Real-time digital holographic microscopy of multiple and arbitrarily oriented planes.

Digital holographic microscopy is used to numerically refocus a recorded hologram at an arbitrary axial distance. However, as a straightforward property of coherent light fields, image reconstruction on an arbitrary tilted plane could be directly obtained by a rotation in k-space. We demonstrate that this property allows the real-time microscopic inspection of particle distribution over three mutually orthogonal planes at the same time. As a straightforward application we use the proposed technique for real-time monitoring of fluid flow over the three cross sections of a microfluidic channel.

[Nephrogenic systemic fibrosis]. / Fibrosi nefrogenica sistemica.

Nephrogenic systemic fibrosis (NSF) is a new, rare, and severe disease occurring in patients with renal failure who have been exposed to gadolinium. The pathogenesis of NSF is not completely known. In fact, the first warning about a significant relationship between NSF and gadolinium (a contrast medium used in magnetic resonance imaging) was only issued in 2006. No cases of NSF have been reported in Italy to date. A nationwide investigation should therefore be carried out to assess the real prevalence of NSF within the Italian uremic population. Furthermore, we need guidelines to reduce the risk of NSF in renal patients undergoing MRI with contrast medium.

Cutaneous plasmacytoma in a hemodialysis patient.

BACKGROUND: Extramedullary plasma cell dyscrasias are rare. CASE REPORT: We report a case of a 56-year-old male Caucasian hemodialysis patient with cutaneous plasmacytoma. The diagnosis was made a few months after surgical removal of his renal graft due to chronic rejection. Investigations for the presence of an associated myeloma were negative. He underwent local radiotherapy with complete resolution of the skin lesion. CONCLUSIONS: Nephrologists should be aware that the frequency of post-transplant lymphoproliferative disorders is increasing in the dialysis population, especially in those previously or currently treated with immunosuppressive drugs.

[Diabetes and atherosclerotic coronary damage in uremia]. / Diabete e danno coronarico negli uremici.

BACKGROUND: Cardiovascular risk factors are common findings in uraemics, but the impact of each single factor on the development of atherosclerosis is still a matter of debate. PATIENTS AND METHODS: In order to evaluate the relationship between diabetes and ischaemic heart disease (IHD) in uraemia, we carried out a retrospective study comparing the results of 33 coronary angiographies performed in non-diabetic patients with those of 13 diabetics (2 had type 1 diabetes, 8 were treated with insulin, 2 with sulfonylureas and 3 received no therapy). Coronary angiography was performed in 29 patients awaiting kidney transplantation and in 17 subjects with IHD. RESULTS: Age, sex, length of time on renal replacement therapy, smoking history, clinical diagnosis of cerebrovascular and peripheral vascular disease, systolic blood pressure (BP), cholesterol, triglycerides, calcium, phosphate, albumin and degree of anaemia were comparable in the two groups. On the contrary, frequency of IHD (77 vs. 30%, p<0.01) and atrial fibrillation (23 vs. 3%, p<0.05) were higher, while diastolic BP (79 +/- 7 vs. 85 +/- 8 mmHg, p<0.05) and calcium phosphate product (47 +/- 10 vs. 57 +/- 15 mg2/dL2, p<0.05) were lower in diabetics than in non-diabetics. Stenotic lesions of the three major coronary arteries were more prevalent in diabetics than in non-diabetics (left anterior descending artery (LAD) 100 vs. 48%, p<0.01; right coronary artery (RCA) 77 vs. 39%, p<0.05; left circumflex artery (LCA) 69 vs. 24%, p<0.01) and in the same way diabetics showed higher narrowing percentage (LAD 74 +/- 30 vs. 30 +/- 36%, p<0.01; RCA 71 +/- 41 vs. 26 +/- 38, p<0.01; LCA 41 +/- 38 vs. 15 +/- 29, p<0.05). CONCLUSIONS: Our study demonstrates that although the uraemic milieu is a risk factor for IHD, diabetes increases the degree of atherosclerotic vascular damage independently of the other cardiovascular risk factors.

Relationship between diabetes mellitus and degree of coronary artery disease in uraemic patients investigated with coronary angiography.

Prevalence of cardiovascular disease is high in diabetic patients on renal replacement therapy (RRT); therefore we examined the role of diabetes mellitus on determining the degree of coronary artery stenosis. Twenty-five patients underwent coronary angiography, 12 were awaiting kidney transplantation and the examination was performed regardless of cardiac symptoms, 13 were affected by ischaemic heart disease (IHD). Diabetic and nondiabetic status together with the other risk factors for cardiovascular disease such as age, sex, length of time on RRT, smoking and elevated phosphorus levels history, clinical diagnosis of IHD, cerebrovascular and peripheral vascular disease, mean blood pressure, cholesterol, triglycerides, calcium, phosphate, albumin, haemoglobin, haematocrit and weekly dose of erythropoietin were derived from clinical records. All investigated parameters were matched in diabetic (group 1, n=10) and nondiabetic patients (group 2, n=15) and showed no differences. Clinical evidence of IHD was detected in 80% of patients in group 1 and 46% in group 2 and the percentage of patients on the renal transplant waiting list was not statistically different in the two groups (30 vs 60%). In 60% of patients in group 1 there were 3 or more stenotic lesions equal or greater than 75% of normal reference segment in the major coronary arteries, whilst in 53% in group 2 there were no haemodynamically significant narrowings. Narrowing percentage of the coronaries in group 1 and 2 were: right coronary artery 83 +/- 30 vs 32 +/- 41 (p<0.05), left anterior descending artery 80 +/- 25 vs 44 +/- 34 (p<0.05), left circumflex artery 46 +/- 37 vs 18 +/- 29 (p=0.05) respectively. Our study confirms that IHD is a clinical feature of uraemic diabetic patients and that diabetes is the main cardiovascular risk factor for determining the degree of coronary stenosis.

The adenosine inhibition of glutamate exocytosis in synaptosomes is removed by the collapse of the vesicle-cytosol deltapH plus the opening of farnesol-sensitive Ca(2+) channels.

Adenosine inhibits synaptosomal exocytosis of glutamate, triggered by KCl or by the K(+) channel inhibitor, 4-aminopyridine (4-AP), without affecting Ca(2+) influx. Its effect is removed by the activation of protein kinase C (PKC). We show that in the presence of the protein kinase inhibitor, staurosporine, the adenosine inhibition is removed also by collapsing deltapH between secretory vesicle and the cytosol with methylamine (MA), provided that exocytosis is triggered by KCl (which activates an initial transient spike of Ca(2+) influx) but not by 4-AP. If KCl is supplied prior to Ca(2+), the spike of Ca(2+) influx is absent and the adenosine inhibition is maintained. MA can remove the adenosine inhibition also with 4-AP, provided that tetraethylammonium (TEA), an inhibitor of a different class of K(+) channels, is supplied together with 4-AP. TEA promotes a further increase of cytosolic free Ca(2+) concentration ([Ca(2+)](i)), which adds to the 4-AP-induced Ca(2+) influx. Farnesol (5-10 microM), a physiological derivative of farnesyl pyrophosphate of the sterol biosynthetic pathway, specifically inhibits the Ca(2+) spike after KCl as well as the TEA-promoted Ca(2+) increase. At the same time, it prevents the removal of the adenosine inhibition by MA. We conclude that the adenosine inhibition is removed by the coincidence of two signals, the alkalinization of secretory vesicles and the opening of a particular class of Ca(2+) channels associated to the TEA-sensitive K(+) channels, equivalent to the Ca(2+) spike after KCl, and sensitive to farnesol.

[Mesenteric ischemia in hemodialysis patients: case report and review of the literature]. / L'ischemia mesenterica negli emodializzati: descrizione di un caso e revisione della letteratura.

Mesenteric infarction is increasingly observed in uremic elderly patients with widespread atherosclerosis. A 77-year-old man on renal replacement therapy since June 1997 was admitted because of abdominal pain. The surgical diagnosis was massive intestinal infarction and the patient died a few hours later. A colonoscopy had been performed a few weeks before and a well-limited necrosis of the caecum mucosa had been detected. Hypotensive episodes were frequent during his hemodialysis sessions. In this work we discuss age, symptoms, laboratory investigations, risk factors and the evolution of case reports published during the last few years. Nephrologists should take into account the possibility of mesenteric ischemia in uremic patients with manifest arterio-occlusive disease, abdominal pain and leukocytosis, especially if hypotension is the major complication of the hemodialysis sessions.

[Coronary angiography in uraemic patients awaiting kidney transplantation]. / L'indagine coronarografica negli uremici in lista di attesa per trapianto renale.

BACKGROUND: Cardiovascular disease is the leading cause of morbidity and mortality in uraemia. Coronary angiography (CA) in patients awaiting kidney transplantation (PAKT) is still a matter of debate. In order to evaluate atherosclerotic coronary damage in PAKT, CAs of 12 PAKT were matched with those of 13 dialysis patients (P) affected by ischaemic heart disease IHD. METHODS: Age sex, length of time on renal replacement therapy, diabetes, smoking and hyperphosphataemia history, clinical diagnosis of IHD, cerebrovascular (CV) and peripheral vascular (PV) disease, mean blood pressure (BP), cholesterol, triglycerides, calcium, phosphate, albumin, haemoglobin, haematocrit and weekly dose of erythropoietin (EPO-dose) were derived from clinical records. RESULTS: PAKT were younger (48 9 vs 63 9 years, p < 0.01) and had higher diastolic BP values (86+/-10 vs 79+/-4 mmHg, p < 0.05) than IHD P. On the contrary all the other parameters investigated were not different in the two groups of P. Prevalence of IHD in PAKT was 16% while frequency of CV and VP disease were not different in the two groups. In 9 of IHD P stenotic lesions >/=75% of normal reference segment were diagnosed in 3 or more vessels whilst in PAKT there were atherosclerotic lesions in right coronary artery, left anterior descending artery and left circumflex artery in 41, 66 and 33% respectively. Narrowing percentage of the coronaries in PAKT and IHD P were: right coronary artery 27+/-42 vs 75+/-35, p < 0.05, left anterior descending artery 29+/-25 vs 86+/-15, p < 0.001, left circumflex artery 11 16 vs 47+/-38, p < 0.05 respectively. CONCLUSIONS: Our study shows that atherosclerotic coronary damage is present in PAKT and, although not hemodynamically significant, it could be an important risk factor for clinical expression of IHD. We conclude that CA should be performed in PAKT especially in those over 45 years.

Do different dialytic techniques have different atherosclerotic and antioxidant activities?

To compare the chronic effect of several dialytic techniques (bicarbonate dialysis, BHD; acetate free biofiltration, AFB; hemodiafiltration, HDF; paired filtration dialysis, PFD) on atherosclerosis and antioxidant activity, three different indices were created. The first (atherosclerotic index = AI) is formed using the sum of three plasma substances: MDA, Hcy, and Cys (malondialdehyde, homocysteine, cysteine). The second (antioxidant activity index = AOAI) is the sum of five erythrocyte (E) parameters: E-GSH, GPx, CAT, SOD, GR (E-glutathione, E-glutathione peroxidase, E-catalase, E-superoxide dismutase, E-glutathione reductase). The third (defense index = DI) is derived from the previous two: (AOAI - AI). The indices were so expressed as AI in mmol/L, AOAI in U/g hemoglobin (Hb), and DI in arbitrary units. These indices were calculated in 20 controls and 51 chronic HD patients (26 female, 25 male) before, during, and after the first session of the week. HD patients were divided according to their dialytic technique: BHD, n = 35; AFB, n = 5 patients; HDF, n = 7 patients; or PFD = 4 patients. All patients had been treated with a given technique for at least 12 months, before entering the study. As expected, HD patients had AI values higher than controls, both before and after the session, with a mean value of 541 (before) and 331 (after), whereas controls had a mean value of 205. The AOAI was lower than controls, both before and after the session, the mean value being 1,122 (before) and 1,582 (after), that of controls being 2,424. In all cases, PFD gave the best "acute" results; at the end of a PFD session, near normal values of AI, AOAI, and DI (defensive index = AOAI - AI) were obtained.

Adenosine inhibits glutamate exocytosis largely without interfering with Ca2+ influx in rat cerebrocortical synaptosomes.

Adenosine is an inhibitor of glutamate release in synaptosomes. The inhibition is removed by the A(1) adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). We monitored the variations of cytoplasmic free Ca(2+) concentrations ([Ca(2+)](i)) in KCl or 4-aminopyridine-stimulated synaptosomes, in the presence of adenosine or adenosine plus DPCPX. The increment of [Ca(2+)](i) upon stimulation was unmodified by adenosine (up to 400-500 microM) while it was strongly decreased when exocytosis was decreased to a similar extent by lowering KCl or 4-aminopyridine. Adenosine also inhibited glutamate release induced by the Ca(2+) ionophore ionomycin. Increasing adenosine to 1.5 mM resulted in a decrease of the stimulus-induced increase of [Ca(2+)](i) and in the further potentiation of the adenosine inhibition of exocytosis from 41+/-3 to 51+/-4%. We conclude that adenosine affects glutamate exocytosis mostly in a Ca(2+) independent mode.

Involvement of nuclear factor-kappa B (NF-kappaB) activation in mitogen-induced lymphocyte proliferation: inhibitory effects of lymphoproliferation by salicylates acting as NF-kappaB inhibitors.

The transcription factor nuclear factor-kappa B (NF-kappaB) is involved in the production of inflammatory cytokines and in the control of the inflammatory response. Some nonsteroidal anti-inflammatory drugs such as acetylsalicylic acid (ASA) or salicylate are known to exert some of their anti-inflammatory pharmacological properties independently of cyclooxygenase inhibition. For ASA and salicylate, an NF-kappaB inhibitory effect at mM concentrations (pharmacological plasma concentrations reached in vivo) has been shown. We studied the action of ASA, salicylate, and several NF-kappaB inhibitors on the mitogen-induced activation of peripheral blood lymphocytes (PBL) and purified T cells. We showed that ASA and salicylate (1-3 mM) (but not indomethacin, a specific cyclooxygenase inhibitor) as well as a group of chemically unrelated inhibitors of NF-kappaB (including the sesquiterpene lactone parthenolide, Bay 11-7082, sulfasalazine, the proteasome inhibitor MG-132 and the peptide SN-50, an inhibitor of the nuclear transfer of the p50 subunit of NF-kappaB), were potent inhibitors of phytohemoagglutinin-activated PBL and T cell proliferation. At the same concentrations, they inhibited NF-kappaB binding to DNA in nuclear extracts. The inhibition of proliferation was not relieved by exogenous interleukin (IL)-2. We concluded that NF-kappaB activation has a fundamental role in T cell proliferation independently of IL-2 production. Some pharmacological actions of ASA may be ascribed to the inhibition of immune cell proliferation via the inhibition of the transcription factor NF-kappaB.

Role of cellulosic and noncellulosic membranes in hyperhomocysteinemia and oxidative stress.

Hyperhomocysteinemia is an independent risk factor for cardiovascular morbidity and mortality in end-stage renal disease (ESRD) with an increased relative risk (RR) of 1% per micromol/L in total homocysteine concentration. In ESRD patients who undergo hemodialysis (HD), the antioxidant system is largely inadequate in correcting the imbalance between generation and scavenging of reactive oxygen species (ROS). To clarify the role of several cellulosic (CMs) and noncellulosic of synthetic membranes (NCMs) upon hyperhomocysteinemia and the oxidative stress, we measured plasma (P) homocysteine (t-HCY), plasma lipid peroxidation (LPO), and erythrocyte (E) concentration of several antioxidant enzymes in 20 normal subjects, in 35 HD patients treated with CMs, and in 29 patients treated with NCMs. Before, during, and after the first session of the week (at times 0', 120', end, 30' after HD end), blood samples were drawn. Plasma (P) homocysteine (t-HCY), cysteine (CYS), malondialdehyde (MDA), erythrocyte (E)-glutathione (GSH), glucose-6-phosphodehydrogenase (G6PD), glutathione reductase (GR), glutathione peroxidase (GPx), catalase (CAT), and superoxide-dismutase (SOD) were determined. The dialytic procedure significantly decreased the three plasma parameters, but none normalized (as a mean). The E-enzymes scavenging ROS (lower than normal before session) increased throughout the session, but the normal range of activity was never reached. Different membranes have shown different effects. When these effects on P and E spaces were pooled, we were able to classify the membranes as follows. In a general sense, cellulosic membranes are less effective than synthetic membranes both on lipoperoxides (LPO) and antioxidant activity (AOA). Among synthetic membranes, PMMA is the best membrane both for plasma values and lesser enzymatic derangement during the session. A practical system for classifying the anti-atherosclerotic action and antioxidant activity of dialytic membranes is proposed.

Oral N-acetyl-cysteome increases the production of anti HIV chemokines in peripheral blood mononuclear cells.

The C-C chemokines MIP-1alpha, MIP-1beta and RANTES are specific and powerful inhibitors of HIV infectivity. They appear to work by blocking the interaction of the virus with the receptor (CCR5). The latter is utilized as a coreceptor for cell penetration by macrophage-tropic (R5) HIV strains responsible for the majority of HIV transmissions. A natural high capability to release such chemokines has been proposed as a protection factor against HIV infection in exposed uninfected individuals. We report that oral administration of N-acetyl-cysteine (NAC) to healthy volunteers increases the capability of their peripheral blood mononuclear cells (PBMC) to release such anti HIV chemokines upon stimulation. The data reported may explain at least in part the mechanism of action of NAC as an anti HIV therapeutic agent: By potentiating chemokine production NAC may decrease susceptibility to infection.

[Extensive cardiac angiosarcoma in the course of immunosuppressive therapy after kidney transplant]. / Esteso angiosarcoma cardiaco in corso di terapia immunosoppressiva post-trapianto renale.

We present a case of a 49-year-old man who underwent a kidney transplant. Treatment with azathioprine and steroid was increased due to an initial rejection of the allograft. Six months later, the patient experienced palpitations and precordial pain: the electrocardiogram recorded atrial fibrillation and the echocardiogram showed moderate pericardial effusion and the presence of a right atrial mass with infiltration into the interatrial septum and right ventricle. Computed tomography imaging confirmed the tumoral mass and magnetic resonance imaging revealed the extensive involvement of the right atrium, right ventricle and superior vena cava, suggesting the presence of an angiosarcoma of the heart. Despite chemotherapy the patient died within 1 month. The diagnosis was confirmed by autopsy, histological section, and electron microscopy examination.

Modulation of glutamate exocytosis by redox changes of superficial thiol groups in rat cerebrocortical synaptosomes.

The treatment of cerebral cortex synaptosomes with the membrane impermeable thiol reagent 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) induces a long-lasting partial inhibition (about 40%) of the KCl-stimulated Ca2+-dependent exocytosis of glutamate. Synaptosomes are not damaged by the treatment. The increase of cytoplasmic free Ca2+ concentration ([Ca2+]i) upon depolarization is not affected by DTNB. The inhibition is observed also if exocytosis is induced with the Ca2+-ionophore ionomycin. In all cases the inhibition is reversed by the impermeable reductant glutathione (GSH). Similarly the inhibition of exocytosis by H2O2 (Zoccarato, F., Valente, M. and Alexandre, A., Hydrogen peroxide induces a long-lasting inhibition of the Ca2+-dependent glutamate release in cerebrocortical synaptosomes without interfering with cytosolic Ca2+. J. Neurochem., 64 (1995) 2552-2558.) is reversed by GSH. It is concluded that redox changes (possibly thiol-disulfide transitions) of superficial groups modulate the exocytotic apparatus directly. In an attempt to identify the protein(s) involved in this novel type of control, we evidenced DTNB (H2O2) reactive bands at 35 and at 85-150 kDa which can be labeled with a monobromotrimethylammoniobimane bromide (qBBr) derivatization.

Ganglioside GM1 protection from apoptosis of rat heart fibroblasts.

Ceramide is involved as a mediator of apoptosis induced by a variety of signaling molecules or stressful events. Ceramide-derived sphingosine 1-phosphate behaves as an antiapoptotic agent. The ganglioside GM1 is known to protect neuronal cell lines from apoptosis induced by serum/growth factor withdrawal and its effect is mediated in part by the direct activation of the trkA NGF receptor [G. Ferrari et al. (1995) J. Biol. Chem. 270, 3074-3080]. We show that GM1, similarly to sphingosine 1-phosphate, protects rat heart fibroblasts from apoptosis induced by the protein kinase C inhibitor staurosporine and by C2-ceramide. Furthermore, we show that GM1 induces the synthesis of sphingosine 1-phosphate and that this effect is partially prevented by the sphingosine kinase inhibitor N,N-dimethylsphingosine. We conclude that the antiapoptotic action of GM1 is largely to be ascribed to an increased sphingosine kinase activity.

The pH-sensitive dye acridine orange as a tool to monitor exocytosis/endocytosis in synaptosomes.

We introduce the use of the pH-sensitive dye acridine orange (AO) to monitor exo/endocytosis of acidic neurotransmitter-containing vesicles in synaptosomes. AO is accumulated exclusively in acidic v-ATPase-dependent bafilomycin (Baf)-sensitive compartments. A fraction of the accumulated AO is rapidly released (fluorescence increase) upon depolarization with KCl in the presence of Ca2+. The release (completed in 5-6 s) is followed by reuptake to values below the predepolarization baseline. The reuptake, but not the release, is inhibited by Baf added 5 s prior to KCl. In a similar protocol, Baf does not affect the initial fast phase of glutamate release measured enzymatically, but it abolishes the subsequent slow phase. Thus, the fast AO release corresponds to the rapid phase of glutamate release and the slow phase depends on vesicle cycling. AO reuptake depends in part on the progressive accumulation of acid-loaded vesicles during cycling. Stopping exocytosis at selected times after KCl by Ca2+ removal with EGTA evidences endocytosis: Its T(1/2) was 12 +/- 0.6 s. The K(A)+, channel inhibitors 4-aminopyridine (100 microM) and alpha-dendrotoxin (10-100 nM) are known to induce glutamate release by inducing the firing of Na+ channels; their action is potentiated by the activation of protein kinase C. Also these agents promote a Ca2+-dependent AO release, which is prevented by the Na+ channel inhibitor tetrodotoxin and potentiated by 4beta-phorbol 12-myristate 13-acetate (PMA). With alpha-dendrotoxin, endocytosis was monitored by stopping exocytosis at selected times with EGTA or alternatively with Cd2+ or tetrodotoxin. The T(1/2) of endocytosis, which was unaffected by PMA, was 12 +/- 0.4 s with EGTA and Cd2+ and 9.5 +/- 0.5 s with tetrodotoxin. Protein kinase C activation appeared to facilitate vesicle turnover.

Dialysis kinetics of homocysteine and reactive oxygen species.

In patients with chronic renal failure who undergo hemodialysis (HD), the antioxidant system is inadequate to correct the imbalance between the generation and scavenging of reactive oxygen species. To clarify the role of six different membranes on oxidative stress, the authors measured plasma lipid peroxidation and erythrocyte (E) concentrations of several antioxidant enzymes in 30 HD patients: 20 on bicarbonate HD, 4 on paired filtration dialysis, 3 on acetate free biofiltration, and 3 on hemodiafiltration. Before, during, and after the first session of the week (at times 0, 30, 60, and 120 min, end, and 30 min after end of HD), several blood samples were drawn. Plasma (P) homocysteine (HCY), cysteine (CYS), malondialdehyde (MDA), E-glutathione (GSH), glucose-6-phosphodehydrogenase, glutathione reductase (GR), glutathione peroxidase (GP), catalase (CAT), and superoxide dismutase (SOD) were determined. All six membranes (Hemophan [HEMO]; cellulose diacetate [DIAC]; acrylonitrile-69 [AN69]; polymethylmethacrylate [PMMA]; cuprammonium rayon [CURAY]; polysulfone plus hemophan [PS + HEMO]) induced a significant decrease in plasma lipid peroxidation (p < 0.001) and an increase in E-GSH, GR, GR + flavinadenine dinucleotide, GP, and SOD (p < 0.001). Some membranes, however, showed some peculiar effects on reactive oxygen species: HEMO is better than DIAC, as far as P-MDA and P-HCY are concerned; PMMA induces higher changes in E-GR and P-CYS than does HEMO; and patients chronically using PMMA and PS + HEMO membranes show the lowest P-HCY levels both before and after dialytic sessions. Based on these changes, implications as to the effects on vascular disorders could be derived.