Neuropeptide Y-induced potentiation of noradrenergic vasoconstriction in the human saphenous vein: involvement of endothelium generated thromboxane

1. We investigated the potentiating effect of low concentrations of neuropeptide Y (NPY) on the vasoconstriction induced by transmural nerve stimulation (TNS) and noradrenaline (NA) in human saphenous veins. The effects of (i) endothelium removal; (ii) the addition of the NO pathway precursor L-arginine; (iii) the ET_A/ET_B endothelin receptor antagonist Ro 47-0203; (iv) the cyclo-oxygenase inhibitor, indomethacin; (v) the selective thromboxane A₂ (TxA₂) receptor antagonists Bay u3405 and ifetroban, and (vi) the TxA₂ synthase inhibitor, UK 38485, were studied in order to gain information about the mechanisms of NPY-induced potentiation.
2. Contractile response curves for TNS (0.5–8 Hz) and for exogenously administered NA (0.1–3 μM) were obtained in superfused saphenous vein rings. The contractions induced by both TNS and NA at all tested frequencies and concentrations, respectively, were significantly potentiated by 50 nM NPY in endothelium intact veins. Conversely, in endothelium-denuded vessel rings the contractile-response curves to TNS and NA overlapped both in the absence and presence of NPY, thus suggesting that a release of vasoactive substances from endothelial cells could account for the noradrenergic NPY-induced potentiation.
3. In vessels with intact endothelium, the potentiating action of NPY on TNS and NA was unaffected by the presence of high concentrations of the NO precursor L-arginine (3–10 mM) or the non-selective ET_A/ET_B endothelin receptor antagonist, Ro 47-0203 (10 μM). These data indicate that the NPY-induced effect does not involve either the endothelium-derived vasodilator nitric oxide or the vasoconstrictor endothelin. Conversely, in the presence of the cyclo-oxygenase inhibitor, indomethacin (30 μM), NPY failed to potentiate the vasoconstrictions produced by either nerve stimulation or by exogenous NA, thus providing evidence that arachidonic acid metabolites through the cyclo-oxygenase pathway are mainly responsible for the potentiation evoked by NPY.
4. When the TxA₂ receptor antagonists, Bay u 3405 (1 μM) and ifetroban (1 μM) were added to the superfusing medium, NPY did not alter either the frequency- or the concentration-response curves for either TNS or NA. Accordingly, both TNS- and NA-induced contractions were not potentiated by NPY in the presence of the TxA₂ synthase inhibitor, UK 38485 (10 μM). This clearly demonstrates the pivotal role of TxA₂ in NPY-induced potentiation.
5. In superfused vein rings with endothelium, a subthreshold concentration (0.2 nM) of the TxA₂ mimetic U 46619 potentiated both TNS- and NA-induced vasoconstrictions. This potentiation was higher at low stimulation frequencies and low NA concentrations, and resembled that produced by NPY.
6. Our results indicate that in the human saphenous vein NPY potentiates the contractions produced by sympathetic nerve stimulation acting at the postjunctional level, primarily on endothelial cells. In particular, the NPY-induced release of a cyclo-oxygenase metabolite, namely TxA₂, may have a synergistic effect on the vasoconstriction induced by the noradrenergic mediator. Thus, such a mechanism may play a key role in the maintenance of the sympathetic tone of large human capacitance vessels.

     

Differential effect of ganglioside GM1 on rat brain phosphoproteins: potentiation and inhibition of protein phosphorylation regulated by calcium/calmodulin and calcium/phospholipid-dependent protein kinases

The monosialoganglioside GM1 displays complex effects on protein phosphorylation of rat cerebral cortex membrane preparations. The exogenous ganglioside at a concentration of 350 microM in absence of calcium only stimulated the phosphorylation of a protein of MW = 64,000. In presence of 1 mM calcium a twofold effect is observed irrespective of the phosphoprotein considered. In particular there is an enhancement of 32P incorporation in four major phosphoproteins of MW = 160,000, 140,000, 64,000 and 50,000 in presence of GM1 compared with that observed with calcium alone. The maximal stimulating effect is achieved with a ganglioside concentration of 35 microM. This effect is inhibited by the addition of 100 microM trifluoperazine (TFP), a phenothiazine known to inhibit calmodulin and protein kinase-C activities. These four proteins represent the major substrates for the calcium/calmodulin-dependent protein kinase with the MW = 64,000 and 50,000 proteins co-migrating with the autophosphorylated subunits of this enzyme. In addition, the ganglioside inhibited the phosphorylation of three proteins with MW = 86,000, 20,000 and 14,000. The electrophoretic properties of these phosphoproteins are similar to the autophosphorylated form of protein kinase-C and to the rat myelin basic proteins, respectively. The effect of the ganglioside on their phosphorylation is not influenced by TFP. Finally, a protein with an apparent molecular weight of 46,000 shows also an increased phosphorylation in presence of GM1. The reported results indicate that exogenous GM1 can have profound effects on different kinases such as the calcium/calmodulin dependent protein kinase, the protein kinase-C and also some unknown calcium-independent protein kinases.     

AgNOR area in interphase nuclei of human tumours correlates with the proliferative activity evaluated by bromodeoxyuridine labelling and Ki-67 immunostaining

The area of silver-stained proteins associated with interphase nucleolar organizer regions (AgNORs) was compared with labelling data obtained by bromodeoxyuridine (BrdU) incorporation and Ki-67 immunostaining in 25 tumours of different origins and two non-neoplastic lesions of the thyroid. Our data demonstrate a highly significant correlation between the mean area occupied by the AgNOR proteins measured by an image processing system and the proliferative indices evaluated by BrdU labelling (r = 0.89, P less than 0.001) and Ki-67 immunostaining (r = 0.86, P less than 0.001). AgNOR protein area measurement is therefore proposed as a simple, inexpensive, and reliable method of evaluating the proliferative activity in routinely processed tumour samples.     

Clinical and ultrasonographic monitoring of response to adalimumab treatment in rheumatoid arthritis

OBJECTIVE: To evaluate by clinical, laboratory, and sonographic assessment the effects of adalimumab therapy in patients with rheumatoid arthritis (RA) over 24 months of treatment. METHODS: Twenty-five patients with RA were commenced on adalimumab therapy. Before the beginning of the therapy (Time 0) and after 3 (T1), 12 (T2), and 24 (T3) months we evaluated erythrocyte sedimentation rate, C-reactive protein, physician and patient visual analog scale for disease activity, number of tender and swollen joints, Health Assessment Questionnaire, and Disease Activity Score in 28 joints. In addition, musculoskeletal ultrasound (US) was performed bilaterally in the 2nd and 5th metacarpophalangeal, 3rd interphalangeal, wrist, and knee joints and in the tendon sheaths and bursae of those areas. A semiquantitative score (0 3) was used to indicate the presence of a localized inflammatory process and/or structural damage. The summed total was used as an indicator of global change in each joint (single joint score). The sum of the single joint scores was used as an indicator of overall polyarticular involvement in each patient (total score). RESULTS: Patients who did not submit to the planned examinations strictly on time were excluded from the study. Then 25 patients were examined at T0 and T1, 20 at T2, and 9 at T3. All clinical and laboratory measures as well as the US scores were significantly reduced during the followup. CONCLUSION: A positive response to treatment with adalimumab was demonstrated by clinical, laboratory, and US evaluation by both short- and longterm followup.     

Induction chemotherapy with carboplatin and taxol followed by radiotherapy and concurrent weekly carboplatin?+?taxol in locally advanced nasopharyngeal carcinoma

Purpose

Aim of this study was the clinical evaluation of carboplatin?Ctaxol combination in a neoadjuvant and concomitant setting with conventional radiotherapy in loco-regionally advanced nasopharyngeal carcinoma (A-NPC).

Methods

Thirty patients were treated with three cycles of carboplatin (AUC6) plus taxol (175?mg/m²) on day 1 every 3?weeks, followed by weekly carboplatin (AUC1) plus Taxol (60?mg/m2) and concomitant radiotherapy (70?Gy).

Results

We observed the objective complete response rates of 33% (after chemotherapy) and 87% (after chemo-radiotherapy). Treatment tolerability and toxicity were controllable. Three- and five-year progression-free survival were 80 and 75%, respectively, and 3- and 5-year overall survival were 85 and 80% (follow-up 49.5?months). Five-year loco-regional control was 90.3%, and five-year distant metastases?Cfree survival was 85%.

Conclusions

Neoadjuvant chemotherapy with such protocol represents a feasible, efficient treatment for patients with A-NPC, ensuring excellent loco-regional disease control and overall survival with low incidence of distant metastases.     

Polarized secretion of lysosomes at the B cell synapse couples antigen extraction to processing and presentation

Engagement of the B cell receptor (BCR) by surface-tethered antigens (Ag) leads to formation of?a synapse that promotes Ag uptake for presentation onto major histocompatibility complex class II (MHCII) molecules. We have highlighted the membrane trafficking events and associated molecular mechanisms involved in Ag extraction and processing at the B cell synapse. MHCII-containing lysosomes are recruited to the synapse where they locally undergo exocytosis, allowing synapse acidification and the extracellular release of hydrolases that promote the extraction of the immobilized Ag. Lysosome recruitment and secretion results from the polarization of the microtubule-organizing center (MTOC), which relies on the cell division cycle (Cdc42)-downstream effector, atypical protein kinase C (aPKCζ). aPKCζ is phosphorylated upon BCR engagement, associates to lysosomal vesicles, and is required for their polarized secretion at the B cell synapse. Regulation of B lymphocyte polarity therefore emerges as?a central mechanism that couples Ag extraction to Ag processing and presentation.     

An overview on the genetic of rheumatoid arthritis: a never-ending story

Rheumatoid arthritis (RA) is a chronic, systemic, inflammatory, multi-factorial disease sustained by environmental and genetic factors. These seem to be necessary but not sufficient in the disease development, nonetheless they can be responsible of different clinical pictures and response to therapy, and they can represent potential therapeutic targets. Several genes have been indicated so far in the pathogenesis of RA. The most important region is the Human Leukocyte Antigen (HLA) that contributes to approximately half of the genetic susceptibility for RA. The association seems to be stronger or specific for anti-citrullinated protein antibodies positive disease. Several alleles in the epitope-recognition part of the HLA molecule that show the highest association with RA susceptibility, also share a common string of amminoacid residues (the so-called shared-epitope hypothesis). Other variants in potentially pathogenic genes located in non-MHC regions have been implicated by recently performed genome wide analysis studies. These genes include PTPN22, TRAF1-C5, PADI4, STAT4. Other polymorphisms seem to be responsible for more aggressive disease phenotype such as those located at TNF, IL-1, IL-6, IL-4, IL-5, OPN, PRF1. However, still nowadays, the genetic background of RA remains to be clearly depicted, and the efforts in the post-genomic era can bring to an estimation of the real likelihood of the genetic effect on RA. Finally, the discovery of new genes associated with the disease can be relevant in finding potential biomarkers, potentially useful in disease diagnosis and treatment.