In vitro assessment of dose-dependent platelet activation by polyclonal antithymocyte globulins: a flow-cytometric analysis

Polyclonal antithymocyte globulins (ATGs) are immunosuppressive drugs widely used in transplantation and hematologic disorders. Treatment with ATGs can induce side effects such as neutropenia and thrombocytopenia because of unspecific antibodies directed against nonmyeloid cells present in these preparations. Depletion, activation, and expression of adhesion molecules on platelets in vitro were studied in the whole blood of healthy volunteers by means of flow cytometry after incubation with different doses of three polyclonal ATGs. Our data show no ATG-mediated cytotoxic activity against platelets. ATGs are able to induce activation of platelets through increased expression of P-selectin and hLAMP-1 and higher percentages of gated thrombocytes expressing these molecules. Furthermore, increased expression of hLAMP-1 presented a dose-dependent pattern. ATGs induced activation and enhanced expression of adhesion molecules in unstimulated platelets. Increased adhesion may be responsible for undesirable side effects such as thrombocytopenia and reticulopenia.     

Is there a specific polysomnographic sleep pattern in children with attention deficit/hyperactivity disorder?

The aim of the study was to characterize the sleep pattern in children with attention deficit/hyperactivity disorder (ADHD). By means of polysomnography (PSG), sleep patterns were studied in 17 unmedicated preadolescent boys rigorously diagnosed with ADHD and 17 control boys precisely matched for age and intelligence. Although ADHD children did not display a general sleep alteration, major PSG data showed a significant increase in the duration of the absolute rapid eye movement (REM) sleep and the number of sleep cycles in ADHD group when compared with controls. In addition, REM sleep latency tended to be shorter in ADHD children. These results suggest that in ADHD children, a forced REM sleep initiation may produce a higher incidence of sleep cycles and may also contribute to an increased duration of the absolute REM sleep. The overall pattern of the findings implies that a forced ultradian cycling appears characteristic for the sleep in ADHD children, which may be related to alterations of brain monoamines and cortical inhibitory control accompanying the ADHD psychopathology.     

Pharmacokinetics and cellular uptake of imatinib and its main metabolite CGP74588

Despite the remarkable clinical response rates to imatinib in the treatment of bcr-abl leukemic patients, pharmacokinetic data on this relatively novel substance are needed to improve our understanding of the emergence of resistance, the interindividual variations of clinical response and the clinical and biologic relevance of its main metabolite N-desmethyl-imatinib. We present here pharmacokinetic data obtained with a newly designed HPLC approach in 97 patients with chronic myeloid leukemia or acute lymphatic leukemia (ALL) under treatment with imatinib that allowed us to calculate the AUC (39.5 g·h/ml for an oral dose of 400 mg daily), the t_1/2 (18.2 h) and the peak concentration (1.92 /ml for an oral dose of 400 mg daily) of imatinib in plasma. In a subgroup of patients, the same parameters were analyzed for N-desmethyl-imatinib. We also provide data on the imatinib concentration in the cerebrospinal fluid (CSF) of ALL patients and demonstrate that oral administration of imatinib resulted only in a marginal flux across the blood-brain barrier. Finally, in an in vitro setting, we determined cellular concentrations of imatinib in HL-60 cells and showed an over-proportional uptake both in RPMI medium and in human plasma. Using an arithmetical approach combining all parameters obtained in imatinib-treated patients, we finally provide a conclusive approximation of basic pharmacokinetic data for both imatinib and its main metabolite N-desmethyl-imatinib.     

Substantial decrease of psychiatric comorbidity in chronic alcoholics upon integrated outpatient treatment - results of a prospective study

It is far from clear how comorbidity changes during alcoholism treatment. This study investigates: (1) the course of comorbid Axis I disorders in chronic alcoholics over 2 years of controlled abstinence in the outpatient long-term intensive therapy for alcoholics (OLITA) and (2) the effect of comorbid Axis I and II disorders in this group of patients on subsequent drinking outcome over a four-year follow-up. This prospective treatment study evaluates psychiatric variables of 89 severely affected chronic alcohol dependent patients on admission (t₁), month 6 (t₂), 12 (t₃) and 24 (t₄). Drinking outcomes have been analyzed from 1998 to 2002. On admission, 61.8% of the patients met criteria for a comorbid Axis I disorder, 63.2% for a comorbid personality disorder. Axis I disorders remit from t₁ (59.0% ill), t₂ (38.5%), t₃ (28.2%) to t₄ (12.8%) (p<0.0001). Anxiety disorders remit more slowly from t₁ (43.6%) to t₃ (20.5%, p=0.0086), whereas mood disorders remit early between t₁ (23.1%) and t₂ (5.1%, p=0.0387) with a slight transient increase at t₃ (10.3%). During the four-year follow-up, the cumulative probability of not having relapsed amounts to 0.59. Two predictors have a strong negative impact on abstinence probability: number of inpatient detoxifications (p=0.0013) and personality disorders (p=0.0106). The present study demonstrates a striking remission of comorbid Axis I disorders upon abstinence during comprehensive long-term outpatient alcoholism treatment. The presence of an Axis II rather than an Axis I disorder on admission strongly predicts drinking outcome over a four-year follow-up.     

Three-amino acid motifs of urocortin II and III determine their CRF receptor subtype selectivity

Corticotropin-releasing factor (CRF) and the CRF-like peptide urocortin I (UcnI) exert their activity through two different CRF receptors, CRF1 and CRF2. Recently, UcnII and UcnIII have been discovered as potential endogenous agonists selective for CRF2 known to be involved in brain functions such as learning and anxiety, as well as in cardiovascular functions. A structure-affinity relationship study using chimeric peptides was designed to characterize mouse UcnII (mUcnII) and mUcnIII further and to investigate the structural basis of their receptor subtype selectivity. In the framework of this study, mUcnII (IC50 = 4.4 nM) but not mUcnIII was identified as high-affinity ligand for the rat CRF binding protein. Such affinity had previously not been observed for the human version of this protein. On the basis of secondary structure predictions, it was hypothesized that the amino acid motifs Pro-Ile-Gly of mUcnII and Pro-Thr-Asn of mUcnIII decrease alpha-helicity and thereby impair binding to CRF1. In support of this hypothesis, binding affinity to CRF1 of the chimeric peptides [Pro11Ile12Gly13]h/rCRF, [Pro11Thr12Asn13]h/rCRF, and the corresponding rUcnI analogs was found to be decreased by three orders of magnitude, whereas binding affinity to CRF2 was much less affected. The dramatic decrease in binding affinity to CRF1 correlated with a decrease in alpha-helicity as indicated by the data of circular dichroism spectroscopy.     

Representation of the standard: stimulus context effects on the process generating the mismatch negativity component of event-related brain potentials

In the auditory oddball paradigm, the frequent occurrence of a sound (the "standard") forms the basis of deviance detection. The incoming sounds are compared with the cortical representation of the standard and those sounds that do not match it elicit the mismatch negativity (MMN) event-related brain potential. Here we address the issue of whether the relative probability of the sounds in a sequence was a critical factor influencing which sounds would be represented as standards in the deviant comparison process. One frequent (F1) and two infrequent (D1 and D2) sounds that differed only in duration were presented in a sequence. D1 occurred proportionally as frequently with respect to D2 as F1 occurred with respect to D1. If the proportional relationship of sounds were critical then D1 could serve as a "standard" to D2 and thus D2 should elicit two MMNs. However, D2 elicited MMN only with respect to F1. This result as well as those obtained in two control conditions suggests that "standards" are not established on the basis of relative probability; they emerge as a result of global characteristics, the longer-term context, of the sound sequence.     

Mouse MHC class I tetramers that are unable to bind to CD8 reveal the need for CD8 engagement in order to activate naive CD8 T cells

Although the role of CD8 as a supplier of lck is unchallenged, its role in contributing to the formation of a stable complex between class I molecules and the TCR, as well as its role as an adhesion molecule, is less clear. To address the role of CD8/MHC-I interactions, we generated tetramers composed of H2-K(b) molecules with mutations in the alpha 3 domain of H2-K(b) that abolish CD8 binding. We show that the ability of tetramers to stain and activate CD8 T cells is strongly dependent on binding of CD8 to the same class I molecule engaged by the TCR. We characterize a mutation in the alpha 3 domain that results in H2-K(b) molecules capable of staining specific CD8 T cells with little ensuing activation. Although CD8 to some extent serves an adhesive function, this contribution is modest and does not substitute for lack of binding of CD8 to the class I molecule engaged by the TCR. We show that CD8 and the TCR associate in a process independent of binding of CD8 to class I. Our data support the notion that CD8 is required to form a stable complex between class I and the TCR.     

Functional regions of the presynaptic cytomatrix protein bassoon: significance for synaptic targeting and cytomatrix anchoring

Exocytosis of neurotransmitter from synaptic vesicles is restricted to specialized sites of the presynaptic plasma membrane called active zones. A complex cytomatrix of proteins exclusively assembled at active zones, the CAZ, is thought to form a molecular scaffold that organizes neurotransmitter release sites. Here, we have analyzed synaptic targeting and cytomatrix association of Bassoon, a major scaffolding protein of the CAZ. By combining immunocytochemistry and transfection of cultured hippocampal neurons, we show that the central portion of Bassoon is crucially involved in synaptic targeting and CAZ association. An N-terminal region harbors a distinct capacity for N-myristoylation-dependent targeting to synaptic vesicle clusters, but is not incorporated into the CAZ. Our data provide the first experimental evidence for the existence of distinct functional regions in Bassoon and suggest that a centrally located CAZ targeting function may be complemented by an N-terminal capacity for targeting to membrane-bounded synaptic organelles.     

Kainate-induced epileptic seizures induce a recruitment of caldendrin to the postsynaptic density in rat brain

Caldendrin defines a novel family of neuronal calcium-sensor proteins, the C-terminal moiety of which displays high similarity to calmodulin. We now report that the protein is recruited to the postsynaptic density (PSD) of cortical and hippocampal neurons in response to kainate-induced epileptic seizures, an animal model of human temporal lobe epilepsy. The translocation of caldendrin to the PSD did not occur in kainate-treated rats that did not develop seizures. The enhanced PSD levels of caldendrin are not due to increased protein synthesis and most likely reflect a recruitment from the soluble caldendrin protein pool. These findings suggest that the transduction of dendritic Ca2+-signals via caldendrin is altered by epileptic seizures and that caldendrin might be involved in the pathophysiology of temporal lobe epilepsy.