Metabolic pathways in immune senescence and inflammaging: Novel therapeutic strategy for chronic inflammatory lung diseases. An EAACI position paper from the Task Force for Immunopharmacology.

The accumulation of senescent cells drives inflammaging and increases morbidity of chronic inflammatory lung diseases. Immune responses are built upon dynamic changes in cell metabolism that supply energy and substrates for cell proliferation, differentiation, and activation. Metabolic changes imposed by environmental stress and inflammation on immune cells and tissue microenvironment are thus chiefly involved in the pathophysiology of allergic and other immune-driven diseases. Altered cell metabolism is also a hallmark of cell senescence, a condition characterized by loss of proliferative activity in cells that remain metabolically active. Accelerated senescence can be triggered by acute or chronic stress and inflammatory responses. In contrast, replicative senescence occurs as part of the physiological aging process and has protective roles in cancer surveillance and wound healing. Importantly, cell senescence can also change or hamper response to diverse therapeutic treatments. Understanding the metabolic pathways of senescence in immune and structural cells is therefore critical to detect, prevent, or revert detrimental aspects of senescence-related immunopathology, by developing specific diagnostics and targeted therapies. In this paper, we review the main changes and metabolic alterations occurring in senescent immune cells (macrophages, B cells, T cells). Subsequently, we present the metabolic footprints described in translational studies in patients with chronic asthma and chronic obstructive pulmonary disease (COPD), and review the ongoing preclinical studies and clinical trials of therapeutic approaches aiming at targeting metabolic pathways to antagonize pathological senescence. Because this is a recently emerging field in allergy and clinical immunology, a better understanding of the metabolic profile of the complex landscape of cell senescence is needed. The progress achieved so far is already providing opportunities for new therapies, as well as for strategies aimed at disease prevention and supporting healthy aging.

The first-generation phosphodiesterase 5 inhibitors and their pharmacokinetic issue.

BACKGROUND: Erectile dysfunction (ED) is a relatively frequent disease that negatively impacts the overall quality of life, well-being, and relationships. Although the use of phosphodiesterase 5 inhibitors (PDE5is) has revolutionized the treatment of ED, a high percentage of ED patients discontinue PDE5i treatment. OBJECTIVES: (i) To analyze the reasons for patient dissatisfaction leading to PDE5i discontinuation; (ii) analyze the pharmacokinetics of new formulations focusing on the time needed to reach an effective plasma concentration of PDE5is (Tonset ) following drug intake; and (iii) summarize the physicochemical properties of sildenafil to understand which excipients may increase the absorption rate. MATERIAL AND METHODS: An online PubMed literature search was conducted to identify English language publications from inception to January 2019. RESULTS: The main reasons for patient dissatisfaction when using PDE5is on demand are the relatively long Tonset after taking vardenafil and sildenafil, including formulations such as film-coated tablets, fine granules, orally disintegrating tablets (ODTs), and oral thin films (ODFs). The relatively long Tonset , further worsened when accompanied by eating, highlights the following: (i) the need for planning intercourse, determining partner-related issues; (ii) issues when having sex before the maximum effect of the drug; and (iii) lower drug-related placebo effects. Some data suggest that sildenafil is a 'difficult' molecule, but Tonset can be improved following absorption by buccal mucosa using appropriate excipients. CONCLUSIONS: We conclude that several ODT and ODF formulations can improve the 'discretion' issue because they are taken without water, but they have similar pharmacokinetics to corresponding film-coated tablet formulations. One ODF formulation of sildenafil was characterized by a shorter Tonset and could potentially increase patient satisfaction following treatment. However, more clinical studies are needed to confirm the findings. Surfactants and ascorbic acid appear to be crucial excipients for achieving a high absorption rate, but more studies are needed.

L-GILZ binds p53 and MDM2 and suppresses tumor growth through p53 activation in human cancer cells.

The transcription factor p53 regulates the expression of genes crucial for biological processes such as cell proliferation, metabolism, cell repair, senescence and apoptosis. Activation of p53 also suppresses neoplastic transformations, thereby inhibiting the growth of mutated and/or damaged cells. p53-binding proteins, such as mouse double minute 2 homolog (MDM2), inhibit p53 activation and thus regulate p53-mediated stress responses. Here, we found that long glucocorticoid-induced leucine zipper (L-GILZ), a recently identified isoform of GILZ, activates p53 and that the overexpression of L-GILZ in p53(+/+) HCT116 human colorectal carcinoma cells suppresses the growth of xenografts in mice. In the presence of both p53 and MDM2, L-GILZ binds preferentially to MDM2 and interferes with p53/MDM2 complex formation, making p53 available for downstream gene activation. Consistent with this finding, L-GILZ induced p21 and p53 upregulated modulator of apoptosis (PUMA) expression only in p53(+/+) cells, while L-GILZ silencing reversed the anti-proliferative activity of dexamethasone as well as expression of p53, p21 and PUMA. Furthermore, L-GILZ stabilizes p53 proteins by decreasing p53 ubiquitination and increasing MDM2 ubiquitination. These findings reveal L-GILZ as a regulator of p53 and a candidate for new therapeutic anti-cancer strategies for tumors associated with p53 deregulation.

Expansion of CD4+CD25-GITR+ regulatory T-cell subset in the peripheral blood of patients with primary Sjögren's syndrome: correlation with disease activity.

OBJECTIVES: CD4+CD25high regulatory T cells (TREG) represent a suppressive T cell subset deeply involved in the modulation of immune responses and eventually in the prevention of autoimmunity. Growing evidence demonstrated that patients with autoimmune and inflammatory chronic diseases display an impairment of TREG cells or activated effector T cells unresponsive to TREG. Glucocorticoid-induced TNFR-related protein (GITR) is a widely accepted marker of murine TREG cells, but little is known in humans. Aim of the present study was to investigate the characteristics of different subsets of TREG cells in Sjögren's syndrome and the potential role of GITR as marker of human TREG cells. METHODS: Fifteen patients with primary Sjogren's syndrome (SS) and 10 sex- and age-matched normal controls (NC) were enrolled. CD4+ T cells were magnetic sorted from peripheral blood by negative selection. Cell phenotype was analyzed through flow-cytometry using primary and secondary antibodies. Disease activity was assessed using the EULAR Sjögren's syndrome disease activity index (ESSDAI). RESULTS: Although the proportion of circulating CD25highGITRhigh subset was similar in SS patients and NC, an expansion of the CD25-GITRhigh cell population was observed in the peripheral blood of SS patients. Interestingly, this expansion was more relevant in patients with inactive rather than active disease. CONCLUSIONS: The number of CD4+CD25-GITRhigh cells is increased in SS as compared to NC. Moreover, the fact that the expansion of this cell subset is prevalently observed in patients with inactive disease suggests that these cells may play a role in counteracting inflammatory response.

The glucocorticoid-induced TNF receptor family-related protein (GITR) is critical to the development of acute pancreatitis in mice.

BACKGROUND AND PURPOSE: Pancreatitis represents a life-threatening inflammatory condition where leucocytes, cytokines and vascular endothelium contribute to the development of the inflammatory disease. The glucocorticoid-induced tumour necrosis factor (TNF) receptor family-related protein (GITR) is a costimulatory molecule for T lymphocytes, modulates innate and adaptive immune system and has been found to participate in a variety of immune responses and inflammatory processes. Our purpose was to verify whether inhibition of GITR triggering results in a better outcome in experimental pancreatitis. EXPERIMENTAL APPROACH: In male GITR knock-out (GITR(-/-)) and GITR(+/+) mice on Sv129 background, acute pancreatitis was induced after i.p. administration of cerulein. Other experimental groups of GITR(+/+) mice were also treated with different doses of Fc-GITR fusion protein (up to 6.25 µg·mouse⁻¹), given by implanted mini-osmotic pump. Clinical score and pro-inflammatory parameters were evaluated. KEY RESULTS: A less acute pancreatitis was found in GITR(-/-) mice than in GITR(+/+) mice, with marked differences in oedema, neutrophil infiltration, pancreatic dysfunction and injury. Co-treatment of GITR(+/+) mice with cerulein and Fc-GITR fusion protein (6.25 µg·mouse⁻¹) decreased the inflammatory response and tissue injury, compared with treatment with cerulein alone. Inhibition of GITR triggering was found to modulate activation of nuclear factor κB as well as the production of TNF-α, interleukin-1ß, inducible nitric oxide synthase, nitrotyrosine, poly-ADP-ribose, intercellular adhesion molecule-1 and P-selectin. CONCLUSIONS AND IMPLICATIONS: The GITR-GITR ligand system is crucial to the development of acute pancreatitis in mice. Our results also suggest that the Fc-GITR fusion protein could be useful in the treatment of acute pancreatitis.

[Glucocorticoid induced TNFR-related protein (GITR) as marker of human regulatory T cells: expansion of the GITR(+)CD25⁻ cell subset in patients with systemic lupus erythematosus]. / Glucocorticoid-induced TNFR-related protein (GITR) come marker di cellule T regolatorie umane: espansione della sottopopolazione cellulare GITR(+)CD25⁻ in pazienti affetti da lupus eritematoso sistemico.

OBJECTIVES: Regulatory T cells (T(REG)) represent a T cell subset able to modulate immune response by suppressing autoreactive T-lymphocytes. The evidence of a reduced number and an impaired function of this cell population in autoimmune/inflammatory chronic diseases led to the hypothesis of its involvement in the pathogenesis of these disorders. Glucocorticoid-induced TNFR-related protein (GITR) is a well known marker of murine T(REG) cells, but little is known in humans. The aim of this study was to investigate the characteristics of T(REG) cells in systemic lupus erythematosus (SLE) and the potential role of GITR as marker of human T(REG). METHODS: Nineteen SLE patients and 15 sex- and age-matched normal controls (NC) were enrolled. CD4(+) T cells were magnetic sorted from peripheral blood by negative selection. Cell phenotype was analyzed through flow-cytometry using primary and secondary antibodies and real time polymerase-chain reaction (PCR) using TaqMan probes. RESULTS: The CD25(high)GITR(high) subset was significantly decreased in SLE patients with respect to NC (0.37±0.21% vs 0.72±0.19%; p<0.05). On the opposite, the CD25⁻GITR(high) cell population was expanded in the peripheral blood of SLE patients (3.5±2.25 vs 0.70±0.32%, p<0.01). Interestingly, FoxP3 at mRNA level was expressed in both CD25⁻GITR(high) and CD25(high)GITR(high) cells, suggesting that both cell subsets have regulatory activity. CONCLUSIONS: CD4(+)CD25⁻GITR(high) cells are increased in SLE as compared to NC. The expression of high level of GITR, but not CD25, on FoxP3+ cells appears to point to a regulatory phenotype of this peculiar T cell subset.

Dietary alpha-linolenic acid reduces COX-2 expression and induces apoptosis of hepatoma cells.

Fatty acid synthetase (FAS) is overexpressed in various tumor tissues, and its inhibition and/or malonyl-CoA accumulation have been correlated to apoptosis of tumor cells. It is widely recognized that both omega-3 and omega-6 polyunsaturated fatty acids (PUFAs) depress FAS expression in liver, although epidemiological and experimental reports attribute antitumor properties only to omega-3 PUFA. Therefore, we investigated whether lipogenic gene expression in tumor cells is differently regulated by omega-6 and omega-3 PUFAs. Morris hepatoma 3924A cells were implanted subcutaneously in the hind legs of ACI/T rats preconditioned with high-lipid diets enriched with linoleic acid or alpha-linolenic acid. Both-high lipid diets depressed the expression of FAS and acetyl-CoA carboxylase in tumor tissue, this effect correlating with a decrease in the mRNA level of their common sterol regulatory element binding protein-1 transcription factor. Hepatoma cells grown in rats on either diet did not accumulate malonyl-CoA. Apoptosis of hepatoma cells was induced by the alpha-linolenic acid-enriched diet but not by the linoleic acid-enriched diet. Therefore, in this experimental model, apoptosis is apparently independent of the inhibition of fatty acid synthesis and of malonyl-CoA cytotoxicity. Conversely, it was observed that apoptosis induced by the alpha-linolenic acid-enriched diet correlated with a decrease in arachidonate content in hepatoma cells and decreased cyclooxygenase-2 expression.

Enhanced expression of hepatic lipogenic enzymes in an animal model of sedentariness.

The hindlimb-suspended rat was used as animal model to investigate the effects induced by immobilization of the skeletal muscle in the expression of the genes encoding hepatic lipogenic enzymes. Following a 14-day period of immobilization, rats were injected intraperitoneally with radioactive acetate, and the labeling of hepatic lipids and cholesterol was evaluated 15 min after the isotope injection. The incorporation of labeled acetate in lipids and cholesterol was almost three times higher in the liver of immobilized rats than in control animals as a consequence of the enhanced transcription of the genes encoding acetyl-CoA synthase, acetyl-CoA carboxylase, fatty acid synthase, and 3-hydroxy-3-methylglutaryl-CoA reductase. The high expression of the key enzymes for fatty acid and cholesterol synthesis induced by immobilization was not paralleled by an increase of the hepatic sterol-regulatory element binding protein (SREBP)-1 and SREBP-2 mRNA content. However, the expression of the mature form of SREBP-1 and SREBP-2 was higher in the nuclear fraction of immobilized rat liver than in controls due to a significant increase of the cleavage of the native proteins. Immobilization also affected the expression of proteins involved in lipid degradation. In fact, the hepatic content of peroxisome proliferator-activated receptor-alpha (PPARalpha) mRNA and of PPARalpha target genes encoding carnitine palmitoyl transferase-1 and acyl-CoA oxidase were significantly increased upon immobilization.

Oxidative stress and apoptosis in immune diseases.

Antigenic stimuli increase ROS that influence T-cell activation by interfering with the oxidant-antioxidant balance. Oxidative stress takes place when excess of ROS production is not counterbalanced by antioxidant mechanisms and bcl-2 gene product that inhibits apoptosis by interacting with mitochondrial superoxide dismutase. ROS Excess induces apoptosis both by activation of NF-kB-dependent genes and DNA damage. The latter has been shown to elicit the activation of poly-ADP-ribose transferase and the accumulation of p53, thus determining apoptosis. Additionally, oxidative stress may induce formation of cell membrane oxidized lipids, potent inducers of apoptosis. Oxidative stress is also involved in immune diseases. In AIDS, ROS excess and deficiency of antioxidants lead to apoptosis and virus activation. ROS produced at sites of chronic inflammation, have genotoxic effects. As a consequence, abnormalities of the p53 genes might explain the conversion from an inflammatory phase into autonomous progression of rheumatoid arthritis or other chronic inflammatory disorders.

Differentiation of Ly49s-positive or -negative natural killer cells is inhibited by anti-H-2b monoclonal antibodies acting at the level of bone marrow progenitors from B6 mice.

To investigate the role of MHC class I on in vitro differentiation of natural killer (NK) cells, a CD44low/-CD2-classlow population was isolated from mouse bone marrow. This population, which lacked expression of NK-1.1, Ly49A, Ly49C/I, and Ly49G, generated populations of NK-1.1+ NK cells expressing Ly49A, Ly49C/I, or Ly49G when cocultured for 13 days with syngeneic supportive stromal cells in the presence of interleukin 2. Ly49A and Ly49C/I were absent on the progeny of progenitors tested after 7 days of culture but were expressed as a late event together with low-level expression of NK-1.1, from day 8 of culture. The addition of anti-H-2b monoclonal antibody to cultures at day 0 inhibited proliferation of progenitors supported by either syngeneic, allogeneic, or H-2b-deficient stromal cells, thus suggesting that the effect was not exerted on stromal cells. Additional analyses demonstrated that class Ilow progenitors generated class I+ cells on which the anti-H-2b monoclonal antibody exerted its inhibitory effect.

Glucocorticoid hormones in the regulation of cell death.

The immune T-cell compartment maintains the capability to respond to a wide variety of antigens (Ag). This whole process is regulated by lymphocyte apoptosis (programmed cell death, PCD) and involves the coordinated expression of a great number of genes including those coding for cytokines and their receptors, such as for example IL-2/IL-2R and the Fas/FasL systems and those coding for transcription factors, including the NF-kB complex, involved in T-cell activation and apoptosis in that they simultaneously activate cell suicide and an anti-death programme. This binary effect, PCD activation and inhibition, is due on one hand to GCH-induced activation of the caspases cascade and on the other to the induction of expression of a new gene that we have named GILZ. In fact, GILZ over-expression in transfected cells inhibits the sequential increase of NF-kB/DNA-binding activity, IL-2 production and IL-2R expression, and transcription of the Fas/FasL complex that follows TCR triggering and plays an important role in the control of T-lymphocyte apoptosis. These results indicate a new mechanism responsible for the GCH-mediated inhibition of T-cell death and activation that could contribute to anti-inflammatory and immunosuppressive efficacy.

Gene structure and chromosomal assignment of mouse GITR, a member of the tumor necrosis factor/nerve growth factor receptor family.

GITR is a type I transmembrane protein that belongs to the tumor necrosis factor/nerve growth factor receptor (TNF/NGFR) family. This receptor is preferentially expressed in activated T lymphocytes and may function as signaling molecule during T-cell development. In the present study, we examined the genomic organization of the entire mouse GITR (mGITR) gene. The gene spans a 2543-bp region and consists of five exons (with a length ranging from 88 bp to 395 bp) and four introns (67 bp to 778 bp). In agreement with GITR expression in activated T cells, consensus elements for transcription factors involved in T-cell development and activation were identified in the 5' flanking region, including a consensus element for NF-kappaB. Two highly significant binding sites for MyoD and one binding site for myogenin were also found, suggesting involvement of GITR in muscle development. The mGITR gene contains 17 transcription initiation sites distributed over a 76-bp region, all used with the same frequency. We localized mGITR to the murine chromosome 4 (E region), where other 4 TNF/NGFR members localize, including m4-1BB and mOX40. These results further indicate that GITR shares several features with OX40, 4-1BB, and CD27, suggesting the existence of a new subfamily of the TNFR family, as also confirmed by the similarity of their cytoplasmic domains.

Angiotensin converting enzyme deletion allele in different kinds of dementia disorders.

In order to verify the association of Angiotensin converting enzyme (ACE) gene with different kinds of dementia, as well as its association with APO-E (genotype), we performed ACE genotyping in subjects with late-onset probable Alzheimer's disease (LOAD, n = 64), early-onset probable Alzheimer's disease (EOAD, n = 32), possible Alzheimer's disease (pAD, n = 44), vascular dementia (VD, n = 12), age-associated memory impairment (AAMI, n = 15) and 40 healthy age-matched controls, who were previously characterized for APO-E. After the principal component analysis ACE D and Apo-Eepsilon4 alleles disclosed the highest prevalence in the cognitively impaired groups of subjects, Apo-Eepsilon4 being more specific for LOAD and pAD. ACE D allele seems to be an unspecific susceptibility factor for mental decline.

TCR kappa, a new splicing of the murine TCR zeta gene locus, is modulated by glucocorticoid treatment.

T-cell receptor (TCR) is a multichain receptor in which the TCRzeta subunit is important for membrane assembly and signal transduction. Four alternative splicings of the murine TCRzeta gene locus have been previously described. We here describe a new alternative splicing of murine TCRzeta gene, TCRkappa, cloned by RT-PCR, that is encoded by exons 1-7, a portion of exon 9 and the whole exon 10 of TCRzeta gene. The protein encoded by TCRkappa mRNA is identical to that encoded by TCReta mRNA, because the stop codon is present in the exon 9 before splicing with exon 10. RNAse protection assays carried out on total RNA from thymocytes indicate that TCRkappa mRNA is 1 half with respect to TCReta mRNA, suggesting that TCRkappa mRNA contributes to determine the TCReta protein levels. The 3' untranslated region of TCRkappa mRNA is different from that of TCReta and this might lead to different t(1/2) for each species in vivo. We also show that dexamethasone (DEX), a synthetic glucocorticoid hormone, increases the amount of TCRkappa in the hybridoma T-cell line 3DO (about 5-fold increase), as indicated by reverse transcriptase-polymerase chain reaction (RT-PCR) and RNAse protection assays. This newly described effect of DEX may constitute a further molecular mechanism that contributes to its immunomodulating activity.

Design and synthesis of modified quinolones as antitumoral acridones.

The bacterial topoisomerase II (DNA gyrase) and the mammalian topoisomerase II represent the cellular targets for quinolone antibacterials and a wide variety of anticancer drugs, respectively. In view of the mechanistic similarities and sequence homologies exhibited by the two enzymes, tentative efforts to selectively shift from an antibacterial to an antitumoral activity was made by synthesizing a series of modified tricyclic quinolones, in which the essential 3-carboxylic function is surrogated by phenolic OH and the classic C-6 fluorine atom is replaced by a NH2 group. The resulting 7-amino-9-acridone derivatives were assayed for their antibacterial as well as cytotoxic activities. No antibacterial activity was found. On the other hand, many derivatives showed significant cytotoxic activity against both HL-60 and P388 leukemias and a wide panel of human and rodent solid tumor cells, derivatives 25 and 26 displaying the best overall antiproliferative activity. Against the LoVo cell line, derivative 25 exhibited higher cytotoxic effects than etoposide.

Antitumor activity of 2,2'-bipyridyl-6-carbothioamide: a ribonucleotide reductase inhibitor.

1. 2,2'-Bipyridyl-6-carbothioamide (BPYTA) is a synthesized compound with chelating properties demonstrating in vitro and in vivo antitumor activity. 2. The BPYTA cytotoxic effect is mainly due to the inhibition of ribonucleotide reductase (RR), a key enzyme in proliferating cells. The active form of BPYTA is its iron chelate [BPYTA-Fe(II), molar ratio 2:1], which destroys the tyrosyl radical of RR small subunit (R2). 3. The copper chelate of BPYTA [BPYTA-Cu(II), molar ratio 2:1] also has antiproliferative activity, but RR is not the only intracellular target. 4. BPYTA potently synergizes in vitro with hydroxyurea, the most widely used R2 inhibitor.

Apolipoprotein-E genotype in normal aging, age-associated memory impairment, Alzheimer's disease and vascular dementia patients.

Apolipoprotein-E (Apo-E) genotype and allele frequencies were evaluated in patients with late-onset probable Alzheimer's disease (LOAD; n = 64), early-onset probable Alzheimer's disease (EOAD; n = 32), possible Alzheimer's disease (pAD; n = 44), vascular dementia (VD; n = 12), age-associated memory impairment (AAMI; n = 15) and 40 healthy age-matched controls. APO-E was performed by polymerase chain reaction products digested by the restriction enzyme HhaI. A statistically significant increase of epsilon4 frequency was found in LOAD as compared to the other groups, and in pAD with respect to controls, while VD and AAMI groups did not disclose any difference as regards to control subjects. Multivariate analysis showed a significant association of epsilon4 with female gender. Our results confirm the increased frequency of epsilon4 in both probable and possible LOAD, failing to show a similar trend in VD and AAMI.