[Substitutive therapies in sepsis and acute renale failure]. / Trattamenti emergenti e/o alternativi nell'nsufficienza renale acuta associata a sepsi.

Substitutive treatment of sepsis associated acute renal failure is an emergent challenge in the intensive care unit due to the number of cases and to the high mortality rate. Standard hemofiltration is unable to improve survival, since a high mortality rate is sustained by the septic process. New therapeutic approaches currently available are based on the increased clearance of molecules ranging 10-30 kDa considered important in the physiopathology of sepsis and multiorgan failure. Clinical experiences in progress are: (1) adsorption resins able to bind bacterial products, cytokines, anaphylotoxins and several inflammation mediators; (2) the bioartificial kidney, that is the addition to hemofilter of human tubular cell culture grown in devices in order to mimic metabolic tubular function to a traditional hemofilter; (3) increased exchange volumes (high volume hemofiltration), up to 0-100 L/24 hr and; (4) increased membrane permeability associated with either discarded ultrafiltrate (high cut-off membranes) or plasma substitution plasmapheresis with regeneration by sorbents technology (C FA). Generally, by applying these new technologies to septic shock patients, the observed survival was higher than that predicted by the gravity score. While these results are encouraging, they are not conclusive and need further study.

Calcein assay for multidrug resistance reliably predicts therapy response and survival rate in acute myeloid leukaemia.

In this study, we evaluated the suitability of the calcein assay as a routine clinical laboratory method for the identification of multidrug-resistant phenotype in acute leukaemia. This study presents the results of the calcein tests obtained in two large haematological centres in Hungary. Assays were performed with blast cells of 93 de novo acute leukaemia patients, including 65 patients with acute myeloid leukaemia (AML). Results were expressed as multidrug resistance activity factor (MAF) values. AML patients were divided into responders and non-responders and MAF values were calculated for each group. In both centres, responder patients displayed significantly lower MAF values than non-responders (P = 0.0045 and P = 0.0454). Cut-off values were established between the MAFR + SEM and MAFNR - SEM values. On the basis of these cut-off levels, multidrug resistance (MDR) negativity showed a 72% predictive value for the response to chemotherapy, whereas MDR positivity was found to have an average predictive value of 69% for therapy failure. MDR activity was a prognostic factor for survival rate and the test was suitable for detecting patients at relapse. The calcein assay can be used as a quantitative, standardized, inexpensive screening test in a routine clinical laboratory setting. The assay detects both P-glycoprotein and multidrug resistance-associated protein activities, and identifies AML patients with unfavourable therapy responses.

Extrahepatic immunological manifestations of hepatitis C virus in dialysis patients.

BACKGROUND: Hepatitis C virus (HCV) infection may be associated with various extrahepatic immunological disorders. Uremic patients on chronic regular dialytic treatment (RDT) frequently develop immunological abnormalities. The aim of this study was to evaluate the probability that HCV infection creates an increased risk for extrahepatic immunological abnormalities in chronic RDT patients. SUBJECTS AND METHODS: In a series of one hundred sixteen chronic RDT patients, HCV status was determined by anti-HCV antibodies, polymerase chain reaction (PCR) RNA and viral genotyping. After excluding four anti-HCV negative/PCRRNA positive patients, a comparison was made between 51 anti-HCV negative/PCR-RNA negative and 61 anti-HCV positive patients, this latter group including seventeen PCR-RNA negative, fifteen genotype 1, thirteen genotype 2, three genotype 3, four genotype 4, four undeterminable genotype and five mixed genotypes. The following investigations were performed: cryoglobulinemia (presence, titer and, when possible, identification), monoclonal gammopathy, antineutrophil cytoplasm antibodies, antidouble stranded DNA antibodies, circulating immunocomplexes and immunoglobulin levels. RESULTS: Cryoglobulinemia was found in 77% of anti-HCV positive versus 29% of anti-HCV negative patients, and cryocrit > 1% in 50% versus 9.8% respectively, p=<0.01. Also cryoglobulin concentration was higher (logarithmic transformation: 4.38 +/- 0.94 vs 3.11 +/- 1.06, p =< 0.001) in anti-HCV positive versus negative patients. Multivariate logistic regression analysis showed a significantly increased odds ratio (12.0, confidence interval 3.0 to 48.3) for having high levels of cryoglobulins (cryocrit >1%) after adjusting for age and dialytic age. The prevalence of this abnormality did not differ significantly among patients infected with different genotypes, but a tendency towards a lower frequency was observed in the anti-HCV positive/PCR negative subgroup. Cryoglobulins were identified as type I (2 anti-HCV positive case), type II (2 anti-HCV positive and 1 anti-HCV negative case) and type 3 (1 anti-HCV negative case). The frequency of monoclonal gammopathy was not significantly different between anti-HCV positive and anti-HCV negative patients (6.5% versus 2%) as well as that of the other parameters evaluated except for IgG concentration which was higher in the anti-HCV positive group (1,685 +/-605 versus 1349 +/- 352 mg/dl, p 0.006). Five events, potentially linked to HCV infection, occurred in our anti-HCV positive patients: 2 cases of porphyria cutanea, 1 case of unexplained peripheral neuropathy, 1 cutaneous leukocytoclastic vasculitis, 1 death for non-Hodgkin's lymphoma. In one anti-HCV positive patient treated with interferon-alpha, the presence of cryoglobulins, monoclonal gammopathy and high IgG levels strictly paralleled that of viremia, disappearing during the recovery phase under treatment and reappearing shortly after stopping treatment. CONCLUSIONS: HCV infection provides a significantly increased risk for developing extrahepatic immunological abnormalities also in chronic RDT patients. It is possible that the clinical relevance of this event might be scant because of the low level of these abnormalities, but an awareness of its possibility should to be taken into account.

Parallel functional and immunological detection of human multidrug resistance proteins, P-glycoprotein and MRP1.

The proper assessment of the expression and drug extrusion activity of multidrug resistance proteins in various tumor cells is a challenging clinical laboratory problem. Recently, we have introduced a fluorescent dye (calcein) accumulation assay for the estimation of the functional expression of both P-glycoprotein (MDR1) and the multidrug resistance-associated protein (MRP1). Since both MDR1 and MRP1 decrease the intracellular accumulation of the fluorescent free calcein, by applying appropriate inhibitors of MDR1 and MRP1, the transport activity of these proteins could be quantitatively and selectively estimated in fluorometry or flow-cytometry assays. In the present work single-cell fluorescence digital imaging has been applied to characterize the kinetics and inhibitor-sensitivity of calcein accumulation in a mixture of HL60 MRP1 and NIH 3T3 MDR1 cells. Subsequent immunofluorescence labeling was performed by the anti-MDR1 monoclonal antibody (mAb) UIC2 in the same cell population. We report that the double labeling approach, based on the single cell calcein accumulation assay and an immunofluorescence detection, provides good sensitivity and selectivity for the simultaneous functional and immunological detection of cellular MDR1 and MRP1.

The multidrug transporters--proteins of an ancient immune system.

The multidrug resistance proteins, discovered as membrane transporters producing chemotherapy-resistance in cancer, are functioning as complex cellular defence systems through recognition and energy-dependent removal of a large variety of toxic agents. The multidrug transporters belong to the ATP-binding cassette (ABC) transporters, present both in prokaryotes and eukaryotes and built from a combination of characteristic membrane-spanning helices and cytoplasmic ATP-binding domains. In mammals the MDR1 (P-glycoprotein) extrudes large hydrophobic compounds and provides the basis of the blood-brain and the blood-testis barrier for such molecules. The multidrug resistance-associated protein (MRP) and its homologues have a major role in the cellular export of large organic anions, including e.g. conjugated bile salts and glutathione-conjugates. The substrate recognition, that is the self and non-self discrimination and the ATP-dependent foreign agent extrusion are directly coupled within the structure of these large plasma membrane proteins. Here we suggest that the multidrug transporters are essential parts of our immune-defence system, working as 'cellular antitoxic' mechanisms.

[Significance of multidrug resistance in the therapy of malignant tumors]. / A multidrog rezisztencia jelentösége a rosszindulatú daganatok terápiájában.

Some human malignant tumours respond poorly to initial chemotherapy, indicating that they possess intrinsic resistance. On the other hand, in a significant portion of tumours after early promising results of therapy, the patients relapse, metastases appear, and acquired resistance to chemotherapy develops. The broad spectrum-resistance against chemotherapy is called multidrug resistance (MDR), and due to its clinical significance, studying of proteins responsible for multidrug resistance has become one of the most active research areas in biomedicine. There are several molecular mechanisms responsible for multidrug resistance. A group of filogenetically conservative plasmamembrane proteins actively extrudes different toxic compounds, xenobiotics, and also cytotoxic drugs from drug-resistant cells by using the energy of ATP. The clinically and biochemically most thoroughly characterized member of these proteins is the P-glycoprotein, which pumps hydrophobic drugs of natural origin and different chemical structure out of the cells. The recently cloned multidrug resistance associated protein (MRP) has also a broad substrate specificity, thus resembling P-glycoprotein. However, besides hydrophobic compounds, organic anions, glucuronide and glutathione conjugates are also excellent substrates of the MRP. The basic and clinically relevant properties of proteins causing multidrug resistance and the state of the art of current diagnostic approaches are summarized in this literature review. The different malignancies are characterized from the point of view of their multidrug resistance and recent clinical and biochemical data concerning therapeutic approaches for reversal of multidrug resistance are also presented.

Membrane topology and glycosylation of the human multidrug resistance-associated protein.

The membrane topology of the human multidrug resistance-associated protein (MRP) was examined by flow cytometry phenotyping, immunoblotting, and limited proteolysis in drug-resistant human and baculovirus-infected insect cells, expressing either the glycosylated or the underglycosylated forms of this protein. Inhibition of N-linked glycosylation in human cells by tunicamycin did not inhibit the transport function or the antibody recognition of MRP, although its apparent molecular mass was reduced from 180 kDa to 150 kDa. Extracellular addition of trypsin or chymotrypsin had no effect either on the function or on the molecular mass of MRP, while in isolated membranes limited proteolysis produced three large membrane-bound fragments. These experiments and the alignment of the MRP sequence with the human cystic fibrosis transmembrane conductance regulator (CFTR) suggest that human MRP, similarly to CFTR, contains a tandem repeat of six transmembrane helices, each followed by a nucleotide binding domain, and that the C-terminal membrane-bound region is glycosylated. However, the N-terminal region of MRP contains an additional membrane-bound, glycosylated area with four or five transmembrane helices, which seems to be a characteristic feature of MRP-like ATP-binding cassette transporters.

Transport properties of the multidrug resistance-associated protein (MRP) in human tumour cells.

In this paper we demonstrate that the expression of the multidrug resistance-associated protein (MRP) in a variety of intact human tumour cells results in the ATP-dependent, mutually exclusive extrusion of both the acetoxymethyl ester and the free anion forms of the fluorescent dye calcein, as well as that of a fluorescent pyrenemaleimide-glutathione conjugate. The MRP-dependent transport of all these three model compounds closely correlates with the expression level of MRP and is cross-inhibited by hydrophobic anticancer drugs, by reversing agents for MDR1, and also by compounds not influencing MDR1, such as hydrophobic anions, alkylating agents, and inhibitors of organic anion transporters. Cellular glutathione depletion affects neither the MRP-dependent extrusion of calcein AM or free calcein, nor its modulation by most hydrophobic or anionic compounds, although eliminating the cross-inhibitory effect of glutathione conjugates. These results suggest that the outward pumping of both hydrophobic uncharged and water-soluble anionic compounds, including glutathione conjugates, is an inherent property of MRP, and offer sensitive methods for the functional diagnostics of this transport protein as well as for the rapid screening of drug-resistance modulating agents.

Drug-stimulated ATPase activity of a deletion mutant of the human multidrug-resistance protein (MDR1).

The baculovirus-insect cell system has been used for the functional expression of the human multidrug resistance protein (MDR1) and a mutant MDR1 variant lacking a twenty amino acid segment from the first extracellular loop (delta aa78-97 MDR1). Both MDR1 proteins were found to be correctly inserted into the insect cell membrane as indicated by their interaction with MRK 16 antibody. The removal of the 78-97 segment from the first extracellular loop dramatically altered drug-stimulated ATPase activity. Rhodamine 123 or vinblastine were not able to stimulate the mutant protein and Calcein AM had also little effect. In contrast, verapamil increased the ATPase activity of the mutant almost to the same maximal level as that of the wild type. However, the verapamil concentration needed for the half maximal stimulation of the ATPase activity was found to be about hundred times higher than that for the wild type MDR1. These results indicate that a partial deletion of an extracellular loop modulates the affinity of MDR1 for its transportable substrates in a variable fashion.

Interaction of bioactive hydrophobic peptides with the human multidrug transporter.

In this report we demonstrate that various biologically active hydrophobic peptide derivatives, e.g., proteinase inhibitors, chemoattractants, ionophores, enkephalins, and immunosuppressants, stimulate a membrane ATPase activity associated with the human multidrug transporter (MDR1). The stimulation of the MDR1-ATPase by these agents does not correlate with their known biochemical or pharmacological activities but rather with their hydrophobicity. The peptides that show high-affinity interaction with the MDR1-ATPase also interfere strongly with fluorescent dye extrusion catalyzed by the multidrug transporter in intact cells and some have been shown to reverse drug resistance in cultured cells. These data suggest that several hydrophobic peptides behave as substrates of the multidrug transporter and may be used to modulate the chemotherapy resistance of tumor cells.

Calcein accumulation as a fluorometric functional assay of the multidrug transporter.

Acetoxymethyl ester (AM) derivatives of various fluorescent indicators (fura-2, fluo-3, indo-1, BCECF, calcein) are actively extruded by the multidrug transporter (MDR1, P-glycoprotein-Homolya, L. et al. (1993) J. Biol. Chem. 268, 21493-21496). In the present paper we show that the measurement of the accumulation of a fluorescent cell viability marker, calcein, can be effectively used as a rapid and sensitive fluorometric and flow cytometric assay for studying P-glycoprotein function. The rate of calcein accumulation in human MDR1-expressing cells is significantly lower than in the control cells, while various drug-resistance reversing agents (verapamil, vinblastine, oligomycin, cyclosporin A and UIC2 monoclonal antibody) greatly increase calcein trapping only in the MDR1-expressing cells. Since calcein-AM is not fluorescent and free calcein is not a substrate of the multidrug transporter, the assay is readily applicable for rapid kinetic studies of the MDR1 function. Calcein has a high fluorescence intensity in the visible range, thus changes in calcein uptake can be easily visualised and MDR1-expressing and control cells separated by conventional flow cytometry.

Fluorescent cellular indicators are extruded by the multidrug resistance protein.

In this report we show that NIH-3T3 mouse fibroblasts stably expressing the human multidrug transporter (MDR1 or P-glycoprotein), in contrast to the control NIH-3T3 cells, actively extrude the hydrophobic acetoxymethyl ester (AM) derivatives used for cellular loading of various fluorescent calcium and pH indicators. This dye extrusion is blocked by competing substrates and inhibitors of the multidrug transporters, e.g. by verapamil, vincristine, sodium orthovanadate, oligomycin, and a monoclonal anti-MDR1 antibody. The hydrophilic free acid forms of the indicators are not exported by MDR1. We also demonstrate that in isolated cell membranes the MDR1-ATPase, similar to that by known substrates of the transporter, is stimulated by the AM derivatives of fluorescent dyes whereas the free acid forms of the dyes are without effect. Since (i) the AM derivatives of the fluorescent indicators rapidly permeate the cell membrane and are readily cleaved by high activity and large capacity cytoplasmic esterases and (ii) the free acid forms are not substrates for export by MDR1, the observations above suggest that dye extrusion by MDR1 may occur without a cytoplasmic appearance of the AM compounds. These data also call attention to the possible interaction of widely used hydrophobic fluorescent indicators with MDR1 and offer an efficient detection of MDR1-expressing tumor cells as well as a screening method for examining drug interactions with the multidrug transporter.

Thapsigargin-induced increase in cytoplasmic Ca2+ concentration and aldosterone production in rat adrenal glomerulosa cells: interaction with potassium and angiotensin-II.

Thapsigargin (Tg), a microsomal Ca2+ pump inhibitor, dose-dependently increases the cytoplasmic Ca2+ concentration and aldosterone production without having any striking effect on the formation of inositol phosphates in isolated rat adrenal glomerulosa cells. The interaction of Tg with the major Ca2(+)-mediated stimuli of glomerulosa cells on aldosterone production was also examined. The effects of Tg and the Ca2(+)-mobilizing angiotensin-II (AII) were additive. The aldosterone production stimulatory effect of potassium, which induces Ca2+ influx via voltage-operated Ca2+ channels, was potentiated by Tg. The positive interaction between Tg and potassium on aldosterone production raises the possibility that stimuli generating Ca2+ signal by depleting intracellular Ca2+ stores, such as Tg or AII, enhance the response of the cell to depolarization. Such an interaction between AII and potassium may have an important role in the physiological control of aldosterone production.

Angiotensin II inhibits K(+)-induced Ca2+ signal generation in rat adrenal glomerulosa cells.

The Ca2(+)-mobilizing hormone angiotensin II (AII) dose-dependently inhibited the K(+)-induced sustained increase of cytoplasmic Ca2+ concentration in adrenal glomerulosa cells and caused a rapid decrease of cytoplasmic Ca2+ when added to cells already stimulated with K+. These effects of AII on the K(+)-induced Ca2+ signal were mimicked, although less effectively, by other Ca2(+)-mobilizing agonists such as [Arg8]vasopressin (AVP) and thapsigargin. Phorbol esters did not show such effects, nor did corticotropin (ACTH), a secretagogue acting via cyclic AMP. The K(+)-stimulated initial 45Ca2+ uptake, a measure of Ca2+ entry into glomerulosa cells, was also prevented by AII pretreatment, and was inhibited by AVP, but not by ACTH. The stimulatory effect of K+ on aldosterone production, however, was not inhibited by AII, and the AII-induced aldosterone production was further increased by increasing K+. These data indicate that AII is able to inhibit static increases in cytoplasmic Ca2+ by inhibiting Ca2+ entry through voltage-sensitive Ca2+ channels and, possibly, by activating Ca2+ extrusion from the cells. It is also concluded that the Ca2+ signal evoked by AII is very efficient in stimulating hormone secretion, and the secretory response of the cells becomes more sensitive to any further increase of Ca2+ entry through voltage-sensitive Ca2+ channels.

Effects of high potassium concentration and dihydropyridine Ca2(+)-channel agonists on cytoplasmic Ca2+ and aldosterone production in rat adrenal glomerulosa cells.

The aldosterone secretory response of isolated rat adrenal glomerulosa cells to potassium was studied in a cell-perifusion system. Increasing the potassium concentration from 3.6 to 5.4 mM in the perifusion medium caused a rapid 40-fold stimulation of aldosterone production which was maintained during the 2 h period of stimulation. A dose of 8.4 mM potassium elicited a 100-fold increase of hormone production with rapid onset and a slowly decreasing plateau. When the potassium concentration was further increased to 18 mM, there was a rapid stimulation of aldosterone production comparable to that evoked by 8.4 mM potassium; however, the response declined very rapidly to levels still above basal. The dihydropyridine-agonist BAY-K 8644 (100 nM) greatly enhanced the aldosterone response to 5.4 mM potassium but did not significantly modify the response evoked by 8.4 mM potassium. The effect of BAY-K 8644 on the aldosterone response was inhibitory at 18 mM potassium concentration, suggesting that the character of dihydropyridine modulation of the secretory response was voltage dependent, showing reversal at relatively negative potentials. When the cytoplasmic Ca2+ concentration was monitored in glomerulosa cells by the fluorescent Ca2(+)-probe Fura-2, potassium evoked a rapid dose-dependent increase in free Ca2+, with elevated steady-state Ca2(+)-levels throughout stimulation, even at potassium concentrations higher than 18 mM. Moreover, BAY-K 8644 (100 nM) enhanced the cytoplasmic Ca2+ response to all potassium concentrations tested up to 30 mM. The initial 30 sec 45Ca2+ uptake, an indicator of potassium-stimulated voltage-sensitive Ca2+ influx into these cells, showed a fast increase and only an initial inactivation in response to 18 mM potassium. This response was enhanced by 100 nM BAY-K 8644, with no sign of enhanced inactivation or inhibition caused by the dihydropyridine agonist. These results indicate that the correlation between Ca2+ influx, cytoplasmic Ca2+ levels, and the secretory response in adrenal glomerulosa cells is lost at potassium concentrations higher than 8 mM, especially in the presence of the dihydropyridine agonist, BAY-K 8644.