Michael Schmitz

Regional citrate versus systemic heparin for anticoagulation in critically ill patients on continuous venovenous haemofiltration: a prospective randomized multicentre trial

BACKGROUNDnContinuous venovenous haemofiltration (CVVH) in the intensive care setting requires anticoagulation to prevent clotting of the extracorporeal circuit. Several protocols avoiding heparin and using regional citrate anticoagulation have been developed to diminish bleeding risks. However, data from randomized trials comparing citrate anticoagulation with systemic heparinization are very limited.nnnMETHODSnOne hundred and seventy-four patients on mechanical ventilation, requiring renal replacement therapy for acute renal failure, were included in this prospective randomized multicentre trial comparing regional citrate with systemic heparin. The study was performed at nine different intensive care units at university or academic teaching hospitals. The participants were randomized to either CVVH using regional citrate anticoagulation or CVVH using systemic anticoagulation with unfractionated heparin. The primary outcome was to compare treatment efficacy represented by the patients acid base status on Day 3 and on each consecutive day. Several parameters of safety and efficacy were analysed as secondary outcomes.nnnRESULTSnComparison of standard bicarbonate from Day 3 to Day 11 revealed no difference between both treatment modalities. Use of citrate resulted in less systemic anticoagulation, a lower risk of bleeding and a longer haemofilter patency. Episodes of hypercalcaemia, hypocalcaemia and the need for additional bicarbonate infusions occurred more often under citrate. The patients high mortality was not influenced by the mode of anticoagulation.nnnCONCLUSIONSnCitrate may be used as a regional anticoagulant and the only buffering agent in CVVH with adequate treatment efficacy and safety. However, neither citrate nor heparin anticoagulation should be regarded as a therapeutic standard, since there is no advantage of one of these substances with regard to patient mortality.

Formation of a thermodynamically metastable structure containing hairpin II is critical for infectivity of potato spindle tuber viroid RNA.

The functional relevance of a hairpin II‐containing structure of viroid RNA was studied by site‐directed mutagenesis, thermodynamic calculations, experimental denaturation curves and infectivity tests. Hairpin II is formed during thermal denaturation of circular viroids or as part of a metastable structure during synthesis of viroid replication intermediates. In potato spindle tuber viroid (PSTVd), eight single‐site mutations were generated in the segments which form hairpin II. From the mutated viroid cDNA clones, linear RNA transcripts of PSTVd unit length were synthesized. The relevance of hairpin II for the mechanism of denaturation was confirmed quantitatively by optical denaturation curves and temperature‐gradient gel electrophoresis. Infectivity tests showed that the mutations in the core region of hairpin II reverted to the wild type sequence whereas the mutations in the peripheral regions of hairpin II remained genetically stable. These data are in accordance with the natural variance of hairpin II in other viroids of the PSTVd class. Thus, the integrity of the core of hairpin II is critical for infectivity. Hairpin II exhibits a strong similarity in sequence as well as in three‐dimensional structure to certain DNA GC‐clusters found in the 5′‐upstream regions of some genes in man, animals, viruses and plants. A hypothesis about a function of hairpin II as a binding site for host cell transcription factors is proposed.

Management of arterial stenosis affecting kidney graft perfusion: a single-centre study in 53 patients.

We assessed clinical and duplex sonographic (CDS) findings, and outcome in patients with stenosis of the transplant renal artery (TRAS) or the aorto‐iliac segment proximal to the graft (Prox‐TRAS) treated with dilatation (PTA), stenting (PTAS) and surgery. From 1988 to 2002, of 1189 patients with renal transplantations, 117 underwent angiography. Fifty‐three patients with TRAS (n = 37)/Prox‐TRAS (n = 16) were found (4.4%).

Base-pair probability profiles of RNA secondary structures

Dynamic programming algorithms are able to predict optimal and suboptimal secondary structures of RNA. These suboptimal or alternative secondary structures are important for the biological function of RNA. The distribution of secondary structures present in solution is governed by the thermodynamic equilibrium between the different structures. An algorithm is presented which approximates the total partition function by a Boltzmann-weighted summation of optimal and suboptimal secondary structures at several temperatures. A clear representation of the equilibrium distribution of secondary structures is derived from a two-dimensional bonding matrix with base-pairing probability as the third dimension. The temperature dependence of the equilibrium distribution gives the denaturation behavior of the nucleic acid, which may be compared to experimental optical denaturation curves after correction for the hypochromicities of the different base-pairs. Similarly, temperature-induced mobility changes detected in temperature-gradient gel electrophoresis of nucleic acids may be interpreted on the basis of the temperature dependence of the equilibrium distribution. Results are illustrated for natural circular and synthetic linear potato spindle tuber viroid RNA respectively, and are compared to experimental data.

Secretion of GM-CSF and M-CSF by human renal cell carcinomas of different histologic types

OBJECTIVESnTo analyze the secretion of hematopoietic growth factors and the expression of their corresponding receptors in 40 newly established renal cell carcinoma (RCC) cell lines of different histologic types. Little is known about the secretion and function of hematopoietic growth factors by human RCCs.nnnMETHODSnThe expression of the hematopoietic growth factors (ie, erythropoietin, interleukin [IL]-3, IL-5, granulocyte colony-stimulating factor [G-CSF], granulocyte-macrophage colony-stimulating factor [GM-CSF], and macrophage colony-stimulating factor [M-CSF]) was determined by enzyme-linked immunosorbent assay analysis under different culture conditions, including suspension culture and monolayer cultures (plastic and Matrigel-coated culture flasks). The expression of their corresponding receptors was defined by fluorescence activated cell scanner analysis and by reverse-transcriptase polymerase chain reaction. The response of the RCC cell lines to exogenous hematopoietic growth factors was analyzed by MTT assay.nnnRESULTSnIn almost all of the cell lines, significant amounts of GM-CSF and M-CSF were secreted, and in four cell lines, a secretion of G-CSF was detected. Fourteen RCC cell lines showed secretion of IL-3, and production of IL-5 and erythropoietin was not observed in any cell line. Secretion of GM-CSF and M-CSF was affected by the substratum offered for cell attachment in the adherent cultures. GM-CSF secretion was more pronounced under culture conditions with a reduced frequency of cell-to-cell contacts. Two cell lines were shown to express receptors for M-CSF, but receptors for G-CSF and GM-CSF could not be detected in any cell line. Exposure to exogenous G-CSF, GM-CSF, and M-CSF did not affect the proliferation of our RCC cell lines.nnnCONCLUSIONSnThe results of our study clearly demonstrate that human RCC cells can secrete significant amounts of G-CSF, GM-CSF, M-CSF, and IL-3, and are thereby theoretically able to modulate the hosts tumor-directed immune response.

Multidrug Resistance Phenotype and Paclitaxel (Taxol®) Sensitivity in Human Renal Carcinoma Cell Lines of Different Histologic Types

Abstract We compared the effects of paclitaxel (Taxol®) in human renal cell carcinoma (RCC) of different histologic types. The growth inhibiotory effects of paclitaxel on 34 human RCC cell lines of strictly defined different histologic types were determined by 3–[4,5-dimethylthiazilyl]-2,5-diphenyltetrazoliumbromide (MTT) assays. Paclitaxel-induced morphologic alterations were visualized by light and immunofluorescence and by transmission electron microscopy. The expression and function of P-glyco-protein arid multidrug resistance-associated protein (MRP) were defined by reverse transcriptase polytnercise chain reaction and fluorescence-activated cell sorting (FACS) analysis, respectively. Modulation of P-glycoprotein function was performed by verapamil or Cretnophor EL. A significant (p < 0.05) dose-dependent paclitaxel-induced growth inhibition could be demonstrated in all cell lines, with the effects of paclitaxel dissolved in Cremophor® EL/ethanol (= Taxol®) exceeding the effects of piclitaxel dissolved in dimethyl sulfoxide. The extent of response markedly varied between the different cell lines, although chromophilic RCCs exhibited a more pronounced response to Taxol (IC50: 0.03–0.38 μM) than clear cell RCCs (IC50,: 0.01–36.69 μM). Exposure to paclitaxe/Taxol® induced an increuse of micro-tubule bundles in the clear cell and the chromophobe RCCs but not in the chromophilic RCCs. The expression of the MRP was low in RCC cell lines and was not found to be related to paclitaxel/Taxol® sensitivity. In contrast, the expression level of P-glycoprotein was much more pronounced and showed a positive correlation (p < 0.05) with the response to paditaxel. Reversal of P-glycoprotein function by verapamil or Cremophor® EL enhanced the growth inhibitory effects of paclitaxel and further supported the role of P-glycoprotein for paclitaxel sensitivity of human RCCs. Paclitaxel/Taxol® effktively inhibits proliferation of human RCCs in vitro, irrespective of their histologic types. Moreover, expression and function of P-glycoprotein markedly contribute to paclitaxel responsiveness, although other as yet undefined drug resistance mechanisms are effective in humun RCCs us well.

Opportunistic infections after renal transplantation.

Opportunistic infection is a serious clinical complication in patients receiving immunosuppressive therapy after kidney transplantation. This article deals with some of the possible infectious agents that were recently encountered at our transplantation centre in Düsseldorf, Germany. Opportunistic organsims such as human herpesviruses 6-8, polyomavirus, parvovirus B19, varicella zoster virus, Nocardia and Listeria monocytogenes are rare but severe complications that are presented in this overview. As a result of the use of new immunosuppresive drugs like tacrolimus and mycophenolate mofetil these infections are now seen more frequently, so they should always be included in differential diagnostic considerations. New diagnostic procedures and new treatment strategies should allow early detection and successful treatment of opportunistic infections in the majority of kidney transplant recipients.

Infection with polyomavirus type BK after renal transplantation.

Tubulointerstitial nephritis caused by polyomavirus of the subtype BK (BK virus nephropathy, BKN) is an important cause of deterioration of renal allograft function after kidney transplantation. In 3 cases of BKN diagnosed at our center, the suspected diagnosis made on the basis of urine cytology and serum PCR was confirmed by electron microscopy and immunohistology of the renal graft biopsy. In 1 patient, stable renal function without further virus detection was seen after reduction of the immunosuppression. In 2 further patients there was loss of graft function. BKN is an important differential diagnosis of unclear deterioration of renal graft function. The risk is particularly high with use of tacrolimus and mycophenolate mofetil (MMF). Urine cytology and serum PCR are suitable screening tests, histology provides conclusive evidence. The only therapeutic option available at present is reduction of immunosuppressive therapy.

Are Prothrombotic Variants of Platelet Glycoprotein Receptor Polymorphisms Involved in the Pathogenesis of Thrombotic Microangiopathies

Thrombotic microangiopathies are life-threatening disorders characterized by vascular microthromboses, schistocytic hemolytic anemia, and thrombocytopenia. Although recent research has partially explained the pathogenesis of these rare entities, the determinants contributing to the onset and modulating the severity of thrombotic microangiopathies are largely unknown. The present study assessed the putative role of prothrombotic platelet receptor polymorphisms in thrombotic microangiopathies that have been found to be associated with premature onset of myocardial infarction in predisposed individuals. Thirty-four consecutive patients admitted with the diagnosis of thrombotic microangiopathy and 759 healthy subjects were enrolled. Genotyping of the human platelet antigen (HPA) 2 an the Kozak sequence polymorphism of GP Ibα of the platelets’ von Willebrand factor receptor glycoprotein (GP) Ib-V-IX, the HPA-1 and the HPA-3 polymorphism of the fibrinogen receptor GP IIb-IIIa (integrin αIIbβ 3) and the HPA-5 and GP Ia 807 C/T polymorphism of the collagen receptor GP Ia-IIa (integrin α2β1) were determined according to standard procedures. As a result, no significant differences in the prevalence of prothrombotic variants of platelet-receptor polymorphisms between patients and healthy control subjects were observed. However, although not significant, the prothrombotic bb genotype of the HPA-1 polymorphism was more prevalent in the patients. The findings do not provide evidence that platelet receptor polymorphisms are determinants for the onset of thrombotic microangiopathies or predispose to a more severe course. Along with this observation, screening for respective platelet-receptor polymorphisms does not appear to contribute to risk stratification of affected patients.

Effects of citrate dialysate in chronic dialysis: a multicentre randomized crossover study

BACKGROUNDnAlthough citrate dialysate (CiDi) is regarded to be safe, dialysis modalities using higher dialysate volumes, like haemodiafiltration (HDF), may expose patients to higher citrate load and thus increase the risk of complications. We investigated the residual risk of CiDi compared with standard dialysate (StDi) in patients on different dialysis modalities and its effect on dialysis dose.nnnMETHODSnIn a multicentre randomized crossover study, 92 dialysis patients (HDF post-dilution: n = 44, HDF pre-dilution: n = 26, haemodialysis: n = 25) were treated for 4 weeks with each dialysate (StDixa0and CiDi). Hypocalcaemia (ionized calcium ≤0.9 mmol/L), alkalosis (pH ≥7.55), post-treatment bicarbonate ≥32 mmol/L, pre-treatment bicarbonate ≥27 mmol/L, intra-dialytic events (IEs) and adverse events (AEs) between dialysis sessions were investigated as primary end points. The secondary objective was dialysis efficacy, i.e. dose and removal ratios of urea, creatinine, phosphate and β-2-microglobulin.nnnRESULTSnPost-dialysis overcorrection of bicarbonate (>32 mmol/L) was less frequent with CiDi (P = 0.008). Other predefined calcium and acid-base disturbances did not vary. There was no significant difference in IE. However, more patients developed AEs such as fatigue, muscle spasms or pain using CiDi (StDi: 41 versus CiDi: 55 patients, P = 0.02), particularly in the first 2 weeks of exposure. Dialysis efficacy was comparable with both dialysates.nnnCONCLUSIONSnIt can be confirmed that CiDi is not associated with the development of severe calcium and acid-base disorders, even when dialysis modalities with higher citrate loads are used. However, a refinement of the CiDi composition to minimize AEs is necessary.