Modulation of the immune response induced by gene electrotransfer of a hepatitis C virus DNA vaccine in nonhuman primates

Induction of multispecific, functional CD4+ and CD8+ T cells is the immunological hallmark of acute self-limiting hepatitis C virus (HCV) infection in humans. In the present study, we showed that gene electrotransfer (GET) of a novel candidate DNA vaccine encoding an optimized version of the nonstructural region of HCV (from NS3 to NS5B) induced substantially more potent, broad, and long-lasting CD4+ and CD8+ cellular immunity than naked DNA injection in mice and in rhesus macaques as measured by a combination of assays, including IFN-gamma ELISPOT, intracellular cytokine staining, and cytotoxic T cell assays. A protocol based on three injections of DNA with GET induced a substantially higher CD4+ T cell response than an adenovirus 6-based viral vector encoding the same Ag. To better evaluate the immunological potency and probability of success of this vaccine, we have immunized two chimpanzees and have compared vaccine-induced cell-mediated immunity to that measured in acute self-limiting infection in humans. GET of the candidate HCV vaccine led to vigorous, multispecific IFN-gamma+CD8+ and CD4+ T lymphocyte responses in chimpanzees, which were comparable to those measured in five individuals that cleared spontaneously HCV infection. These data support the hypothesis that T cell responses elicited by the present strategy could be beneficial in prophylactic vaccine approaches against HCV.     

IL-21 counteracts the regulatory T cell-mediated suppression of human CD4+ T lymphocytes

High expression of IL-21 and/or IL-21R has been described in T cell-mediated inflammatory diseases characterized by defects of counterregulatory mechanisms. CD4(+)CD25(+) regulatory T cells (Treg) are a T cell subset involved in the control of the immune responses. A diminished ability of these cells to inhibit T cell activation has been documented in immune-inflammatory diseases, raising the possibility that inflammatory stimuli can block the regulatory properties of Treg. We therefore examined whether IL-21 controls CD4(+)CD25(+) T cell function. We demonstrate in this study that IL-21 markedly enhances the proliferation of human CD4(+)CD25(-) T cells and counteracts the suppressive activities of CD4(+)CD25(+) T cells on CD4(+)CD25(-) T cells without affecting the percentage of Foxp3(+) cells or survival of Treg. Additionally, CD4(+)CD25(+) T cells induced in the presence of IL-21 maintain the ability to suppress alloresponses. Notably, IL-21 enhances the growth of CD8(+)CD25(-) T cells but does not revert the CD4(+)CD25(+) T cell-mediated suppression of this cell type, indicating that IL-21 makes CD4(+) T cells resistant to suppression rather than inhibiting CD4(+)CD25(+) T cell activity. Finally, we show that IL-2, IL-7, and IL-15, but not IL-21, reverse the anergic phenotype of CD4(+)CD25(+) T cells. Data indicate that IL-21 renders human CD4(+)CD25(-) T cells resistant to Treg-mediated suppression and suggest a novel mechanism by which IL-21 could augment T cell-activated responses in human immune-inflammatory diseases.     

Hydroxytyrosol glucuronides protect renal tubular epithelial cells against H(2)O(2) induced oxidative damage

Hydroxytyrosol (2-(3′,4′-dihydroxyphenyl)ethanol; HT), the most active ortho-diphenolic compound, present either in free or esterified form in extravirgin olive oil, is extensively metabolized in vivo mainly to O-methylated, O-sulfated and glucuronide metabolites. We investigated the capacity of three glucuronide metabolites of HT, 3′-O-β-d-glucuronide and 4′-O-β-d-glucuronide derivatives and 2-(3′,4′-dihydroxyphenyl)ethanol-1-O-β-d-glucuronide, in comparison with the parent compound, to inhibit H₂O₂ induced oxidative damage and cell death in LLC-PK1 cells, a porcine kidney epithelial cell line. H₂O₂ treatment exerted a toxic effect inducing cell death, interacting selectively within the pro-death extracellular-signal relate kinase (ERK 1/2) and the pro-survival Akt/PKB signaling pathways. It also produced direct oxidative damage initiating the membrane lipid peroxidation process. None of the tested glucuronides exhibited any protection against the loss in renal cell viability. They also failed to prevent the changes in the phosphorylation states of ERK and Akt, probably reflecting their inability to enter the cells, while HT was highly effective. Notably, pretreatment with glucuronides exerted a protective effect at the highest concentration tested against membrane oxidative damage, comparable to that of HT: the formation of malondialdehyde, fatty acid hydroperoxides and 7-ketocholesterol was significantly inhibited.     

A Novel Spore Protein,ExsM, Regulates Formation of the Exosporium in Bacillus cereus and Bacillus anthracis and Affects Spore Size and Shape

Bacillus cereus spores are assembled with a series of concentric layers that protect them from a wide range of environmental stresses. The outermost layer, or exosporium, is a bag-like structure that interacts with the environment and is composed of more than 20 proteins and glycoproteins. Here, we identified a new spore protein, ExsM, from a β-mercaptoethanol extract of B. cereus ATCC 4342 spores. Subcellular localization of an ExsM-green fluorescent protein (GFP) protein revealed a dynamic pattern of fluorescence that follows the site of formation of the exosporium around the forespore. Under scanning electron microscopy, exsM null mutant spores were smaller and rounder than wild-type spores, which had an extended exosporium (spore length for the wt, 2.40 ± 0.56 μm, versus that for the exsM mutant, 1.66 ± 0.38 μm [P < 0.001]). Thin-section electron microscopy revealed that exsM mutant spores were encased by a double-layer exosporium, both layers of which were composed of a basal layer and a hair-like nap. Mutant exsM spores were more resistant to lysozyme treatment and germinated with higher efficiency than wild-type spores, and they had a delay in outgrowth. Insertional mutagenesis of exsM in Bacillus anthracis ΔSterne resulted in a partial second exosporium and in smaller spores. In all, these findings suggest that ExsM plays a critical role in the formation of the exosporium.Bacillus cereus and Bacillus anthracis are closely related members of the Bacillus cereus group (47). Although B. cereus is mainly an apathogenic organism, certain isolates can cause two different types of food poisoning, emetic syndrome and diarrheal disease (18). The emetic syndrome is caused by ingestion of cereulide, a heat-resistant toxin produced by vegetative cells contaminating the food (30), while the diarrheal disease occurs when spores germinate in the intestinal tract. Spores are also the infective agent in anthrax, a disease caused by B. anthracis (64).B. cereus and B. anthracis differentiate into spores when faced with nutrient deprivation. The spore is a dormant cell type that can remain viable for decades until favorable conditions induce germination and the resumption of vegetative growth. The remarkable resistance properties of the spore result from its unique architecture, consisting of a series of concentric protective layers (51). The spore core contains the genetic material and is surrounded by the cortex, a thick layer of modified peptidoglycan that promotes a highly dehydrated state. Encasing the core and the cortex, the coat is a multilayer protein shell that provides mechanical and chemical resistance. In addition, both the cortex and coat contribute to spore germination (17). Separated from the coat by an interspace, the exosporium encloses the rest of the spore, and it is composed of an inner basal layer and an outer hair-like nap (25).Being the most external layer of the spore, the exosporium interacts directly with the environment and as such provides a semipermeable barrier that may exclude large molecules, like antibodies and hydrolytic enzymes (3, 23, 24, 54). However, the exosporium does not appear to contribute to the typical resistance properties of the spore (6, 35, 60). Also, the exosporium is not necessary in anthrax pathogenesis when tested under laboratory conditions (7, 27, 59), although it is able to down-modulate the innate immune response to spores and mediate adhesion to host tissues (4, 8, 43, 44). The exosporium may also help the spore avoid premature germination in unsustainable environments, since it contains two enzymes, alanine racemase (Alr) and inosine hydrolase (Iunh), that can inactivate low quantities of the germinants l-alanine and inosine, respectively (6, 48, 55, 61). However, regulation of germination by the exosporium is poorly understood. Mutation of exosporial proteins has resulted in only negligible and inconsistent germination phenotypes (2, 5, 27, 28, 52, 54).The exosporium is composed of at least 20 proteins and glycoproteins in tight or loose association (48, 53, 57, 61, 65). These proteins are synthesized in the mother cell and always start self-assembly at the forespore pole near the middle of the mother cell, concurrently with the cortex and coat formation (42). Exosporium assembly is discontinuous and starts with a synthesis of a substructure known as the cap, which likely contains only a subset of the proteins present in the exosporium (55). After cap formation, construction of the rest of the exosporium requires the expression of ExsY (6). BclA is the main component of the hair-like nap on the external side of the exosporium, and it is linked to the basal layer through interaction with ExsFA/BxpB (54, 58). In addition, CotE participates in the correct attachment of the exosporium to the spore (27).Despite these findings, exosporium assembly continues to be a poorly understood process, and many questions remain regarding its composition and the regulation of its synthesis. In this study, we characterized a new spore protein, ExsM, which plays a key role in assembly of the exosporium. In B. cereus, inactivation of exsM resulted in spores with an unusual double-layer exosporium, and a similar phenotype was also observed in B. anthracis exsM null mutant spores. Finally, double-layer exosporium spores allowed us to study the role of the exosporium in germination and outgrowth.     

Increased kynurenine-to-tryptophan ratio in the serum of patients infected with SARS-CoV2: An observational cohort study.

Immune dysregulation is a hallmark of patients infected by SARS-CoV2 and the balance between immune reactivity and tolerance is a key determinant of all stages of infection, including the excessive inflammatory state causing the acute respiratory distress syndrome. The kynurenine pathway (KP) of tryptophan (Trp) metabolism is activated by pro-inflammatory cytokines and drives mechanisms of immune tolerance. We examined the state of activation of the KP by measuring the Kyn:Trp ratio in the serum of healthy subjects (n = 239), and SARS-CoV2-negative (n = 305) and -positive patients (n = 89). Patients were recruited at the Emergency Room of St. Andrea Hospital (Rome, Italy). Kyn and Trp serum levels were assessed by HPLC/MS-MS. Compared to healthy controls, both SARS-CoV2-negative and -positive patients showed an increase in the Kyn:Trp ratio. The increase was larger in SARS-CoV2-positive patients, with a significant difference between SARS-CoV2-positive and -negative patients. In addition, the increase was more prominent in males, and positively correlated with age and severity of SARS-CoV2 infection, categorized as follows: 1 = no need for intensive care unit (ICU); 2 ≤ 3 weeks spent in ICU; 3 ≥ 3 weeks spent in ICU; and 4 = death. The highest Kyn:Trp values were found in SARS-CoV2-positive patients with severe lymphopenia. These findings suggest that the Kyn:Trp ratio reflects the level of inflammation associated with SARS-CoV2 infection, and, therefore, might represent a valuable biomarker for therapeutic intervention.     

Regulation of phospholipid synthesis inMycobacterium smegmatis by cyclic adenosine monophosphate

Forskolin, an adenylate cyclase activator and a cyclic AMP analogue, dibutyryl cyclic AMP have been used to examine the relationship between intracellular levels of cyclic AMP and lipid synthesis inMycobacterium smegmatis. Total phospholipid content was found to be increased in forskolin grown cells as a result of increased cyclic AMP levels caused by activation of adenylate cyclase. Increased phospholipid content was supported by increased [¹⁴C] acetate incorporation as well as increased activity of glycerol-3-phosphate acyltransferase. Pretreatment of cells with dibutyryl cyclic AMP had similar effects on lipid synthesis. Taking all these observations together it is suggested that lipid synthesis is being controlled by cyclic AMP in mycobacteria.     

Adenoviral Delivery of Angiotensin-(1-7) or Angiotensin-(1-9) Inhibits Cardiomyocyte Hypertrophy via the Mas or Angiotensin Type 2 Receptor

The counter-regulatory axis of the renin angiotensin system peptide angiotensin-(1-7) [Ang-(1-7)] has been identified as a potential therapeutic target in cardiac remodelling, acting via the mas receptor. Furthermore, we recently reported that an alternative peptide, Ang-(1-9) also counteracts cardiac remodelling via the angiotensin type 2 receptor (AT₂R). Here, we have engineered adenoviral vectors expressing fusion proteins which release Ang-(1-7) [RAdAng-(1-7)] or Ang-(1-9) [RAdAng-(1-9)] and compared their effects on cardiomyocyte hypertrophy in rat H9c2 cardiomyocytes or primary adult rabbit cardiomyocytes, stimulated with angiotensin II, isoproterenol or arg-vasopressin. RAdAng-(1-7) and RAdAng-(1-9) efficiently transduced cardiomyocytes, expressed fusion proteins and secreted peptides, as demonstrated by western immunoblotting and conditioned media assays. Furthermore, secreted Ang-(1-7) and Ang-(1-9) inhibited cardiomyocyte hypertrophy (Control = 168.7±8.4 µm; AngII = 232.1±10.7 µm; AngII+RAdAng-(1-7) = 186±9.1 µm, RAdAng-(1-9) = 180.5±9 µm; P<0.05) and these effects were selectively reversed by inhibitors of their cognate receptors, the mas antagonist A779 for RAdAng-(1-7) and the AT₂R antagonist PD123,319 for RAdAng-(1-9). Thus gene transfer of Ang-(1-7) and Ang-(1-9) produces receptor-specific effects equivalent to those observed with addition of exogenous peptides. These data highlight that Ang-(1-7) and Ang-(1-9) can be expressed via gene transfer and inhibit cardiomyocyte hypertrophy via their respective receptors. This supports applications for this approach for sustained peptide delivery to study molecular effects and potential gene therapeutic actions.     

Osteogenesis promoted by calcium phosphate N,N-dicarboxymethyl chitosan

The effects of N,N-dicarboxymethyl chitosan (DCMC) on the precipitation of insoluble calcium salts, namely phosphate, sulfate, oxalate, carbonate, bicarbonate and fluoride, and magnesium salts, namely phosphate and carbonate, were studied. Results indicated that the chelating ability of DCMC interfered effectively with the well-known physico-chemical behaviour of magnesium and calcium salts. Dicarboxymethyl chitosan formed self-sustaining gels upon mixing with calcium acetate, as a consequence of calcium chelation. DCMC mixed with calcium acetate and with disodium hydrogen phosphate in appropriate ratios (molar ratio Ca/DCMC close to 2.4) yielded a clear solution, from which, after dialysis and freeze-drying, an amorphous material was isolated containing an inorganic component about one half its weight. This compound was used for the treatment of bone lesions in experimental surgery and in dentistry. Bone tissue regeneration was promoted in sheep, leading to complete healing of otherwise non-healing surgical defects. Radiographic evidence of bone regeneration was observed in human patients undergoing apicectomies and avulsions. The DCMC–CaP chelate favoured osteogenesis while promoting bone mineralization.