Rapidly progressive hepatic alveolar echinococcosis in an ABO-incompatible renal transplant recipient.

We report on the case of an ABO-incompatible renal re-transplant recipient maintained on an intensified immunosuppressive regimen for recurrent cellular rejection episodes and transplant glomerulopathy who presented with rapidly growing hepatic tumors, radiologically suggestive of hemangiosarcoma. Upon resection and pathological work-up, the lesions revealed alveolar echinococcosis, a rare but potentially life-threatening parasitosis. Usually infection with Echinococcus multilocularis remains asymptomatic for extended periods of time and can go unrecognized for years. In the case presented, we observed an atypically rapid growth pattern of E. multilocularis that might have been due to the extent of the immunosuppressive regimen, which included repetitive anti-CD20 treatments. Retrospectively performed serological studies with enzyme-linked immunosorbent assays known to provide high sensitivity and specificity for the detection of echinococcosis in the general population, yielded ambiguous results in our immunocompromised host, which could be, in part, explained by B-cell depletion and its effects on antibody production and indirect actions on cellular immunity. In conclusion, this is the first report to our knowledge of hepatic alveolar echinococcosis in a renal transplant recipient. This case documents an altered clinical course of the parasitosis and the challenge of serological diagnostic tools under an intensified regimen of immunosuppressive agents, including rituximab.

ABO-incompatible kidney transplantation with antigen-specific immunoadsorption and rituximab - insights and uncertainties.

Several protocols have been developed to effectively overcome the blood group barrier in renal transplantation. In the evolution of these protocols, one of the latest steps was the combination of anti-CD20 treatment with antigen-specific immunoadsorptions. Over the last years we have learned that these relatively new protocols carry very promising short-term and intermediate-term results which compare favorably to the outcome of ABO-compatible living donor transplantations. Latest reports suggest that combining immunoadsorptions with rituximab does not result in an increased risk of infectious complications or tumors in the first years after transplantation compared to ABO-compatible living donor transplantations. We recently demonstrated that a majority of patients with isoagglutinin titers >1:128 can be safely transplanted using rituximab and immunoadsorptions without an added risk of early antibody-mediated rejections. We have also shown that a cost saving 'on-demand strategy' of postoperative immunoadsorptions based on careful titer monitoring can be used as an alternative to preemptively scheduled immunoadsorptions. Although rituximab and antigen-specific immunoadsorptions are significantly less invasive than splenectomy and plasma-pheresis, long-term follow-up of patients treated with a combination of anti-CD20 antibody and antigen-specific immunoadsorption will be needed to benchmark this therapeutic option in relation to more established protocols.

Paraneoplastic cerebellar degeneration and nephrotic syndrome preceding Hodgkin's disease: case report and review of the literature.

A patient presented with symptoms of cerebellar degeneration and nephrotic syndrome. A work-up at that time failed to reveal an underlying disease; however, 20 months later Hodgkin's disease was diagnosed. Hodgkin's lymphadenopathy developed 2 wk after prednisone therapy for the nephrotic syndrome had been discontinued. Systemic polychemotherapy resulted in complete remission of both Hodgkin's disease and nephrotic syndrome, while the neurological deficit persisted. Patients with unexplained cerebellar degeneration and/or nephrotic syndrome demand extensive evaluation for the presence of Hodgkin's disease, and steroid therapy may delay diagnosis.

Extracellular nucleotides regulate cellular functions of podocytes in culture.

Extracellular nucleotides are assumed to be important regulators of glomerular functions. This study characterizes purinergic receptors in podocytes. The effects of purinergic agonists on electrophysiological properties and the intracellular free Ca(2+) concentration of differentiated podocytes were examined with the patch-clamp and fura 2 fluorescence techniques. mRNA expression of purinergic receptors was investigated by RT-PCR. Purinergic agonists depolarized podocytes. Purinergic agonists similarly increased intracellular free Ca(2+) concentration of podocytes. The rank order of potency of various nucleotides on membrane voltage and free cytosolic calcium concentration was UTP approximately UDP > [adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S)] > ATP > 2-methylthioadenosine 5'-triphosphate (2-MeS-ATP) > 2'- and 3'-O-(4-benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP) > ADP-beta-S. alpha,beta-Me-ATP was without effect. In the presence of UTP, BzATP did not cause an additional depolarization of podocytes. Incubation of cells with ATP or BzATP did not induce lactate dehydrogenase release. In RT-PCR studies, mRNAs of the P2Y(1), P2Y(2), P2Y(6), and P2X(7) receptors were detected within glomeruli and podocytes. The data indicate that extracellular nucleotides modulate podocyte function mainly by an activation of both P2Y(2) and P2Y(6) receptors.

Acidic pH inhibits non-MHC-restricted killer cell functions.

Immunotherapeutic strategies in advanced stages of solid tumors have generally met with little success. Various mechanisms have been discussed permitting the escape of tumor cells from an effective antitumoral immune response. Solid tumors are known to develop regions with acidic interstitial pH. In a recent study performed in the human system, we were able to demonstrate that non-MHC-restricted cytotoxicity is inhibited by an acidic microenvironment. To get more insight into the mechanisms leading to this reduced cytotoxic activity, we have now investigated the influence of an acidic extracellular pH (pH(e)) on the killing process in detail. Unstimulated PBMC and LAK cells were used as effector cells. Both populations are able to kill tumor cells in a MHC-independent manner via perforin/granzymes or TNFalpha, whereas only IL-2-activated cells can use the killing pathway via Fas/FasL. We studied the influence of a declining pH(e) on the different killing pathways against TNFalpha-sensitive and -resistant, as well as Fas-positive and -negative, target cells. Experiments in the absence of extracellular Ca(2+) were used to discriminate the Ca(2+)-dependent perforin-mediated killing. Here we show that the release of perforin/granzyme-containing granules, the secretion of TNFalpha, and also the cytotoxic action of Fas/FasL interaction or of membrane-bound TNFalpha were considerably inhibited by declining pH(e). Furthermore, the secretion of the activating cytokine IFNgamma, as well as the release of the down-regulating cytokines IL-10 and TGF-beta(1), was strictly influenced by surrounding pH. As a pH(e) of 5.8 resulted in a nearly complete loss of cytotoxic effector cell functions without affecting their viability, we investigated the influence of pH(e) on basic cellular functions, e.g. , mitochondrial activity and regulation of intracellular pH. We found an increasing inhibition of both functions with declining pH(e). Therefore, an acidic pH(e) obviously impairs fundamental cellular regulation, which finally prevents the killing process. In summary, our data show a strict pH(e) dependence of various killer cell functions. Thus, an acidic microenvironment within solid tumors may contribute to the observed immunosuppression in vivo, compromising antitumoral defense and immunotherapy in general, respectively.

Amino acid transport in podocytes.

It has recently been shown that formation of podocyte foot processes is dependent on a constant source of lipids and proteins (Simons M, Saffrich R, Reiser J, and Mundel P. J Am Soc Nephrol 10: 1633-1639, 1999). Here we characterize amino acid transport mechanisms in differentiated cultured podocytes and investigate whether it may be disturbed during podocyte injury. RT-PCR studies detected mRNA for transporters of neutral amino acids (ASCT1, ASCT2, and B(0/+)), cationic AA (CAT1 and CAT3), and anionic AA (EAAT2 and EAAT3). Alanine (Ala), asparagine, cysteine (Cys), glutamine (Gln), glycine (Gly), leucine (Leu), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), glutamic acid (Glu), arginine (Arg), and histidine (His) depolarized podocytes and increased their whole cell conductances. Depletion of extracellular Na(+) completely inhibited the depolarization induced by Ala, Gln, Glu, Gly, Leu, and Pro and decreased the depolarization induced by Arg and His, indicating the presence of Na(+)-dependent amino acid transport. Incubation of podocytes with 100 microg/ml puromycin aminonucleoside for 24 h significantly attenuated the effects induced by the various amino acids by approximately 70%. The data indicate the existence of different amino acid transporter systems in podocytes. Alteration of amino acid transport may participate in podocyte injury and disturbed foot process formation.

Dopamine depolarizes podocytes via a D1-like receptor.

BACKGROUND: Dopamine influences glomerular haemodynamics and dopamine receptors have been demonstrated in the glomerulus, but little is known about the cellular effects of dopamine in glomerular cells. The aim of this study was to investigate the influence of dopamine on the cellular functions of podocytes. METHODS: The effect of dopamine on membrane voltage was investigated in differentiated mouse podocytes. The membrane voltage was measured using the patch clamp technique. Reverse transcribed-polymerase chain reaction (RT-PCR) studies were performed to investigate the expression of dopamine receptor mRNA in mouse glomeruli and podocytes. RESULTS: The addition of dopamine (100 nM-1000 microM) caused a concentration-dependent depolarization of podocytes (EC50 is approximate to 10 microM). Like dopamine, the selective agonist of the D1-like receptor, SKF 82958, depolarized podocytes in a concentration-dependent manner. (EC50 is approximate to 50 microM). SKF 82958 stimulated a time-and concentration-dependent accumulation of cyclic adenosine 3',5'-monophosphate (cAMP) in podocytes (EC50 is approximate to microM). RT-PCR studies with primers derived from mouse sequences amplified mouse mRNA for the D1-like and the D2-like receptor in glomeruli, which were obtained by the sieve technique, whereas only mRNA for the D1-like receptor was detected in cultured mouse podocytes. CONCLUSION: The data indicate that dopamine induces a cAMP-dependent depolarization via a D1-like receptor in podocytes.

Agonist-induced activation of a non-selective ion current in glomerular endothelial cells.

The control of intracellular calcium activity ([Ca2+]i) and membrane voltage (Vm) play an important role in regulating functions of glomerular endothelial cells (GEC). We investigated the effect of extracellular ATP on the intracellular [Ca2+]i, Vm and ion conductances in GEC. ATP (100 mumol/liter) induced a rapid increase of [Ca2+]i in GEC from 20 +/- 6 to 442 +/- 84 nmol/liter, which was followed by a sustained Ca2+ plateau of 112 +/- 29 nmol/liter. In a bath solution with a low extracellular Ca2+ concentration the ATP-induced [Ca2+]i peak was still present, but the [Ca2+]i plateau was completely prevented. In 186 experiments with the patch clamp technique the addition of ATP (1 to 100 mumol/liter) to GEC induced a transient small hyperpolarization, which was followed by a depolarization. During the ATP-induced depolarization an increase of the whole cell conductance was found. The Ca2+ ionophore A23187 (10 mumol/liter) mimicked the effect of ATP on Vm. Reduction of the extracellular Ca2+ to 1 mumol/liter itself depolarized GEC reversibly from -88 +/- 2 to -60 +/- 12 mV and increased the ATP-induced depolarization to -18 +/- 3 mV. In the absence of Na+ in the bathing solution (replacement by NMDG+) ATP induced only an attenuated depolarization and no inward current was activated. Flufenamate (100 mumol/liter), a blocker of non-selective ion channels inhibited the ATP-induced depolarization of Vm significantly by 58 +/- 13%, whereas nicardipine (10 mumol/liter) or amiloride (10 mumol/liter) had no effect. Our data indicate that the resting Vm of GEC cells is almost completely dominated by K+ conductances and that ATP activates a Ca2+ dependent non-selective ion conductance in GEC.

UTP and ATP induce different membrane voltage responses in rat mesangial cells.

UTP and ATP induce different membrane voltage responses in rat mesangial cells. Recent studies have indicated that UTP and ATP might modulate mesangial cell function in a different manner. Here we compared the effect of UTP and ATP on membrane voltage (Vm) and ion currents in mesangial cells in primary culture, and we examined whether different nucleotide receptors are involved. In patch-clamp experiments in the fast whole cell configuration, UTP (in contrast to ATP) caused a sustained and concentration-dependent depolarization (half-maximal effective dose, 10(-5) M), but ATP caused only a transient depolarization. During the depolarization, UTP induced a sustained increase of the whole cell conductance (Gm), whereas ATP induced only a transient increase of Gm. When cells were dialyzed with Cs2SO4 and extracellular Cl- was replaced by 145 mM sodium gluconate, addition of UTP or ATP (both 10(-4) M) did not significantly increase Gm. Addition of ATP in the presence of UTP caused an additional depolarization by 5 mV, which was followed by a hyperpolarization by 21 mV. Repetitive application of ATP led to an attenuation of the ATP-induced depolarization. Then, in the presence of ATP, UTP still induced a significant depolarization by 10 mV. Suramine and reactive blue 2 did not inhibit the depolarization induced by UTP, but these inhibited the Vm response to ATP. In microfluorescence experiments, UTP and ATP caused a concentration-dependent increase of the intracellular calcium activity ([Ca2+]i) in mesangial cells. Application of both UTP and ATP had no additive effect on [Ca2+]i. The results suggest that mesangial cells possess, in addition to P2y purinoceptors, separate nucleotide receptors for UTP.

Attenuation of stimulated Ca2+ influx in colonic epithelial (HT29) cells by cAMP.

In HT29 colonic epithelial cells agonists such as carbachol (CCH) or ATP increase cytosolic Ca2+ activity ([Ca2+]i) in a biphasic manner. The first phase is caused by inositol 1,4,5-trisphophate-(Ins P3-) mediated Ca2+ release from their respective stores and the second plateau phase is mainly due to stimulated transmembraneous Ca2+ influx. The present study was undertaken to examine the effect of increased adenosine 3',5'-cyclic monophosphate (cAMP) (forskolin 10 micromol/l = FOR) on the Ca2+ transient in the presence of CCH (100 micromol/l). In unpaired experiments it was found that FOR induced a depolarization and reduced cytosolic Ca2+ ([Ca2+]i, measured as the fura-2 fluorescence ratio 340/380 nm) significantly. Dideoxyforskolin had no such effect. The effect of FOR was abolished when the cells were depolarized by a high-K+ solution. In further paired experiments utilizing video imaging in conjunction with whole-cell patch-clamp, [Ca2+]i was monitored separately for the patch-clamped cell and three to seven neighbouring cells. In the presence of CCH, FOR reduced [Ca2+]i uniformly from a fluorescence ratio (345/380) of 2.9 +/- 0.12 to 1.8 +/- 0.07 in the patch-clamped cell and its neighbours (n = 48) and depolarized the membrane voltage (Vm) of the patch-clamped cells significantly and reversibly from -54 +/- 7.4 to -27 +/- 5.9 mV (n = 6). In additional experiments Vm was depolarized by 15-54 mV by various increments in the bath K+ concentration. This led to corresponding reductions in [Ca2+]i. Irrespective of the cause of depolarization (high K+ or FOR) there was a significant correlation between the change in Vm and change in [Ca2+]i. These data indicate that the cAMP-mediated attenuation of Ca2+ influx is caused by the depolarization produced by this second messenger.

Ca2+ influx induced by store release and cytosolic Ca2+ chelation in Ht29 colonic carcinoma cells.

Cl- secretion in HT29 cells is regulated by agonists such as carbachol, neurotensin and adenosine 5'-triphosphate (ATP). These agonists induce Ca2+ store release as well as Ca2+ influx from the extracellular space. The increase in cytosolic Ca2+ enhances the Cl- and K+ conductances of these cells. Removal of extracellular Ca2+ strongly attenuates the secretory response to the above-mentioned agonists. The present study utilises patch-clamp methods to characterise the Ca2+ influx pathway. Inhibitors which have been shown previously to inhibit non-selective cation channels, such as flufenamate (0.1 mmol.l-1, n = 6) and Gd3+ (10 micromol.l-1, n=6) inhibited ATP (0.1 mmol.l-1) induced increases in whole-cell conductance (Gm). When Cl- and K+ currents were inhibited by the presence of Cs2SO4 in the patch pipette and gluconate in the bath, ATP (0.1 mmol.l-1) still induced a significant increase in Gm from 1.2 +/- 0.3 nS to 4.7 +/- 1 nS (n = 24). This suggests that ATP induces a cation influx with a conductance of approximately 3-4 nS. This cation influx was inhibited by flufenamate (0.1 mmol.l-1, n = 6) and Gd3+ (10 micromol.l-1, n = 9). When Ba2+ (5 mmol.l-1) and 4,4'-diisothiocyanato-stilbene-2-2'-disulphonic acid (DIDS, 0.1 mmol.l-1) were added to the KCl/K-gluconate pipette solution to inhibit K+ and Cl- currents and the cells were clamped to depolarised voltages, ATP (0.1 mmol.l-1) reduced the membrane current (Im) significantly from 86 +/- 14 pA to 54 +/- 11 pA (n = 13), unmasking a cation inward current. In another series, the cation inward current was activated by dialysing the cell with a KCl/K-gluconate solution containing 5-10 mmol.l-1 1,2-bis-(2-aminoethoxy)ethane-N,N,N',N'-tetraacetic acid (EGTA) or 1,2-bis-(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA). The zero-current membrane voltage (Vm) and Im (at a clamp voltage of +10 mV) were monitored as a function of time. A new steady-state was reached 30-120 s after membrane rupture. Vm depolarised significantly from -33 +/- 2 mV to -12 +/- 1 mV, and Im fell significantly from 17 +/- 2 pA to 8.9 +/- 1.0 pA (n = 71). This negative current, representing a cation inward current, was activated when Ca2+ stores were emptied and was reduced significantly ( Im) when Ca2+ and/or Na+ were removed from the bathing solution: removal of Ca2+ in the absence of Na+ caused a Im of 5.0 +/- 1.2 pA (n = 12); removal of Na+ in the absence of Ca2+ caused a Im of 12.8 +/- 3.5 pA (n = 4). The cation inward current was also reduced significantly by La3+, Gd3+, and flufenamate. We conclude that store depletion induces a Ca2+/Na+ influx current in these cells. With 145 mmol.l-1 Na+ and 1 mmol.l-1 Ca2+, both ions contribute to this cation inward current. This current is an important component in the agonist-regulated secretory response.

pH regulation in HT29 colon carcinoma cells.

The pH regulation in HT29 colon carcinoma cells has been investigated using the pH-sensitive fluorescent indicator 2',7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). Under control conditions, intracellular pH (pHi) was 7.21 +/- 0.07 (n = 22) in HCO3(-)-containing and 7.21 +/- 0.09 (n = 12) in HCO3(-)-free solution. HOE-694 (10 mumol/l), a potent inhibitor of the Na+/H+ exchanger, did not affect control pHi. As a means to acidify cells we used the NH4+/NH3 (20 mmol/l) prepulse technique. The mean peak acidification was 0.37 +/- 0.07 pH units (n = 6). In HCO3(-)-free solutions recovery from acid load was completely blocked by HOE-694 (1 mumol/l), whereas in HCO3(-)-containing solutions a combination of HOE-694 and 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS, 0.5 mmol/l) was necessary to show the same effect. Recovery from acid load was Na(+)-dependent in HCO3(-)-containing and HCO3(-)-free solutions. Removal of external Cl- caused a rapid, DIDS-blockable alkalinization of 0.33 +/- 0.03 pH units (n = 15) and of 0.20 +/- 0.006 pH units (n = 5), when external Na+ was removed together with Cl-. This alkalinization was faster in HCO3(-)-containing than in HCO3(-)-free solutions. The present observations demonstrate three distinct mechanisms of pHi regulation in HT29 cells: (a) a Na+/H+ exchanger, (b) a HCO3-/Cl- exchanger and (c) a Na(+)-dependent HCO3- transporter, probably the Na(+)-HCO3-/Cl- antiporter.(ABSTRACT TRUNCATED AT 250 WORDS)

Voltage-dependent Ca2+ influx in the epithelial cell line HT29: simultaneous use of intracellular Ca2+ measurements and nystatin perforated patch-clamp technique.

Indirect evidence has accumulated indicating a voltage dependence of the agonist-stimulated Ca2+ influx into epithelial cells. Manoeuvres expected to depolarise the membrane voltage during agonist stimulation resulted in: (1) a decrease of the sustained phase of the adenosine triphosphate (ATP, 10(-5) mol/l)-induced intracellular Ca2+ transient, (2) a reduced fura-2 Mn(2+)-quenching rate, and (3) prevention of the refilling of the agonist-sensitive store. To quantify the change in intracellular Ca2+ as a function of membrane voltage, we measured simultaneously the intracellular Ca2+ activity ([Ca2+]i) with fura-2 and the electrical properties using the nystatin perforated patch-clamp technique in single HT29 cells. Ca2+ influx was either stimulated by ATP (10(-5) mol/l) or thapsigargin (TG, 10(-8) mol/l). After [Ca2+]i reached the sustained plateau phase we clamped the membrane voltage in steps of 10 mV in either direction. A stepwise depolarisation resulted in a stepwise reduction of [Ca2+]i. Similarly a stepwise hyperpolarisation resulted in a stepwise increase of [Ca2+]i (ATP: 27.5 +/- 10 nmol/l per 10 mV, n = 6; TG: 19 +/- 7.9 nmol/l per 10 mV, n = 12). The summarised data show a linear relationship between the delta fluorescence ratio 340/380 nm change and the applied holding voltage. In unstimulated cells the same voltage-clamp protocol did not change [Ca2+]i (n = 9). Under extracellular Ca(2+)-free conditions [Ca2+]i remained unaltered when changing the membrane voltage. These data provide direct evidence that the Ca2+ influx in epithelial cells is membrane voltage dependent. Our data indicate that small changes in membrane voltage lead to substantial changes in [Ca2+]i.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for agonist-induced export of intracellular Ca2+ in epithelial cells.

There is increasing evidence that some agonists not only induce intracellular Ca2+ increases, due to store release and transmembranous influx, but also that they stimulate Ca2+ efflux. We have investigated the agonist-stimulated response on the intracellular Ca2+ activity ([Ca2+]i) in the presence of thapsigargin (10(-8) mol/l, TG) in HT29 and CFPAC-1 cells. For CFPAC-1 the agonists ATP (10(-7)-10(-3) mol/l, n = 9), carbachol (10(-6)-10(-3) mol/l, n = 5) and neurotensin (10(-10)-10(-7) mol/l, n = 6) all induced a concentration-dependent decrease in [Ca2+]i in the presence of TG. Similar results were obtained with HT29 cells. This decrease of [Ca2+]i could be caused by a reduced Ca2+ influx, either due to a reduced driving force for Ca2+ in the presence of depolarizing agonists or due to agonist-regulated decrease in Ca2+ permeability. Using the fura-2 Mn2+ quenching technique we demonstrated that ATP did not slow the TG-induced Mn2+ quench. This indicates that the agonist-induced [Ca2+]i decrease in the presence of TG was not due to a reduced influx of Ca2+ into the cell, but rather due to stimulation of Ca2+ export. We used the cell attached nystatin patch clamp technique in CFPAC-1 cells to examine whether, in the presence of TG, the above agonists still led to the previously described electrical changes. The cells had a mean membrane voltage of -49 +/- 3.6 mV (n = 9). Within the first 3 min ATP was still able to induce a depolarization which could be attributed to an increase in Cl- conductance.(ABSTRACT TRUNCATED AT 250 WORDS)