals. Deletion of SGLT1 resulted also in reduced blood glucose elevations and abolished GIP and GLP-1 secretion in response to glucose. In mice lacking GLUT2, glucose-induced insulin but not incretin secretion was impaired. Western blot analysis revealed unchanged protein levels of SGLT1 after glucose gavage. GLUT2 detected in apical membrane fractions mainly resulted from contamination with basolateral membranes but did not change in density after glucose administration. SGLT1 is unequivocally the prime intestinal glucose transporter even at high luminal glucose concentrations. Moreover, SGLT1 mediates glucose-induced incretin secretion. Our studies do not provide evidence for GLUT2 playing any role in either apical glucose influx or incretin secretion.
This longitudinal study was performed to evaluate the feasibility of detecting the interaction between wall shear stress (WSS) and plaque development. 20 ApoE\(^{-/-}\)mice were separated in 12 mice with Western Diet and 8 mice with Chow Diet. Magnetic resonance (MR) scans at 17.6 Tesla and histological analysis were performed after one week, eight and twelve weeks. Allin vivoMR measurements were acquired using a flow sensitive phase contrast method for determining vectorial flow. Histological sections were stained with Hematoxylin and Eosin, Elastica van Gieson and CD68 staining. Data analysis was performed using Ensight and a Matlab-based "Flow Tool". The body weight of ApoE\(^{-/-}\)mice increased significantly over 12 weeks. WSS values increased in the Western Diet group over the time period; in contrast, in the Chow Diet group the values decreased from the first to the second measurement point. Western Diet mice showed small plaque formations with elastin fragmentations after 8 weeks and big plaque formations after 12 weeks; Chow Diet mice showed a few elastin fragmentations after 8 weeks and small plaque formations after 12 weeks. Favored by high-fat diet, plaque formation results in higher values of WSS. With wall shear stress being a known predictor for atherosclerotic plaque development, ultra highfield MRI can serve as a tool for studying the causes and beginnings of atherosclerosis.