Original Article

The pancreas taken from the frog (Rana nigromaculata) was fixed in 1% OsO_4 and sliced into ultrathin sections for electron microscopic studies. The following observations were made: 1. A great \u27number of minute granules found in the cytoplasm of a pancreatic cell were called the microsomes, which were divided into two types, the C-microsome and S-microsome. 2. Electron microsopic studies of the ergastoplasm showed that it is composed of the microsome granules and A-substance. The microsomes were seen embedded in the A-substance which was either filamentous or membranous. The membranous structure, which was called the Am-membrane, was seen to form a sac, with a cavity of varying sizes, or to form a lamella. 3. The Am-membrane has close similarity to α-cytomembrane of Sjostrand, except that the latter is rough-surfaced. It was deduced that the Am-membrane, which is smooth-surfaced, might turn into the rough-surfaced α-cytomembrane. 4. There was the Golgi apparatus in the supranuclear region of a pancreatic cell. It consisted of the Golgi membrane, Golgi vacuole and. Golgi vesicle. 5. The mitochondria of a pancreatic cell appeared like long filaments, and some of them were seen to ramify. 6. The membrane of mitochondria, i. e. the limiting membrane, consisted of the Ammembrane. The mitochondria contained a lot of A-substances, as well as the C-microsomes and S-microsomes. When the mitochondria came into being, there appeared inside them chains of granules, which appeared like strips of beads, as the outgrowths of the A-substance and the microsome granules attached to the Am-membrane. They are the so-called cristae mitochondriales. 7. The secretory granules originate in the microsomes. They came into being when the microsomes gradually thickened and grew in size as various substances became adhered to them. Some of the secretory granules were covered with a membrane and appeared like what they have called the intracisternal granule of Palade.It seemed that this was a phenomenon attendant upon the dissolution and liqutefaction of the secretory granule. 8. Comparative studies were made of the ergastoplasm of the pancreatic cells from the frogs in hibernation, the frogs artificially hungered, the frogs which were given food after a certain period of fasting, the frogs to which pilocarpine was given subcutaneously, and the very young, immature frogs. The studies revealed that the ergastoplasm of the pancreatic cells greatly varied in form with the difference in nutritive condition and with different developmental stages of the cell. The change in form and structure occured as a result of transformation of the microsomes and A-substance. The ergastoplasm, even after it has come into being, might easily be inactivated if nutrition is defective. The ergastoplasm is concerned in the secretory mechanism, which is different from the secretory phenomenon of the secretory granules. It would seem that structurally the mitochondria have no direct relation to this mechanism

Extracellular vesicle-mediated intercellular communication in HIV-1 infection and its role in the reservoir maintenance

HIV-1 infection is efficiently controlled by combination anti-retroviral therapy (cART). However, despite preventing disease progression, cART does not eradicate virus infection which persists in a latent form for an individual's lifetime. The latent reservoir comprises memory CD4+ T lymphocytes, macrophages, and dendritic cells; however, for the most part, the reservoir is generated by virus entry into activated CD4+ T lymphocytes committed to return to a resting state, even though resting CD4+ T lymphocytes can be latently infected as well. The HIV-1 reservoir is not recognized by the immune system, is quite stable, and has the potential to re-seed systemic viremia upon cART interruption. Viral rebound can occur even after a long period of cART interruption. This event is most likely a consequence of the extended half-life of the HIV-1 reservoir, the maintenance of which is not clearly understood. Several recent studies have identified extracellular vesicles (EVs) as a driving force contributing to HIV-1 reservoir preservation. In this review, we discuss recent findings in the field of EV/HIV-1 interplay, and then propose a mechanism through which EVs may contribute to HIV-1 persistence despite cART. Understanding the basis of the HIV-1 reservoir maintenance continues to be a matter of great relevance in view of the limitations of current strategies aimed at HIV-1 eradication

Thermal and microchemical characterisations of CaSO4-SiO2 investment materials for casting jewellery alloys

Differential thermal analysis (DTA) and thermogravimetry (TG), X-ray photoelectron spectroscopy (XPS), and scanning electron microscopy (SEM) and energy dispersive spectrometry (EDS) were used to study the thermal decomposition of calcium sulphate in the CaSO4 (25 wt%)-bonded silica investment which is the commonly used material for casting jewellery gold-based alloys. The thermal decomposition of CaSO4 generates sulphur dioxide, leads to gas porosity in the molten gold-based alloys and therefore, defective jewellery products. This latter reaction was studied as a function of the temperature and atmosphere (air, argon and argon-5% hydrogen). in order to simulate, as for as possible, different casting conditions used by the jewellery industry. Furthermore, special attention was given to the effect of the presence of Zn, Cu2O. CuO and Ag2O on the thermal decomposition of CaSO4. DTG-TG results confirmed that the temperature of the thermal decomposition of CaSO4 bonded with silica was lower with respect to the nearly pure CaSO4. Unfortunately, it was very close to the casting temperature of some typical gold alloys. in addition, the temperature of the decomposition was further lowered when inert and reducing atmospheres are used, as well as in the presence of ZnO, Cu2O. CuO and Ag2O. (C) 1998 Elsevier Science B.V

N-terminal fatty acids of NEFmut are required for the CD8+ t-cell immunogenicity of in vivo engineered extracellular vesicles

We recently described a cytotoxic CD8+ T lymphocyte (CTL) vaccine platform based on the intramuscular (i.m.) injection of DNA eukaryotic vectors expressing antigens of interest fused at the C-terminus of HIV-1 Nefmut, i.e., a functionally defective mutant that is incorporated at quite high levels into exosomes/extracellular vesicles (EVs). This system has been proven to elicit strong CTL immunity against a plethora of both viral and tumor antigens, as well as inhibit both transplantable and orthotopic tumors in mice. However, a number of open issues remain regarding the underlying mechanism. Here we provide evidence that hindering the uploading into EVs of Nefmut-derived products by removing the Nefmut N-terminal fatty acids leads to a dramatic reduction of the downstream antigen-specific CD8+ T-cell activation after i.m. injection of DNA vectors in mice. This result formally demonstrates that the generation of engineered EVs is part of the mechanism underlying the in vivo induced CD8+ T-cell immunogenicity. Gaining new insights on the EV-based vaccine platform can be relevant in view of its possible translation into the clinic to counteract both chronic and acute infections as well as tumors

Cell activation and HIV-1 replication in unstimulated CD4+ T lymphocytes ingesting exosomes from cells expressing defective HIV-1

Background: A relevant burden of defective HIV-1 genomes populates PBMCs from HIV-1 infected patients, especially during HAART treatment. These viral genomes, although unable to codify for infectious viral particles, can express viral proteins which may affect functions of host cells as well as bystander ones. Cells expressing defective HIV-1 have a lifespan longer than that of cells producing infectious particles. Hence, their interaction with other cell types, including resting lymphocytes, is expected to occur frequently in tissues where HIV actively replicates. We investigated the effects of the expression of a prototype of functionally defective HIV-1 on bystander, unstimulated CD4(+) T lymphocytes. Results: We observed that unstimulated human primary CD4(+) T lymphocytes were activated and became permissive for HIV-1 replication when co-cultivated with cells expressing a functionally defective HIV-1 (F12/Hut-78 cells). This effect depended on the presence in F12/Hut-78 supernatants of nanovesicles we identified as exosomes. By inspecting the underlying mechanism, we found that ADAM17, i.e., a disintegrin and metalloprotease converting pro-TNF-alpha in its mature form, associated with exosomes from F12/Hut-78 cells, and played a key role in the HIV-1 replication in unstimulated CD4(+) T lymphocytes. In fact, the treatment with an inhibitor of ADAM17 abolished both activation and HIV-1 replication in unstimulated CD4(+) T lymphocytes. TNF-alpha appeared to be the downstream effector of ADAM17 since the treatment of unstimulated lymphocytes with antibodies against TNF-alpha or its receptors blocked the HIV-1 replication. Finally, we found that the expression of Nef(F12) in exosome-producing cells was sufficient to induce the susceptibility to HIV-1 infection in unstimulated CD4(+) T lymphocytes. Conclusions: Exosomes from cells expressing a functionally defective mutant can induce cell activation and HIV-1 susceptibility in unstimulated CD4(+) T lymphocytes. This evidence highlights the relevance for AIDS pathogenesis of the expression of viral products from defective HIV-1 genomes

Tumor cells endowed with professional antigen-presenting cell functions prime PBLs to generate antitumor CTLs

Abstract: Intrinsic genetic instability of tumor cells leads to continuous production of mutated proteins referred to as tumor-specific neoantigens. Generally, they are recognized as nonself products by the host immune system. However, an effective adaptive response clearing neoantigen-expressing cells is lost in tumor diseases. Most advanced therapeutic strategies aim at inducing neoantigen-specific immune activation through personalized approaches. They include tumor cell exome sequencing, human leukocyte antigen (HLA) typing, synthesis, and injection of peptides/RNA with adjuvants. Here, we propose an innovative method to induce a CD8+ T cytotoxic lymphocyte (CTL) immune response against tumor neoantigens bypassing the steps needed in current therapeutic strategies of personalized vaccination. We assumed that tumor cells can be the most efficient and precise factory of major histocompatibility complex (MHC) class I-associated, tumor neoantigen-derived peptides. Hence, endowing tumor cells with professional antigen-presenting functions would prime CD8+ T lymphocytes towards a response against nonself tumor antigens. To explore this possibility, both adenocarcinoma and melanoma human cells were engineered to express both CD80 and CD86 costimulatory molecules. HLA-matched lymphocytes were then primed through cocultivation with the engineered tumor cells. The generation of tumor-specific CD8+ T lymphocytes was tested through the combined analysis of cell activation markers, formation of immunologic synapses, generation of tumor antigen-specific CD8+ T lymphocytes, and cytotoxic activity. Our data consistently indicate that tumor cells endowed with professional antigen-presenting functions can generate an effective tumor-specific CTL immune response. This finding may open avenues towards the development of innovative antitumor immunotherapies. Key messages: We established a novel method to induce antitumor CTLs without a need to identify TAAs and/or tumor neoantigens.This strategy relies on transducing tumor cells with a retroviral vector expressing both CD80 and CD86.In this way, tumor cells prime naïve CD8+ T lymphocytes in a way that CTLs killing the same tumor cells are generated.These findings open the way towards preclinical assays in the perspective to introduce this antitumor immunotherapy strategy in clinic