Procalcitonin in hemodialysis patients presenting with fever or chills to the emergency department.

We sought to assess the role of procalcitonin in discriminating severe bacterial infections requiring antibiotic treatment from non-bacterial causes of fever or chills in chronic dialysis patients. Chronic hemodialysis patients who were admitted to the emergency room due to fever and/or chills were recruited to the study. The presence or absence of bacterial infection was defined after recruitment conclusion by an infectious disease specialist who was blinded to procalcitonin results. Procalcitonin levels were compared between infected and non-infected patients. Out of 54 patients recruited, 22 (41%) patients eventually diagnosed with infection. Mean (± SD) procalcitonin values were 4.3 (± 5.5) ng/ml among cases, 1.0 (± 2.0) ng/ml among controls with no infection (p = 0.02). A cutoff PCT value of 1 ng/ml or higher had 77% sensitivity and 59% specificity for the diagnosis of severe infection. Procalcitonin cannot usefully identify hemodialysis patient with bacterial infection.

Human melanoma cells expressing the alphavbeta3 integrin are partially protected from necrotic cell death induced by dynamic matrix detachment.

Anchorage-independence is a hallmark of metastatic cancer cells. In previous studies we characterized a novel model for anchorage-independence employing dynamic matrix detachment (DMD) using rotation in low shear stress conditions. We observed that in contrast to the classical apoptosis-inducing static matrix detachment (SMD) model, the venous circulation-mimicking DMD model induced necrosis in transformed cells. In the current study we revisited the mechanism of DMD-induced cell death and evaluated the contribution of alphavbeta3 integrin overexpression in human melanoma cells to anchorage-independence in DMD. DMD cell culture induced primarily necrosis in the melanoma cells studied. alphavbeta3, but not the control related alphaIIbbeta3 integrin, could confer survival advantage in DMD. While apoptosis was unaffected, constitutive, unligated alphavbeta3 overexpression was associated with attenuation of necrosis in DMD. alphavbeta3 overexpressing melanoma cells manifested AKT activation that was independent of DMD conditions. Furthermore, while a small molecular inhibitor of AKT phosphorylation induced apoptosis in adherent cells, in DMD conditions it had no effect on cell outcome. Thus, alphavbeta3-overexpressing melanoma cells are partially protected from DMD-induced cell death in an apoptosis-independent mechanism. This finding may be one of the factors accounting for anchorage-independence in circulating metastatic melanoma cells.

A novel system to study adenovirus tropism to normal and malignant colon tissues.

We describe here a new organ culture system for the evaluation of viral tropism to colon carcinomas and normal colon tissues. Organ cultures of mouse and human colon retained viability for several days and thus facilitated studies of viral tropism. Two adenoviral vectors (AD) were compared in the study: AD5, that utilizes the CAR receptor, demonstrated poor infectivity to both normal and carcinoma tissues, while a capsid-modified-AD, recognizing haparan-sulfate receptor, demonstrated efficient infectivity of both tissues. Immunohistochemistry analysis demonstrated different viral tropism; while AD5 infected only the colon epithelia, the capsid-modified-adeno infected both the epithelia and mesothelial layers. To investigate other determinants in the tissue that influence viral tropism, human cancer tissues were pretreated with collagenase and infected with the AD viruses. Increased infectivity and altered tropism were noted in the treated tumor tissue. Taken together, this ex vivo system indicated that receptor utilization and extracellular-matrix components influence AD viral tropism in solid tissues.

Fibrin microbeads (FMB) as a 3D platform for kidney gene and cell therapy.

Cell and gene therapy may alter the outcome of renal diseases, such as hereditary nephropathies, acute and chronic glomerulonephritis and allograft nephropathy. However, owing to blockade of many viral and cellular vehicles by the complex glomerular architecture, the exact nature of gene and cell delivery into specific renal compartments remains currently unknown. To study the interaction of viral vectors with a variety of renal cells and mesenchymal stem cells (MSCs), we employed a novel biological three-dimensional (3D) matrix comprised of fibrin microbeads (FMB) in comparison to monolayer cell culture. Our studies showed that renal cells of both established and primary lines can grow efficiently on FMB and differentiate into epithelial structures, as shown by electron microscopy. Gene delivery into renal cells in 3D was observed for several viral vectors and growth in 3D on FMB conferred resistance to renal cancer cells in the context of oncolytic adenoviruses. Finally, MSCs from various rodent species attached to FMB, grew robustly, survived for several weeks and could efficiently be transduced on FMB. Thus, on the basis of growth, differentiation and transduction of renal cells in 3D, FMB emerge as a novel 3D cellular microenvironment that differs substantially from monolayer cell cultures.

Heparin-induced recurrent anaphylaxis.

BACKGROUND: Heparin-related immediate-type hypersensitivity reactions like urticaria, angio-oedema or bronchospasm are very rare, and only a few cases of anaphylaxis-like responses because of heparin have been described. However, the mechanisms underlying these reactions and the role of mast cells in their pathogenesis have not been elucidated. OBJECTIVES: We report a patient with end-stage renal disease who presented with recurrent anaphylaxis after receiving heparin during haemodialysis. The underlying aetiology was obscured by the initiation of haemodialysis with its known anaphylactic-like side-effects. The diagnosis of hypersensitivity to heparin was confirmed by the clinical picture, positive skin tests and elevated serum tryptase levels. MATERIALS AND METHODS: We performed prick and intradermal skin tests with heparin, enoxaparin and danaparoid heparinoid. Total and mature tryptase levels were measured in serum by ELISAs at 1, 24 and 36 h following the reaction. RESULTS: An elevated mature tryptase level was found at 1 h, which returned to normal levels at 24 and 36 h. A high total tryptase level was detected at 1 h, but remained somewhat elevated at 24 h. Prick tests were negative with the three compounds. Intradermal skin tests with heparin and enoxaparin were both positive, while with danaparoid negative. Following negative skin test results, danaparoid was used as an anticoagulant during dialysis for the next 3 years without any adverse effects. CONCLUSIONS: In conclusion, we report the first case of heparin-induced anaphylaxis confirmed by an elevated level of mature tryptase in serum. Following skin tests, the patient was treated with danaparoid during haemodialysis sessions three times a week without any adverse effects. Because of increasing use of heparin in daily medical practice, physicians should be aware of possible immediate hypersensitivity reactions to this medication and know how to diagnose and treat them.

Shortening and migration of Wallstents after stenting of central venous stenoses in hemodialysis patients.

PURPOSE: To report our results for the placement of central venous stents in patients undergoing hemodialysis. METHODS: Ten Wallstents (Schneider, Bülach, Switzerland) were placed in 10 patients with shunt thrombosis, shunt dysfunction or arm swelling associated with central vein stenosis or occlusion. Technical success, patency and complications were evaluated. RESULTS: Stent deployment was successful in all cases. In seven cases (70%) there was significant delayed stent shortening. In two of these cases there was also stent migration. All these cases required additional stents. Primary patency rates at 6, 12 and 24 months were 66%, 25% and 0. Twenty-three additional procedures (percutaneous transluminal angioplasty or stenting) were required to achieve secondary patency rates at 6, 12 and 24 months of 100%, 75% and 57%. CONCLUSION: Stent placement in the central veins of dialysis patients has a high technical success rate resulting in symptomatic relief and preservation of access. Repeat interventions are required to maintain patency. Significant delayed shortening of the Wallstent occurred in 70% of patients which may have affected the patency rates. Strategies are suggested to avoid this problem.

The common mutations C677T and A1298C in the human methylenetetrahydrofolate reductase gene are associated with hyperhomocysteinemia and cardiovascular disease in hemodialysis patients.

BACKGROUND: Plasma total homocysteine (tHcy) level might be an important risk factor for the development of cardiovascular disease (CVD) in dialysis patients. While both renal failure and mutations of the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene may result in hyperhomocysteinemia and CVD, the distinct roles of the thermolabile MTHFR mutation at nucleotide C677T and the more recently described mutation at nucleotide A1298C have not been evaluated concurrently in patients on hemodialysis. METHODS: A cross-sectional study was performed in 120 maintenance HD patients to determine the prevalence of MTHFR C677T and A1298C mutations and their relative association to hyperhomocysteinemia and CVD. RESULTS: Both mutations, the C677T and the A1298C, were highly prevalent in HD patients with allele frequencies of 0.41 and 0.27, respectively. The prevalence of CVD in HD patients was 55% and its significant risk factors included, in descending order, hyperhomocysteinemia, MTHFR C677T mutation, low serum folate levels, diabetes mellitus, hypertension, and double heterozygote state for both MTHFR mutations (677CT/1298AC). MTHFR A1298C mutation alone and gender were not associated with either hyperhomocysteinemia or increased CVD risk, but the HD patients with homozygotes 1298CC and wild alleles 677CC (677CC/1298CC) have significant increase of tHcy (37.7 +/- 12) and high prevalence of CVD. CONCLUSIONS: Hyperhomocysteinemia, serum folate levels and both C677T and A1298C MTHFR mutations are associated with CVD in HD patients.

Conditional gene targeting for cancer gene therapy.

Current treatment of solid tumors is limited by severe adverse effects, resulting in a narrow therapeutic index. Therefore, cancer gene therapy has emerged as a targeted approach that would significantly reduce undesired side effects in normal tissues. This approach requires a clear understanding of the molecular biology of both the malignant clone and the biological vectors that serve as vehicles to target cancer cells. In this review we discuss novel approaches for conditional gene expression in cancer cells. Targeting transgene expression to malignant tissues requires the use of specific regulatory elements including promoters based on tumor biology, tissue-specific promoters and inducible regulatory elements. We also discuss the regulation of both replication and transgene expression by conditionally-replicative viruses. These approaches have the potential to restrict the expression of transgenes exclusively to tissues of interest and thereby to increase the therapeutic index of future vectors for cancer gene therapy.

Heat shock and heat shock protein 70i enhance the oncolytic effect of replicative adenovirus.

Replication-competent viruses are currently being evaluated for their cancer cell-killing properties. These vectors are designed to induce tumor regression after selective viral propagation within the tumor. However, replication-competent viruses have not resulted heretofore in complete tumor eradication in the clinical setting. Recently, heat shock has been reported to partially alleviate replication restriction on an avian adenovirus (Ad) in a human lung cancer cell line. Therefore, we hypothesized that heat shock and overexpression of heat shock protein (hsp) would support the oncolytic effect of a replication-competent human Ad. To this end, we tested the oncolytic and burst kinetics of a replication-competent Ad after exposure to heat shock or to inducible hsp 70 overexpression by a replication-deficient Ad (Adhsp 70i). Heat-shock resulted in augmentation of Ad burst and oncolysis while decreasing total intracellular Ad DNA. Overexpression of hsp 70i also enhanced Ad-mediated oncolysis but did not decrease intracellular Ad DNA levels. We conclude that heat shock and Adhsp 70i enhance the Ad cell-killing potential via distinct mechanisms. A potential therapeutic implication would be the use of local hyperthermia to augment oncolysis by increasing the burst of replication-competent Ad. The role of hsp in Ad-mediated oncolysis should be additionally explored.

Late-onset downregulation of NaPi-2 in experimental Fanconi syndrome.

The pathogenesis of renal phosphate (Pi) leak in Fanconi syndrome is unknown. Disorders of apical membrane transporters, leaky apical membrane, depleted cellular Pi and ATP, and impaired sodium (Na) pumps have been proposed as underlying defects. The present study examined the role of type II Na-Pi cotransport system (NaPi-2) in experimental Fanconi syndrome in rats. Following a single injection of maleic acid (MA), 75 mg/kg body weight i.p., rats were sacrificed after 90 min, 4 h, and 24 h. Renal cortical expression of NaPi-2 mRNA was determined by Northern blotting, and brush border membrane (BBM) NaPi-2 protein by Western blotting. Increased urinary excretion of phosphate was demonstrated as soon as 90 min after MA injection, and was sustained at 4 and 24 h, NaPi-2 mRNA expression and NaPi-2 protein were not decreased after 90 min. NaPi-2 mRNA decreased after 4 h, while NaPi-2 protein decreased only at 24 h. Hence, the immediate phosphaturia in experimental Fanconi syndrome may be independent of NaPi-2 downregulation, possibly resulting from energy depletion or membrane dysfunction. The decrease in NaPi-2 mRNA expression and the subsequent NaPi-2 protein decrease may account for the second-phase phosphaturia.

Human immunodeficiency virus type 1-mediated syncytium formation is compatible with adenovirus replication and facilitates efficient dispersion of viral gene products and de novo-synthesized virus particles.

Conditionally replicative adenovirus (CRAd) vectors are designed for specific oncolytic replication in tumor tissues with concomitant sparing of normal cells. As such, CRAds offer an unprecedented level of anticancer potential for malignancies that have been refractory to previous cancer gene therapy interventions. CRAd efficacy may, however, be compromised by inefficient dispersion of the replicating vector within the tumor tissue. To address this issue, we evaluated the utility of a fusogenic membrane glycoprotein (FMG), which induces the fusion of neighboring cellular membranes to form multinucleated syncytia. We hypothesized that the FMG-mediated syncytia would facilitate dispersion of the adenovirus (Ad) gene products and viral progeny. To test this, human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins, which induce syncytia in the presence of CD4+ target cells, were expressed by an Ad (Ad5HIVenv) in permissive (CD4-positive) and nonpermissive (CD4-negative) cell lines. After validating this Ad-FMG model, the efficiency of Ad replication in the presence or absence of syncytia was evaluated. The results demonstrated that syncytium formation was compatible with Ad replication and dramatically increased the dispersion of virus gene products within the cytoplasm of the syncytia as well as viral particles in the nuclei of the syncytial mass. Moreover, progeny virions were released more efficiently from syncytia compared with nonsyncytial cells. These data demonstrate the utility of FMGs as a dispersion agent and suggest that FMGs can improve the efficacy of CRAd gene therapy.

Association of anticardiolipin antibodies with vascular access occlusion in hemodialysis patients: cause or effect?

BACKGROUND: Uremic patients on hemodialysis (HD) often display occlusive disorders despite their uremic thromboasthenia. The role of anticardiolipin antibodies (aCL) and the recently identified anti-beta2-glycoprotein I antibodies (abetaGPI) in these phenomena is unknown. METHODS: The prevalence of IgG and IgM aCL and abetaGPI was documented in 54 HD patients and 19 nondialyzed patients with chronic renal failure (CRF). The HD patients were further divided into arteriovenous (AV) access occluders and nonoccluders. RESULTS: HD patients had a higher mean IgG aCL level than CRF patients (14.68 +/- 3 vs. 2.96 +/- 1 IU/ml, respectively, p < 0.0007). IgM aCL did not differ significantly. AV access occluders had a higher mean IgG and IgM aCL levels than nonoccluders (24.47 and 8.39 IU/ml in occluders vs. 8.45 and 3.59 IU/ml in nonoccluders, p < 0.0226 and p < 0.05, respectively). IgM and IgG abetaGPI antibody levels were below the cutoff point and did not differ significantly between the various groups. Cardiovascular episodes were not correlated with the titer of aCL or abetaGPI. CONCLUSION: These results indicate that HD, especially in patients with recurrent access occlusion episodes, is associated with elevated levels of IgG aCL, and that IgG aCL level may predict the occlusive status of HD patients.

Hypoglycemia in patients with renal failure.

Of a total of 1545 admissions of end stage renal failure (ESRD) patients, fifty-six (3.6%) were admitted during a ten-year period with hypoglycemia. Thirty-eight of them were diabetic while the others were non-diabetic patients. The most common etiology found to be drug-induced hypoglycemia in 26 (46%) patients. In 22 (39%) cases, sepsis was the contributing cause of hypoglycemia. Severe malnutrition caused 7% of hypoglycemic episodes. Of the patients, 18 (32%) with ESRD eventually died, none of them were from the drug induced group. However, mortality rate in the sepsis induced hypoglycemia group was 66%, and of the malnutrition group 17% of the deaths. Thus, hypoglycemia is frequent in ESRD patients, and is fatal if associated with either sepsis or malnutrition.

Pancreatitis following hepatitis A vaccination.

We describe a 23-year-old male patient who presented with epigastric abdominal pain, 8 days following vaccination with inactivated hepatitis A virus (Haverix(R)). Clinical and laboratory data confirmed the diagnosis of pancreatitis. Repeat polymerase chain reaction (PCR) for hepatitis A replication was negative. A comprehensive evaluation ruled out other etiologies for pancreatitis. IgM Hepatitis A antibodies did not develop even after 3 months. Pancreatitis following Hepatitis A is a well-known complication of the viremia, but the exact mechanism is controversial. We suggest that the pancreatitis may have been a cellular immunlogical reaction to one of the antigens of hepatitis A virus vaccine, or it might have been caused by the release of mediators of anaphylaxis such as histamine and leucotriens, induced by HAV antigens, resulting in pancreatitis without development of humoral immunization.