Drug‐induced cardiomyopathy: Characterization of a rat model by [18F]FDG/PET and [99mTc]MIBI/SPECT

BackgroundDrug‐induced cardiomyopathy is a significant medical problem. Clinical diagnosis of myocardial injury is based on initial electrocardiogram, levels of circulating biomarkers, and perfusion imaging with single photon emission computed tomography (SPECT). Positron emission tomography (PET) is an alternative imaging modality that provides better resolution and sensitivity than SPECT, improves diagnostic accuracy, and allows therapeutic monitoring. The objective of this study was to assess the detection of drug‐induced cardiomyopathy by PET using 2‐deoxy‐2‐[¹⁸F]fluoro‐D‐glucose (FDG) and compare it with the conventional SPECT technique with [^99mTc]‐Sestamibi (MIBI).MethodsCardiomyopathy was induced in Sprague Dawley rats using high‐dose isoproterenol. Nuclear [¹⁸F]FDG/PET and [^99mTc]MIBI/SPECT were performed before and after isoproterenol administration. [¹⁸F]FDG (0.1 mCi, 200‐400 µL) and [^99mTc]MIBI (2 mCi, 200‐600 µL) were administered via the tail vein and imaging was performed 1 hour postinjection. Isoproterenol‐induced injury was confirmed by the plasma level of cardiac troponin and triphenyltetrazolium chloride (TTC) staining.ResultsIsoproterenol administration resulted in an increase in circulating cardiac troponin I and showed histologic damage in the myocardium. Visually, preisoproterenol and postisoproterenol images showed alterations in cardiac accumulation of [¹⁸F]FDG, but not of [^99mTc]MIBI. Image analysis revealed that myocardial uptake of [¹⁸F]FDG reduced by 60% after isoproterenol treatment, whereas that of [^99mTc]MIBI decreased by 45%.ConclusionWe conclude that [¹⁸F]FDG is a more sensitive radiotracer than [^99mTc]MIBI for imaging of drug‐induced cardiomyopathy. We theorize that isoproterenol‐induced cardiomyopathy impacts cellular metabolism more than perfusion, which results in more substantial changes in [¹⁸F]FDG uptake than in [^99mTc]MIBI accumulation in cardiac tissue.     

Effect of aerobic training on 99mTc-methoxy isobutyl isonitrile (99mTc-sestamibi) uptake by myocardium and skeletal muscle: implication for noninvasive assessment of muscle metabolic profile

Aim: The effect of long‐term endurance training on skeletal muscle and myocardial uptake of ^99mTc‐sestamibi, a radiopharmaceutical accumulating in the mitochondria, was investigated. Methods: Twenty‐six Wistar rats were divided into a trained (5 days week^?1 endurance running for 14 weeks) and an untrained group. On completion of training, ^99mTc‐sestamibi was administered and, 2 h post‐injection, the myocardium and the soleus, extensor digitorum longus (EDL) and medial gastrocnemius (MG) muscles were removed for the measurement of cytochrome c oxidase (CCO) activity and ^99mTc‐sestamibi uptake. Tissue ^99mTc‐sestamibi kinetics was preliminarily studied in 16 other rats for up to 2 h post‐injection. Results: Two hours post‐injection ^99mTc‐sestamibi uptake was either stable (myocardium) or still rising (skeletal muscles). Both CCO activity and ^99mTc‐sestamibi uptake decreased in the same order (myocardium, soleus, EDL, MG) in the tissues examined. The CCO activity of the EDL and MG muscles was higher (P < 0.05) in the trained compared to the untrained group. ^99mTc‐sestamibi uptake in the soleus and EDL muscles was higher (P < 0.05) in the trained compared to the untrained rats, whereas the difference in MG was marginally significant (P = 0.06) in favour of the trained group. Conclusions: Long‐term endurance training, resulting in elevated skeletal muscle CCO activity, is also associated with a similar increase in ^99mTc‐sestamibi uptake. This finding suggests that ^99mTc‐sestamibi could be used in imaging assessment of skeletal muscle metabolism with possible applications in both clinical and sports medicine settings.     

Tumor targeting HPMA-porphyrin-99mTc copolymer molecular imaging agent

Porphyrins typically show preferential uptake and retention by tumor tissues via receptor-mediated endocytosis of low-density lipoproteins. To investigate the relative importance of active and passive targeting strategies, the synthesis, characterization, in vitro uptake, and in vivo biodistribution of specific targeting porphyrin HPMA [HPMA: N-(2-hydroxypropyl)methacrylamide] copolymer tracer poly(HPMA)-porphyrin-DTPA-^99mTc (DTPA: diethylenetriaminepentaacetic acid), nonspecific targeting HPMA copolymer tracer poly(HPMA)-DTPA-^99mTc, and nontargeting tracer DTPA-^99mTc are described in this study. The results showed that the cellular accumulation of poly(HPMA)-porphyrin-DTPA-^99mTc complex was found to be time-dependent. The uptake of poly(HPMA)-porphyrin-DTPA-^99mTc was significantly higher than that of poly(HPMA)-DTPA-^99mTc, indicating that uptake of the poly(HPMA)-porphyrin-DTPA-^99mTc was active binding. The uptake of poly(HPMA)-DTPA-^99mTc was significantly higher than that of DTPA-^99mTc, suggesting that uptake of the poly(HPMA)-DTPA-^99mTc was passive binding. Twenty-four hour necropsy data in the hepatocellular carcinoma tumor model showed significantly higher (p < 0.001) tumor localization for poly(HPMA)-porphyrin-DTPA-^99mTc (5.18 ± 0.50% ID/g [percentage injected dose per gram tissue]) compared with poly(HPMA)-DTPA-^99mTc (2.69 ± 0.15% ID/g) and DTPA-^99mTc (0.83 ± 0.03% ID/g). Moreover, higher T/B for poly(HPMA)-porphyrin-DTPA-^99mTc indicated reduced extravasation of the targeted polymeric conjugates in normal tissues. Thus, the poly(HPMA)-porphyrin-DTPA-^99mTc is a potential macromolecular tumor targeting molecular agent.     

SPECT/CT imaging of radiolabeled cubosomes and hexosomes for potential theranostic applications

We have developed a highly efficient method for the radiolabeling of phytantriol (PHYT)/oleic acid (OA)-based hexosomes based on the surface chelation of technetium-99m (^99mTc) to preformed hexosomes using the polyamine 1, 12-diamino-3, 6, 9-triazododecane (SpmTrien) as chelating agent. We also report on the unsuccessful labeling of cubosomes using the well-known chelating agent hexamethylpropyleneamine oxime (HMPAO). The ^99mTc-labeled SpmTrien-hexosomes (^99mTc-SpmTrien-hexosomes) were synthesized with good radiolabeling (84%) and high radiochemical purity (>90%). The effect of radiolabeling on the internal nanostructure and the overall size of these aqueous dispersions was investigated by using synchrotron small angle X-ray scattering (SAXS), dynamic light scattering (DLS), and transmission electron cryo microscopy (cryo-TEM). Further, we show the utility of ^99mTc-SpmTrien-hexosomes for the in vivo imaging of healthy mice using single photon emission computed tomography (SPECT) in combination with computed tomography (CT), i.e. SPECT/CT. SPECT/CT experiments of subcutaneously administered ^99mTc-SpmTrien-hexosomes to the flank of mice showed a high stability in vivo allowing imaging of the distribution of the radiolabeled hexosomes for up to 24 h. These injected ^99mTc-SpmTrien-hexosomes formed a deposit within the subcutaneous adipose tissue, displaying a high biodistribution of ∼343% injected dose/g tissue (%ID/g), with negligible uptake in other organs and tissues. The developed ^99mTc labeling method for PHYT/OA-based hexosomes could further serve as a useful tool for investigating and imaging the in vivo performance of cubosomal and hexosomal drug nanocarriers.     

Passive and active hepatoma tumor targeting of new N-(2-hydroxypropyl)methacrylamide copolymer conjugates: synthesis,characterization, and evaluation in vitro and in vivo

Human hepatocellular carcinoma (HCC) is one of the major causes of death worldwide. To investigate the relative importance of active and passive targeting strategies, the synthesis, characterization, in vitro uptake, and in vivo biodistribution of specific sulfapyridine HPMA (HPMA: N-(2-hydroxypropyl methacrylamide)) copolymer (sulfapyridine: SPD) conjugates, nonspecific HPMA copolymer conjugates, and DTPA are described in this study. The poly(HPMA)-SPD-DTPA (DTPA: diethylenetriaminepentaacetic acid), poly(HPMA)-DTPA, and DTPA conjugates were radiolabeled with the radionuclide ^99mTc and tested for uptake by cultured H22 cells. The cellular accumulation of poly(HPMA)-SPD-DTPA-^99mTc complex was found to be time-dependent. The poly(HPMA)-SPD-DTPA-^99mTc tracer exhibited rapid uptake kinetics in cell culture with a t _1/2 of ～5?min. The uptake of poly(HPMA)-SPD-DTPA-^99mTc was significantly higher than that of poly(HPMA)-DTPA-^99mTc, indicating that the uptake of the poly(HPMA)-SPD-DTPA-^99mT was active binding. The uptake of poly(HPMA)-DTPA-^99mTc was significantly higher than that of DTPA-^99mTc, suggesting that the uptake of the poly(HPMA)-DTPA-^99mT was passive binding. Twenty-four hour necropsy data in the hepatocellular carcinoma tumor model showed significantly higher (p?<?0.001) tumor localization for poly(HPMA)-SPD-DTPA-^99mTc (4.98?±?0.48%ID/g [percentage injected dose per gram tissue]) compared with poly(HPMA)-DTPA-^99mTc (2.69?±?0.15% ID/g) and DTPA-^99mTc (0.83?±?0.03%ID/g). Moreover, higher T/B for poly(HPMA)-SPD-DTPA-^99mTc indicated reduced extravazation of the targeted polymeric conjugates in normal tissues. Specific molecular targeting and nonspecific vascular permeability are both significant in the relative tumor localization of poly(HPMA)-SPD-DTPA-^99mTc. Extravascular leak in nonspecific organs appears to be a major factor in reducing the T/B for the sulfapyridine molecules. Thus, the poly(HPMA)-SPD-DTPA is expected to be used as the potential macromolecular targeting carrier for hepatoma carcinoma in mice.     

Size Control of 99mTc-tin Colloid Using PVP and Buffer Solution for Sentinel Lymph Node Detection

Colloidal particle size is an important characteristic that allows mapping sentinel nodes in lymphoscintigraphy. This investigation aimed to introduce different ways of making a ^99mTc-tin colloid with a size of tens of nanometers. All agents, tin fluoride, sodium fluoride, poloxamer-188, and polyvinylpyrrolidone (PVP), were mixed and labeled with ^99mTc. Either phosphate or sodium bicarbonate buffers were used to adjust the pH levels. When the buffers were added, the size of the colloids increased. However, as the PVP continued to increase, the size of the colloids was controlled to within tens of nanometers. In all samples, phosphate buffer added PVP (30 mg) stabilized tin colloid (^99mTc-PPTC-30) and sodium bicarbonate solution added PVP (50 mg) stabilized tin colloid (^99mTc-BPTC-50) were chosen for in vitro and in vivo studies. ^99mTc-BPTC-50 (<20 nm) was primarily located in bone marrow and was then secreted through the kidneys, and ^99mTc-PPTC-30 (>100 nm) mainly accumulated in the liver. When a rabbit was given a toe injection, the node uptake of ^99mTc-PPTC-30 decreased over time, while ^99mTc-BPTC-50 increased. Therefore, ^99mTc-BPTC-50 could be a good candidate radiopharmaceutical for sentinel node detection. The significance of this study is that nano-sized tin colloid can be made very easily and quickly by PVP.

Graphical Abstract

相似文献   

Detection and quantification of experimental joint inflammation in mice by measurement of99mTc-pertechnetate uptake

We adapted the method of^99mTc-pertechnetate (^99mTc) uptake measurements to the mouse knee for detection and quantification of arthritis because clinical assessment of mouse knee-joint arthritis is not reliable. The main points to ensure reproducibility of measurements were proper fixation and positioning of the knee and careful shielding of the rest of the body from the gamma-radiation detector.^99mTc uptake was calculated as the mean of three countings. The variation coefficient of these countings ranged from 0.007 to 0.082 in non-arthritic joints and from 0.025 to 0.081 in arthritic joints. Arthritis was scored as the ratio of the^99mTc uptake in the right knee versus that in the left knee. This ratio averated 1.06 (S.D. 0.05) in non-arthritic mice 20 minutes after^99mTc administration i.p. On the second day after induction of arthritis in the right knee, this ratio ranged from 1.44 to 1.96; this was significantly higher (P<0.005) than in non-arthritic mice, and the increase remained significant during at least 20 days.^99mTc uptake measurements seem to be a useful method to detect and quantify arthritis of the knee joint in mice.     

Functional investigation of bone implant viability using radiotracers in a new model of osteonecrosis

OBJECTIVES:Conventional imaging methods are excellent for the morphological characterization of the consequences of osteonecrosis; however, only specialized techniques have been considered useful for obtaining functional information. To explore the affinity of radiotracers for severely devascularized bone, a new mouse model of isolated femur implanted in a subcutaneous abdominal pocket was devised. To maintain animal mobility and longevity, the femur was harvested from syngeneic donors. Two technetium-99m-labeled tracers targeting angiogenesis and bone matrix were selected.METHODS:Medronic acid and a homodimer peptide conjugated with RGDfK were radiolabeled with technetium-99m, and biodistribution was evaluated in Swiss mice. The grafted and control femurs were evaluated after 15, 30 and 60 days, including computed tomography (CT) and histological analysis.RESULTS:Radiolabeling achieved high (>95%) radiochemical purity. The biodistribution confirmed good blood clearance 1 hour after administration. For ^99mTc-hydrazinonicotinic acid (HYNIC)-E-[c(RGDfK)₂, remarkable renal excretion was observed compared to ^99mTc-methylene diphosphonate (MDP), but the latter, as expected, revealed higher bone uptake. The results obtained in the control femur were equal at all time points. In the implanted femur, ^99mTc-HYNIC-E-[c(RGDfK)₂ uptake was highest after 15 days, consistent with early angiogenesis. Regarding ^99mTc-MDP in the implant, similar uptake was documented at all time points, consistent with sustained bone viability; however, the uptake was lower than that detected in the control femur, as confirmed by histology.CONCLUSIONS:1) Graft viability was successfully diagnosed using radiotracers in severely ischemic bone at all time points. 2) Analogously, indirect information about angiogenesis could be gathered using ^999mTc-HYNIC-E-[c(RGDfK)₂. 3) These techniques appear promising and warrant further studies to determine their potential clinical applications.     

An experimental model to study the effects of a senna extract on the blood constituent labeling and biodistribution of a radiopharmaceutical in rats

ABSTRACT Cassia angustifolia Vahl (senna) is a natural product that contains sennosides, which are active components that affect the intestinal tract and induce diarrhea. Authors have shown that senna produces DNA (deoxyribonucleic acid) lesions in Escherichia coli cultures and can act as an antifungal agent. Natural drugs can alter the labeling of blood constituents with technetium-99m (^99mTc) and can affect the biodistribution of radiopharmaceuticals. In this work, we have evaluated the influence of a senna extract on the radiolabeling of blood constituents and on the biodistribution of the radiopharmaceutical sodium pertechnetate (Na^99mTcO₄) in Wistar rats. Twelve animals were treated with senna extract for 7 days. Blood samples were withdrawn from the animals and the radiolabeling procedure was carried out. The senna extract did not modify the radiolabeling of the blood constituents. A biodistributional assay was performed by administering Na^99mTcO₄ and determining its activity in different organs and in blood. The senna extract altered the biodistribution of Na^99mTcO₄ in the thyroid, liver, pancreas, lungs and blood. These results are associated with properties of the chemical substances present in the aqueous senna extract. Although these assays were performed in animals, our findings suggest that caution should be exercised when nuclear medicine examinations using Na^99mTcO₄ are conducted in patients who are using senna extract.     

Quantitation of arthritis by99mTc-uptake measurements in the mouse knee-joint: correlation with histological joint inflammation scores

A method is described to detect and to quantitate inflammation in knee-joints of mice. Approximately 10 Ci^99mTechnetium pertechnetate (^99mTc) is injected subcutaneously in the neck region and the accumulation of the short-lived isotope in the knee-joint is detected by external gamma counting.^99mTc-uptake values correlate well with histological grading of joint inflammation at various intervals after induction of the inflammation. The method can be used to detect changes in activity of unilateral as well as bilateral joint inflammation using either the ratio of^99mTc uptake in the right knee versus that in the left knee or absolute^99mTc-uptake values.     

Biosynthesis of [7-3H]16α-hydroxy-dehydroepiandrosterone having high specific activity

Biosynthesis of [7-³H]16α-hydroxy-dehydroepiandrosterone in high specific activity has been studied. [7-³H] dehydroepiandrosterone (13.9 C/mM) in trace quantity was oxidized by Streptomyces roseochromogenes (NRRLB-1233) for 5 min at 27 °C. The radioactive products were chromatographically separated, identified and their radiochemical purity established by isotopic dilution analysis. [7-³H]16α-hydroxy-dehydroepiandrosterone (2.5 × 10⁷ dpm) was obtained by microbial hydroxylation of substrate (1.9 × 10⁹ dpm). In some cases [7-³H]5-androstene-3β, 16α, 17β-triol in a small amount of radioactivity could be found at the prolonged reaction for 30 hr.     

Evaluation of 99mTc-HYNIC-βAla-Bombesin(7-14) as an agent for pancreas tumor detection in mice

Pancreatic adenocarcinoma is important in oncology because of its high mortality rate. Deaths may be avoided if an early diagnosis could be achieved. Several types of tumors overexpress gastrin-releasing peptide receptors (GRPr), including pancreatic cancer cells. Thus, a radiolabeled peptide derivative of gastrin-releasing peptide (GRP) may be useful as a specific imaging probe. The purpose of the present study was to evaluate the feasibility of using^99mTc-HYNIC-βAla-Bombesin_(7-14)as an imaging probe for Capan-1 pancreatic adenocarcinoma. Xenographic pancreatic tumor was developed in nude mice and characterized by histopathological analysis. Biodistribution studies and scintigraphic images were carried out in tumor-bearing nude mice. The two methods showed higher uptake by pancreatic tumor when compared to muscle (used as control), and the tumor-to-muscle ratio indicated that^99mTc-HYNIC-βAla-Bombesin_(7-14)uptake was four-fold higher in tumor cells than in other tissues. Scintigraphic images also showed a clear signal at the tumor site. The present data indicate that^99mTc-HYNIC-βAla-Bombesin_(7-14)may be useful for the detection of pancreatic adenocarcinoma.     

How regenerating lymphatics function: Lessons from lizard tails

Rational treatment of lymphoedema may be improved in the future with a better understanding of the physiological processes involved in the regeneration of new lymphatic vessels (lymphangiogenesis). Many lizard species undergo tail autotomy as a predator escape response and subsequently regenerate nonlymphoedematous tails. Such species may offer novel models for examining lymphangiogenesis. In this lymphoscintigraphic evaluation, three radioactive tracers were employed, ^99mTc‐antimony trisulphide colloid (～ 10 nm diameter), ^99mTc‐tin fluoride colloid (～2,000 nm; ^99mTc‐TFC), and ^99mTc‐diethylenetriaminepentaacetic acid (soluble; ^99mTc‐DTPA), to examine lymphatic function in regenerating tails of the Australian marbled gecko, Christinus marmoratus. Rate of local clearance and velocity of migration were determined in geckos with original tails and at 6, 9, 12, and >24 weeks after autotomy. In original‐tailed geckos, the smaller radiocolloid was cleared to a greater extent and had a faster lymph velocity than in geckos with regenerated tails. The same parameters measured for larger particles were greater in early regeneration than later. ^99mTc‐TFC did not migrate from the injection site in fully regenerated and original gecko tails, which indicates that larger particles are increasingly impeded as tail regeneration progresses. Soluble ^99mTc‐DTPA diffused from the injection site extremely rapidly via venous capillaries in all tails, confirming that the slower clearance of the colloids is solely via the lymphatics. Differences in clearance and lymph velocity between differently sized colloids throughout tail regeneration may be influenced by changes in surrounding tissue structure density and the lymphatic vessel porosity. Anat Rec, 290:108–114, 2007. © 2006 Wiley‐Liss, Inc.     

Der autoradiographische Nachweis von99mTe

Zusammenfassung ^99mTc läßt sich autoradiographisch nachweisen, obwohl es keine primäre-Strahlung emittiert. Die autoradiographische Darstellung gelingt durch Konversionselektronen, die in 8,7% der Zerfälle auftreten. Infolge des relativ hohen autoradiographischen Auflösungsvermögens konnte eine celluläre Speicherung in einem Neurofibrom bei einer 32jährigen Patientin nachgewiesen werden.

---

Summary ^99mTc can be detected by high-resolution autoradiography, although it does not emit any primary-radiation. The autoradiographic detection succeeds by conversion-electrons, which arise in 8.7% of decays. In a neurofibroma of a 32 years old femal patient a cellular accumulation was demonstrated.

     

Changes in the Electrochemical Potential Difference for HCO3-- between Blood and Cerebrospinal Fluid and in Cerebrospinal Fluid Lactate Concentration during Isocarbic Hypoxia1

The changes in csf [HCO₃^-] and [lactate] were followed in dogs whose arterial pH, Pco₂, and [HCO₃^-_-] were kept constant during 6 hrs hypoxia or normoxia. The results confirm that hypoxia causes a lowering of [HCO₃^-]csf. We believe it is reasonable to assume that an increased anaerobic glycolysis in the brain is responsible for titrating HCO₃^-_- out of csf during isocarbic hypoxia although the lactate concentration in csf increased only about one third as much as the [HCO₃-_-] decreased. The action of hypoxia on [HCO₃^-] esf is expressed in terms of its effect on the electrochemical potential difference for HCO₃^-_- between mean capillary plasma water and csf.     

An Association of Prior Therapeutic Chest Irradiation and 99mTc Pyrophosphate Uptake in the Heart

The effect of ionizing radiation on the heart is well known, however, the exact magnitude of cardiac involvement in patients undergoing therapeutic radiation directed to or near the heart is not known. Current tests (ie, serum enzymes [SGPT, SGOT, CPK]) to assess cardiac damage are quite insensitive. With the availability of ^99mTc pyrophosphate for myocardial imaging, a population of 70 subjects who had prior irradiation, 32 to the left chest, 38 elsewhere to the body, were evaluated by pyrophosphate imaging with bone scans.     

Polymeric chiral crown ethers, 7. Synthesis of polymers incorporating methyl glycosides of α-D-altrose, α-D-galactose, α-D-glucose and α-D-mannose residues via copolymerization of divinyl ethers

Polymers with chiral asymmetric crown ether units ( 5, 6, 7 and 8 ) were synthesized via cationic cyclopolymerization of methyl 2,3-bis{O-[2-(2-vinyloxyethoxy)ethyl]}-4,6-O-benzylidene α-D -altro-, α-D -galacto-, α-D -gluco- and α-D -manhopyranosides ( 1, 2, 3 and 4 ), respectively. The enantioselective transport of the methyl ester of phenylglycine (PhGlyOCH₃) and phenylalanine (PhAlaOCH₃) was examined through a bulk chloroform solution of chiral polymers from one aqueous solution to another. The transport rate of PhAlaOCH₃ was larger than that of PhGlyOCH₃ for every host polymer. For polymer 7 , the optical purity of PhAlaOCH₃ transported from one to the other phase was 12,6%, and the ratio of rate constants for the faster moving enantiomer A and the slower moving enantiomer B (k_A^*/k_B^*) was 1,48. The faster moving enantiomer was the L -isomer except for the systems polymer 7 ? PhAlaOCH₃ and polymer 8 ? PhAlaOCH₃. This enantioselectivity is caused by the diastereotopic faces of the crown ether units in the host polymers.     

Na+-dependent regulation of the free Mg2+ concentration in neuropile glial cells and P neurones of the leech Hirudo medicinalis

 To investigate the Mg²⁺ regulation in neuropile glial (NG) cells and pressure (P) neurones of the leech Hirudo medicinalis the intracellular free Mg²⁺ ([Mg²⁺]_i) and Na⁺ ([Na⁺]_i) concentrations, as well as the membrane potential (E _m), were measured using Mg²⁺- and Na⁺-selective microelectrodes. The mean steady-state values of [Mg²⁺]_i were found to be 0.91 mM (mean E _m=–63.6 mV) in NG cells and 0.20 mM (mean E _m=–40.6 mV) in P neurones with a [Na⁺]_i of 6.92 mM (mean E _m=–61.6 mV) and 7.76 mM (mean E _m=–38.5 mV), respectively. When the extracellular Mg²⁺ concentration ([Mg²⁺]_o) was elevated, [Mg²⁺]_i in P neurones increased within 5–20 min whereas in NG cells a [Mg²⁺]_i increase occurred only after long-term exposure (6 h). After [Mg²⁺]_o was reduced back to 1 mM, a reduction of the extracellular Na⁺ concentration ([Na⁺]_o) decreased the inwardly directed Na⁺ gradient and reduced the rate of Mg²⁺ extrusion considerably in both NG cells and P neurones. In P neurones Mg²⁺ extrusion was reduced to 15.4% in Na⁺-free solutions and to 6.0% in the presence of 2 mM amiloride. Mg²⁺ extrusion from NG cells was reduced to 6.2% in Na⁺-free solutions. The results suggest that the major [Mg²⁺]_i-regulating mechanism in both cell types is Na⁺/ Mg²⁺ antiport. In P neurones a second, Na⁺-independent Mg²⁺ extrusion system may exist. Received: 11 August 1998 / Received after revision: 14 October 1998 / Accepted: 15 October 1998     

Altered peptide ligands of myelin basic protein (MBP87–99) conjugated to reduced mannan modulate immune responses in mice

Mutations of peptides to generate altered peptide ligands, capable of switching immune responses from T helper 1 (Th1) to T helper 2 (Th2), are promising candidates for the immunotherapy of autoimmune diseases such as multiple sclerosis (MS). We synthesized two mutant peptides from myelin basic protein 87–99 (MBP_87–99), an immunodominant peptide epitope identified in MS. Mutations of residues K⁹¹ and P⁹⁶, known to be critical T-cell receptor (TCR) contact sites, resulted in the mutant peptides [R⁹¹, A⁹⁶]MBP_87–99 and [A⁹¹, A⁹⁶]MBP_87–99. Immunization of mice with these altered peptide ligands emulsified in complete Freund’s adjuvant induced both interferon-γ (IFN-γ) and interleukin-4 (IL-4) responses compared with only IFN-γ responses induced to the native MBP_87–99 peptide. It was of interest that [R⁹¹, A⁹⁶]MBP_87–99 conjugated to reduced mannan induced 70% less IFN-γ compared with the native MBP_87–99 peptide. However, [A⁹¹, A⁹⁶]MBP_87–99 conjugated to reduced mannan did not induce IFN-γ-secreting T cells, but elicited very high levels of interleukin-4 (IL-4). Furthermore, antibodies generated to [A⁹¹, A⁹⁶]MBP_87–99 peptide conjugated to reduced mannan did not cross-react with the native MBP_87–99 peptide. By molecular modelling of the mutant peptides in complex with major histocompatibility complex (MHC) class II, I-A^s, novel interactions were noted. It is clear that the double-mutant peptide analogue [A⁹¹, A⁹⁶]MBP_87–99 conjugated to reduced mannan is able to divert immune responses from Th1 to Th2 and is a promising mutant peptide analogue for use in studies investigating potential treatments for MS.     

Further studies of the ethanol-induced changes in pancreatic lipid metabolism

Previous in vitro studies have shown that ethanol increased de novo triglyceride synthesis in the rat pancreas. The present study extends these observations on the effects of ethanol on pancreatic lipid metabolism. Ethanol significantly stimulated [1-¹⁴C]acetate incorporation into pancreatic lipids at concentrations as low as 0.068 mM, as well as at 3.4 and 34 mM. This suggests that known metabolic pathways of ethanol oxidation are not involved in these changes. Ethanol also stimulated the incorporation of [1-¹⁴C]oleate into pancreatic triglyceride and cholesteryl ester, but not into phospholipids. These changes were less marked than those obtained with [1-¹⁴C]acetate. Furthermore, incorporation of [1-¹⁴C]palmitate into pancreatic lipid was affected even less by ethanol. Thus, ethanol-induced changes in pancreatic lipid metabolism are unlikely to be due to fatty acid esterification alone.