Immunological therapy of sepsis: experimental therapies.
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Anticoagulants↗
Biomedical subjects
P Arndt
Publications and source records attributed to P Arndt.
Interaction of cations, anions, and weak base quinine with rat renal cation transporter rOCT2 compared with rOCT1.
The rat organic cation transporter (rOCT)-2 was characterized by electrical and tracer flux measurements compared with rOCT1. By applying choline gradients to voltage-clamped Xenopus oocytes expressing rOCT2, potential-dependent currents could be induced in both directions. Tracer flux measurements with seven organic cations revealed similar Michaelis-Menten constant values for both transporters, with the exception of guanidine. In parallel experiments with rOCT2 and rOCT1, inhibition of tetraethylammonium transport by 12 cations, 2 weak bases, corticosterone, and the anions para-amminohippurate, alpha-ketoglutarate, and probenecid was characterized. The IC(50) values of many inhibitors were similar for both transporters, whereas others were significantly different. Mepiperphenidol and O-methylisoprenaline showed an approximately 70-fold lower and corticosterone a 38-fold higher affinity for rOCT2. With the use of these inhibitors together with previous information on cation transporters, experimental protocols are proposed to dissect out the individual contributions of rOCT2 and rOCT1 in intact proximal tubule preparations. Inhibition experiments at different pH levels strongly suggest that the weak base quinine passively permeates the plasma membrane at physiological pH and inhibits rOCT2 from the intracellular side.
Animals↗
A two-stage model for visual-auditory interaction in saccadic latencies.
In two experiments, saccadic response time (SRT) for eye movements toward visual target stimuli at different horizontal positions was measured under simultaneous or near-simultaneous presentation of an auditory nontarget (distractor). The horizontal position of the auditory signal was varied, using a virtual auditory environment setup. Mean SRT to a visual target increased with distance to the auditory nontarget and with delay of the onset of the auditory signal relative to the onset of the visual stimulus. A stochastic model is presented that distinguishes a peripheral processing stage with separate parallel activation by visual and auditory information from a central processing stage at which intersensory integration takes place. Two model versions differing with respect to the role of the auditory distractors are tested against the SRT data.
Adult↗
The transport modifier RS1 is localized at the inner side of the plasma membrane and changes membrane capacitance.
Previously we cloned membrane associated (M(r) 62000-67000) polypeptides from pig (pRS1), rabbit (rbRS1) and man (hRS1) which modified transport activities that were expressed in Xenopus laevis oocytes by the Na(+)-D-glucose cotransporter SGLT1 and/or the organic cation transporter OCT2. These effects were dependent on the species of RS1 and on the target transporters. hRS1 and rbRS1 were shown to be intronless single copy genes which are expressed in various tissues and cell types. Earlier immunohistochemical data with a monoclonal IgM antibody suggested an extracellular membrane association of RS1. In the present paper antibodies against recombinant pRS1 were raised and the distribution and membrane localization of RS1 reevaluated. After subcellular fractionation of renal cortex RS1 was found associated with brush border membranes and an about 1:200 relation between RS1 and SGLT1 protein was estimated. Also after overexpression in X. laevis oocytes RS1 was associated with the plasma membrane, however, at variance to the kidney it was also observed in the cytosol. Labeling experiments with covalently binding lipid-permeable and lipid-impermeable biotin analogues showed that RS1 is localized at the inner side of the plasma membrane. Western blots with plasma membranes from Xenopus oocytes revealed that SGLT1 protein in the plasma membrane was reduced when hRS1 was coexpressed with human SGLT1 which leads to a reduction in V(max) of expressed glucose transport. Measurements of membrane capacitance and electron microscopic inspection showed that the expression of hRS1 leads to a reduction of the oocyte plasma membrane surface. The data suggest that RS1 is an intracellular regulatory protein that associates with the plasma membrane. Overexpression of RS1 may effect the incorporation and/or retrieval of transporters into the plasma membrane.
Animals↗
Polyspecific cation transporters in the proximal tubule.
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Animals↗
Multichannel auditory brainstem implant: US clinical trial results.
Since 1994, a US Food and Drug Administration clinical trial evaluated the multichannel auditory brainstem implant (ABI) on 92 subjects with neurofibromatosis type 2 (NF2). The trial has shown that 85 per cent of patients receive auditory sensations. A small number of patients demonstrate a clinically significant degree of open-set sentence recognition in the sound-alone condition; however, when the ABI is combined with lip-reading cues, 93 per cent of patients demonstrate improved sentence understanding at three to six months. In addition, the majority of recipients report daily use of their devices, and satisfaction with the decision to receive the ABI.
Adolescent↗
Molecular pharmacology of organic cation transporters in kidney.
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Amino Acid Sequence↗
A chemiluminescence test for predicting the outcome of transfusing incompatible blood.
A chemiluminescent test (CLT) which measures the metabolic response of human monocytes to sensitized red cells was developed to distinguish antibodies capable of causing the increased destruction of transfused incompatible red cells from antibodies which are clinically benign. Thirty sera containing IgG antibodies to high-frequency antigens were tested; 27 of these sera were also tested using the monocyte monolayer assay (MMA). The clinical significance of antibodies in 14 of the sera was known: three (anti-Ata (two), -JMH) caused accelerated clearance of 51Cr-labelled cells, five (anti-'MiIII', -Yta, three unidentified) caused haemolytic transfusion reactions and six (anti-Yta, -Ge, -JMH, -Xga, -Kna (two)) did not appear to affect red cell survival. Overall, results from the MMA and CLT showed good agreement; seven sera were negative in both assays, 18 sera were positive in both assays and two sera were positive in the MMA but negative in the CLT. There was no clear relationship between the activity of different antibodies and the level of sensitization as determined by flow cytometry. Antibody activity could be either increased or decreased by incubation of sensitized red cells with fresh serum. MMA results were in concordance with the clinical significance of antibodies where known in eight of 10 cases. CLT results were in concordance with clinical significance in 12 of 14 cases. Both assays gave false-positive results with serum from a patient with anti-Kna who had received red cell transfusions without adverse effect. This appeared to be due to the ability of anti-Kna to cross-link complement receptor 1 (CR1) on red cells to CR1 on monocytes; negative results were obtained using autologous monocytes.
Blood Group Antigens↗
Selectivity of the polyspecific cation transporter rOCT1 is changed by mutation of aspartate 475 to glutamate.
After site-directed mutagenesis, the organic cation transporter rOCT1 was expressed in Xenopus laevis oocytes or human embryonic kidney cells and functionally characterized. rOCT1 belongs to a new family of polyspecific transporters that includes transporters for organic cations and anions and the Na(+)-carnitine cotransporter. When glutamate was substituted for Asp475 (middle of the proposed 11th transmembrane alpha-helix), the V(max) values for choline, tetraethylammonium (TEA), N(1)-methylnicotinamide, and 1-methyl-4-phenylpyridinium were reduced by 89 to 98%. The apparent K(m) values were also decreased (choline by 15-fold, TEA by 8-fold, N(1)-methylnicotinamide by 4-fold) or remained constant (1-methyl-4-phenylpyridinium). After the mutation, the membrane potential dependence of the K(m) value for [(3)H]choline uptake was abolished. The affinity of n-tetraalkyl ammonium compounds to inhibit TEA uptake was increased. This affinity and its increase by the D475E mutation were increased with the length of the n-alkyl chains. After expression in X. laevis oocytes, the IC(50) ratios of wild-type and D475E mutant were 1.7 (tetramethylammonium), 4.3 (TEA), 5.0 (tetrapropylammonium), 5.0 (tetrabutylammonium), and 65 (tetrapentylammonium). Cationic inhibitors with ring structures were differentially affected: the IC(50) value for TEA inhibition by cyanine 863 remained unchanged, whereas it was increased for quinine. The data suggest that rOCT1 contains a large cation-binding pocket with several interaction domains that may be responsible for high-affinity binding of structurally different cations and that Asp475 is located close to one of these interaction domains.
Amino Acid Substitution↗
Mycoplasma pneumoniae-associated bronchiolitis causing severe restrictive lung disease in adults: report of three cases and literature review.
STUDY OBJECTIVES: To characterize adult Mycoplasma pneumoniae-induced bronchiolitis requiring hospitalization. DESIGN: We encountered an adult patient with severe bronchiolitis in the absence of pneumonia due to M. pneumoniae. To determine the relative frequency of such a condition, we retrospectively reviewed the medical records of adults over a 4-year period with a hospital discharge diagnosis of "bronchiolitis" from a university hospital. SETTING: University Hospital of the University of Colorado Health Sciences Center, Denver, CO. STUDY SUBJECTS: From 1994 to 1998, 10 adult inpatients were identified with a diagnosis of bronchiolitis. There were two with respiratory bronchiolitis, one with panbronchiolitis, one patient with bronchiolitis obliterans organizing pneumonia (BOOP), and six with acute inflammatory bronchiolitis. Including the initial patient, three had a definitive clinical diagnosis of Mycoplasma-associated bronchiolitis. RESULTS: The three adult patients with bronchiolitis due to M. pneumoniae are unusual because they occurred in the absence of radiographic features of a lobar or patchy alveolar pneumonia. Hospital admission was occasioned by the severity of symptoms and gas exchange abnormalities. One patient had bronchiolitis as well as organizing pneumonia (BOOP) that responded favorably to corticosteroid treatment. The other two had high-resolution CT findings diagnostic of an acute inflammatory bronchiolitis. One of the patients with inflammatory bronchiolitis had an unusual pattern of marked ventilation and perfusion defects localized predominantly to the left lung. All three had restrictive ventilatory impairment on physiologic testing. CONCLUSIONS: In adults, Mycoplasma-associated bronchiolitis without pneumonia is rarely reported, but in hospitalized patients, it may be more common than expected and may be associated with severe physiologic disturbances.
Adult↗
Testicular abnormalities in male rats after lactational exposure to nonylphenols.
Lactational exposure of male rat pups to nonylphenols (NPs) decreased the size of their testes and male accessory glands. At 31 d of age, NP-treatment of male rats resulted in less cellular differentiation of the seminiferous tubules (STs) and increased intertubular space compared to controls. At maturity, NP-treated males showed varying degrees of abnormalities in the affected testes. In the moderately affected ones, about 20-30% of their STs had poorly differentiated germinal elements. Cell lineage was less organized. In extreme cases, all STs of the affected testis failed to differentiate into germinal elements. These abnormalities in germinal element differentiation might be the primary cause for a number of the NP-treated males having a lower epididymal sperm count and a lower percentage of motile sperm compared to age-matched control males. Zymogram analysis of testis homogenates by sodium dodecyl sulfate gelatin gels revealed two major forms (64-66 kDa and 50-52 kDa) of gelatinases. Only the 50-52-kDa form was greatly reduced or absent in the affected testis. Lactational exposure of male pups to NPs thus leads to various testicular abnormalities including lack of differentiation of STs, lowering of sperm count, and reduction in the percentage of motile sperm and modulation of a specific form of testicular proteinases.
Animals↗
Membrane localization of the electrogenic cation transporter rOCT1 in rat liver.
The polyspecific cation transporter rOCT1 in the rat was the first identified member of a new protein family with 12 presumed membrane-spanning alpha-helices and two large hydrophilic loops. Previous studies showed that rOCT1 is mainly expressed in liver and mediates electrogenic uptake of small organic cations into cells. Antibodies against partial sequences of rOCT1 were raised and their specificity was verified. Immunohistochemistry with rat liver and Western blots with isolated membranes showed that rOCT1 is localized within sinusoidal membranes of hepatocytes. Antibody reactions were also performed with intact and permeabilized human embryonic kidney cells that were stably transfected with rOCT1. They showed that the large hydrophilic loop after the first alpha-helix of rOCT1 is located extracellularly, while the C-terminus is located intracellularly. Translational regulation is suggested since the message of rOCT1 was distributed throughout the liver lobuli, whereas rOCT1 protein was observed only in hepatocytes surrounding the central veins.
Animals↗
Human neurons express the polyspecific cation transporter hOCT2, which translocates monoamine neurotransmitters, amantadine, and memantine.
Recently, we cloned the human cation transporter hOCT2, a member of a new family of polyspecific transporters from kidney, and demonstrated electrogenic uptake of tetraethylammonium, choline, N1-methylnicotinamide, and 1-methyl-4-phenylpyridinium. Using polymerase chain reaction amplification, cDNA sequencing, in situ hybridization, and immunohistochemistry, we now show that hOCT2 message and protein are expressed in neurons of the cerebral cortex and in various subcortical nuclei. In Xenopus laevis oocytes expressing hOCT2, electrogenic transport of norepinephrine, histamine, dopamine, serotonin, and the antiparkinsonian drugs memantine and amantadine was demonstrated by tracer influx, tracer efflux, electrical measurements, or a combination. Apparent Km values of 1.9 +/- 0.6 mM (norepinephrine), 1.3 +/- 0.3 mM (histamine), 0.39 +/- 0.16 mM (dopamine), 80 +/- 20 microM (serotonin), 34 +/- 5 microM (memantine), and 27 +/- 3 microM (amantadine) were estimated. Measurement of trans-effects in depolarized oocytes and human embryonic kidney cells expressing hOCT2 suggests that there were different rates and specificities for cation influx and efflux. The hypothesis is raised that hOCT2 plays a physiological role in the central nervous system by regulating interstitial concentrations of monoamine neurotransmitters that have evaded high affinity uptake mechanisms. We show that amantadine does not interact with the expressed human Na+/Cl- dopamine cotransporter. However, concentrations of amantadine that are effective for the treatment of Parkinson's disease may increase the interstitial concentrations of dopamine and other aminergic neurotransmitters by competitive inhibition of hOCT2.
Amantadine↗
Severe autoimmune hemolytic anemia associated with IgM warm autoantibodies directed against determinants on or associated with glycophorin A.
BACKGROUND AND OBJECTIVES: Autoimmune hemolytic anemiA (AIHA) is rarely caused by IgM warm autoantibodies, and is sometimes difficult to diagnose. We describe three patients with severe AIHA caused by IgM warm autoantibodies; in two of the three, the hemolysis was fatal. MATERIALS AND METHODS: Conventional hematologic and serologic procedures were used. RESULTS: The IgM autoantibodies in all three cases were directed against determinants on or associated with glycophorin A (Ena, Wrb, and Pr). The IgM autoantibodies and unusual serological characteristics, in that the agglutinins were detected or greatly enhanced only by the presence of albumin or a low pH, and/or the agglutinins reacted optimally at 20-30 degrees C. CONCLUSIONS: IgM warm autoantibodies directed at determinants on or associated with glycophorin A appear to cause severe (often fatal) hemolytic anemia.
Aged↗
Clinical significance of an anti-Dib assessed by flow cytometry.
Although antibodies to the Dib antigen are generally considered to be of potential clinical significance, we know of no reports assessing the clinical significance of anti-Dib (in vivo or in vitro). We report on an 88-year-old Japanese male gastrectomy patient who had alloanti-Dib. After transfusion of two Di(b-) units, three Di(b+) units had to be transfused, and there were no clinical signs of acute hemolysis. Di(b+) RBC survival was followed retrospectively by flow cytometry. On days 1, 7, and 10, the percent of circulating Di(b+) RBCs was determined to be 39, 30, and 11 percent, respectively, compared to an expected 49, 43, and 41 percent based on calculations. The Di(b+) RBCs appear to have been tolerated for about 6 days, then were removed from the circulation. Direct anti-IgG tests were 1-2+ mixed field with all posttransfusion samples. Monocyte monolayer assays (MMAs), which have been reported to predict the clinical significance of alloantibodies, gave borderline positive results. MMA results using sera from days 0, 3, and 9 were 2.7 and 5.5, 0.8 and 4.8, and 3.0 and 3.7 percent, respectively, without and with added fresh normal serum as a source of complement (clinical significance = > 3% reactivity). The subclass of the anti-Dib was IgG1. This is the first documentation of the clinical significance of an anti-Dib.
Journal Article↗
Electrogenic properties and substrate specificity of the polyspecific rat cation transporter rOCT1.
The previously cloned rat cation transporter rOCT1 detected in renal proximal tubules and hepatocytes (Gründemann, D., Gorboulev, V., Gambaryan, S., Veyhl, M., and Koepsell, H. (1994) Nature 372, 549-552) was expressed in Xenopus oocytes, and transport properties were analyzed using tracer uptake studies and electrophysiological measurements. rOCT1 induced highly active transport of a variety of cations, including the classical substrates for cation transport, such as N-1-methylnicotinamide, 1-methyl-4-phenylpyridinium (MPP), and tetraethylammonium (TEA), but also the physiologically important choline. In oocytes rOCT1 also mediated efflux of MPP, which could be trans-stimulated by MPP and TEA. Cation transport via rOCT1 was electrogenic. In voltage-clamped oocytes, transport of TEA and choline via rOCT1 produced inwardly directed currents, which were independent of extracellular ion composition or pH. The choline- and TEA-induced currents were voltage-dependent at nonsaturating concentrations, and the apparent affinity of these cations was decreased at depolarized voltages. Other substrates transported by rOCT1 were the polyamines spermine and spermidine. Interestingly, the previously described potent inhibitors of rOCT1, cyanine 863, quinine, and D-tubocurarine were substrates themselves. The data indicate that rOCT1 is an effective transport system that is responsible for electrogenic uptake of a wide variety of organic cations into epithelial cells of renal proximal tubules and hepatocytes.
Animals↗
Fatal hemolytic transfusion reaction resulting from ABO mistyping of a patient with acquired B antigen detectable only by some monoclonal anti-B reagents.
BACKGROUND: Some monoclonal anti-B reagents are prepared exclusively from an anti-B clone, ES4, that is known to detect acquired B antigens that are not detectable by other anti-B clones or polyclonal anti-B reagents. CASE REPORT: A 92-year-old group A, Rh-negative man with diverticulitis was mistyped as group AB with the use of a monoclonal anti-B. The hospital did not detect anti-B in the patient's serum. After a negative antibody screen, blood was issued through an abbreviated crossmatch (i.e., immediate-spin crossmatch). The patient was given 3 units of group AB blood and 1 unit of group A blood, and no problems were reported. After the transfusion of a ¿fourth unit of AB blood the patient had a severe hemolytic transfusion reaction which resulted in kidney failure and death 10 days later. After the transfusion reaction, the patient's pretransfusion red cells were found to be group A with an acquired B antigen. The monoclonal anti-B used the hospital was formulated from the ES4 clone. A sample of the patient's serum taken before the transfusion was later found to contain a weak anti-B, detectable most obviously by the antiglobulin test, which was not performed at the crossmatch stage. The manufacturers of monoclonal anti-B reagents prepared from ES4 have since modified their reagents (i.e., lowered the pH) so that they now detect only the strongest examples of acquired B antigen. CONCLUSION: A fatal hemolytic transfusion reaction resulted because a monoclonal anti-B that detected acquired B antigen was used to type red cells from an elderly man whose serum had weak anti-B that was not detected by abbreviated compatibility testing.
ABO Blood-Group System↗
Applications of flow cytofluorometry to transfusion science.
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Blood Transfusion↗