Micro and nanoparticles as possible pathogenetic co-factors in mixed cryoglobulinemia.

BACKGROUND: Mixed cryoglobulinemia (MC) is a rare multisystem disease whose aetiopathogenesis is not completely understood. Hepatitis C virus (HCV) infection may have a causative role, and genetic and/or environmental factors may also contribute. AIMS: To investigate the presence and possible role of environmental agents in MC. METHODS: We recruited 30 HCV-infected MC patients with different clinical manifestations and a control group of 30 healthy, sex-/age-matched volunteers. We collected serum samples from each patient and incubated at 4°C for 7 days to obtain cryoprecipitate samples. We used environmental scanning electron microscopy (ESEM) and energy dispersive X-ray spectroscopy microanalysis to verify the presence of microparticles (MPs) and nanoparticles (NPs) in serum and cryoprecipitate samples. We evaluated environmental exposure using a medical and occupational history questionnaire for each subject. RESULTS: MC patients had a significantly higher risk of occupational exposure (OR 5.6; 95% CI 1.84-17.50) than controls. ESEM evaluation revealed a significantly higher concentration, expressed as number of positive spots (NS), of serum inorganic particles in MC patients compared with controls (mean NS 18, SD = 16 versus NS 5.4, SD = 5.1; P < 0.05). Cryoprecipitate samples of MC patients showed high concentrations of inorganic particles (mean NS 49, SD = 19). We found a strong correlation between NS and cryocrit (i.e. percentage of cryoprecipitate/total serum after centrifugation at 4°C) levels (P < 0.001). CONCLUSIONS: In addition to HCV infection, MPs and NPs might play an important role in the aetiopathogenesis of MC.

Carcinogenic potential of metal nanoparticles in BALB/3T3 cell transformation assay.

Metal-based nanoparticles (NPs), are currently used in many application fields including consumer products, pharmaceuticals, and biomedical treatments. In spite to their wide applications, an in-depth study of their potential toxic effects is still lacking. The aim of the present research was to investigate the potential initiator or promoter-like activity of different metallic NPs such as gold, iron, cobalt, and cerium using the Balb/3T3 two-stage transformation assay. The results indicated that all the selected metallic NPs, except for cobalt, when used as initiators did not induce any transformation in Balb/3T3 cell line. Moreover, Au and Fe3 O4 NPs, when used in place of the tumor promoter treatment TPA, increased significantly the number of Foci/dish as compared to the MCA treatment alone. The number of Foci/dish was 2.6 for Au NPs and 2.13 for Fe3 O4 ones, similar to those obtained by the positive control treatment (MCA + TPA), whereas 1.27 for MCA treatment alone. On the contrary, CeO2 NPs did not show any difference in the number of Foci/dish, as compared to MCA alone, but it decreased the number of foci by 65% in comparison to the positive control (MCA + TPA). As expected, cobalt NPs showed an increased cytotoxicity and only a few surviving cells were found at the time of analysis showing a number of Foci/dish of 0.13. For the first time, our data clearly showed that Au and Fe3 O4 NPs act as promoters in the two stage transformational assay, suggesting the importance to fully investigate the NPs carcinogenic potential with different models.

Acquired immunodeficiency similar to Gulf War illness in a dead former serviceman.

A 38-year-old non-commissioned officer was certified unfit for military duty several months before his death. The forensic autopsy revealed a severe bone marrow aplasia and a pulmonary angioinvasive aspergillosis. Moreover, the presence of inorganic foreign particles in the pulmonary macrophages and intestinal endothelia was observed. The microanalysis implemented on these last selected specimens revealed the presence of silica particles microimpregnated by lanthanides and steel. The patient's acquired immunodeficiency appears comparable with that of Iraqi civilians suffering from Gulf War illness. This is the first report in the literature of the presence of intestinal endothelia engulfed by foreign war particulates; the silica particles may have entered the intestinal endothelia via the blood stream or by ingestion of impregnated fruit and vegetable foodstuffs. This finding provides new perspectives in the assessment of war-associated diseases and includes electron probe microanalysis among the new techniques of military and forensic medicine.

Metallic nanoparticles exhibit paradoxical effects on oxidative stress and pro-inflammatory response in endothelial cells in vitro.

Particulate matter is associated with different human diseases affecting organs such as the respiratory and cardiovascular systems. Very small particles (nanoparticles) have been shown to be rapidly internalized into the body. Since the sites of internalization and the location of the detected particles are often far apart, a distribution via the blood stream must have occurred. Thus, endothelial cells, which line the inner surface of blood vessels, must have had direct contact with the particles. In this study we tested the effects of metallic nanoparticles (Co and Ni) on oxidative stress and pro-inflammatory response in human endothelial cells in vitro. Exposure to both nanoparticle types led to a concentration-dependent cytotoxic effect. However, the effects on oxidative stress and pro-inflammatory response differed dramatically. Due to the nanoparticle-induced effects, a comparison between metallic nanoparticle- and metal ion-treatment with the corresponding ions was made. Again, divergent effects of nanoparticles compared with the ions were observed, thus indicating differences in the signaling pathways induced by these compounds. These paradoxical responses to different metallic nanoparticles and ions demonstrate the complexity of nanoparticle-induced effects and suggest the need to design new strategies for nanoparticle toxicology.

Retrieval analysis of clinical explanted vena cava filters.

Vena cava filters are the most commonly used mechanical devices to prevent pulmonary embolism. A retrievable permanent filter has been available since 1999. That has allowed the direct study of thrombi captured in humans and the punctual interaction of blood and device at long and short term. Through traditional histologic methods, captured thrombi and the tissues formed around the filter were observed. An innovative environmental scanning electron microscopy technique allowed detection of micro- and nanosized foreign bodies inside thrombi and tissues, and chemical analysis could be carried out by means of energy dispersive spectroscopy. All specimens contained different quantities of foreign debris ranging from few tens of microns to 50 nanometers; their chemistry was not homogeneous when patients were compared, and also differed considerably within the same filter. The constant presence of debris deeply embedded in all thrombi observed may mean that they are the cause that triggered the formation of those thrombi as a result of the interaction between foreign bodies and blood components.

Environmental nanoparticles are significantly over-expressed in acute myeloid leukemia.

The increase in the incidence of acute myeloid leukemia (AML) may suggest a possible environmental etiology. PM2.5 was declared by IARC a Class I carcinogen. No report has focused on particulate environmental pollution together with AML. The study investigated the presence and composition of particulate matter in blood with a Scanning Electron Microscope coupled with an Energy Dispersive Spectroscope, a sensor capable of identifying the composition of foreign bodies. 38 peripheral blood samples, 19 AML cases and 19 healthy controls, were analyzed. A significant overload of particulate matter-derived nanoparticles linked or aggregated to blood components was found in AML patients, while almost absent in matched healthy controls. Two-tailed Student's t-test, MANOVA and Principal Component Analysis indicated that the total numbers of aggregates and particles were statistically different between cases and controls (MANOVA, P<0.001 and P=0.009 respectively). The particles detected showed to contain highly-reactive, non-biocompatible and non-biodegradable metals; in particular, micro- and nano-sized particles grouped in organic/inorganic clusters, with statistically higher frequency of a subgroup of elements in AML samples. The demonstration, for the first time, of an overload of nanoparticles linked to blood components in AML patients could be the basis for a possible, novel pathogenetic mechanism for AML development.

Mobilization and transplantation of Philadelphia-negative peripheral-blood progenitor cells early in chronic myelogenous leukemia.

PURPOSE: Mobilization of Philadelphia (Ph) chromosome-negative progenitors is now possible in many Ph1-positive chronic myelogenous leukemia (CML) patients who had received interferon alfa (IFN-alpha) with no cytogenetic response. In this pilot study, we used this approach in patients without prior IFN-alpha therapy to determine if the number and quality of mobilized progenitors would be increased and to evaluate the potential effect of these cells as autografts. PATIENTS AND METHODS: Twenty-two untreated patients were mobilized within 12 months of diagnosis. The treatment regimen consisted of the mini-ICE protocol. Beginning on day +8, granulocyte colony-stimulating factor (G-CSF) was used in all patients. Leukophoresis was performed as the patients were recovering from aplasia, when WBC count exceeded 0.8 x 10(9)/L. RESULTS: In 14 patients, (63%) the leukophoresis product was entirely Ph1-negative and in four patients the Ph1-positive cell rate was < or = 7%. Significant numbers of long-term culture-initiating cells (LTC-IC) and CD34+ Thy1+Lin- cells were found in most of the Ph1-negative collections that were tested. Twelve patients underwent autografting with their mobilized peripheral-blood progenitor cells (PBPC) (Ph1-negative collections, 10 patients; major cytogenetic response, two patients). All patients engrafted and are alive; six have Ph1-negative marrow 7 to 15 months after autografting. Posttransplant treatment was IFN-alpha combined with interleukin (IL)-2 because of the recent demonstration of synergistic activity in augmenting cytolytic activity. CONCLUSION: Intensive chemotherapy given in early chronic phase of CML is well tolerated and results in high numbers of circulating Ph1-negative precursor cells.

Long-term behaviour of active glasses in sheep mandibular bone.

Granules of a glass (A) prepared according to Hench's formula and a new vitreous material for biological applications (AKRA 15) were used for repair of bone defects in the dental field. The behaviour of these materials implanted in holes drilled in sheep's mandibular bone was examined 4, 8, 12 month after implantation. Microradiographic analyses, optical and scanning electron microscopic observations, and X-ray microprobe evaluations were carried out using undecalcified, methacrylate-embedded sections of the jaw containing the granules. After one year the granules of A disappeared, but not important bone growth was observed also in the holes containing AKRA 15. SEM and microprobe showed: disappearance of Na and Si ions at different stages; increase of P and Ca up to 4 month and then decrease, but in different ways in the two glasses; unexpected appearance of K ions after 4 month only in AKRA 15.

Short-term behaviour of two similar active glasses used as granules in the repair of bone defects.

The bioactivity of two similar vitreous materials used in the form of granules of 'critical' size was investigated in bone defects in jaws of two sheep. The granules consisted of Hench's Bioglass and another glass with the same chemical composition made in Italy. Two months after implantation, the sheep were killed and elemental analyses carried out on sections of the embedded jaws. The microanalyses for both the glasses showed a diffusion from the granules towards the surrounding tissue of silicon and sodium, and an inverse diffusion (from the surrounding tissue towards the granules) of calcium and phosphorus. The degradation for the Italian glass was slower than for the Bioglass. No significant osteoinduction was seen after that time at the interface of the glass granules or in the bone pocket.

Behaviour of tricalcium phosphate and hydroxyapatite granules in sheep bone defects.

Granules of hydroxyapatite (HA) and tricalcium phosphate (TCP) were implanted in separate holes drilled in mandibular bone of sheep to check the bone growth and in vivo behaviour of the materials. The experiment was performed in three sheep, killed respectively at 4, 8, 12 month. Samples of bone with the materials were explanted, microradiographed and sectioned to evaluate the interface under optical and electron scanning electron microscope. The hole, left empty as a reference, showed no full repair; whereas 4 month after implantation the TCP granules induce total repair of the hole. HA granules crumbled and no new bone induction was seen even 12 month after implantation.

Analysis of the in vivo reactions of a bioactive glass in soft and hard tissue.

A bioactive glass, S53P4, was implanted as granules subcutaneously in muscles and connective tissue of rabbits, as well as in the mandibular bone of a sheep. After the implantation period of 2-3 months, cross-sections were prepared and studied by scanning electron microscopy and energy dispersive X-ray analysis. The glass reacted essentially in the same way in all types of tissue. The granules consisted of an unreacted core and a reacted layer with a silica-rich and calcium phosphate-rich zone. Large hydroxyapatite crystals were occasionally found on top of the calcium phosphate surface of the granules implanted in soft tissue. On the basis of elemental analysis of the reaction layers it was found that the release of calcium from inside the glass is sufficient to account for the formation of the calcium phosphate surface layer, whereas the release of phosphate from the glass is not sufficient.

High-dose chemo-radiotherapy followed by autologous Philadelphia chromosome-negative blood progenitor cell transplantation in patients with chronic myelogenous leukemia.

Twenty-three patients with chronic myelogenous leukemia in early chronic phase (ECP) and not previously treated with alpha-interferon (IFN-alpha) (10 patients), in ECP but pretreated with IFN-alpha (<12 months) (seven patients) and in late chronic phase (LCP) pretreated with IFN-alpha (>12 months) (six patients) underwent autografting with Philadelphia (Ph) chromosome-negative blood progenitor cells (BPCs) (20 patients), or partially/totally Ph-positive BPCs (three patients), previously mobilized during the early phase of recovery after aplasia induced by intensive chemotherapy. The conditioning regimen consisted of high-dose chemotherapy alone or followed by total body irradiation (TBI). Recombinant G-CSF was given after BPCs infusion on day +8. All patients in ECP not pretreated with IFN-alpha are alive and five of them are Ph-negative in the marrow after autografting. Six of seven patients autografted with Ph-negative BPCs in the group of ECP pretreated with IFN-alpha (<12 months) are alive and two of them are still Ph-negative in the marrow. In the same group, the only patient transplanted with partially Ph-positive BPCs, died of blastic transformation 2 months after reinfusion. Three patients (two patients autografted with Ph-negative BPCs and one patient with Ph-positive BPC) in the group of LCP pretreated with IFN-alpha >12 months are alive but Ph-positive after autografting. The other three patients of the same group died of procedure-related toxicity (two patients) and blastic transformation (one patient). Seventeen patients (10/10 ECP not pretreated with IFN-alpha; 5/7 ECP pretreated with IFN-alpha and 2/6 LCP pretreated with IFN-alpha) of 23 autografted patients were treated with IFN-alpha +/- IL-2. Toxicities after autografting were mostly related to myelosuppression, particularly thrombocytopenia. All patients of the two groups pretreated with IFN-alpha developed febrile episodes during the aplastic phase following BPCs reinfusion. No patient autografted in ECP and those not pretreated with IFN-alpha developed febrile episodes. This is also probably due to the use of i.v. antibiotic and antimicotic prophylaxis when neutrophils were < or = 1 x 10(9)/l after autografting. Greater toxicity was observed in patients pretreated with IFN-alpha, being lethal in two cases in LCF. In conclusion, the "in vivo' manipulation approach employed in our institution is a safe procedure and it results in a high collection of Ph-negative cells in the blood if the cells are harvested: (1) in early chronic phase; (2) in early phase of recovery after chemotherapy-inducing aplasia; (3) in patients not extensively pretreated with IFN-alpha. The data presented here have shown encouraging trends in chronic phase of CML and offer new perspective for patients without an HLA-identical donor or for patients who do not respond to IFN-alpha.

Twelve years experience with high-dose therapy and autologous stem cell transplantation for high-risk Hodgkin's disease patients in first remission after MOPP/ABVD chemotherapy.

High-dose therapy followed by autografting can cure patients with aggressive Hodgkin's disease (HD) refractory or with early relapse to first-line combination chemotherapy. On the other hand, the eradication of the disease is rarely achieved in heavily pretreated patients. It has been suggested that patients with HD with very high risk characteristics at diagnosis, often relapse despite appropriate therapy with 7-8 drugs combination. Thus it seems to us that such patients are potential candidates for early autografting during first remission. Twelve years ago, we initiated a pilot study to investigate whether patients with very high risk characteristics, would benefit from early autografting. The application of early autografting was compared with our historical group of patients in complete remission after receiving MOPP/ABVD, who had the same negative prognostic characteristics, refused autografting and who did not receive other treatment after achieving complete remission. Among the 22 consecutive patients entered into the pilot study and autografted, 18 are alive and 17 (77%) remain alive in unmaintained remission at a median of 86 months. One patient (4%) died of interstitial pneumonitis in the transplantation group. Only 8/24 (33%) patients, who did not receive an autograft, are currently alive and disease free at a median of 89 months. In conclusion, the early application of autografting appears to improve the outcome in patients with very high risk HD who achieved remission with MOPP/ABVD.

Detoxified glutaraldehyde cross-linked pericardium: tissue preservation and mineralization mitigation in a subcutaneous rat model.

BACKGROUND AND AIM OF THE STUDY: Glutaraldehyde is considered a promoter of calcification by the action of toxic aldehyde group residuals from cross-linking. Post-fixation treatment with homocysteic acid (HA), besides bonding aldehyde groups and neutralizing toxicity, should enhance biocompatibility due to the strongly electronegative sulfonic group. The aim of this investigation was to evaluate HA efficacy on tissue preservation and dystrophic calcification mitigation in glutaraldehyde cross-linked bovine pericardium (BP) using a subcutaneous rat model. METHODS: Four samples of BP, two with glutaraldehyde-HA and two with glutaraldehyde treatment, were implanted in each of 24 male Sprague-Dawley rats. Three rats were killed at 14 days, eight at 28 days, eight at 56 days and five at 84 days. Unimplanted glutaraldehyde-HA- and glutaraldehyde-treated samples served as controls. All samples were studied by gross examination, mammography, light transmission and scanning electron microscopy, and atomic absorption spectroscopy. The nature of mineralization was investigated by coupling techniques of scanning electron microscopy, electron microprobe analysis and X-ray powder diffraction. RESULTS: No histological and ultrastructural differences were found between glutaraldehyde-HA- and glutaraldehyde-treated BP, whether implanted or unimplanted. In both groups, calcification progressed with time, but significantly less after glutaraldehyde-HA treatment than after glutaraldehyde alone and at all time intervals (14.63 +/- 21.34 versus 43.17 +/- 15.99 at 28 days, p = 0.003; 56.42 +/- 40.20 versus 90.59 +/- 32.90 at 56 days, p = 0.008; 91.68 +/- 67.68 versus 156.23 +/- 17.85 at 84 days, p = 0.01). Differences were evident by mammography and histology (von Kossa stain). Electron microprobe analysis in both groups showed the composition of calcified nuclei to be calcium phosphate, stoichiometrically close to apatite (Ca5(PO4)3(OH)). The occurrence of crystallized apatite was supported by X-ray powder diffraction findings, the amount of crystallized apatite being higher in glutaraldehyde-treated samples. CONCLUSIONS: Post-fixation treatment with HA preserves BP structural properties and significantly mitigates mineralization of long-term subcutaneous implants.

Is the tricuspid position suitable for testing replacement bioprosthetic valves in the sheep model?

BACKGROUND AND AIM OF THE STUDY: Glutaraldehyde may promote calcification in xenograft tissue by the action of toxic aldehyde group residues involved in the cross-link process. Post-fixation treatment with homocysteic acid (HA) neutralizes this toxicity by bonding aldehyde groups, and enhances biocompatibility on the basis of strongly electronegative sulfonic groups. Previous studies in a rat subcutaneous model showed significant long-term mitigation of mineralization of glutaraldehyde-fixed pericardium treated with HA. This study aimed to assess the anticalcific efficacy of HA in a valvular implant in growing sheep, and establish if the tricuspid position is suitable for testing replacement bioprosthetic valves. METHODS: Eleven stented 25 mm Pericarbon bioprostheses (seven HA-treated, four standard) were implanted in the tricuspid position of growing sheep. Infective endocarditis occurred in four prostheses. Among the remaining seven, three (two HA-treated, one standard) were explanted at 91 days (mid-term), and four (two HA-treated, two standard) at 140-141 days (long-term). All explants were studied by gross, X-ray, light, transmission and scanning electron microscopy, as well as by atomic absorption spectroscopy. RESULTS: No histological and ultrastructural difference in tissue preservation were observed between HA-treated and standard Pericarbon bioprostheses, either in the mid or long term. The mean calcium content of mid-term HA-treated explants was 9.55 mg/g compared with 16.26 mg/g in mid-term standard explants. Only one late standard explant failed as a result of severe stenosis caused by massive dystrophic calcification. Among four late explants, two showed significant increase in mineralization (HA-treated, 87.45 mg/g; standard, 181.20 mg/g), while two showed calcium contents similar to those in mid-term explants (HA-treated, 11.96 mg/g; standard, 17.32 mg/g). CONCLUSION: Post-fixation treatment with HA preserves structural properties after tricuspid implantation in growing sheep. The tricuspid implant in the sheep model failed to reproduce remarkable accelerated progressive calcification in all xenografts so as to demonstrate a significant difference between HA and standard explants. The tricuspid position for testing replacement bioprosthetic valves should be abandoned, and investigations repeated with the prosthesis in the mitral position.

Cytogenetic and fluorescence in situ hybridization monitoring in Ph+ Chronic Myeloid Leukemia patients treated with imatinib mesylate.

Imatinib mesylate determines a favorable clinical course in most Ph positive Chronic Myeloid Leukemia (CML) patients in the chronic phase. Cytogenetic response is usually evaluated by analyzing 20-25 bone marrow metaphases using standard banding techniques. Since this methodology has very low sensitivity, we compared the results obtained by standard banding techniques to the ones obtained by fluorescent in situ hybridization (FISH). This was also done to identify any possible discrepancies between the two techniques. We analyzed 40 Ph+ CML patients in the chronic phase who had previously been treated with interferon alpha (IFNalpha) and who were receiving imatinib. The studies were performed by utilizing the same BM cell samples fixed in acetic acid/methanol, before imatinib therapy and then quarterly. Comparison of cytogenetic results to FISH results at 3 and 6 months of imatinib treatment showed that some patients who had achieved major cytogenetic response (i.e.<35% of examined metaphases showing Ph), showed retention of a higher number of persisting Ph+ cells when examined by FISH, and they did not achieve major FISH response (i.e. <35% of examined interphase cells show the BCR-ABL fusion signal). The discrepancy we found between the results that were obtained by analyzing metaphases and interphase cells disappeared in the subsequent examinations. Moreover, we found that 4 patients (10%) were still Ph+ in all the metaphases we examined even though they achieved excellent clinical response. On the basis of this small series of patients, we suggest that cytogenetic evaluation of patients on imatinib therapy should be performed by utilizing the classic banding technique (metaphase examination), but also by using the FISH technique (interphase examination), since the two methodologies may provide different results.

Physico-chemical observations on a failed Greenfield vena cava filter.

A Greenfield vena cava filter for the prevention of thromboembolism failed in vivo due to the displacement of 2 of the 6 legs and their subsequent break. The explanted stainless steel filter was analyzed with an electron microscope and energy dispersion system in order to assess the reason for the break. Fatigue and corrosion of the metal were responsible for the failure.

Calcification of chemically treated bovine pericardium.

One of the most important problems arising in cardiac bioprostheses made with bovine pericardium and, more generally, with biologically-derived tissues is tissue calcification. The present study assessed four chemical treatments on patches of bovine pericardium, intended to avoid or minimize calcification. Pericardium specimens were treated with: A) 0.5% glutaraldehyde; B) 0.5% glutaraldehyde + 4% formaldehyde; C) same as A, but with a further neutralization treatment; D) acylation of fresh bovine pericardium. Circular samples of 1 cm diameter were subcutaneously implanted in the abdominal region of three groups of six rats. The explants were retrieved after 2, 4 and 8 weeks. The calcium content and the histological results showed better behaviour for C and D samples than with the commonly used fixation methods (A and B). The lowest calcification was observed with treatment D, even though its morphological structures were somewhat modified with homogenation of collagen bundles. Among the glutaraldehyde-based treatments, treatment C appears to be the most promising because the pericardium shows slower calcium accumulation with a diffusive pattern.

[Therapeutic aspects of hairy cell leukemia]. / Aspetti terapeutici dell'hairy cell leukemia.

This work considers the new advances in hairy cell leukemia therapy. During the last decades the only useful treatments were splenectomy, or, in case of failure or relapse, various chemotherapeutic approaches. Sometimes leukapheresis, radiotherapy, androgens, allogenic bone marrow transplantation, corticosteroids and lithium salts were used with few good results. Interferon and 2-deoxicoformycin recently introduced for the treatment of HCL have determined a dramatic change in the outlook of this disease, producing a high percentage of complete and partial remission.