Chronic granulomatous disease. Diagnosis by the dihydrorhodamine assay. / Enfermedad granulomatosa crónica. Diagnóstico mediante el ensayo de dihidrorodamina.

INTRODUCTION: Chronic granulomatous disease (CGD) is characterized by an alteration of the neutrophil oxidative function. Its inheritance patterns are linked to the X chromosome (X-linked CGD) and autosomal recessive (AR CGD). The dihydrorhodamine (DHR) assay is used for the diagnosis and detection of carriers and provides information on inheritance patterns. OBJECTIVE: To detect CGD cases in chil dren with recurrent infections and to evaluate their female relatives through the DHR assay to iden tify carriers and obtain information about possible inheritance patterns. PATIENTS AND METHOD: 107 patients (<18 years of age) with clinical suspicion of CGD such as pneumonia, lymphadenopathies, and abscesses were included, referred by physicians from public hospitals between 2014 and 2017. Six female relatives of children with CGD were also included. The DHR assay was performed on all patient samples and the results were expressed as neutrophils stimulation index (SI). RESULTS: The median age of patients was 3 years and 62/107 of them were male. The average SI was 39.7±13.8 and a complete shift of DHR was found in 101/107 children. In 2/107 children, no DHR shift was observed (SI=1.0) indicating possible X-linked CGD, and a third child showed a slight DHR shift (SI=4.8) compatible with AR CGD. 5/6 female relatives presented a bimodal pattern, showing a carrier status. CONCLUSIONS: Three cases of CGD and five female carriers were detected through the DHR assay, being the first time that this technique was used in Paraguay. Information on the most likely inheri tance patterns, two X-linked CGD, and one AR CGD case was also obtained.

Enfermedad granulomatosa crónica: diagnóstico mediante el ensayo de dihidrorodamina / Chronic granulomatous disease: diagnosis by the dihydrorhodamine assay

Resumen: Introducción: La enfermedad granulomatosa crónica (EGC) se caracteriza por una alteración de la función oxidativa de neutrófilos, presentando herencia ligada al cromosoma X (EGC LX) y autosómica recesiva (EGC AR). El ensayo de dihidrorodamina (DHR) es utilizado para el diagnóstico y detección de portadoras, además proporciona información sobre patrones de herencia. Objetivo: Detectar casos de EGC en niños con infecciones recurrentes y evaluar a sus familiares femeninos mediante el ensayo de DHR, para identificar portadoras y obtener información acerca de posibles patrones de herencia. Pacientes y Método: Fueron incluidos 107 pacientes (<18 años de edad) con sospecha clínica de EGC como neumonías, linfadenopatías y abscesos, remitidos por médicos de hospitales públicos, del 2014 al 2017. Además, se incluyeron seis mujeres, familiares de los niños con EGC. A las muestras de los pacientes se aplicó el ensayo DHR, expresando los resultados como índice de estimulación de neutrófilos (IE). Resultados: La mediana de edad de los pacientes fue de 3 años y 62/107 fueron varones. El IE promedio fue 39,7 ± 13,8 y 101/107 niños exhibieron un cambio completo de fluorescencia de DHR. En 2/107 niños no se observó dicho cambio (IE = 1,0), lo cual indica posible EGC LX, y un tercer niño mostró un leve cambio (IE = 4,8), compatible con EGC AR. En 5/6 mujeres se encontró un patrón bimodal, indicando un estado de portadora. Conclusiones: Fueron detectados tres casos de EGC y cinco portadoras mediante el ensayo de DHR, realizado por primera vez en Paraguay. También se obtuvo información sobre los posibles patrones de herencia, EGC LX en dos familias y un caso probable de EGC AR.

Abstract: Introduction: Chronic granulomatous disease (CGD) is characterized by an alteration of the neutrophil oxidative function. Its inheritance patterns are linked to the X chromosome (X-linked CGD) and autosomal recessive (AR CGD). The dihydrorhodamine (DHR) assay is used for the diagnosis and detection of carriers and provides information on inheritance patterns. Objective: To detect CGD cases in chil dren with recurrent infections and to evaluate their female relatives through the DHR assay to iden tify carriers and obtain information about possible inheritance patterns. Patients and Method: 107 patients (<18 years of age) with clinical suspicion of CGD such as pneumonia, lymphadenopathies, and abscesses were included, referred by physicians from public hospitals between 2014 and 2017. Six female relatives of children with CGD were also included. The DHR assay was performed on all patient samples and the results were expressed as neutrophils stimulation index (SI). Results: The median age of patients was 3 years and 62/107 of them were male. The average SI was 39.7±13.8 and a complete shift of DHR was found in 101/107 children. In 2/107 children, no DHR shift was observed (SI=1.0) indicating possible X-linked CGD, and a third child showed a slight DHR shift (SI=4.8) compatible with AR CGD. 5/6 female relatives presented a bimodal pattern, showing a carrier status. Conclusions: Three cases of CGD and five female carriers were detected through the DHR assay, being the first time that this technique was used in Paraguay. Information on the most likely inheri tance patterns, two X-linked CGD, and one AR CGD case was also obtained.

Plasmonic Paper Microneedle Patch for On-Patch Detection of Molecules in Dermal Interstitial Fluid.

Minimally invasive devices to detect molecules in dermal interstitial fluid (ISF) are desirable for point-of-care diagnostic and monitoring applications. In this study, we developed a microneedle (MN) patch that collects ISF for on-patch biomarker analysis by surface-enhanced Raman scattering (SERS). The micrometer-scale MNs create micropores in the skin surface, through which microliter quantities of ISF are collected onto plasmonic paper on the patch backing. The plasmonic paper was prepared by immobilizing poly(styrenesulfonate) (PSS) coated gold nanorods (AuNRs) on a thin strip of filter paper using plasmonic calligraphy. Negatively charged PSS was used to bind positively charged rhodamine 6G (R6G), which served as a model compound, and thereby localize R6G on AuNR surface. R6G bound on the AuNR surface was detected and quantified by acquiring SERS spectra from the plasmonic paper MN patch. This approach was used to measure pharmacokinetic profiles of R6G in ISF and serum from rats in vivo. This proof-of-concept study indicates that a plasmonic paper MN patch has the potential to enable on-patch measurement of molecules in ISF for research and future medical applications.

An upconversion nanocomposite for fluorescence resonance energy transfer based cholesterol-sensing in human serum.

Upconversion nanophosphors (UCNPs) are extremely useful for analytical applications, since they display a high signal-to-noise ratio, and their photobleaching can be ignored. Herein, a novel upconversion nanocomposite composed of ß-cyclodextrin (ß-CD) derivative modified UCNPs and rhodamine B (RB) was prepared for the detection of cholesterol (Cho). The upconversion luminescence (UCL) emission can serve as a Cho-sensing signal by an effective fluorescence resonance energy transfer (FRET) process, using UCNPs as the donor and RB as the quencher. The sensor for Cho detection in human serum shows excellent sensitivity and selectivity, which has the potential for clinical applications in the analysis of other biological and environmental samples.

Attenuation of responses to endotoxin by the triggering receptor expressed on myeloid cells-1 inhibitor LR12 in nonhuman primate.

BACKGROUND: The triggering receptor expressed on myeloid cells-1 is an immunoreceptor that amplifies the inflammatory response mediated by toll-like receptors engagement. Triggering receptor expressed on myeloid cells-1 inhibitory peptides such LR12 have been shown to prevent hyperresponsiveness and death in several experimental models of septic shock. METHODS: Twelve adult male Cynomolgus (Macaca fascicularis) monkeys exposed to an intravenous bolus of endotoxin (10 µg/kg) were randomized to receive LR12 or placebo (n = 6 per group) as an initial intravenous bolus followed by an 8-h continuous intravenous infusion. An additional group of four only received vehicle infusion. Vital signs were monitored for 8 h. Blood was sampled at H0, 1, 2, 4, and 8 for analysis of clinical chemistries, leukocyte count, coagulation parameters, and cytokine plasma concentration. RESULTS: LR12 showed no effect on heart rate and body temperature. By contrast to the placebo group, which experienced a 25 to 40% blood pressure decrease after endotoxin administration, LR12-treated monkeys remained normotensive. Endotoxin induced leukopenia at 2 h (mean leukocyte count, 7.62 g/l vs. 21.1 at H0), which was attenuated by LR12. LR12 also attenuated cytokine production. CONCLUSIONS: The triggering receptor expressed on myeloid cells-1 inhibitor LR12 is able to mitigate endotoxin-associated clinical and biological alterations, with no obvious side effects. This study paves the way for future phases Ia and Ib trials in humans.

Seeing citrulline: development of a phenylglyoxal-based probe to visualize protein citrullination.

Protein arginine deiminases (PADs) catalyze the hydrolysis of peptidyl arginine to form peptidyl citrulline. Abnormally high PAD activity is observed in a host of human diseases, but the exact role of protein citrullination in these diseases and the identities of specific citrullinated disease biomarkers remain unknown, largely because of the lack of readily available chemical probes to detect protein citrullination. For this reason, we developed a citrulline-specific chemical probe, rhodamine-phenylglyoxal (Rh-PG), which we show can be used to investigate protein citrullination. This methodology is superior to existing techniques because it possesses higher throughput and excellent sensitivity. Additionally, we demonstrate that this probe can be used to determine the kinetic parameters for a number of protein substrates, monitor drug efficacy, and identify disease biomarkers in an animal model of ulcerative colitis that displays aberrantly increased PAD activity.

Assessment of P-glycoprotein activity in the Blood-Brain Barrier (BBB) using Near Infrared Fluorescence (NIRF) imaging techniques.

PURPOSE: To examine functional activity of P-glycoprotein (P-gp) in the blood-brain barrier (BBB) using near infrared fluorescence (NIRF) imaging techniques. METHODS: Cellular accumulation and bi-directional permeability of the NIRF probe, rhodamine 800 (R800) was determined in MDCKMDR1 and MDCKwt monolayers under normal conditions and following P-gp inhibition with GF120918. Functional P-gp activity was also assessed in mice following administration of R800 alone and with GF230918. Quantitative analysis of R800 fluorescence in brain tissue and blood was measured ex-vivo using Odyssey Near Infrared imaging. RESULTS: R800 accumulation was reduced in MDCKMDR1 compared to MDCKwt monolayers. Addition of GF120918, resulted in increased R800 accumulation in MDCKMDR1 monolayers. Permeability of R800 in MDCKMDR1 monolayers was significantly enhanced (4-fold) in the basolateral to apical direction under control conditions and was abolished following treatment with GF120918. With the exception of the choriod plexus, there was very little penetration of R800 into the brain under control conditions. Treatment of mice with GF120918 resulted in a nearly 4-fold increase in R800 fluorescence in the brain. In contrast, GF120918 had no effect on brain penetration of a vascular permeability marker. CONCLUSIONS: In vitro studies demonstrate the P-gp transporter properties of the NIRF probe R800. Preliminary in vivo studies confirm the P-gp transporter liabilities of R800 and suggest this probe may be useful as a molecular imaging agent for examining P-gp activity in the BBB.

Peroxynitrite is an important mediator in thermal injury-induced lung damage.

OBJECTIVE: Intestinal ischemia and reperfusion injury was known to cause postinjury multiple organ failure by neutrophil and unclear nonneutrophil factors. Peroxynitrite formed by the rapid reaction between superoxide and nitric oxide, is a toxic substance that contributes to tissue injury in a number of biological systems. In this study, the role of nitric oxide and neutrophils on lung damage after burn was investigated. DESIGN: Prospective, experimental study. SETTING: Research laboratory at a university hospital. SUBJECTS: Thermal injury models in the rat. INTERVENTIONS: In experiment 1, specific pathogen-free Sprague-Dawley rats underwent 35% total body surface area burn. At 4, 8, 16, and 24 hrs after burn, intestinal mucosa and lung tissue were harvested for myeloperoxidase (MPO) assay, blood was collected for measurement of peroxynitrite-mediated oxidation of dihydrorhodamine 123, and pulmonary microvascular dysfunction was quantified by measuring the extravasation of Evans blue dye. In experiment 2, polymorphonuclear granulocyte antibody (0.12 mL/100 g administered intraperitoneally 16 hrs before burn), S-methylisothiourea (7.5 mg/kg, intraperitoneally, immediately after burn), a specific inducible nitric oxide synthase inhibitor, and sterile saline (15 mL/kg, intraperitoneally, immediately after burn) were given to different groups of thermally injured animals individually. The plasma dihydrorhodamine 123 oxidation level, intestinal and lung MPO activity, lung permeability, and lung histology were evaluated at 8 hrs after burn. The cellular localization of nitrotyrosine, a marker for peroxynitrite reactivity, was also examined by immunostaining. In experiment 3, 3-morpholinosydnonimine (10 mM, intraperitoneally), a peroxynitrite donor, was given to nonburned rats to examine the peroxynitrite effect on lung inducible nitric oxide synthase expression. MEASUREMENTS AND MAIN RESULTS: The level of MPO activity in intestine and lung, blood dihydrorhodamine 123 oxidation, and lung permeability were increased up to 2-fold, 2.5-fold, 2-fold, and 2-fold of normal, respectively, at 8 hrs after burn. S-methylisothiourea injection significantly decreased (p <.05) 31% of the lung MPO activity, 41% of the blood peroxynitrite level, 54% of the lung permeability, and the lung peroxynitrite production in burned rats. Polymorphonuclear granulocyte antibody pretreatment significantly decreased 60% of the intestinal MPO, 92% of the blood peroxynitrite level, and 56% the lung MPO activity in burned rats, but the lung permeability was only slightly decreased by polymorphonuclear granulocyte antibody pretreatment. Furthermore, 3-morpholinosydnonimine increased the lung inducible nitric oxide synthase messenger RNA levels. CONCLUSIONS: Thermal injury induces blood dihydrorhodamine 123 oxidation, intestinal and lung neutrophil deposition, lung nitrotyrosine production, and lung damage. Both specific inhibition of inducible nitric oxide synthase and polymorphonuclear granulocyte antibody pretreatment decrease blood dihydrorhodamine 123 oxidation and intestinal and lung neutrophil deposition, but only inducible nitric oxide synthase inhibition with S-methylisothiourea reduces lung peroxynitrite production and thermal injury-induced lung damage. Nitric oxide and the ensuing peroxynitrite production in lung play a more important role than neutrophil in contributing to thermal injury-induced lung damage.

Sulfadimethoxine and rhodamine B as oral biomarkers for European badgers (Meles meles).

A field study was carried out on Little Island (County Waterford, Ireland) in June 2000 to evaluate the potential of a bait-marking system for use in European badgers (Meles meles). Two oral biomarkers, sulfadimethoxine (SDM) and rhodamine B, were incorporated into fishmeal baits and distributed by hand at main sets in five test territories for 3 consecutive days. In parallel, non-biomarked baits were distributed at a single control territory. The objectives of the study were to: (1) assess the effects of SDM and rhodamine B on palatability and thus bait acceptance, and (2) investigate the marking capacity of SDM and rhodamine B in serum and hair samples taken from badgers. Trapping was carried out in each territory for 5 consecutive days immediately after bait distribution. Analysis of data revealed that 90-100% of baits were removed in four of the test territories and from the control territory. In the fifth test territory, 61% of baits were removed. Of the badgers (n = 26) trapped in the test territories, 18 (69%) were positive when tested for both biomarkers. In contrast, the remaining eight animals and those captured in the control territory (n = 6 badgers) were negative. In the marked animals, the highest levels of SDM were recorded in serum samples taken soon after bait distribution. Thereafter, the levels declined in each badger over the course of the study. In contrast, rhodamine B was readily detectable by fluorescence microscopy of hair samples throughout the period of study. The results indicate that SDM and rhodamine B act as systemic markers in badgers and have potential future applications for monitoring of oral vaccine uptake.

Ultrasensitive detection and identification of fluorescent molecules by FCS: impact for immunobiology.

An experimental application of fluorescence correlation spectroscopy is presented for the detection and identification of fluorophores and auto-Abs in solution. The recording time is between 2 and 60 sec. Because the actual number of molecules in the unit volume (confocal detection volume of about 1 fl) is integer or zero, the fluorescence generated by the molecules is discontinuous when single-molecule sensitivity is achieved. We first show that the observable probability, N, to find a single fluorescent molecule in the very tiny space element of the unit volume is Poisson-distributed below a critical bulk concentration c*. The measured probability means we have traced, for example, 5 x 10(10) fluorophore molecules per ml of bulk solution. The probability is related to the average frequency, C, that the volume of detection contains a single fluorescent molecule and to the concentration, c, of the bulk solution. The analytical sensitivity of an assay is calculated from the average frequency C. In the Goodpasture experiment, we determined as analytical sensitivity a probability of 99.1% of identifying one single immune complex. Under these conditions, a single molecule event is proven. There exist no instrumental assumptions of our approach on which the experiment itself, the theoretical background, or the conclusion are based. Our results open up a broad field for analytics and diagnostics in solution, especially in immunology.

Inducible nitric oxide synthase knockout mice exhibit resistance to the multiple organ failure induced by zymosan.

In the present study, by comparing the responses in wild-type mice (+/+) and mice lacking (-/-) the inducible (or type 2) nitric oxide synthase (iNOS), we investigated the role played by iNOS in the development of non-septic shock. A severe inflammatory response characterized by peritoneal exudation, high peritoneal levels of nitrate/nitrite, and leukocyte infiltration into peritoneal exudate was induced by zymosan administration in iNOS +/+ mice. This inflammatory process coincided with the damage of lung, liver, and small intestine, as assessed by histological examination. Lung, small intestine, and liver myeloperoxidase (MPO) activity, indicative of neutrophil infiltration and lipid peroxidation, were significantly increased in zymosan-treated iNOS +/+ mice. Peritoneal administration of zymosan in the iNOS +/+ mice induced also a significant increase in the plasma levels of nitrite/nitrate and in the levels of peroxynitrite at 18 h after zymosan challenge. Immunohistochemical examination demonstrated a marked increase in the immunoreactivity to nitrotyrosine and to poly ADP-ribose synthetase (PARS) in the lung, liver, and intestine of zymosan-treated iNOS +/+ mice. The intensity and degree of nitrotyrosine and PARS were markedly reduced in tissue section from zymosan-iNOS -/- mice. Zymosan-treated iNOS -/- mice showed a significantly decreased mortality and inhibition of the development of peritonitis. In addition, iNOS -/- mice showed a significant protection on the development of organ failure since tissue injury and MPO were reduced in lung, small intestine, and liver. Furthermore, a significant reduction of suppression of mitochondrial respiration, DNA strand breakage, and reduction of cellular levels of NAD+ was observed in ex vivo macrophages harvested from the peritoneal cavity of iNOS -/- mice subjected to zymosan-induced non-septic shock. In vivo treatment with aminoguanidine (300 mg/kg 1 and 6 h after zymosan administration) significantly prevents the inflammatory process. Taken together, our results clearly demonstrate that iNOS plays an important role in zymosan-induced non-septic shock.

Inhibition of inducible nitric oxide synthase (iNOS) prevents lung neutrophil deposition and damage in burned rats.

This study was designed to investigate the role of NO and effect of iNOS inhibitor on the lung neutrophil deposition and damage after burn. In Experiment 1, specific pathogen-free (SPF) Sprague-Dawley rats underwent 35% total body surface area (TBSA) burn. On the 4th, 8th, 16th, and 24th h after burn, blood was collected for peroxynitrite-mediated dihydrorhodamine 123 (DHR 123) oxidation assay, and lung tissues were harvested for myeloperoxidase (MPO) test and histologic study. Pulmonary microvascular dysfunction was quantitated by measuring the extravasation of Evans blue dye (EBD). In Experiment 2, S-methylisothiourea (SMT) was given (7.5 mg/kg, intraperitoneal immediately post-burn) to suppress iNOS activity. On the 8th h after burn, the effect of SMT on blood DHR 123 oxidation, lung MPO, lung damage, and lung iNOS expression were evaluated. Lung MPO activity increased up to a maximum of 2-fold 8 h after burn. Blood DHR 123 oxidation increased up to a maximum of 2-fold 8 h after burn. Lung permeability increased up to a maximum of 2.5-fold 4 h after burn. SMT significantly decreased lung MPO activity, blood DHR 123 oxidation, and lung permeability by 31%, 41%, and 54%, respectively. SMT markedly decreased the thermal injury-induced perivascular and interstitial inflammatory cell infiltration and iNOS staining in bronchiolar epithelium, endothelial cells, and perivascular and interstitial inflammatory cells. In conclusion, thermal injury induces blood DHR 123 oxidation, lung neutrophil deposition, lung iNOS expression, and lung damage. Peroxynitrite might play an important role in thermal injury-induced lung neutrophil deposition and damage. Specific inhibition of lung iNOS expression and blood DHR 123 oxidation with SMT on thermal injury not only attenuated the lung neutrophil deposition, but also reduced lung damage.

Beneficial effects of peroxynitrite decomposition catalyst in a rat model of splanchnic artery occlusion and reperfusion.

The aim of the present study was to investigate the protective effect of the peroxynitrite decomposition catalyst 5,10,15, 20-tetrakis(2,4,6-trimethyl-3,5-disulfonatophenyl)-porphyrinato iron (III) (FeTMPS) in a model of splanchnic artery occlusion shock (SAO). SAO shock was induced in rats by clamping both the superior mesenteric artery and the celiac trunk for 45 min, followed by release of the clamp (reperfusion). At 60 min after reperfusion, animals were killed for histological examination and biochemical studies. There was a marked increase in the oxidation of dihydrorhodamine 123 to rhodamine (a marker of peroxynitrite-induced oxidative processes) in the plasma of the SAO-shocked rats after reperfusion, but not during ischemia alone. Immunohistochemical examination demonstrated a marked increase in the immunoreactivity to nitrotyrosine, an index of nitrogen species such as peroxynitrite, in the necrotic ileum in shocked rats. SAO-shocked rats developed a significant increase of tissue myeloperoxidase and malonaldehyde activity, and marked histological injury to the distal ileum. SAO shock was also associated with a significant mortality (0% survival at 2 h after reperfusion). Reperfused ileum tissue sections from SAO-shocked rats showed positive staining for P-selectin localized mainly in the vascular endothelial cells. Ileum tissue sections obtained from SAO-shocked rats and stained with antibody to ICAM-1 showed a diffuse staining. Administration of FeTMPS significantly reduced ischemia/reperfusion injury in the bowel, and reduced lipid and the production of peroxynitrite during reperfusion. Treatment with PN catalyst also markedly reduced the intensity and degree of P-selectin and ICAM-1 staining in tissue sections from SAO-shocked rats and improved survival. Our results clearly demonstrate that peroxynitrite decomposition catalysts exert a protective effect in SAO and that this effect may be due to inhibition of the expression of adhesion molecules and the tissue damage associated with peroxynitrite-related pathways.

Fluorescence properties of rhodamine 800 in whole blood and plasma.

We have characterized the fluorescence spectral properties of rhodamine 800 (Rh800) in plasma and blood in order to test the possibility of making clinical fluorescence measurements in whole blood without separation steps. Rh800 was used because of its absorption at red/near-infrared wavelengths away from the absorption bands of hemoglobin. We utilized the front-face illumination and detection to minimize the effects of absorption and/or scatter during measurements. The presence of Rh800 was detected in plasma and blood using steady-state fluorescence measurements. Absorption due to hemoglobin reduced the Rh800 intensity from the blood. Fluorescence lifetime measurements in plasma and blood showed that it is possible to recover lifetime parameters of Rh800 in these media. We obtained mean lifetimes of 1.90 and 1.86 ns for Rh800 in plasma and blood, respectively. Using the recently described modulation sensing method, we quantified the concentrations of Rh800 in plasma and blood. Rh800 was detected at a concentration of as low as 2 microM in both media. High anisotropy values were obtained for Rh800 in plasma and blood using steady-state and anisotropy decay measurements, implying the tight binding of this probe to the contents of these media. This binding can be exploited to monitor the concentrations of different blood components using already existing or new red-emitting probes that will be specially designed to bind to these components with high specificity. To test this possibility of direct measurements in blood, we used Rh800 to monitor albumin in the presence of red blood cells. Increase in the polarization of Rh800 as the concentration of albumin was increased in the presence of the red cells showed the feasibility of such measurements.

Zonal compartmentation of perfused rat liver: plasma reappearance of rhodamine B explained.

Rhodamine B (RB) fluorescence reappears in perfusion medium of a cyclically perfused rat liver after a rapid initial removal phase. At the same time the compound redistributes in the liver from acinar zone 1 toward zone 3. By analysis of the metabolic profile of RB, and by inhibition of glucuronidation (the main metabolic route) with salicylamide, we show in this paper that formation and secretion of RB-conjugates from liver into perfusate is not involved in the reappearance and redistribution phenomena. We therefore sought the explanation in a kinetic model, in which the acinar heterogeneity of the liver was simulated by several sequential liver compartments. Most kinetic parameters we used in the simulation were calculated from previous experiments with RB (Braakman et al., Hepatology 7: 849-855, 1987). This led to an accurate simulation of the measured RB curves in bile, medium and the acinar zones of the liver. In this study we show that a secondary rise in medium concentration of an injected compound is not necessarily caused by metabolism, but can be easily explained by considering the liver a sequence of compartments, instead of one well-stirred compartment. The conditions for the reappearance as well as for the intrahepatic redistribution are: a fast uptake into the liver, combined with a fast sinusoidal secretion and a slow biliary excretion of the injected substance.

Radioiodinated Rhodamine-123: a potential cationic hepatobiliary imaging agent.

The labelling of the cationic dye Rhodamine-123 with 125I is described. The biodistribution of the iodinated Rhodamine-123 has been determined at different time intervals after intravenous injection into fasted rats. It turned out that the dye is predominantly cleared by the liver and discharged into the bile. The bile acid taurocholate did not enhance the rate of excretion of 125I-Rhodamine-123.

Inhibitory effect of rhodamine 123 on the growth of the rodent malaria parasite, Plasmodium yoelii.

The effect of the cationic permeant fluorescent dye, rhodamine 123 (R123), on the in vivo growth of Plasmodium yoelii was examined. Plasmodium yoelii-infected mouse erythrocytes were incubated in vitro with R123 and injected intravenously into mice. Examination of daily parasitemias showed that R123 delayed parasite growth whereas rhodamine 110, a neutral compound, and fluorescein, a negatively charged fluorescent dye, did not. Infected erythrocytes treated with R123 were not cleared from the circulation even 7 h after injection. Quantitation of cell-associated R123 by spectrophotometry revealed that infected cells with increased levels of R123 considerably prolonged the 2% prepatent period, the time required for the parasite to develop a 2% parasitemia. Degenerating parasites within and outside the host erythrocytes were observed on day 1 of infection in the mice. Thus it follows that R123, which accumulated in infected erythrocytes, inhibits the growth of P. yoelii; moreover, when R123-labeled infected erythrocytes were treated with 1-10 microM carbonylcyanide m-chlorophenylhydrazone (CCCP), a proton ionophore, to release R123 from the cells, the inhibitory effect on the growth rate of P. yoelii was partially reversed.