Long-term neurobehavioral and histological damage in brain of mice induced by L-cysteine

We investigated whether structural central neural damage and long-term neurobehavioral deficits after L-cysteine (L-Cys) administration in mice is caused by hypoglycemia. Neonatal ICR mice were injected subcutaneously with L-Cys (0.5-1.5 mg/g body weight [BW]) or saline (control). Blood glucose was measured. At 50 days of age, mice were introduced individually into an eight-arm maze for evaluation of spatial memory (hippocampal-related behavior). Times for visiting all eight arms and number of entries until completion of the eight-arm visits (maze criteria) were measured. The test was repeated once daily for 5 days. In situ terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay was used for detection of brain damage. As early as 20 min and up to 2 h postinjection, animals treated with L-Cys doses higher than 1.2 mg/g BW developed hypoglycemia and looked ill. Several animals convulsed. Long-term survivors required more time, in a dose-dependent manner, to assimilate the structure of the maze, and animals treated with L-Cys (1.5 mg/g BW) exhibited TUNEL-positive changes in the hippocampal regions. All these changes were reversible by coadministration of glucose. We conclude that L-Cys injection can cause pronounced hypoglycemia associated with long-term neurobehavioral changes and central neural damage in mice. Since L-Cys is chemically different from the other excitatory amino acids (glutamate and aspartate), the long-reported L-Cys-mediated neurotoxicity may be connected to its hypoglycemic effect.     

beta-Phenylpyruvate induces long-term neurobehavioral damage and brain necrosis in neonatal mice

Administration of beta-phenylpyruvate at high concentrations reduces blood glucose levels and causes neurophysiological deterioration in insulin-deprived mice. We investigated whether beta-phenylpyruvate administration would cause long-term neurobehavioral and structural central neural damage in mice. Neonatal ICR mice were injected with beta-phenylpyruvate (0.5-2.5mg/g body weight (BW)) or saline (control). Blood glucose was measured. At 43 days of age, the animals were put on a 1-week regimen of restricted water supply, after which the mice were introduced into an eight-arm maze for evaluation of spatial-memory abilities (hippocampal-related behavior). Times for visiting all eight arms and number of entries until completion of the eight-arm visits (maze criteria) were measured. The test was repeated once daily for 5 days. TUNEL assay was used for detection of brain apoptosis. beta-Phenylpyruvate-treated animals (except the 0.5mg/g group) developed hypoglycemia. Treated mice required more time to assimilate the maze structure. Mice treated with 2.5mg/g beta-phenylpyruvate did not meet the maze criteria as compared with control (P<0.001) and suffered from necrotic changes in the hippocampal regions. The above-mentioned neurobehavioral damage was abrogated by coadministration of glucose. We conclude that beta-phenylpyruvate is able to produce necrotic neural damage accompanied by structurally related neurobehavioral dysfunction. Together with its hypoglycemic effect, these findings may explain the neurodegenerative process that occurs in phenylketonuria (PKU), insofar as beta-phenylpyruvate is a metabolite of phenylalanine known to accumulate in vast amounts in this inherited disorder.     

Ligands for the common allosteric site of acetylcholine M2-receptors: development and application

Ligands for the allosteric site of acetylcholine M2 receptors are able to retard the dissociation of simultaneously bound ligands for the orthosteric site. This effect promotes receptor occupation by the orthosteric ligand. The allosteric effect opens various therapeutic perspectives, e.g., in organophosphorus poisoning. The aim of our studies was to optimize the affinity of the modulators for the common allosteric binding site of muscarinic M2 receptors, the orthosteric site of which was liganded with the N-methylscolopamine. The phthalimido substituted hexane-bisammonium compound W84 served as a starting point. Previous molecular modelling studies revealed two positive charges and two aromatic imides in a sandwich-like arrangement to be essential for a high allosteric potency. A three-dimensional quantitative structure activity relationship (3D QSAR) analysis predicted compounds with substituents of increasing size on the lateral imide moieties to enhance the affinity for the allosteric binding site. Thus, we synthesized and pharmacologically evaluated compounds bearing "saturated" phthalimide moieties as well as phthalimidines with substituents of systematically increasing size in position 3 or on the aromatic ring at one or both ends of the molecule. Within each series, QSAR could be derived: 1. "Saturation" of the aromatic ring of the phthalimide moiety results in less potent compounds. 2. Increasing the size of the substituents in position 3 of the phthalimide enhances the potency. 3. Putting substituents on the aromatic part of the phthalimide increases the potency more effectively: the introduction of a methyl group in position 5 gave a compound with a potency in the nanomolar concentration range which was subsequently developed as the first radioligand for the allosteric binding site.     

Structure-activity relationships in a series of bisquaternary bisphthalimidine derivatives modulating the muscarinic M(2)-receptor allosterically

Hexane-bisammonium-type compounds containing lateral phthalimide moieties are well-established ligands of the common allosteric binding site of muscarinic M(2) receptors. Previous structure-activity relationships (SAR) revealed two positively charged centers and two lateral phthalimide moieties in a defined arrangement to be essential of a high allosteric potency. The purpose of this study was to replace one carbonyl group of the phthalimides with hydrogens, hydroxy, alkoxy, phenyl, benzyl, and benzylidene groups in order to check the influence of these substituents on the allosteric activity in antagonist-linked receptors. The analysis of the quantitative SAR indicated that a high allosteric potency is related to a certain amount of rigidity as well as polarizibility and the ability to form hydrophobic interactions.     

CTLA‐4 (CD152) enhances the Tc17 differentiation program

Although CD8⁺ T cells that produce IL‐17 (Tc17 cells) have been linked to host defense, Tc17 cells show reduced cytotoxic activity, which is the characteristic function of CD8⁺ T cells. Here, we show that CTLA‐4 enhances the frequency of IL‐17 in CD8⁺ T cells, indicating that CTLA‐4 (CD152) specifically promotes Tc17 differentiation. Simultaneous stimulation of CTLA‐4^+/+ and CTLA‐4^?/? T cells in cocultures and agonistic CTLA‐4 stimulation unambiguously revealed a cell‐intrinsic mechanism for IL‐17 control by CTLA‐4. The quality of CTLA‐4‐induced Tc17 cells was tested in vivo, utilizing infection with the facultative intracellular bacterium Listeria monocytogenes (LM). Unlike CTLA‐4^+/+ Tc17 cells, CTLA‐4^?/? were nearly as efficient as Tc1 CTLA‐4^+/+ cells in LM clearance. Additionally, adoptively transferred CTLA‐4^?/? Tc17 cells expressed granzyme B after rechallenge, and produced Tc1 cytokines such as IFN‐γ and TNF‐α, which strongly correlate with bacterial clearance. CTLA‐4^+/+ Tc17 cells demonstrated a high‐quality Tc17 differentiation program ex vivo, which was also evident in isolated IL‐17‐secreting Tc17 cells, with CTLA‐4‐mediated enhanced upregulation of Tc17‐related molecules such as IL‐17A, RORγt, and IRF‐4. Our results show that CTLA‐4 promotes Tc17 differentiation that results in robust Tc17 responses. Its inactivation might therefore represent a central therapeutic target to enhance clearance of infection.     

Anatomie,Physiologie, path. Anatomie,allg. und exper. Pathologie und Therapie

Ohne Zusammenfassung     

Influence of clinical status on the association between plasma total and unbound bilirubin and death or adverse neurodevelopmental outcomes in extremely low birth weight infants

Objectives: To assess the influence of clinical status on the association between total plasma bilirubin and unbound bilirubin on death or adverse neurodevelopmental outcomes at 18–22 months corrected age in extremely low birth weight infants. Method: Total plasma bilirubin and unbound bilirubin were measured in 1101 extremely low birth weight infants at 5 ± 1 days of age. Clinical criteria were used to classify infants as clinically stable or unstable. Survivors were examined at 18–22 months corrected age by certified examiners. Outcome variables were death or neurodevelopmental impairment, death or cerebral palsy, death or hearing loss, and death prior to follow‐up. For all outcomes, the interaction between bilirubin variables and clinical status was assessed in logistic regression analyses adjusted for multiple risk factors. Results: Regardless of clinical status, an increasing level of unbound bilirubin was associated with higher rates of death or neurodevelopmental impairment, death or cerebral palsy, death or hearing loss and death before follow‐up. Total plasma bilirubin values were directly associated with death or neurodevelopmental impairment, death or cerebral palsy, death or hearing loss, and death before follow‐up in unstable infants, but not in stable infants. An inverse association between total plasma bilirubin and death or cerebral palsy was found in stable infants. Conclusions: In extremely low birth weight infants, clinical status at 5 days of age affects the association between total plasma bilirubin and death or adverse neurodevelopmental outcomes at 18–22 months of corrected age. An increasing level of UB is associated a higher risk of death or adverse neurodevelopmental outcomes regardless of clinical status. Increasing levels of total plasma bilirubin are directly associated with increasing risk of death or adverse neurodevelopmental outcomes in unstable, but not in stable infants.