Effects of Sirolimus treatment on patients with β-Thalassemia: Lymphocyte immunophenotype and biological activity of memory CD4+ and CD8+ T cells

Inhibitors of the mammalian target of rapamycin (mTOR) have been proposed to improve vaccine responses, especially in the elderly. Accordingly, testing mTOR inhibitors (such as Sirolimus) and other geroprotective drugs might be considered a key strategy to improve overall health resilience of aged populations. In this respect, Sirolimus (also known as rapamycin) is of great interest, in consideration of the fact that it is extensively used in routine therapy and in clinical studies for the treatment of several diseases. Recently, Sirolimus has been considered in laboratory and clinical studies aimed to find novel protocols for the therapy of hemoglobinopathies (e.g. β-Thalassemia). The objective of the present study was to analyse the activity of CD4⁺ and CD8⁺ T cells in β-Thalassemia patients treated with Sirolimus, taking advantages from the availability of cellular samples of the NCT03877809 clinical trial. The approach was to verify IFN-γ releases following stimulation of peripheral blood mononuclear cells (PBMCs) to stimulatory CEF and CEFTA peptide pools, stimulatory for CD4⁺ and CD8⁺ T cells, respectively. The main results of the present study are that treatment of β-Thalassemia patients with Sirolimus has a positive impact on the biological activity and number of memory CD4⁺ and CD8⁺ T cells releasing IFN-γ following stimulation with antigenic stimuli present in immunological memory. These data are to our knowledge novel and in our opinion of interest, in consideration of the fact that β-Thalassemia patients are considered prone to immune deficiency.     

Tetanus toxin selectively impairs anti-tumoral but not anti-microbial macrophage-mediated effector functions

Abstract The present study was designed to establish the susceptibility of macrophage-mediated effector functions to tetanus toxin (TT). Using the murine macrophage cell line, GG2EE, generated in vitro by v- raf /v- myc oncogenes, we have previously provided evidence that TT selectively inhibits interferon gamma (IFN-γ), but not basal, lysozyme activity. Here we show that while neither phagocytic nor candidacidal activities are affected by TT treatment, antitumoral activity is significantly impaired after exposure to TT. This phenomenon, which is dose-dependent, is fully ascribed to the holotoxin, as heat inactivated TT, C or A-B fragments result ineffective. Furthermore, C but not A-B fragment competes with TT in abrogating its inhibitory effects. Overall, these data indicate that TT is not a broad-spectrum, down-regulating signal on macrophage-mediated functions, thus implying that its toxic action is exerted on specific molecular targets.     

Immunocytochemical analysis of phosphatidylinositol-specific phospholipase C in PC12 cells: predominance of the δ isoform during neural differentiation

The rat pheochromocytoma PC12 cell line, which differentiates into sympathetic neurons under nerve growth factor (NGF) treatment, contains at least three phosphoinositidase C (PIC) isozymes, PIC , PIC , PIC . These isozymes have been previously shown to display a different subcellular localization. To determine whether or not NGF induces changes in the presence and/or distribution of PIC isozymes during PC12 neural differentiation, studies were carried out by means of in situ immunocytochemistry. After NGF administration the proliferative activity was progressively reduced to very low levels, as measured by bromodeoxyUridine incorporation, and a neuron-like morphology was displayed by almost all cells. In unstimulated PC12 cells, PIC was detected in the nucleus whereas PIC was only cytoplasmic; PIC was found in both cell compartments. In cells treated with NGF for 3 days, neural processes extended to twice the diameter of the cell body; the isoform was concentrated near the nucleus, while the immunoreactivity of the form remained constant and the form was increased. After 10 days of treatment with NGF, PIC was hardly detectable and PIC immunostaining was considerably decreased. On the contrary, PIC progressively increased and, after 14 days of NGF exposure, fully differentiated cells displayed an intense labelling of cell body and neurites. In the same cells, PIC and PIC were almost negative. These results suggest that NGF dependent neural differentiation is related to the selective down regulation of PIC and and the increase of PIC isozyme associated with the decrease of cell proliferation.     

Cyclodextrins enhance the aerobic degradation and dechlorination of low-chlorinated biphenyls

Summary The aerobic dechlorination of 4-chlorobiphenyl and of Aroclor 1221 by Pseudomonas sp. CPE1 strain was enhanced by the presence of hydroxypropyl--cyclodextrin in the medium. The best dechlorination results were obtained when 4-chlorobiphenyl or Aroclor 1221 and the cyclodextrin were used at the molar ratios 1:1 and 1:1.5. This agent can be adopted to enhance the efficiency of PCB degradation tests.     

Aqueous plant extracts as stimulators of laccase production in liquid cultures of Lentinus edodes

Summary Total and specific activities of extra-cellular laccases from Lentinus edodes were enhanced by adding corn straw and chestnut juice to the liquid growth medium. The aqueous extracts were chemically characterized and revealed the presence of several phenolic and non-phenolic compounds. Extensive extraction of these components from the tested extracts completely annulled their stimulating properties on laccase production, suggesting that these compounds can act at micromole levels.     

Poplar (Populus nigra L.) plants transformed with aBacillus thuringiensis toxin gene: insecticidal activity and genomic analysis

Insect-resistant poplar (Populus nigra L.) plants have been produced by infecting leaves withAgrobacterium tumefaciens strains carrying a binary vector containing different truncated forms of aBacillus thuringiensis (B.t.) toxin gene under a duplicated CaMV 35S promoter. Putative transgenic plants were propagated by cuttings at two experimental farms (in Beijing and Xinjiang, China). At 2–3 years after transformation, 17 of them were selected on the bases of insect-tolerance and good silvicultural traits, and evaluated for insect resistance, for the presence of theB.t. toxin DNA fragment (Southern blots and PCR) and for the expression of the transgene (western and northern blots). Somaclonal variation, as suggested by the appearance of permanent changes in the shape of the leaves, was also investigated with molecular tools (RFLP (restriction fragment length polymorphism), RAPD (random amplified polymorphic DNA) and microsatellite DNA).Bioassays withApochemia cineraius andLymantria dispar on the leaves of the selected clones showed different and, in some cases, high levels of insecticidal activity. The molecular analysis demonstrated integration and expression of the foreign gene. Somatic changes were correlated to extensive genomic changes and were quantified in dendrograms, in terms of genomic similarity. The analysis of control plants suggested that genomic changes were correlated to thein vitro culture step necessary forA. tumefaciens-mediated gene transfer, rather than to the integration of the foreign genes.Three transgenic clones (12, 153 and 192), selected for insect resistance, reduced morphological changes and promising silvicultural traits, are now under large-scale field evaluation in six different provinces in China.     

An amino-terminal domain of the hepatitis C virus NS3 protease is essential for interaction with NS4A.

Hepatitis C virus (HCV) genomic RNA is translated into a large polyprotein that is processed into structural and nonstructural proteins. Processing at the N termini of several nonstructural proteins requires sequences contained in both NS3 and NS4A. NS3 contains a serine protease, whereas the function of NS4A in proteolysis is yet to be determined. By using the vaccinia virus-T7 hybrid expression system to transiently express HCV polypeptides in HeLa cells, we studied the effect of several N-terminal and C-terminal deletions of HCV NS3 on the processing activity at all the downstream cleavage sites. In this way, we have delineated the minimal domain of NS3 required for the serine protease activity associated with this protein. In addition, we demonstrate the formation of a stable complex between NS3 and NS4A: analysis of the deletion mutants reveals a region at the N terminus of NS3 that is necessary for both complex formation and modulation of the proteolytic activity by NS4A but not for the NS4A-independent serine protease activity of NS3.     

Insertion mutations at the maizeOpaque2 locus induced by transposable element familiesAc, En/Spm andBg

Eight independently isolated unstable alleles of theOpaque2 (O2) locus were analysed genetically and at the DNA level. The whole series of mutations was isolated from a maize strain carrying a wild-typeO2 allele and the transposable elementActivator (Ac) at thewx-m7 allele. Previous work with another unstable allele of the same series has shown that it was indeed caused by the insertion of anAc element. Unexpectedly, the remaining eight mutations were not caused by the designatedAc element, but by other insertions that are structurally similar or identical to one of two different autonomous transposable elements. Six mutations were caused by the insertion of a transposable element of theEnhancer/Suppressor-Mutator (En/Spm) family. Two mutations were the result of the insertion of a transposable element of theBergamo (Bg) family. Genetic tests carried out with plants carrying the unstable mutations demonstrated that all were caused by the insertion of an autonomous transposable element.