Requirement for diverse, low-abundance peptides in positive selection of T cells

Whether a single major histocompatibility complex (MHC)-bound peptide can drive the positive selection of large numbers of T cells has been a controversial issue. A diverse population of self peptides was shown to be essential for the in vivo development of CD4 T cells. Mice in which all but 5 percent of MHC class II molecules were bound by a single peptide had wild-type numbers of CD4 T cells. However, when the diversity within this 5 percent was lost, CD4 T cell development was impaired. Blocking the major peptide-MHC complex in thymus organ culture had no effect on T cell development, indicating that positive selection occurred on the diverse peptides present at low levels. This requirement for peptide diversity indicates that the interaction between self peptides and T cell receptors during positive selection is highly specific.     

Establishing the etiology of childhood hearing loss

The cause of hearing loss in children is often difficult to identify. We evaluated a cohort of 114 children (47 boys, 67 girls) referred with newly diagnosed hearing loss (non-otitis media) to identify factors predictive of etiology and type of hearing loss. Clinical (history and physical examination), laboratory, and radiographic data were collected. One hundred children (87.7%) had sensorineural hearing loss, and 14 (12.3%) had conductive or mixed hearing loss. The cause of hearing loss was identified in 54 children (48%). Patients with isolated aural atresia (n = 7) or with a known diagnosis of congenital cytomegalovirus infection (n = 21) were excluded from further data analysis. We conducted statistical analysis to identify factors predictive of the cause and type of hearing loss. Clinical factors that aided in identifying a cause included abnormal physical examination findings (p = 0.001) and craniofacial anomalies (p = 0.006). Computed tomography of the temporal bones was the only diagnostic test predictive of cause (p < 0.001). Factors predictive of the type of hearing loss detected (sensorineural vs. conductive or mixed) were abnormal physical examination findings (p = 0.01) and craniofacial anomalies (p = 0.004). An exhaustive laboratory or radiographic workup did not prove beneficial in identifying the etiology of hearing loss in our series.     

Empirical solvent-mediated potentials hold for both intra-molecular and inter-molecular inter-residue interactions

Whether knowledge-based intra-molecular inter-residue potentials are valid to represent inter-molecular interactions taking place at protein-protein interfaces has been questioned in several studies. Differences in the chain connectivity effect and in residue packing geometry between interfaces and single chain monomers have been pointed out as possible sources of distinct energetics for the two cases. In the present study, the interfacial regions of protein-protein complexes are examined to extract inter-molecular inter-residue potentials, using the same statistical methods as those previously adopted for intra-molecular residue pairs. Two sets of energy parameters are derived, corresponding to solvent-mediation and "average residue" mediation. The former set is shown to be highly correlated (correlation coefficient 0.89) with that previously obtained for inter-residue interactions within single chain monomers, while the latter exhibits a weaker correlation (0.69) with its intra-molecular counterpart. In addition to the close similarity of intra- and inter-molecular solvent-mediated potentials, they are shown to be significantly more residue-specific and thereby discriminative compared to the residue-mediated ones, indicating that solvent-mediation plays a major role in controlling the effective inter-residue interactions, either at interfaces, or within single monomers. Based on this observation, a reduced set of energy parameters comprising 20 one-body and 3 two-body terms is proposed (as opposed to the 20 x 20 tables of inter-residue potentials), which reproduces the conventional 20 x 20 tables with a correlation coefficient of 0.99.     

A motor neuron-specific epitope and the low-affinity nerve growth factor receptor display reciprocal patterns of expression during development, axotomy, and regeneration

Somatic motor neurons begin to express the transmitter synthesizing enzyme, choline acetyltransferase (ChAT) and the low-affinity nerve growth factor receptor (NGFR) during embryonic development. However, as motor neurons mature in postnatal life, they lose immunoreactivity for NGFR and acquire a motor neuron-specific epitope that is recognized by the monoclonal antibody, MO-1. The present study was undertaken to examine the effect of nerve injury in adult rats on these three developmentally regulated markers in two populations of somatic motor neurons. Unilateral transection, ligation, or crushing of the sciatic nerve resulted in a loss of MO-1 binding and a concomitant rise in immunoreactivity for NGFR within axotomized motor neurons in lumbar levels of the spinal cord. These changes, detectable within 5 days following nerve injury, are reversed with reinnervation, but persist if reinnervation is prevented by chronic axotomy. Thus, regulation of the expression of NGFR and the MO-1 epitope appears to be critically dependent upon interactions between motor neurons and target muscles. These observations are also consistent with the idea that during regeneration, neurons may revert to a developmentally immature state; in motor neurons, this state is characterized by the presence of NGFRs and the absence of the MO-1 epitope. Transection of the hypoglossal nerve, a purely motor nerve, resulted in a similar loss of MO-1 binding and a selective rise in NGFR immunoreactivity in neurons within the ipsilateral hypoglossal motor nucleus. In addition, immunoreactivity for ChAT was also lost in axotomized hypoglossal motor neurons. In contrast, injury to the sciatic nerve, which bears both sensory and motor axons, did not result in any detectable change in ChAT immunoreactivity in spinal motor neurons.     

Human glutamate dehydrogenase is immunologically distinct from other mammalian orthologues

Five monoclonal antibodies (mAbs) that recognize human glutamate dehydrogenase (GDH) have been selected and designated as monoclonal antibodies hGDH60-6, hGDH60-8, hGDH63-10, hGDH63-11, and hGDH91-14. A total of five mAbs recognizing different epitopes of the enzyme were obtained, two of which inhibited human GDH activity. When total proteins of human homogenate separated by SDS- PAGE, were probed with mAbs, a single reactive protein band of 55 kDa, which co-migrated with purified recombinant human GDH was detected. When the purified GDH was incubated with each of the mAbs, its enzyme activity was inhibited by up to 58%. Epitope mapping analysis identified, two subgroups of mAbs recognizing different peptide fragments. Using the individual anti-GDH antibodies as probes, the cross reactivities of brain GDH obtained from human and other animal brain tissues were investigated. For the human and animal tissues tested, immunoreactive bands on Western blots appeared to have the same molecular mass of 55 kDa when hGHD60-6, hGHD60-8, or hGHD91-14 mAbs were used as probes. However, the anti-human GDH mAbs immunoreactive to bands on Western blots reacted differently on the immunoblots of the other animal brains tested, i.e., the two monoclonal antibodies hGDH63-10 and hGDH63-11 only produced positive results for human. These results suggest that human brain GDH is immunologically distinct from those of other mammalian brains. Thorough characterization of these anti-human GDH mAbs could provide potentially valuable tool as immunodiagnostic reagents for the detection, identification and characterization of the various neurological diseases related to the GDH enzyme.     

Stress-induced tau phosphorylation in mouse strains with different brain Erk 1 + 2 immunoreactivity

BACKGROUND: To our knowledge, the contribution of prothrombotic conditions to cerebral thromboembolism has never been prospectively studied in a large series of pediatric patients. METHODS: The Hospital for Sick Children, Toronto, Ontario, established a program in January 1992 to diagnose and treat children (term newborn to 18 years old) with arterial ischemic stroke or sinovenous thrombosis. The routine evaluation for prothrombotic conditions included plasminogen, antithrombin, protein C, free protein S, activated protein C resistance, IgG and IgM anticardiolipin antibody, and lupus anticoagulant. We analyzed samples taken within 2 years of the event. We report results on patients seen from January 1, 1992, to January 1, 1997. RESULTS: Ninety-two patients (47 males and 45 females) entered the program during the study interval. Patients ranged from newborn to 18 years in age. Arterial ischemic stroke occurred in 78% of patients while sinovenous thrombosis occurred in 22%. All were tested for prothrombotic disorders. One or more abnormal results were present in 35 (38%) of the 92 patients. The majority (21/35) had multiple abnormal test results. The abnormal test results were anticardiolipin antibody (33%), plasminogen (9.5%), activated protein C resistance (9%), protein C (7%), antithrombin (12.5%), lupus anticoagulant (8%), and free protein S (11.5%). Male sex predicted the presence of prothrombotic abnormalities (relative risk, 1.7; 95% confidence interval, 1.2-2.5), but stroke type (relative risk, 0.8; 95% confidence interval, 0.7-1.1), age group, and presence of other risk factors did not predict abnormal testing. CONCLUSIONS: A significant proportion (38%) of children with cerebral thromboembolism had evidence of prothrombotic conditions. In particular, there was a predominance of children with anticardiolipin antibody (33%). These data support a recommendation that children with cerebral thromboembolism be evaluated for prothrombotic disorders.     

Alcohol-membrane interaction: calcium uptake in mitochondria

A biphasic effect of ethanol was observed on Ca2+-uptake activity, Ca2+-uncoupled respiratory control ratio (CURCR), and State 3 oxygen uptake. At low concentrations (0.1-0.5%) ethanol enhanced these activities, probably as the result of an increase in membrane fluidity. At higher levels (0.7-4.0%) ethanol had an inhibitory influence on mitochondrial activities as the membrane macromolecules were probably disturbed due to the hydrophobic alcohol-membrane interaction.     

Is impaired cerebral vasomotor reactivity a predictive factor of stroke in asymptomatic patients?

BACKGROUND AND PURPOSE: Identification of the subgroup of asymptomatic patients with severe internal carotid artery stenosis and high risk of stroke has important clinical implications. Cerebral vasomotor reactivity provides information regarding intracranial hemodynamic features and might have a prognostic value in predicting cerebrovascular ischemic events, especially in patients with carotid stenosis. The aim of our study was to assess the cerebral vasomotor reactivity in asymptomatic patients with carotid stenosis and evaluate its role in stroke occurrence. METHODS: Cerebral vasomotor reactivity was assessed using transcranial Doppler ultrasonology and the Diamox test (intravenous administration of 1.0 g acetazolamide) in 44 asymptomatic patients with severe (> 70%) internal carotid artery stenosis. Patients were followed up prospectively (mean, 2 years). RESULTS: Cerebral vasomotor reactivity was estimated as good (> 40% increase of blood flow velocity in the middle cerebral artery ipsilateral to the carotid stenosis after undergoing the Diamox test) in 23 patients; it was impaired in the other 21. During the follow-up period, the overall annual rate for ipsilateral stokes was 2.3%; it was 7.9% for all ischemic cerebral events. No strokes or transient ischemic attacks occurred in the former group, but there were 7 cerebral ischemic events (2 strokes [1 fatal] and 5 transient ischemic attacks) in the latter group. There was a statistically significant correlation between cerebral ischemic events and impaired cerebral vasomotor reactivity (P = .009). CONCLUSIONS: The data of this preliminary study suggest an important role of impaired cerebral vasomotor reactivity in predicting ischemic cerebral events. Preventive vascular surgery might be considered in this high-risk subgroup of asymptomatic patients with severe carotid stenosis.