A stochastic view on surface inhomogeneity of nanoparticles.

The interactions between and with nanostructures can only be fully understood when the functional group distribution on their surfaces can be quantified accurately. Here we apply a combination of direct stochastic optical reconstruction microscopy (dSTORM) imaging and probabilistic modelling to analyse molecular distributions on spherical nanoparticles. The properties of individual fluorophores are assessed and incorporated into a model for the dSTORM imaging process. Using this tailored model, overcounting artefacts are greatly reduced and the locations of dye labels can be accurately estimated, revealing their spatial distribution. We show that standard chemical protocols for dye attachment lead to inhomogeneous functionalization in the case of ubiquitous polystyrene nanoparticles. Moreover, we demonstrate that stochastic fluctuations result in large variability of the local group density between particles. These results cast doubt on the uniform surface coverage commonly assumed in the creation of amorphous functional nanoparticles and expose a striking difference between the average population and individual nanoparticle coverage.

iPAINT: a general approach tailored to image the topology of interfaces with nanometer resolution.

Understanding interfacial phenomena in soft materials such as wetting, colloidal stability, coalescence, and friction warrants non-invasive imaging with nanometer resolution. Super-resolution microscopy has emerged as an attractive method to visualize nanostructures labeled covalently with fluorescent tags, but this is not amenable to all interfaces. Inspired by PAINT we developed a simple and general strategy to overcome this limitation, which we coin 'iPAINT: interface Point Accumulation for Imaging in Nanoscale Topography'. It enables three-dimensional, sub-diffraction imaging of interfaces irrespective of their nature via reversible adsorption of polymer chains end-functionalized with photo-activatable moieties. We visualized model dispersions, emulsions, and foams with ∼20 nm and ∼3° accuracy demonstrating the general applicability of iPAINT to study solid/liquid, liquid/liquid and liquid/air interfaces. iPAINT thus broadens the scope of super-resolution microscopy paving the way for non-invasive, high-resolution imaging of complex soft materials.

Psychological stress symptoms before and after autologous bone marrow transplantation in patients with solid tumors.

This study sought to examine patterns of changes of psychological stress symptoms in autologous bone marrow transplantation (ABMT) recipients. Forty-nine patients affected by solid tumors were assessed using the Symptom Questionnaire on admission to hospital (before high-dose chemotherapy and ABMT) and before discharge. Symptoms of anxiety and anger tended to decrease and relaxation of improve over time. Nevertheless, on admission to hospital 30-50% of the patients reported severe to moderate symptoms of anxiety and depression. Before discharge, the prevalence was still high (20-35%). The implications of these findings are discussed in terms of the need to monitor the evolution of emotional functioning of cancer patients undergoing ABMT.

High dose chemotherapy with carboplatin, VP 16 +/- ifosfamide in germ cell tumors: the Italian experience.

This schedule has shown an interesting activity with nearly 40% of the patients achieving CR. Moreover 4 patients experienced an inversion rate (CR with ABMT when they never achieved this status before). In terms of toxicity, this schedule seems feasible with 2/28 toxic deaths, which is in the lower part of the range of major solid tumors ABMT programs. But even if the rationale is proper, a better patients' selection is required. The CCR (continuous Complete Remission) rate is overimposable to other main studies previously published, but all our CCR were obtained in responding patients (Sensitive Relapses or unresectable PR). We may suggest that earlier transplantation is advisable when less tumor bulky is present and less clonal eterogeneity. The exact maximum tolerated dose of Carboplatin/VP 16/Ifosfamide programs has not yet clearly pointed out. The lack of major life-threatening episodes and neuro/nephrotoxicity may allow us to explore higher Carboplatin doses. Anyway the ultimate answer to the utility of ABMT trials must come from a randomized study in responding patients.

Allogeneic bone marrow transplantation for multiple myeloma.

Seventeen patients received marrow transplants from their HLA-matched, MLC-negative, sibling donors. Nine patients had progressive disease not responding to conventional treatments, while the other 8 patients were rated as responders. The most frequently used conditioning regimen consisted of total body irradiation and high-dose multi-agent chemotherapy with cyclophosphamide plus either oral melphalan (5 cases) or BCNU (1 case) on both these drugs (7 cases). Twelve patients were valuable for response to BTM: 7 of them (6 responders and 1 with advanced refractory MM) entered complete remission, while 5 had sustained decrease in tumor mass that ranged between 72% and 93%. Eleven patients died of transplant-related causes, 1 of them with signs of progressive disease. The remaining 6 patients are alive and 5 of them maintain a complete remission status 4 to 67 (median 36) months after BMT.

An Italian experience of high-dose chemotherapy and autologous bone marrow transplantation (ABMT) in germ cell tumours. Suggestions for future direction.

In this paper an Italian cooperative trial investigates the role of a high-dose regimen with carboplatin, etoposide and ifosfamide in germ cell tumours. Twenty-eight patients underwent one or two transplants. Seventeen with progressive disease. Nine in sensitive relapse and two with stable disease after salvage therapy. Toxicity was generally moderate: two deaths occurred at day 15 from ABMT (one from VOD and one from tumour growth). Five patients are alive and disease free at least 10 months follow-up. In highly pre-treated patients this high-dose combination seems to give an option of cure for relapsed patients. Early transplantation is suggested.

Do Italian small cell lung cancer (SCLC) specialists share a common language? An analysis based on 549 questionnaires.

Different specialists are involved in the treatment of SCLC: medical oncologists, pneumologists, radiotherapists, and thoracic surgeons; only in large institutions the therapeutic policy is the result of a multidisciplinary approach. In order to investigate the opinions of the Italian physicians about the state of the art in the diagnosis and treatment of SCLC, 2369 questionnaires have been sent to an equal number of specialists. Each questionnaire contained 16 topics addressing what we consider major open questions. The analysis is based on 549 interpretable questionnaires received back (23.1%). The general attitude of responding physicians is quite pessimistic on the present state of the art; the large majority considering insufficient the current knowledge of both clinical and basic research. Some differences have been registered, among different specialists, regarding the role of surgery and radiation therapy in prolonging the expected survival; while a nearly unanimous consensus has been reached on the role of radiation therapy for local control. Optimism merges about the possibilities of ameliorating the survival in the next decades: 48% have confidence in new drugs, 45% in the development of integrated modalities, and 41% in the application of basic research.

The use of granulocyte colony-stimulating factors following peripheral blood progenitor cell rescue after high-dose chemotherapy for advanced breast cancer: a prospective study.

The use of high-dose chemotherapy followed by hematopoietic rescue is increasing worldwide for solid tumors. Several studies have suggested that the period of absolute neutrophil count (ANC, < 500/ml) may be shortened in patients who receive peripheral blood progenitor cells (PBPC). To estimate the clinical value of granulocyte-colony-stimulating factor, we examined a cohort of 26 consecutive patients with advanced breast cancer who received one or two cycles of high-dose chemotherapy with PBPC rescue with or without filgrastim. Thirty-five courses of high-dose ICE (ifosfamide, carboplatin, etoposide) chemotherapy were administered and evaluated. All patients received PBPC rescue. Sixteen patients (21 courses) received subcutaneous filgrastim (5 mg/kg) following PBPC infusion. Recovery to > or = 500 ANC occurred at a median time of 7 days post PBPC infusion among patients who received filgrastim versus 10 days among patients who received standard support care only (P < 0.01). The administration of filgrastim was not associated with a reduction in the duration of hospitalization, in the total number of days on nonprophylactic antibiotics, number of red blood cell transfusions, time to platelet engraftment, or number of febrile days. This could be the consequence of the high hematopoietic cell dose administered in the study. Therefore, any effect of filgrastim was probably masked by the use of a large number of PBPC. Larger prospective randomized studies, specifically focused on the utility of the administration of growth factors following high-dose chemotherapy and PBPC rescue, may be warranted to know whether the administration of filgrastim after PBPC transplantation is really necessary.

[Incidence of neoplasm recurrence after colorectal resection for cancer]. / Incidenza delle recidive neoplastiche dopo resezione rettocolica per cancro.

The Authors report their experience on the incidence of anastomotic recurrence in 122 patients surgically treated with colorectal resection for cancer. The number of local recurrences (3 cases, that is 2.5%) is in the inferior range of what reported in Literature. Parameters that influence the local failure are: depth of tumor invasion, extension to adjacent organs and structures, presence of lymph node metastases (valuable with Dukes' modified staging). Type of operation and histologic grade do not seem to influence local recurrence. The Authors report the therapeutic choices adopted in the cases considered.

Preparation of stable dispersion of barium titanate nanoparticles: Potential applications in biomedicine.

Nanoscale structures and materials have been explored in many biological applications because of their extraordinary novel properties. Here we propose a study of cellular interactions with barium titanate nanoparticles, an interesting ceramic material that has received a lot of interest in the nanotechnology research, but without any attention about its biological potential. We introduced for the first time an efficient method for the preparation of stable aqueous dispersions of barium titanate nanoparticles, characterized with FIB, TEM and AFM imaging, light scattering, Z-potential and UV/vis analysis. Finally, we presented a systematic study of short-term cytotoxicity of the prepared dispersion based both on quantitative (metabolism, proliferation) and qualitative (apoptosis, viability, differentiation) assays.

Angiotensin II protects fibroblast-like synoviocytes from apoptosis via the AT1-NF-kappaB pathway.

OBJECTIVE: To evaluate the effects of angiotensin II (Ang II) treatment on apoptosis of fibroblast-like synoviocytes (FLS) from patients with osteoarthritis (OA) and rheumatoid arthritis (RA). METHODS: AT1 receptor expression was detected by western blotting and flow cytometry. Apoptosis induction was quantified by nucleosome ELISA and by TUNEL; cell proliferation was determined by a bromodeoxyuridine (BrdU) incorporation assay. Silencing of p65 NF-kappaB was obtained by using a specific siRNA. Caspase 3 activation was evaluated by a colorimetric assay and its cleavage by western blotting. RESULTS: AT1 expression resulted comparable in FLS from OA and RA patients. Ang II pre-treatment reduced FLS apoptotic response to serum starvation and nitric oxide (NO) exposure. This protective effect was reverted in the presence of the AT1 receptor antagonist losartan as well as after silencing the expression of NF-kappaB. Moreover, FLS treatment with the caspase inhibitor z-VAD-fmk cancelled this Ang II effect on apoptosis. Caspase 3 activation was reduced in presence of Ang II. CONCLUSIONS: Ang II could represent an important mediator involved in FLS expansion, reducing their capacity to undergo apoptosis, through the activation of NF-kappaB and the blockage of caspase cascade.

Abnormal intracellular level of Bax in CD3+ cells from untreated B-cell chronic lymphocytic leukemia patients.

B-cell chronic lymphocytic leukemia (B-CLL) is a lymphoproliferative disease caused by impaired apoptosis regulation that leads to an abnormal survival and an accumulation of B-lymphocytes. Anti-apoptotic Bcl-2 and proapoptotic Bax proteins are involved in the highly regulated mechanism of cell death. Bax and Bcl-2 intracellular levels were analyzed both in CD19+ and CD3+ cells from 28 B-CLL de novo patients and compared with cells from healthy donors. Our results were expressed as a ratio (Bax/Bcl-2) obtained by dividing Bax mean fluorescence intensity (MFI) and Bcl-2 MFI; obviously, a lower ratio is associated with an anti-apoptotic status, while a higher index correlates to apoptosis activation. In CD19+ B-CLL cells, the Bax/Bcl-2 ratio was lower than in the CD19+ normal counterpart (1.3 versus 3.51; P<.05), mainly due to a Bcl-2 over expression (17.65 versus 9.02; P<.001). In CD3+ cells from B-CLL patients, the Bax/Bcl-2 ratio was lower than in normal CD3+ cells (7.89 versus 8.96; P<.005), most importantly as a result of Bax suppression (77.22 versus 96.63; P<.001). These study data show an apoptosis inhibition not only in CD19+ cells, but also in CD3+ cells, suggesting a pivotal role of T-cells in B-CLL pathogenesis.

Absence of apolipoprotein B-48 in the chick, Gallus domesticus.

This study was designed to investigate: a) whether multiple forms of apoB are present in chick plasma lipoproteins; and b) which forms of apoB are produced in vitro by liver and intestine at various stages of pre- and post-natal development. Plasma lipoproteins of d less than 1.019 g/ml, isolated from fasted and nonfasted chicks, contained exclusively the high molecular weight apoB form (apoB-100) that comigrated with human and rat apoB-100 on SDS-PAGE gel. No apoB-48 was detected either in overloaded Coomassie blue-stained gels or after immunoblotting. ApoB-100 but no apoB-48 was found in portomicrons, the triglyceride-rich lipoproteins equivalent to chylomicrons, that in the chick are transported via the porto-mesenteric venous system. To ascertain whether a minute amount of apoB-48 was present in chick plasma, [35S]methionine was injected intraduodenally and the 35S-labeled d less than 1.019 g/ml plasma lipoproteins were isolated 45 min later from the systemic and the porto-mesenteric circulation. Only apoB-100 was found to be labeled in these lipoproteins. Cholesterol feeding did not induce the appearance of apoB-48 in plasma despite a marked accumulation of cholesterol-rich d less than 1.040 g/ml lipoproteins in the plasma. In vitro synthesis of apoB forms was studied in liver and intestinal slices isolated from chick embryos (8 and 5 days before hatching), newly hatched chicks (2 and 7 days after hatching), and young chicks (21 days old) that were incubated in the presence of [35S]methionine. At each stage of development, liver slices secreted predominantly apoB-100. Intestinal slices of newly hatched and young chicks secreted two forms of apoB: apoB-100 and an additional form with an electrophoretic mobility similar to rat plasma apoB-95. No apoB-48 was synthesized or secreted by the intestine. Our results indicate that the absence of apoB-48 in chick plasma reflects the lack of synthesis of this peptide in the intestine. It is conceivable that in chick intestine the recently described molecular mechanism responsible for the co/posttranscriptional modification of apoB mRNA leading to the formation of apoB-48 is lacking or defective.

Dyslipoproteinemia in an inbred rat strain with spontaneous chronic progressive nephrotic syndrome.

Rats of the Milan Normotensive Strain (MNS) develop a dyslipoproteinemia that is associated with a spontaneous, age-dependent and slowly progressive nephropathy characterized by proteinuria and hypoalbuminemia (nephrotic syndrome). We assumed that the MNS strain might be a suitable model for studying the features of nephrotic dyslipoproteinemia and its relationship with proteinuria, hypoalbuminemia, and hepatic apolipoprotein production. Plasma lipoproteins were investigated in MNS rats at various ages (4-48 weeks) and in another rat strain (Milan Hypertensive Strain, MHS), genetically related to MNS but free of nephropathy, that was used as control. In MNS rats, abnormal proteinuria was detectable at 20 weeks and increased 2-fold up to 34 weeks with no reduction of plasma albumin (compensated stage). During this stage we found increased levels of plasma cholesterol (+ 34%), high density lipoprotein-1 (HDL1) (+ 73%), and HDL2 (+ 31%) that were positively correlated with proteinuria but not with plasma albumin. The later stage (34-48 weeks) (nephrotic stage) was characterized by a further increase of proteinuria, moderate hypoalbuminemia (- 25%), a 2-fold increase of plasma cholesterol, triacylglycerols, low density lipoprotein (LDL), and HDL1, and a 1.2-fold increase of HDL2. In this stage the levels of LDL, HDL1, and HDL2 were positively correlated with proteinuria, and negatively correlated with plasma albumin. The most striking change in apolipoproteins was a progressive increase of the relative content of apoA-I in HDL (in 48-week-old MNS rats the A-I/E ratio was 3-fold that found in MHS rats) that was associated with a similar increase of plasma apoA-I. None of these lipoprotein changes were observed in age-matched MHS rats. At the end of the compensated stage, the hepatic levels of A-I, B, A-II, and albumin mRNA were 5.3-, 3.5-, 1.3-, and 2.0-fold, respectively, those found in age-matched MHS rats. During the nephrotic stage, albumin mRNA continued to increase, whereas A-I, B, and A-II mRNAs decreased toward the levels found in age-matched MHS rats. Thus, nephrotic dyslipoproteinemia in MNS rats starts to develop in the compensated stage before the onset of hypoalbuminemia, is characterized by an early elevation of HDL1 + HDL2, and is associated with an increased content of hepatic mRNAs of some apolipoproteins, especially apoA-I. The slow progression of nephrotic syndrome with the long-standing proteinuria and no reduction in plasma albumin renders the MNS strain the most suitable animal model for the study of the effect of proteinuria on plasma lipoprotein metabolism.

Antibody response to human immunodeficiency virus after infected bone marrow transplant.

An antibody response to human immunodeficiency virus (HIV) is described in a young woman with T-lymphoblastic leukemia, who received a bone marrow transplant from a donor retrospectively found to be HIV positive.

Effect of heparin-like compounds on the in vitro proliferation and protein synthesis of various cell types.

Previous studies have shown that heparin and heparin-like compounds inhibit the proliferation of arterial smooth muscle cells (SMC) both in vivo and in vitro. This anti-proliferative effect seems to be exerted almost exclusively on arterial SMC and related cell types. In the present study the effect of heparin (HTh) is compared with that of two sulfated glycosaminoglycans with low anticoagulant activity, sulodexide (SDX) and low molecular weight heparin (OP/LMWH) on cell proliferation and protein synthesis of 3 cell types: human arterial smooth muscle cells (SMC), fibroblast-like cells (BHK-21) and epithelial cells (rat hepatoma cells, FAO). HTh, SDX and OP/LMWH (5-100 micrograms/ml) are equally effective in reducing the proliferation of human arterial SMC. This inhibition is dose dependent and reversible. BHK-21 and FAO cells are even more sensitive than SMC to heparin-like compounds. For example 1 microgram/ml of heparin-like compounds is sufficient to produce 40-60% inhibition of FAO cell proliferation. In all types of cells HTh, SDX and OP/LMWH do not reduce the incorporation of 35S-methionine into cellular and medium proteins; they increase the radioactivity incorporated into some proteins secreted into the medium. In the case of SMC this effect is dependent on the concentration and the length of exposure to heparin-like compounds. These findings demonstrate that several cell types are sensitive to the anti-proliferative effect of heparin-like compounds.

The effect of a thromboxane A2 synthase inhibitor on the dyslipoproteinemia of an inbred rat strain with spontaneous age-related nephrotic syndrome.

We have previously shown that the administration of a thromboxane A2 (TXA2) synthase inhibitor (FCE 22178) reduced the progression of glomerular lesions and proteinuria in MNS rats, an inbred strain which develops an age-related nephrotic syndrome. In the present study we investigated the effect of FCE 22178 on the plasma lipoproteins of MNS rats at 28 weeks of age (with mild proteinuria and moderate dyslipoproteinemia) and at 48 weeks of age (with heavy proteinuria and severe dyslipoproteinemia). Drug treatment reduced proteinuria (by 70% and 36% at 28 and 48 weeks of age, respectively) plasma cholesterol (by 36% and 27% at 28 and 48 weeks of age, respectively) and prevented the decrease of plasma albumin observed in untreated rats (C-MNS) 48 weeks old. In treated rats (T-MNS), the decrease of proteinuria was positively correlated with that of plasma cholesterol. FCE 22178 reduced the elevation in plasma HDL1 (by 17.4%) and HDL2 levels (by 30%), a key feature of nephrotic dyslipoproteinemia in the rat. From 28 to 48 weeks of age plasma apo A-I and apo E increased 217% and 128%, respectively, in C-MNS rats and 191% and 121%, respectively, in T-MNS rats. A significant increase of apo A-I/apo E ratio was found in C-MNS rats from 28 (2.28 +/- 0.36) to 48 weeks of age (3.84 +/- 0.9) but not in T-MNS rats. FCE 22178 altered the lipid composition of VLDL and HDL2 by reducing the content of cholesteryl esters and increasing that of free cholesterol and phospholipids. These findings suggest that the beneficial effect of FCE 22178 on the dyslipoproteinemia of nephrotic MNS rats is secondary to the amelioration in kidney function and to the reduction of proteinuria produced by this drug.

Synthesis and secretion of apolipoprotein A-I by chick skin.

Chick skin slices were incubated with [35S]methionine and labeled apoA-I was immunoprecipitated from incubation medium and tissue homogenate. ApoA-I accounted for approximately 13 and 2.5% of radioactive medium and cell proteins, respectively. After ultracentrifugation of the medium, 55% of labeled apoA-I was found as a constituent of lipoproteins (d less than 1.210 g/ml) and 45% in a lipid-poor form (1.210-1.260 g/ml). To ascertain whether this large proportion of lipid-poor apoA-I was due to a dissociation of this peptide from medium lipoproteins during ultracentrifugation, labeled incubation medium was applied to an anti-chick apoA-I immunoaffinity column. The material bound to the column was analyzed by nondenaturing polyacrylamide gradient gel electrophoresis and found to contain three subpopulations of lipoproteins with a particle size of 12, 11, and 9 nm, respectively. The radioactivity of these subpopulations accounted for 82% of total radioactive medium apoA-I. ApoA-I was localized by immunohistochemistry in the viable cells of the epidermis and in the stratum corneum. Rat skin slices were found to synthesize and secrete apoE but no apoA-I. ApoA-I and apoE secreted by chick and rat skin, respectively, may play a role in the secretion of lipids from the differentiating keratinocytes and thus contribute to the formation of the hydrophobic barrier of the skin.

Modulation of the synthesis of apolipoproteins in rat hepatoma cells.

The present study was designed to investigate whether plasma lipoproteins and albumin can affect the basal synthetic rate of apolipoproteins in differentiated rat hepatoma cells (Fao) incubated in serum-free medium. The synthesis of apolipoproteins was measured by the incorporation of [35S]methionine into medium lipoproteins isolated by density gradient ultracentrifugation. Under all the experimental conditions used, Fao cells synthesized almost exclusively apolipoprotein E. When cells were incubated in the presence of 5-10% rat plasma the synthesis of apolipoprotein E increased 2-3-fold; lipoprotein-deficient serum had a negligible effect. Fatty acid-poor bovine serum albumin (BSA), which had been found to reduce very-low-density lipoprotein secretion in isolated rat hepatocytes, did not modify the synthesis of apolipoprotein E. When Fao cells were incubated in medium containing rat plasma lipoprotein fractions, the synthesis of apolipoprotein E increased. The d less than 1.090 g/ml plasma lipoprotein fraction had the major stimulatory effect. Increased apolipoprotein E synthesis was observed when cells were incubated in the presence of lipids extracted from rat plasma lipoproteins. These results suggest that the intracellular accumulation of lipoprotein-lipids plays an important role in regulating apolipoprotein E synthesis in Fao cells.