Utilizing the nanosecond pulse technique to improve antigen intracellular delivery and presentation to treat tongue squamous cell carcinoma

BACKGROUND: Tongue squamous cell carcinoma is the most common squamous cell carcinoma of the head and neck. Immunotherapy has great potential in the treatment of tongue squamous cell carcinoma because of its unique advantages. However, the efficacy of immunotherapy is limited by the efficiency of antigen phagocytosis by immune cells. MATERIAL AND METHODS: We extracted dendritic cells (DCs) from human peripheral blood. Utilizing a nanosecond pulsed electric field (nsPEF), we deliver the tumour lysate protein into DCs and then incubate the DCs with PBMCs to obtain specific T cells to kill tumour cells. The biosafety of nsPEF was evaluated by the ANNEXIN V-FITC/PI kit. The efficacy of lysate protein delivery was evaluated by flow cytometry. The antitumour efficacy was tested by CCK-8 assay. RESULTS: The nsPEF of the appropriate field strength can significantly improve the phagocytic ability of DCs to tumour lysing proteins and have good biosafety. The tumour cell killing rate of the nsPEF group was higher than the other group (p< 0.05). CONCLUSIONS: Utilizing nsPEF to improve the phagocytic and presenting ability of DCs could greatly activate the adaptive immune cells to enhance the immunotherapeutic effect on tongue squamous cell carcinoma

Chlamydia pneumoniae CPj0783 interaction with Huntingtin-protein14

Chlamydia pneumoniae is a Gram-negative, obligate intracellular pathogen that causes community-acquired respiratory infections. After C. pneumoniae invades host cells, it disturbs the vesicle transport system to escape host lysosomal or autophagosomal degradation. By using a yeast mis-sorting assay, we found 10 C. pneumoniae candidate genes involved in aberrant vesicular trafficking in host cells. One of the candidate genes, CPj0783, was recognized by antibodies from C. pneumoniae-infected patients. The expression of CPj0783 was detected at mid to late-cycle time points and increased during the inclusion maturation. Two-hybrid screening in yeast cells revealed that CPj0783 interacted with Huntingtin-interacting protein 14 (HIP14). The specific interaction between CPj0783 and HIP14 could be demonstrated by an in vivo co-immunoprecipitation assay and an in vitro GST pull-down assay. It was also demonstrated that HIP14 was localized in the Golgi apparatus and colocalized with CPj0783. HIP14 has a palmitoyl transferase activity that is involved in the palmitoylation-dependent vesicular trafficking of several acylated proteins. These findings suggest that CPj0783 might cause abnormal vesicle-mediated transport by interacting with HIP14 (AU)

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Mechanisms associated to impaired activity of cardiac P-type ATPases in endothelial nitric oxide synthase knockout mice

The effect of long-lasting in vivo restriction of nitric oxide (NO) bioavailability on cardiac and renal P-type ATPases critical for intracellular ion homeostasis is controversial. Previous work has shown in eNOS knockout (eNOS−/−) mice hearts that Na+/K+- and Ca2+-ATPase activities were depressed but the underlying mechanisms are still unclear. The goal of this study was to characterize potential alterations responsible for impaired enzyme activity in eNOS−/− mice. Na+/K+-ATPase activity from crude preparations of adult male eNOS−/− mice hearts and kidneys was reduced compared with wild-type animals (32 %, p < 0.05 and 16 %, p < 0.0001, respectively). Immunoblot analysis showed that although the expression of the predominant (or exclusive, for the kidney) Na+/K+-ATPase á1 isoform was not significantly changed, there was an important downregulation of the less abundant á2 isoform in the heart (57 %, p < 0.0001). In addition, although cardiac Ca2+-ATPase activity was unaltered, the expression of sarco/endoplasmic reticulum Ca2+-ATPase 2 protein in eNOS−/− mice was very high (290 % compared with wild-type animals, p < 0.0001) without any significant change in phospholamban expression. Consistent with these findings, the content of cardiac and renal free sulfhydryl groups, essential for the catalytic function of such ATPases, was decreased (23 %, p < 0.01 and 35 %, p < 0.05, respectively). Altogether, the present results suggest that the absence of eNOS promotes a compartmentalized altered redox balance that affects the activity and expression of ion transport ATPases (AU)

The hidden side of the prokaryotic cell: rediscovering the microbial world

How many different forms of life exist and how they are evolutionarily related is one of the most challenging problems in biology. In 1962, Roger Y. Stanier and Cornelis B. van Niel proposed «the concept of a bacterium» and thus allowed (micro)biologists to divide living organisms into two primary groups: prokaryotes and eukaryotes. Initially, prokaryotes were believed to be devoid of any internal organization or other characteristics typical of eukaryotes, due to their minute size and deceptively simple appearance. However, the last few decades have demonstrated that the structure and function of the prokaryotic cell are much more intricate than initially thought. We will discuss here two characteristics of prokaryotic cells that were not known to Stanier and van Niel but which now allow us to understand the basis of many characteristics that are fully developed in eukaryotic cells: First, it has recently become clear that bacteria contain all of the cytoskeletal elements present in eukaryotic cells, demonstrating that the cytoskeleton was not a eukaryotic invention; on the contrary, it evolved early in evolution. Essential processes of the prokaryotic cell, such as the maintenance of cell shape, DNA segregation, and cell division, rely on the cytoskeleton. Second, the accumulation of intracellular storage polymers, such as polyhydroxyalkanoates (a property studied in detail by Stanier and colleagues), provides a clear evolutionary advantage to bacteria. These compounds act as a «time-binding» mechanism, one of several prokaryotic strategies to increases survival in the Earth's everchanging environments (AU)

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TGN1412: Superagonist Dr. Jekyll and Superantigen Mr. Hyde

Los anticuerpos anti-CD28 superagonistas inducen activación de los linfocitos T en ausencia de estimulación del TCR. Los ensayos clínicos de uno de tales anticuerpos, TGN1412, han resultado en severas complicaciones en los voluntarios que lo recibieron. El análisis de las características y modo de acción de los superagonistas de CD28 sugiere que dichos efectos son similares a los previamente observados con el uso terapeútico de anticuerpos anti-CD3 y al síndrome tóxico inducido por superantígenos bacterianos

Superagonistic anti-CD28 antibodies induce T cell activation in the absence of TCR triggering. Clinical trials of one of such antibodies, TGN1412, resulted in severe adverse effects in the volunteers receiving the drug. Analysis of characteristics and mechanism of action suggests that those effects are similar to those previously reported for the therapeutic use of anti-CD3 antibodies and the toxic shock syndrome triggered by bacterial superantigens

Intracellular transport of a heterologous membrane protein, the human transferrin receptor, in Saccharomyces cerevisiae / Transporte intracelular de una proteína de membrana heteróloga, el receptor de transferrina humano, en Saccharomyces cerevisiae

We have analyzed the intracellular behavior of the human transferrin receptor (TfR) in Saccharomyces cerevisiae. The major part of the heterologously expressed TfR, which has previously been used as a model for heterologous expression of membrane proteins in yeast, is localized in the endoplasmic reticulum (ER) membranes; a minor fraction is present in the plasma membrane (PM). The stability of the TfR depends on vacuolar proteases, implying that it is degraded in the vacuolar compartment. Degradation is further dependent on favorable transport conditions to this compartment. The main bottleneck of transport seems to be the transition from the ER to the PM. The chaperone Cne1p, which is involved in quality control in the ER, plays a role in regulating the amount of heterologous TfR, as deletion of CNE1 leads to significant accumulation of the protein. This is the first demonstration of the involvement of CNE1 in regulating the level of heterologous membrane proteins (AU)

Hemos analizado el comportamiento intracelular del receptor de la transferrina humano (TfR) en Saccharomyces cerevisiae. La mayor parte del TfR expresado heterólogamente, usado previamente como modelo de expresión heteróloga de proteínas de membrana en levaduras, se localiza en las membranas del retículo endoplásmico (RE); una fracción menor se encuentra en la membrana plasmática. La estabilidad del TfR depende de proteasas vacuolares, lo cual implica que es degradado en el compartimento vacuolar. La degradación depende asimismo de condiciones favorables de transporte a este compartimento. El factor limitante del transporte parece ser la transición desde el RE a la membrana plasmática. La chaperona Cne1p, implicada en el control de calidad en el RE, juega un papel importante en la regulación de los niveles de TfR heterólogo, ya que la deleción de CNE1 provoca una acumulación significativa de la proteína. Se trata de la primera demostración de la implicación de CNE1 en la regulación de los niveles de proteínas de membrana heterólogas (AU)

Enzymatic systems of inorganic pyrophosphate bioenergetics in photosynthetic and heterotrophic protists: remnants or metabolic cornerstones?

An increasing body of biochemical and genetic evidence suggests that inorganic pyrophosphate (PPi) plays an important role in protist bioenergetics. In these organisms, two types of inorganic pyrophosphatases [EC 3.6.1.1, namely soluble PPases (sPPases) and proton-translocating PPases (H+-PPases)] that hydrolyse the PPi generated by cell anabolism, thereby replenishing the orthophosphate pool needed for phosphorylation reactions, are present in different cellular compartments. Photosynthetic and heterotrophic protists possess sPPases located in cellular organelles (plastids and mitochondria), where many anabolic and biosynthetic reactions take place, in addition to H+-PPases, which are integral membrane proteins of the vacuolysosomal membranes and use the chemical energy of PPi to generate an electrochemical proton gradient useful in cell bioenergetics. This last category of proton pumps was considered to be restricted to higher plants and some primitive photosynthetic bacteria, but it has been found recently in many protists (microalgae and protozoa) and bacteria, thus indicating that H+-PPases are much more widespread than previously thought. No cytosolic sPPase (in bacteria, fungi and animal cells) has been shown to occur in these lower eukaryotes. The widespread occurrence of these key enzymes of PPi metabolism among evolutionarily divergent protists strongly supports the ancestral character of the bioenergetics based on this simple energy-rich compound, which may play an important role in survival under different biotic and abiotic stress conditions (AU)

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