12-months prospective Pentraxin-3 and metabolomic evaluation in multiple sclerosis patients treated with glatiramer acetate.

BACKGROUND: Pentraxin-3 (PTX-3) is involved in acute immunological responses and it is a pro-inflammatory protein and a novel biomarker of inflammatory diseases. It is demonstrated that PTX-3 is higher in cerebrospinal fluid (CSF) of aggressive Multiple Sclerosis (MS). Metabolomics, the identification of small endogenous molecules, offers a molecular profile of MS. Glatiramer acetate (GA) is a widely used treatment for (MS) but its mechanism of action is not completely defined. The aim of our study is to analyze PTX-3 and metabolomic profile in MS patients compared to controls and to investigate the effect of GA on PXT-3 and metabolic molecules during treatment in responder and not responder MS patients. METHODS: 28 unrelated MS patients and 27 age-and sex-matched controls were recruited. In serum, PTX-3 levels were measured by ELISA and Metabolomic panel was evaluated trough Nuclear Magnetic Resonance (NMR). According to clinical practice patients started GA treatment; PTX-3 and metabolomic identification were performed before and during treatment. Responders to treatment were identified if no evidence of instrumental, clinical relapses and disability progression (NEDA) occurred during follow up. RESULTS: Serum PTX-3 levels were higher in MS patients compared to matched controls (7,85 ± 2,19 vs 6,20 ± 1,63 ng/ml) (p = 0,03); metabolomic evaluation shows higher levels of lactate and lower levels of valine, tyrosine and tryptophan in MS patients compared to controls. During therapy, PTX-3 levels have been reduced statistically significant (p = 0,001) at six months and one year of treatment. After one year, of the twenty patients that completed the study, 55% were considered fully responders to treatment; in these patients the mean reduction of PTX-3 at one year was higher respect to not responders (-3,82 ± 1,24 ng/ml vs -2,32 ± 1,03 ng/ml p = 0,02) and we observed a higher reduction of lactate, tyrosine and hypoxanthine and an increase of hydroxyproline and ADP as well as of three oxidative phosphorylation markers, citrulline, ornithine and tryptophan approaching the metabolic profile of healthy subjects. DISCUSSION AND CONCLUSIONS: We demonstrated a metabolomic imbalance with mitochondrial dysfunction detected by higher levels of lactate and lower levels of tryptophan, tyrosine and valine in MS patients compared to healthy controls. The reduction of PTX-3 levels and the restoring of mitochondrial function, reducing oxidative stress by GA, allows to identify responder patients. Further and larger studies are needed to understand the predictive role of PTX-3 and metabolomic pattern in the identification of responder patients to GA.

Adenylate cyclase/cAMP pathway downmodulation counteracts apoptosis induced by IFN-alpha in human epidermoid cancer cells.

We have reported previously that interferon-alpha (IFN-alpha) induces apoptosis that is counteracted by an epidermal growth factor (EGF) --> Ras --> extracellular signal-regulated kinase (ERK)-dependent survival response in human epidermoid cancer KB cells. We have studied the effects of the cytokine on the cAMP-dependent pathway in these cells. A decrease in the intracellular cAMP levels was recorded in KB cells treated with IFN-alpha, whereas forskolin induced an increase in the production of cAMP that was reduced in the presence of IFN-alpha, suggesting a reduction in the activity of adenylate cyclase (AC) induced by IFN-alpha. These effects were paralleled by significant change in the expression of some AC catalytic subunit(s) and by reduction in the activity of protein kinase A (PKA). 8-Br-cAMP completely antagonized the reduction of PKA activity induced by IFN-alpha, whereas PKA inhibitor KT5720 enhanced the reduction of the enzyme activity induced by IFN-alpha. We have found that IFN-alpha induced a decrease in cAMP response element binding protein (CREB) phosphorylation without changes in its total expression. The concomitant treatment with IFN-alpha and 8-Br-cAMP potentiated and KT5720 counteracted apoptosis induced by IFN-alpha alone. In conclusion, these data suggest that the decrease in AC/cAMP pathway activity is a survival response to the apoptosis induced by IFN-alpha. Therefore, this pathway could represent a target to enhance the antitumor activity of IFN-alpha.

Differentiation of H9c2 cardiomyoblasts: The role of adenylate cyclase system.

The adenylate cyclase (AC)/cAMP/cAMP-dependent protein kinase pathway controls many biological phenomena. The molecular mechanisms by which cAMP induces alternative commitment towards differentiation or proliferation are not still completely known. The differentiation of myoblast cell lines into myocytes/myotubes represents a well-established model of skeletal muscle differentiation. We analyzed the AC/cAMP pathway during terminal differentiation of H9c2 myoblasts. When cultured in low-serum containing medium, H9c2 myoblasts exit the cell cycle and differentiate into myocytes/myotubes. A key step of this process is the expression of myogenin, an essential transcription factor for the terminal differentiation into myocytes. During this phenomenon we observed a decrease in both cAMP levels and AC activity, which suggests a functional negative role of cAMP on the differentiation process of H9c2 cells. 8-Br-cAMP and other cAMP-elevating agents, such as forskolin, IBMX, and isoproterenol, negatively affected skeletal muscle differentiation of H9c2 myoblasts. Both AC activity down-regulation and intracellular cAMP reduction were accompanied by significant variations in the levels of membrane proteins belonging to the AC system (AC catalytic subunit, G(alphai-1), G(alphas)). The functional relationship between intracellular cAMP content and protein levels of AC system is discussed.

Treatment of v-Ki-ras-transformed SVC1 cells with low retinoic acid induces malignancy reversion associated with ras p21 down-regulation.

The effect of nontoxic, low concentrations (10(-8) M) of retinoic acid (RA) for a relatively long time (28 days) on a Kirsten ras-virus transformed cell line (Ki-SVC1), derived from the rat seminal vesicle epithelium, was investigated. In these experimental conditions, the cell treatment with RA induced a decrease of the proliferation rate, apoptosis and a marked reduction of both anchorage-independent growth and tumorigenicity. These biological responses were either preceded or associated with important changes in adenylate cyclase/protein kinase C signaling pathways, the activation of important apoptosis-linked genes and a marked decrease of the v-Ki-ras p21 protein. The significance of these findings is discussed.

GTPase and transglutaminase are associated in the secretion of the rat anterior prostate.

We have found that in the secretion of rat anterior prostate, a hydrolyzing activity on GTP is present with a high affinity for the substrate; ATP, GDP, and ADP are not substrates for enzymatic activity. At the same time we have shown that GTP is a negative modulator for the well-known type IV transglutaminase activity present in the prostatic secretion. The hydrolyzing activity on GTP appears to be due to two molecular species: a high-molecular-weight GTPase, having electrophoretical mobility higher than 100 kDa, and a low-molecular-weight GTPase, of about 30 kDa. The two enzymatic activities are associated in the prostatic secretion with the transglutaminase (type IV). We describe an experimental procedure to separate them.

tTGase/G alpha h protein expression inhibits adenylate cyclase activity in Balb-C 3T3 fibroblasts membranes.

Stably transfected Balb-C 3T3 fibroblasts (clone 5), overexpressing a catalytically active tissue transglutaminase, showed a basal adenylate cyclase activity lower than control cells (clone 1). Several modulators of the adenylate cyclase activity (forskolin, Mn2+ and pertussis toxin) showed the existence of a marked negative control on the adenylate cyclase activity present in clone 5 cells. Very interestingly, this same marked negative control was also found in a Balb-C 3T3 fibroblast clone stably transfected with a mutagenized human tissue transglutaminase (mut277 cys > ser) virtually devoid of transglutaminase catalytic activity (clone Ser). Conversely, a significant increase of the adenylate cyclase activity was observed in bovine aortic endothelial cells after the lowering of tissue transglutaminase expression levels by the transfection of an eukaryotic expression vector containing the gene for tissue transglutaminase in antisense orientation. All these findings suggest a possible role for type II tissue transglutaminase as a negative modulator of the adenylate cyclase activity in different cell types, beside its transglutaminase enzyme activity.

ras oncogene-induced transformation of a rat seminal vesicle epithelial cell line produces a marked increase of adenylate cyclase and protein kinase C activities.

Cells transformed by Kirsten murine sarcoma virus (Ki-MSV) have basal adenylate cyclase activity (AC) higher than control cells and comparable level of forskolin-stimulated AC activity. Moreover, a higher protein kinase C (PKC) activity was found to be present in the transformed cells. The molecular mechanism underlying the increase of AC activity was investigated. Our findings strongly suggest that this biochemical event is due to a marked decrease of the alpha i negative control of the enzyme, even though the alpha i of transformed cells appears to possess fully functional domains interacting with both the effector enzyme and the agonist-activated receptor.

Role of calcium in chloride secretion mediated by cAMP pathway activation in rabbit distal colon mucosa.

Effects of Ca2+ on adenosine 3',5'-cyclic monophosphate (cAMP)-mediated Cl- secretion were investigated in intact mucosa and isolated crypt cells of rabbit descending colon. Addition of 10 microM prostaglandin (PG)E2 or forskolin to tissues incubated in Ca(2+)-free medium increased the size of short-circuit current (Isc) and Cl- secretion as estimated by unidirectional 36Cl flux measurements (net flux = -2.31 +/- 0.24 vs. -1.22 +/- 0.10 mueq.h-1.cm-2, n = 4, P < 0.001). Addition of 10 microM PGE2 to tissues incubated in 1.2 mM Ca2+ Ringer induced a 7-fold increase in mean cAMP level, whereas it produced an 11-fold increase in tissues exposed to Ca(2+)-free medium. Membrane preparations from whole mucosa incubated in Ca(2+)-free medium displayed a cyclic nucleotide phosphodiesterase activity significantly lower than controls (18.76 +/- 0.54 vs. 31.20 +/- 0.39 pmol cAMP. mg protein-1.min-1, means +/- SE, n = 4, P < 0.001). Ca2+ removal also affected adenylate cyclase (AC) responsiveness to agonists; AC activity increased in controls by 54 and 226% after stimulation with 10 microM PGE2 and forskolin, respectively, but it increased more (77 and 325%, respectively) after incubation in Ca(2+)-free solutions.(ABSTRACT TRUNCATED AT 250 WORDS)

Surgical treatment and desensitization therapy of giant papillary allergic conjunctivitis.

We found that three cases of giant papillary conjunctivitis responded well to local specific desensitization therapy and transplantation of the conjunctiva with saphenous vein tissue.

[Brown's syndrome]. / Le syndrome de Brown.

Having observed a very tight adhesion between the trochlea and the tendon of the obliquus superior bulbi muscle or an increase in tendon thickness posterior to the trochlea in operated patients, the author tried to correct this abnormality using partial trochleotomy and the lysis of the adhesions, followed by the application of Scott-Knapp sutures. The morphological basis of Brown's syndrome, consisting of an obstruction at the level of the trochlea, was demonstrated experimentally by means of an eye motion simulator.

Protein kinase C activities are increased in rat thyroid epithelial cells expressing v-ras genes.

Both cytoplasmic and membrane-bound protein kinase C activities are increased in: Harvey-Sarcoma Virus, infected thyroid epithelial cells. The cytoplasmic kinase C increase is found in the chromatographic fraction eluted at lower salt concentration (100 mM NaCl-S100), while the more acidic protein fraction eluted at higher salt concentration (35 mM NaCl-S350) is virtually absent. Although the cytoplasmic S100 fraction from the control and ras-virus infected cells display a comparable PBt2 binding activity, they are different in the Ca+2-dependence and the TPA down regulation. In addition, the membranes from the control and ras-virus infected cells are different phosphate acceptors in place of the H1 histones.

The Annette von Droste-Hulshoff syndrome. Pseudostrabismus due to macular ectopia in retinopathy of prematurity.

Four cases of macular ectopia are described. Pseudostrabismus, caused by an increased angle alpha, is only one of the signs common to macular ectopia. A correct diagnosis is of fundamental importance because the treatment of macular ectopia is different from that of strabismus.

Inheritance of Brown's syndrome.

Two families are described with members affected by Brown's syndrome. Another case of Brown's syndrome is described in a dizygotic twin. The authors suggest a hypothesis for the influence of heredity in Brown's syndrome.

A stimulating effect of guanyl nucleotides on the rat-liver soluble cyclic GMP high-affinity phosphodiesterase activity.

The high affinity (low Km) cyclic GMP phosphodiesterase (PDE) is activated by GTP, while the cyclic AMP PDE is not. GTP and its hydrolysis-resistant analogue, guanylylimidodiphosphate (GppNHp), display a half-maximal stimulating effect at almost the same concentration (5 X 10(-6) M). The GTP stimulating effect is not observed when the socalled cyclic GMP low affinity (high Km) PDE is operative. GTP cooperates with the increase of the substrate concentration on removing the IBMX inhibitory effect. The isolation through a classical chromatographic procedure on a DEAE-cellulose column, of a PDE fraction specific for cyclic GMP, results in the loss of the GTP stimulating effect.

Low insulin concentrations stimulate in vitro the soluble guanylate cyclase activity of rat liver.

The soluble guanylate cyclase activity of rat liver appears to be stimulated in VITRO by insulin at pMolar concentrations, while proinsulin, denaturated insulin or desoctapeptide insulin, are not able to stimulate the studied enzymic activity. Corresponding concentrations of other peptide hormones such as corticotropin (ACTH) or glucagon, either in the absence or in the presence of bacitracin, do not show any effect on the investigated enzymic system. Insulin stimulation of the soluble guanylate cyclase is characterized by a significant increase in the Vmax together with a decrease of the apparent Km. Insulin at low concentrations doesn't affect the cyclic GMP hydrolyzing activity; conversely higher concentrations of the hormone, while exerting a less marked effect on the guanylate cyclase activity, inhibit the cyclic GMP hydrolyzing activity.

Relationship between the fluidity of the membrane lipids and the activity of the membrane-bound proteins: the effects of lidocaine on the adenylate cyclase activity of rat myocardium.

The lidocaine effects on the basal adenylate cyclase activity of the studied membranes depend on the substrate concentration: the drug has an inhibiting effect on non-saturating ATP concentrations, but displays a stimulating effect on the saturating ones. In the presence of maximally stimulating NaF concentrations, lidocaine further stimulates the adenylate cyclase activity through all the ATP concentrations used. In the presence of ATP saturating concentrations, the adenylate cyclase activity stimulated by various Gpp(NH)p concentrations, is inhibited in the presence of lidocaine as well as the enzymic activity stimulated by several l-isoproterenol concentrations in the presence of a fixed Gpp(NH)p concentration.

Hydrolyzing activities on cAMP in E. coli B cells incubated in the presence of polyamines.

1. Hydrolyzing activities on cAMP were investigated in E. coli B cells incubated in the presence or absence of 10(-5) M spermine, spermidine or putrescine. 2. Bacterial cells incubated in the presence of each of the investigated polyamines show an increase in the PDE (phosphodiesterase) activities already evident after 3 min of incubation, reaching the maximum between 5 and 10 min. disappearing between 15 and 25 min. 3. The stimulating effect is higher in the presence of lower (10(-6) M) substrate concentration (high affinity PDE activities). 4. Kinetic analyses carried out for the "high affinity" PDE activity, indicate that spermidine and putrescine are the most effective on increasing both the Vmax or the apparent Km. 5. Kinetic analysis carried out for the "low affinity" PDE activity, indicate that putrescine is the most active on increasing either the Vmax or the apparent Km and that spermine and spermidine have both quantitatively and qualitatively comparable effects. 6. Analyses, by TLC, of the products of the hydrolytic activities on cyclic AMP (5'-adenosine monophosphate (5'-AMP), adenosine, inosine) indicate that the incubation in the presence of each of three polyamines, results in an increase also of the 5' nucleotidase and adenosine deaminase activity.