Reduction of autophagy and increase in apoptosis correlates with a favorable clinical outcome in patients with rheumatoid arthritis treated with anti-TNF drugs.

BACKGROUND: Autophagy has emerged as a key mechanism in the survival and function of T and B lymphocytes, and its activation was involved in apoptosis resistance in rheumatoid arthritis (RA). To investigate whether the relationship between autophagy and apoptosis may impact the response to the therapy, we analyzed ex vivo spontaneous autophagy and apoptosis in patients with RA subjected to treatment with anti-tumor necrosis factor (TNF) drugs and in vitro the effects of TNFα and anti-TNF drugs on cell fate. METHODS: Peripheral blood mononuclear cells (PBMCs) from 25 RA patients treated with anti-TNF drugs were analyzed for levels of autophagy marker LC3-II by western blot and for the percentage of annexin V-positive apoptotic cells by flow cytometry. The same techniques were used to assess autophagy and apoptosis after in vitro treatment with TNFα and etanercept in both PBMCs and fibroblast-like synoviocytes (FLS) from patients with RA. RESULTS: PBMCs from patients with RA responsive to treatment showed a significant reduction in LC3-II levels, associated with an increased apoptotic activation after 4 months of therapy with anti-TNF drugs. Additionally, the expression of LC3-II correlated with DAS28. TNFα was able to induce autophagy in a dose-dependent manner after 24 h of culture in RA PBMCs and FLS. Moreover, etanercept caused a significant reduction of autophagy and of levels of citrullinated proteins. CONCLUSIONS: Our results show how the crosstalk between autophagy and apoptosis can sustain the survival of immune cells, thus influencing RA progression. This suggests that inhibition of autophagy represents a possible therapeutic target in RA.

Association of fission proteins with mitochondrial raft-like domains.

It was shown that receptor-mediated apoptosis involves a cascade of subcellular events including alterations of mitochondria. Loss of mitochondrial membrane potential that follows death receptor ligation allows the release of apoptogenic factors that result in apoptosis execution. Further important mitochondrial changes have been observed in this regard: mitochondrial remodeling and fission that appear as prerequisites for the occurrence of the cell death program. As it was observed that lipid rafts, glycosphingolipid-enriched structures, can participate in the apoptotic cascade being recruited to the mitochondria under receptor-mediated proapoptotic stimulation, we decided to analyze the possible implication of these microdomains in mitochondrial fission. We found that molecules involved in mitochondrial fission processes are associated with these domains. In particular, although hFis1 was constitutively included in mitochondrial raft-like domains, dynamin-like protein 1 was recruited to these domains on CD95/Fas triggering. Accordingly, the disruption of rafts, for example, by inhibiting ceramide synthase, leads to the impairment of fission molecule recruitment to the mitochondria, reduction of mitochondrial fission and a significant reduction of apoptosis. We hypothesize that under apoptotic stimulation the recruitment of fission-associated molecules to the mitochondrial rafts could have a role in the morphogenetic changes leading to organelle fission.

Lipid microdomains contribute to apoptosis-associated modifications of mitochondria in T cells.

Plasma membrane lipid microdomains have been considered as a sort of 'closed chamber', where several subcellular activities, including CD95/Fas-mediated proapoptotic signaling, take place. In this work we detected GD3 and GM3 gangliosides in isolated mitochondria from lymphoblastoid CEM cells. Moreover, we demonstrated the presence of microdomains in mitochondria by immunogold transmission electron microscopy. We also showed that GD3, the voltage-dependent anion channel-1 (VDAC-1) and the fission protein hFis1 are structural components of a multimolecular signaling complex, in which Bcl-2 family proteins (t-Bid and Bax) are recruited. The disruption of lipid microdomains in isolated mitochondria by methyl-beta-cyclodextrin prevented mitochondria depolarization induced by GD3 or t-Bid. Thus, mitochondrion appears as a subcompartmentalized organelle, in which microdomains may act as controllers of their apoptogenic programs, including fission-associated morphogenetic changes, megapore formation and function. These results disclose a new scenario in which mitochondria-associated lipid microdomains can act as regulators and catalysts of cell fate.

Intranasal and intramuscular human calcitonin in female osteoporosis and in Paget's disease of bones: a pilot study.

It has been shown that human calcitonin (hCT) is absorbed through the nasal mucosa when administered together with promoters like sodium glycocholate (SGC) or dihydrofusinate. The aim of this study was to compare the clinical and metabolic effect of intranasal (in) and intramuscular (im) hCT in patients with osteoporosis or with Paget's disease of bones. Fifteen women with postmenopausal or with senile osteoporosis entered a randomized six months trial with in hCT (plus SGC) or with im hCT 100 U on alternate days. Six women in each group were treated for 2 months, and only four women in each group continued treatment for an additional 4 months period. In hCT, but not im hCT, reduced subjective pain, while urinary cAMP increased to a similar extent in the 2 groups. Other metabolic indexes and bone mineral content (BMC) were unchanged, no new fractures took place, and side effects were fewer with in than with im hCT. To confirm the analgesic effect of in hCT, twelve patients with Paget's disease of bone were randomly treated for 20 days with in or im hCT 100 U/day: during the short period of treatment, pain was reduced by in, not by im hCT, and urinary cAMP excretion similarly increased in the two groups of patients.

Clinical pharmacokinetics of cefotaxime and penetration in sputum, bone, and prostatic tissue.

Cefotaxime is a new powerful methoxycephalosporin with a broad anti-microbial spectrum, suitable for parenteral administration. In the present study, the concentrations of cefotaxime in serum and in bronchial secretion were determined after intramuscular injection of 1 gm every eight hours for seven days. Subjects were patients suffering from an exacerbation of chronic bronchitis. Serum levels versus time curve were interpreted in terms of a one-compartment open model. Pharmacokinetic parameters after single and multiple doses were investigated. No evidence of significant accumulation was found. Furthermore, a type of in vivo rate of killing with cefotaxime was investigated by evaluating the decrease in the number of colonies in bronchial mucus cultures daily for seven days. In two groups of volunteers who had to undergo surgery, bone and prostatic concentrations of cefotaxime were determined and correlated with serum levels.