Incorporation of the Tat cell‐penetrating peptide into nanofibers improves the respective antitumor immune response

A major challenge for the development of anticancer vaccines is the induction of a safe and effective immune response, particularly mediated by CD8+ T lymphocytes, in an adjuvant‐free manner. In this respect, we present a simple strategy to improve the specific CD8+ T cell responses using KFE8 nanofibers bearing a Class I (Kb)‐restricted peptide epitope (called E. nanofibers) without the use of adjuvant. We demonstrate that incorporation of Tat, a cell‐penetrating peptide (CPP) of the HIV transactivator protein, into E. nanofibers remarkably enhanced tumor‐specific CD8+ T cell responses. E. nanofibers containing 12.5% Tat peptide (E.Tat_12.5 nanofiber) increased antigen cross‐presentation by bone marrow‐derived dendritic cells as compared with E. nanofibers, or E. nanofibers containing 25 or 50% the Tat peptide. Uptake of KFE8.Tat_12.5 nanofibers by dendritic cells (DCs) was significantly increased compared with KFE8 nanofiber lacking Tat. Peritoneal and lymph node DCs of mice immunized with E.Tat_12.5 nanofibers exhibited increased presentation of the H2kb‐epitope (reminiscent for cross‐presentation) compared with DCs obtained from E. nanofiber vaccinated mice. Tetrameric and intracellular cytokine staining revealed that vaccination with E.Tat_12.5 triggered a robust and specific CD8+ T lymphocyte response, which was more pronounced than in mice vaccinated with E. nanofibers alone. Furthermore, E.Tat_12.5 nanofibers were more potent than E. nanofiber to induce antitumor immune response and tumor‐infiltrating IFN‐γ CD8 T lymphocyte. In terms of cancer vaccine development, we propose that harnessing the nanofiber‐based vaccine platform with incorporated Tat peptide could present a simple and promising strategy to induce highly effective antitumor immune response.     

Correction to: Ecological modeling and distribution analysis of digger scorpions: Odontobuthus doriae,Odontobuthus bidentatus (Scorpiones: Buthidae) and Scorpio maurus (Scorpiones: Scorpionidae) in Iran using the maximum entropy method

Applied Entomology and Zoology - The word “Odonthubutus” should be replaced with “Odontobuthus” throughout the article.     

Immunogenicity of Salmonella enterica serovar Enteritidis virulence protein,InvH, and cross-reactivity of its antisera with Salmonella strains

Acellular vaccines containing bacterial immunodominant components such as surface proteins may be potent alternatives to live attenuated vaccines in order to reduce salmonellosis risk to human health. invH gene, an important part of needle complex in type three secretion system (TTSS) plays important role in efficient bacterial adherence and entry into epithelial cells. In this work we hypothesize that use of a 15 kDa recombinant InvH as Salmonella enterica serovar Enteritidis surface protein could provoke antibody production in mouse and would help us study feasibility of its potential for diagnosis and/or a recombinant vaccine. The purified InvH provoked significant rise of IgG in mice. Active protection induced by immunization with InvH against variable doses of S. enterica serovar Enteritidis, indicated that the immunized mice were completely protected against challenge with 10⁴ LD₅₀. The immunoreaction of sera from immunized mice with other Salmonella strains or cross reaction with sera of Salmonella strains inoculated mice is indicative of possessing by Salmonella strains of the surface protein, InvH, that can be employed in both prophylactic and diagnostic measures against S. enterica. Bacteria free spleen and ileum of the immunized mice in this study indicate that the invH gene affects bacterial invasion. Efficacy of the virulence protein, InvH, in shuttling into host cells in injectisome of S. enterica serovar Enteritidis and inhibition of this phenomenon by active immunization was shown in this study. In conclusion immunization with InvH protein can develop protection against S. enterica serovar Enteritidis infections. InvH in Salmonella strains can be exploited in protective measures as well as a diagnostic tool in Salmonella infections.     

Measurement variability analysis for fermentation process assays

The paper reports the variability of replicate measurements for soluble protein and some enzyme activities in batch and continuous culture of S. cerevisiae. The measurement variability in these assays depended on the measured concentration and could be represented as a standard deviation proportional to the measured value.The support of the ESPRC and BBSRC through the Interdisciplinary Research Centre for Process Systems Engineering, Imperial College of Science, Technology and Medicine, and through the Advanced Centre for Biochemical Engineering, University College London is gratefully acknowledged.     

Generation of an enriched pool of DNA aptamers for an HER2-overexpressing cell line selected by Cell SELEX

Overexpression of human epidermal growth factor receptor 2 (HER2) occurs in a large percentage of breast cancers. Monoclonal antibodies targeting HER2 are vastly used for both diagnostic and therapeutic aims. However, identifying a new molecular probe against HER2 with improved diagnostic and therapeutic features is of great importance. In this report, we have applied the cell systematic evolution of ligands by exponential enrichment (SELEX) strategy for 16 selection rounds to generate an enriched pool of aptamers that specifically recognize the HER2 positive cell line. During the Cell SELEX procedure, a human HER2-overexpressing breast cancer cell line and a human HER2 negative breast cancer cell line were used. Our results reveal that polymerase chain reaction (PCR) amplification of random DNA libraries and the selected single-stranded DNA pool in different Cell SELEX rounds are different from what we expect from PCR amplification of homologous DNA. Our results also confirmed previous studies describing positive HER2 status of SK-BR3 and the absence of the HER2 expression in the MDA-MB468. We also developed a new method, Cell enzyme-linked assay, to monitor the enrichment of aptamers in a given round of Cell SELEX. This method would also be useful in other experiments using live cell enzyme-linked immunosorbent assay on adherent cells.     

Using Immunoinformatics and Structural Approaches to Design a Novel HHV8 Vaccine

International Journal of Peptide Research and Therapeutics - Kaposi’s sarcoma (KS)-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV8) has caused infection in different parts of the...     

The serine protease Omi/HtrA2 is involved in XIAP cleavage and in neuronal cell death following focal cerebral ischemia/reperfusion

Omi/HtrA2 is a pro-apoptotic mitochondrial serine protease involved in both forms of apoptosis, caspase-dependent as well as caspase-independent cell death. However, the impact of Omi/HtrA2 in the apoptotic cell machinery that takes place in vivo under pathological conditions such as cerebral ischemia remains unknown. The present study was monitored in order to examine whether Omi/HtrA2 plays a decisive role in apoptosis observed after focal cerebral ischemia in rats. Male adult rats were subjected to 90min of focal cerebral ischemia followed by reperfusion and treated with vehicle or ucf-101, a novel and specific Omi/HtrA2 inhibitor, prior reperfusion. Focal cerebral ischemia/reperfusion induced a mitochondrial up-regulation of Omi/HtrA2 and significantly increased cytosolic accumulation of Omi/HtrA2. Furthermore, ischemia led to activation of caspase-3 and degradation X-linked inhibitor of apoptosis protein (XIAP). Treatment of animals prior ischemia with ucf-101, the specific inhibitor of Omi/HtrA2, was able to (1) reduce the number of TUNEL-positive cells, to (2) attenuate the XIAP-breakdown and to (3) reduce the infarct size. This study shows for the first time that focal cerebral ischemia in rats results in Omi/HtrA2 translocation from the mitochondria to the cytosol, where it participates in neuronal cell death. Blocking the proteolytic activity of Omi/HtrA2 with specific inhibitors, such as the ucf-101, could be a novel way to afford neuroprotection and minimize cellular damage in cerebral ischemia/reperfusion.     

Chemotactically directed redistribution of alpha-actinin precedes morphological polarization and reversal of polarity in human polymorphonuclear leucocytes (PMNs)

We have determined the temporal and spatial relationship between cell polarization and alpha-actinin localization by analysing the redistribution of alpha-actinin and F-actin in spherical PMNs developing polarity and in polarized cells reversing polarity following localized stimulation with chemotactic peptide using micropipettes. Initially spherical PMNs develop a one-sided accumulation of alpha-actinin before lamellipodia enriched in alpha-actinin are formed. In polarized cells, alpha-actinin is concentrated at the leading front. When polarity is reversed, alpha-actinin redistribution to the uropod precedes reversal of morphological polarity and formation of new lamellipodia at the uropod. Later, lamellipodia enriched in F-actin and alpha-actinin develop at the former uropod to form a new front. The data document that redistribution of alpha-actinin is a very early event in the development of polarity, which precedes formation of lamellipodia.     

Peripheral chemoreceptor modulation of the pulmonary vasculature in the cat.

The present study was undertaken to determine whether stimulation of the carotid and aortic bodies (cb and ab) could affect the pulmonary vasculature. Our hypothesis was that each promoted vasodilation and thus could modulate the pulmonary vasoconstrictor response to hypoxia. The experimental design of the first set of experiments took advantage of the facts that 1) the ab, but not the cb, increases its neural output in response to CO, whereas both respond to a decreased arterial PO2 (hypoxic hypoxia, HH) and 2) the aortic nerves in cats are easily transected. Hence, both cb and ab sent neural activity to the brain stem when the intact cat was exposed to 10% O2 in N2. Only the ab sent information during CO hypoxia (COH intact). Only the cb did so during HH in the cat in which the aortic nerves had been transected, removing the aortic body (HH abr); neither ab nor cb did so during COH abr. Fifteen anesthetized paralyzed artificially ventilated cats were fit with catheters in the femoral artery and vein, right and left atria, left ventricle, and pulmonary artery and with an aortic flow probe. In the HH intact and HH abr conditions, there was a significant rise in cardiac output, whereas pulmonary arterial pressure (Ppa) rose initially but then leveled off while cardiac output continued to rise. During the 15-min exposure to HH, pulmonary vascular resistance [PVR = (Ppa - Pla)/cardiac output, where Pla is left atrial pressure] rose initially and then decreased significantly at 2-3 min. In response to COH, PVR showed only a significant decrease. In the second set of experiments, seven cats were instrumented as above and had loops placed in the common carotid arteries for selectively perfusing the cbs. In response to a brief infusion of venous blood mixed with 0.3-0.5 micrograms NaCN, which selectively stimulated only the cb, aortic flow remained relatively constant while heart rate and Ppa - alveolar pressure difference decreased significantly; so also did PVR. These data are consistent with the hypothesis that stimulation of the ab and cb singly or together can provoke a significant pulmonary vasodilation in the anesthetized paralyzed artificially ventilated cat.     

Tissue slice cultures from humans or rodents: a new tool to evaluate biological effects of heavy ions

The aim of this interdisciplinary project is to establish slice culture preparations from rodents and humans as a new model system for studying effects of X-rays and heavy ions within normal and tumor tissues. The advantage of such slice cultures relies on the conservation of an organotypic environment, the easy treatment and observation by live-imaging microscopy, and the independence from genetic immortalization strategies used to generate cell lines. Rat brains as well as human tumors were cut into 300-μm-thick sections and cultivated in an incubator in a humidified atmosphere at 37°C. This is realized by a membrane-based culture system with a liquid–air interface. With this system, it is possible to keep rodent slices viable for several months. Human brain tumor slices remained vital for at least 21 days. Slices were irradiated with X-rays at the radiation facility of the University Hospital in Frankfurt/Main at doses up to 40 Gy. Heavy ion irradiations were performed at GSI (Darmstadt) with different ions, energies, and doses. The irradiated slices were analyzed by 3D-confocal microscopy following immunostaining for DNA damage, microglia, and proliferation markers. The phosphorylated histone γH2AX proved to be suitable for the detection of ion traversals in this system.