Mesenchymal stem cells as delivery vehicle of porphyrin loaded nanoparticles: effective photoinduced in vitro killing of osteosarcoma.

Mesenchymal stem cells (MSC) have the unique ability to home and engraft in tumor stroma. These features render them potentially a very useful tool as targeted delivery vehicles which can deliver therapeutic drugs to the tumor stroma. In the present study, we investigate whether fluorescent core-shell PMMA nanoparticles (FNPs) post-loaded with a photosensitizer, namely meso-tetrakis (4-sulfonatophenyl) porphyrin (TPPS) and uploaded by MSC could trigger osteosarcoma (OS) cell death in vitro upon specific photoactivation. In co-culture studies we demonstrate using laser confocal microscopy and time lapse imaging, that only after laser irradiation MSC loaded with photosensitizer-coated fluorescent NPs (TPPS@FNPs) undergo cell death and release reactive oxygen species (ROS) which are sufficient to trigger cell death of all OS cells in the culture. These results encourage further studies aimed at proving the efficacy of this novel tri-component system for PDT applications.

["Terzo fuoco", lead poisoning and chronic renal failure]. / "Terzo fuoco", intossicazione da piombo e insufficienza renale cronica.

BACKGROUND: In ceramics, "Terzo fuoco" (Third fire) means a third firing of clay to fix colors on tiles or pottery. The low firing temperatures (800-900 degrees C) and the use of a spray gun are risk factors for lead poisoning. Because of their small size, handicraft companies often fail to implement the preventive measures that are utilized efficiently in large tile factories. We report a case of chronic lead poisoning with special emphasis on diagnostic tools and treatment goals. CASE REPORT: A 38-year-old woman was hospitalized in 2005 because of grade 3 chronic renal failure (serum creatinine 1.69 mg%, Cockroft-Gault glomerular filtration rate [GFR] 45 mL/min), hypertension and elevated serum uric acid (13.4 mg%) without gout. She had been previously hospitalized elsewhere and diagnosed as suffering from hypertensive nephropathy. Her occupational history included acute lead poisoning 12 years before, which was treated with a short leave from work. She subsequently continued her job, using a spray gun for decorative drawing in a small tile company until 2004. Because of a low GFR she underwent a 3-day chelation test with 750 mg CaNaEDTA i.v., and excreted 1056 microg Pb (n.v < 600 microg) -- (PbU/EDTA ratio 1.41; n.v < 0.6). A renal biopsy showed chronic interstitial nephropathy with severe arteriolosclerosis. The patient was treated with 5 courses of EDTA, resulting in a final Pb excretion of 517 microg/72 h (PbU/EDTA 0.69). Her serum creatinine fell to 1.32 mg% (CFR 58 mL/min). A further course of chelation therapy is planned. DISCUSSION AND CONCLUSIONS: The EDTA chelation test allows to determine the lead body burden (PbBB) and to titrate subsequent chelation therapy. Recent papers have shown that PbBB is a major factor in the progression of chronic renal failure besides pressure control, and have indicated a PbBB safety level of less than 100 microg/test (PbU/EDTA < 0.1). In order to prevent the development of chronic renal failure, it is important that not only occupational but also environmental lead exposure be identified and adequately treated.

Liver and kidney foreign bodies granulomatosis in a patient with malocclusion, bruxism, and worn dental prostheses.

Granulomatous reactions caused by foreign bodies have been described in drug abusers, in subjects exposed to occupational pollutants, and more rarely, in association with the use of prosthetic devices. We describe a 62-year-old patient with multiorgan parenchymal granulomatosis caused by inorganic debris of unknown origin. The patient presented with fever, hepatosplenomegaly, progressive cholestasis, and acute renal failure. Liver and kidney biopsies showed the presence of noncaseating epithelioid giant-cell granulomas containing scattered polarizable particles. Similar particles were also present in stools. Studies by innovative scanning electron microscopy and energy-dispersive microanalytical techniques showed that the particles isolated in liver, kidney, and stools were made by feldspars, the main component of porcelain. No occupational or environmental exposure to these materials could be identified in this patient and the only reliable source of the porcelain debris turned out to be constituted by 2 dental bridges evidently worn because of a possible inappropriate construction, malocclusion, and bruxism. The porcelain of the dental prostheses had the same elemental spectrum of the particles isolated from stool specimens and liver-kidney granuloma. After identification of the dental prostheses as the most likely source of ceramic debris, and after their removal, the particles from stool specimens disappeared. The patient was then treated with steroids leading to a remission of the clinical symptoms and a decrease in granulomatous inflammatory reaction in both liver and kidney. This is the first report suggesting that a foreign body systemic granulomatosis can be associated with worn dental prostheses.

Dialysate flow distribution in hollow fiber hemodialyzers with different dialysate pathway configurations.

UNLABELLED: The efficiency of a hemodialyzer is largely dependent on its ability to facilitate diffusion, since this is the main mechanism by which small solutes are removed. The diffusion process can be impaired if there is a mismatch between blood and dialysate flow distribution in the dialyzer. The objective of the paper was to study the impact of different dialysate compartment designs on dialysate flow distribution and urea clearances. Eighteen hollow fiber 1.3 m2 hemodialyzers were studied, 6 each of 3 designs: Type A--standard fiber bundle (PAN 65DX Asahi Medical, Tokyo, Japan); Type B--spacing filaments external to the fibers (PAN 65SF Asahi Medical, Tokyo, Japan); Type C--fibers waved to give Moiré structure (FB130 Nissho-Nipro, Osaka, Japan). IN VITRO STUDIES: 3 dialyzers of each type were studied following dye injection into the dialysate compartment. Dynamic sequential imaging of longitudinal sections of the dialyzer were undertaken, using a new generation helical CT scanner (X-Press/HS1 Toshiba Corporation, Tokyo, Japan). In vivo studies: 3 dialyzers of each type were studied, in randomized sequence, in 3 different patients under standardized dialysis conditions. Blood- and dialysate-side urea clearances were measured at 30 and 150 minutes of treatment. Macroscopic and densitometrical analysis revealed that flow distribution was most homogeneous in the dialyzer with Moiré structure (Type C) and least homogeneous in the standard dialyzer (Type A). Space yarns (Type B) gave an intermediate dialysate flow distribution. Significantly increased urea clearances (p<0.001) were seen with Types B and C, compared to the standard dialyzer. Type C (Moiré) had the highest clearances although these were not significantly greater than Type B (space yarns). In conclusion, more homogeneous dialysate flow distribution and improved small solute clearances can be achieved by use of spacing yarns or waved (Moiré structure) patterns of fiber packing in the dialyzer. These effects are achieved probably as a result of reduced dialysate channeling resulting in a lower degree of mismatch between blood and dialysate flows. The new radiological technique using the helical CT scanner allows detailed flow distribution analysis and has the potential for testing future modifications to dialyzer design.

Biofilms invade nephrology: effects in hemodialysis.

Bacteria attach to surfaces and aggregate in a biopolymer matrix to form biofilm. Studies on biofilm have shown its presence in many prosthetic devices used in nephrology as well as in fluid pathways of hemodialysis plants and monitors. Once present, this community of bacteria increases resistance to biocide due to slime production and, as a result, chemical products for dialysis monitor disinfection and descaling procedures do not result in an effective treatment. Ultrapure dialysate is a goal in modern hemodialysis, and ultrafiltration is used to obtain sterile and apyrogen fluids. Microbial colonisation of ultrafilters may occur if, due to inadequate disinfection protocols, membrane is exposed to persistent bacterial contamination, and biofilm is allowed to form and to grow. As more and more data link final dialysate microbial contamination to clinical effects of bioincompatibility from chronic inflammation in dialysis patients, attention has to be focused on possibilities of biofilm avoidance.

Influence of different hemodialysis membranes on red blood cell susceptibility to oxidative stress.

Oxidative stress is crucial in red blood cell (RBC) damage induced by activated neutrophils in in vitro experiments. The aim of the study was to evaluate whether the bioincompatibility phenomena occurring during hemodialysis (HD) (where neutrophil activation with increased free radical production is well documented) may have detrimental effects on RBC. We evaluated RBC susceptibility to oxidative stress before and after HD in 15 patients using Cuprophan, cellulose triacetate, and polysulfone membrane. RBC were incubated with t-butyl hydroperoxide as an oxidizing agent both in the presence and in the absence of the catalase inhibitor sodium azide. The level of malonaldehyde (MDA), a product of lipid peroxidation, was measured at 0, 5, 10, 15, and 30 min of incubation. When Cuprophan membrane was used, the MDA production was significantly higher after HD, indicating an increased susceptibility to oxidative stress in comparison to pre-HD. The addition of sodium azide enhanced this phenomenon. Both cellulose triacetate and polysulfone membranes did not significantly influence RBC susceptibility to oxidative stress. Neither the level of RBC reduced glutathione nor the RBC glutathione redox ratio changed significantly during HD with any of the membranes used. The RBC susceptibility to oxidative stress was influenced in different ways according to the dialysis membrane used, being increased only when using the more bioincompatible membrane Cuprophan, where neutrophil activation with increased free radical production is well documented. The alterations found in this study might contribute to the reduced RBC longevity of HD patients where a bioincompatible membrane is used.

In vitro and in vivo evaluation of a new polysulfone membrane for hemodialysis. Reference methodology and clinical results. (Part 1: in vitro study).

Different high flux membranes have been recently developed. The present study is aimed at describing the technical features and the clinical performances of a new high flux polysulfone membrane (T-sulfone, Toray, Japan). The study has been carried out on two different dialyzers (surface area = 1.3 and 1.8 m2). The filters have been tested in vitro under definite experimental conditions. The hydraulic flow resistance, the pressure drop in the blood compartment and the hydraulic permeability have been determined in a wide range of in vitro experimental conditions. The in vitro sieving coefficients for various solutes have also been determined utilizing human blood. Hydraulic permeability was found in the range of 28.4 ml/h/mm Hg/m2 and sieving coefficients were between 0.96 and 1.0 for all low molecular weight solutes. The sieving coefficient for inulin was 0.95. The pressure drop in the filter at 300 ml/min of blood flow was 95 mm Hg for the 1.3 m2 and 57 mm Hg for the 1.8 m2. The filters are then designed to operate in the presence of high blood flows without excessive resistance in the blood compartment. The blood compartment analyzed by means of a special radiological sequence obtained with a helical scanner after dye injection confirmed the homogeneous distribution of the blood flow in several cross sections of the bundle. Adequate distribution of dialysate was confirmed with a similar method applied to the dialysate compartment. The new imaging techniques utilized were greatly helpful to determine adequacy of filter design and flows distribution.

In vitro and in vivo evaluation of a new polysulfone membrane for hemodialysis. Reference methodology and clinical results. (Part. 2: in vivo study).

Different high flux membranes have been recently developed. The present study is aimed at describing the technical features and the clinical performances of a new high flux polysulfone membrane (T-sulfone, Toray, Japan). The study has been carried out on two different dialyzers (surface area = 1.3 and 1.8 m2). The filters have been tested in vivo during hemodialysis and hemodiafiltration. The in vivo study was carried out on 12 ESRD patients on regular hemodialysis treatment. The protocol was reviewed and approved by the local ethical committee. The in vivo clearances (K) at 300 ml/min of blood flow are reported in the following Table: [Table in text]. Beta-2-m reduction ratio exceeded 50% in all sessions. Beta-2-m mass balance executed by collection of spent dialysate and elution from the used filters evidenced that removal is obtained mostly by filtration while absorption is negligible. Excellent tolerance and hemocompatibility was observed in all the studied sessions.

Cellulose triacetate: another membrane for continuous renal replacement therapy.

The evolution of technology and biomaterials has permitted a parallel development of renal replacement therapies in the acute, critically ill patient. From the original continuous artero-venous hemofiltration method new techniques such as continuous veno-venous hemofiltration, hemodiafiltration and high-flux dialysis have been developed and are clinically used. Similar progress has been made with artificial membranes. We investigated the possibility of using a modified cellulosic membrane for continuous therapies, assessing the hydraulic characteristics and clearance performances of high-flux cellulose triacetate hemodiafilter (0.7 m2) in vitro and in vivo. The flowdynamic characteristics of the filter suggest its optimal use in veno-venous pump-drive techniques. Efficiency was excellent, with urea daily clearances as high as 50 liters or more. The high permeability and porosity of the membrane also increased the clearances of larger solutes such as creatinine and inulin. No side effects occurred during treatment and we conclude that cellulose triacetate may be considered a good alternative to synthetic membranes in continuous renal replacement therapies.

Acute renal failure of medical type in an elderly population.

One hundred and nine unselected patients with Acute Renal Failure (ARF) of medical aetiology were hospitalized at the Nephrological Unit of Policlinico University Hospital (Modena) during a 30-month period. ARF was considered as a rapid increase of serum creatinine > 2mg/dl over the baseline level or the doubling of pre-existing value in chronic renal failure. Mean age of patients was 67+/-17 years and median age was 72; 64.2% needing dialytic treatment. Four main causes of ARF were identified: 33 patients had reduced renal perfusion by dehydration, hypotension etc.; 20 multifactorial aetiology; 14 biopsy-investigated renal parenchymal diseases and 39 had drug-related acute renal failure (D-ARF). The clinical outcome was significantly worse in elderly patients as regard mortality (P < 0.02), chronic dialytic treatment (P < 0.04) and complete recovery (P < 0.004). The mean age of D-ARF patients was significantly greater than remaining ARF patients (72.6+/-12.8 vs 63.2+/-18.5: P < 0.004. Nonsteroidal antiinflammatory drugs (NSAIDs) and ACE-inhibitors (Ace-i) caused ARF in 24 and 8 patients respectively. Elderly age, vascular disease and monoclonal gammopathy represented the main risk factors and were significantly more frequent in D-ARF patients (P<001, <0.01, <0.04 respectively). Our data confirm the high susceptibility of ageing kidneys to nephrotoxic damage caused by drugs affecting glomerular autoregulation by microvascular mechanisms. Greater attention to renal changes in ageing and an increased dissemination of preventative measures among nephrologists, could reduce the incidence of these serious and potentially lethal diseases.

Which level of cytokine production is critical in haemodialysis?

Monocyte activation with cytokine production is a well known event in the course of dialysis treatment but its relation to symptoms of haemodialysis or long-term pathological changes in chronic dialysis patients is still under discussion. Cytokine production depends on the balance between inducers and inhibitors while effects rely on the peculiar uraemic environment and cell metabolism. 'Foot-prints' for monocyte activation have been found, but no marker for clinical symptoms has been demonstrated clearly. In this scenario it is almost impossible to link a specific symptom to a definite stimulus such as dialysate microbial contamination or membrane complement generation. The topics discussed in this paper include cytokines synthesis modulation factors, levels in haemodialysis patients, and results of finding markers of clinical relevance. Special attention is paid to microbial contamination of dialysis fluid with analysis of cytokine inducing substances in commercial sterile solutions. Data on cytokine synthesis and activity in the aged are also discussed, with special regard to the haemodialysis setting.

Protective effects of delapril, indapamide and their combination chronically administered to stroke-prone spontaneously hypertensive rats fed a high-sodium diet.

1. Stroke-prone spontaneously hypertensive rats (SHRsp) have been used widely to test agents putatively capable of vascular protection. These animals present an accelerated time course of hypertension and a reduced life-span. When fed a high-sodium diet from the eighth week of life, a further acceleration in blood pressure increase is obtained, and rats start to die after 5 weeks of diet as a consequence of cerebral haemorrhage. In this model, angiotensin-converting enzyme (ACE) inhibitors were repeatedly proved to prevent vascular lesions and death. Notably, this effect was independent of any hypotensive effect. On the contrary, diuretics were shown not to be equally effective. A combination of ACE inhibitors and diuretics, although known to have synergistic effects in the therapy of hypertension, has never previously been tested. 2. Our aim was to study the effects of long-term treatment with the ACE inhibitor delapril (12 mg day-1 kg-1), the thiazide-like diuretic indapamide (1 mg day-1 kg-1), and their combination (12 and 1 mg day-1 kg-1 respectively), on the survival of SHRsp rats fed a high-sodium diet from the eighth week of life onwards. The effects of the treatments on blood pressure, body weight, food and fluid intake, diuresis, proteinuria and the appearance of lesion signs and death were assessed weekly. When control rats reached 50% mortality, they were killed, together with some drug-treated rats, to compare lesions in brain and kidney. The other drug-treated rats continued treatments until 50% mortality was reached in two treatment groups. 3. All drug treatments were able to delay death significantly when compared with control rats, which reached 50% mortality after 6 weeks of salt loading. This event was preceded by a highly significant increase in proteinuria, diuresis and fluid intake that took place 3 weeks after the increase in blood pressure over the initial range. In delapril- or indapamide-treated SHRsp these changes were never seen, even when animals started to die. In the combination-treated group, a significant increase (P < 0.01) in fluid intake and diuresis, but not proteinuria, was observed from the third week of treatment onwards. 4. Treatment with delapril or indapamide did not block the progressive increase in blood pressure as observed in control animals. However, the increase in blood pressure was markedly retarded with respect to control rats. At variance with this, in combination-treated animals blood pressure levels were maintained until the end of the experiment within the 99% confidence interval initially observed in control animals. 5. Infarctual and haemorrhagic cerebral lesions were observed in 38% of control rats; no lesions were noted in brains of age-matched rats receiving a drug treatment. Kidneys from control animals presented major degenerative lesions of glomeruli and arteries, characterized by fibrinoid necrosis. This condition was absent in drug-treated animals, which presented minor signs of ischaemic lesion. Heart hypertrophy, when heart weight was expressed as a percentage of body weight, was similar in saline-, delapril- or indapamide-treated rats. At variance with this, in combination-treated animals the heart weight to body weight ratio was significantly (P < 0.01) lower than in the other groups. 6. In conclusion, the diuretic indapamide showed similar protective effects as the ACE inhibitor delapril on acute vascular lesions and survival of SHRsp. Moreover, their combination synergized in preventing heart hypertrophy consequent to longterm hypertension. This results is probably related to the enhanced diuresis and the better control of blood pressure levels selectively found in combination-treated animals.

[Treatment of post-transplant erythrocytosis: comparison of lisinopril and theophylline]. / Trattamento dell'eritrocitosi post-trapianto: confronto tra lisinopril e teofillina.

OBJECTIVE: The proposal of this study is to compare the efficacy of lisinopril and theophylline, alone or in association, on erythrocytosis in renal-transplanted patients. DESIGN: Prospectic, case-control study. PATIENTS/ENVIRONMENT: 15 inpatients meeting 3 main criteria: 1) ACE therapy for past erythrocytosis, 2) actual erythrocytosis, 3) symptomatic increase of haematocrit (Hct). INTERVENTION: The treatment has been divided into 3 consecutive phases of 30 days each: 1) lisinopril (5 mg/die), 2) theophylline (4 mg/kg/die), 3) association of 2 drugs. MEASUREMENTS: The evaluations were scheduled at the beginning and every month and consisted of renal function control, haemochromocytometric exam, serum level of folates, B12 vitamin and erythropoietin (EPO), iron level, cyclosporinemia, as well as clinic control and adverse events detection. RESULTS: A significative decrease of Hct values and a decrease of serum erythropoietin values was observed in patients treated with lisinopril. Patients treated with theophylline showed a significant reduction of Hct values causing a reduction of erythropoietin serum level in 8/13 patients. Lisinopril and theophylline administered in combination presented a significant decrease of Hct values, while EPO values diminished compared to basal values. CONCLUSIONS: These data showed that lisinopril is a valid therapy for the treatment of posttransplanted patients affected by erythrocytosis and, moreover, has the benefit of antihypertensive action. Theophylline remains an alternative therapy when ACEi are contraindicated. The combination of the 2 drugs doesn't produce additional benefits.

Influence of urinary calcium concentration on erythrocyte morphology.

The effect of the urinary calcium concentration (CaU) on erythrocyte morphology was studied by incubating erythrocytes in urine with prefixed CaUs of 5, 10, 20 and 40 mmol/l by addition of CaCl2. The same experiment was carried out on erythrocytes preincubated with levo-verapamil (l-V) at 10, 100 and 200 mumol/l. Phase contrast microscopy observations were performed at 0, 30, 60, 120, and 240 min by 2 experienced investigators. At 0 min the erythrocytes showed a clear extraglomerular pattern. At 60 min marked morphological and volumetric alterations were evident when the CaU was > or = 10 mmol/l. On the contrary, no change was found when red cells were treated with > or = 100 mumol/l l-V, independent of the CaU. Dysmorphic erythrocyturia has been related to transglomerular passage even if it was sporadically observed in hypercalciuric or lithiasic patients. This work suggests a role for a high CaU in causing the formation of microcytic and warped erythrocytes. In our opinion, in hypercalciuric urine the appearance of dysmorphic or mixed hematuria does not necessarily indicate transglomerular passage.