Opposing contributions of NR1 and NR2 to protein kinase C modulation of NMDA receptors

N-Methyl-D-aspartate (NMDA) receptors mediate increases in intracellular calcium that can be modulated by protein kinase C (PKC). As PKC modulation of NMDA receptors in neurons is complex, we studied the effects of PKC activation on recombinant NMDA receptor-mediated calcium rises in a nonneuronal mammalian cell line, human embryonic kidney 293 (HEK-293). Phorbol 12-myristate 13-acetate (PMA) pretreatment of HEK-293 cells enhanced or suppressed NMDA receptor-mediated calcium rises based on the NMDA receptor subunit composition. NR2A or NR2B, in combination with NR1(011), conveyed enhancement whereas NR2C and NR2D conveyed suppression. The PKC inhibitor bisindolylmaleimide blocked each of these effects. The region on NR2A that conveyed enhancement localized to a discrete segment of the C terminus distal to the portion of NR2C that is homologous to NR2A. Calcium-45 accumulation, but not intracellular calcium store depletion, matched PMA effects on NMDA receptor-mediated calcium changes, suggesting that these effects were not due to effects on intracellular calcium stores. The suppression of intracellular calcium transients seen with NR2C was eliminated when combined with NR1 splice variants lacking C-terminal cassette 1. Thus, the intracellular calcium effects of PMA were distinguishable based on both the NR1 splice variant and the NR2 subunit type that were expressed. Such differential effects resemble the diversity of PKC effects on NMDA receptors in neurons.     

Primary blasts from infants with acute lymphoblastic leukemia cause overt leukemia in SCID mice

The establishment of an in vivo animal model system for infant acute lymphoblastic leukemia (ALL) would allow the testing of new agents against primary leukemic cells from infant ALL patients. We have demonstrated previously that growth of B-lineage leukemic cells in mice with severe combined immunodeficiency (SCID) was a significant prognostic factor for children with high risk ALL. We now have examined the significance of this prognostic variable for 13 infants with newly diagnosed ALL treated at participating institutions of the Children's Cancer Group (CCG). Chromosomal translocations were detected in 10/12 evaluated cases, including five with t(4;11), one each with t(7;9) and t(7;11), t(1;19), and t(9;22), and two with t(11;19). Twelve of the thirteen infants with ALL achieved remissions following induction chemotherapy. Primary leukemic cells from 8 of the 13 infants caused overt leukemia in SCID mice. Among these 8 SCID+ infants, 7 were CD10- and seven had cytogenetic or molecular evidence of an 11q23 rearrangement. Six of the 8 SCID+ infants have relapsed; only 2 remain in remission following chemotherapy or bone marrow transplant. However, among the 5 SCID- infants there were also two relapses. These data are suggestive of a poorer outcome for SCID+ infants, but larger numbers of patients must be analyzed to assess their statistical significance. In summary, we have established a SCID mouse model for human infant ALL that will be useful for 1) predicting short-term and long-term outcome of patients, 2) testing pharmacokinetics, efficacy, and toxicity of new agents, and 3) elucidating the in vivo mechanisms of chemotherapeutic drug resistance in infant ALL.     

How to evaluate suspected immunodeficiency

The periacetabular osteotomy is a strategy for the treatment of residual hip dysplasia in young adults with the aim of preventing secondary coxarthrosis. This polygonal, juxta-articular osteotomy respects the vascular blood supply to the acetabular fragment and thus facilitates an extensive acetabular reorientation with improvement of the insufficient coverage of the femoral head, including medial displacement of the fragment. All osteotomies are performed by one approach, the modified Smith-Petersen, which allows an anterior capsulotomy. Inspection of the joint not only provides information on acetabular rim pathologies, but also facilitates the control of impingement-free range of motion after the correction. The posterior column remains partially intact, allowing minimal internal fixation of the acetabular fragment and early mobilization similar to that after an intertrochanteric osteotomy. The dimensions of the true pelvis are unchanged, providing the capacity to have an unimpeded delivery in women. This paper describes the preoperative evaluation, current indications, surgical technique, postoperative care, and the results of this osteotomy.     

Conditioning heat stress reduces excitotoxic and apoptotic components of oxygen-glucose deprivation-induced neuronal death in vitro

Both accidental and experimental lesions of the spinal cord suggest that neuronal processes occurring in the spinal cord modify the relay of information through the dorsal column-lemniscal pathway. How such interactions might occur has not been adequately explained. To address this issue, the receptive fields of mechanosensory neurons of the dorsal column nuclei were studied before and after manipulation of the spinal dorsal horn. After either a cervical or lumbar laminectomy and exposure of the dorsal column nuclei in anesthetized cats, the representation of the hindlimb or of the forelimb was defined by multiunit recordings in both the dorsal column nuclei and in the ipsilateral spinal cord. Next, a single cell was isolated in the dorsal column nuclei, and its receptive field carefully defined. Each cell could be activated by light mechanical stimuli from a well-defined cutaneous receptive field. Generally the adequate stimulus was movement of a few hairs or rapid skin indentation. Subsequently a pipette containing either lidocaine or cobalt chloride was lowered into the ipsilateral dorsal horn at the site in the somatosensory representation in the spinal cord corresponding to the receptive field of the neuron isolated in the dorsal column nuclei. Injection of several hundred nanoliters of either lidocaine or cobalt chloride into the dorsal horn produced an enlargement of the receptive field of the neuron being studied in the dorsal column nuclei. The experiment was repeated 16 times, and receptive field enlargements of 147-563% were observed in 15 cases. These data suggest that the dorsal horn exerts a tonic inhibitory control on the mechanosensory signals relayed through the dorsal column-lemniscal pathway. Because published data from other laboratories have shown that receptive field size is controlled by signals arising from the skin, we infer that the control of neuronal excitability, receptive field size and location for lemniscal neurons is determined by tonic afferent activity that is relayed through a synapse in the dorsal horn. This influence of dorsal horn neurons on the relay of mechanosensory information through the lemniscal pathways must modify our traditional views concerning the relative independence of these two systems.     

Lipoprotein lipase greatly enhances the retention of lipoprotein(a) to endothelial cell-matrix

Proinflammatory cytokines exert a number of important effects on vascular reactivity. At one end of the spectrum, certain cytokines may induce vascular paresis leading to profound vasodilatation and hyporesponsiveness to constrictor stimuli. This may be relevant to the pathogenesis of septic shock and other types of inflammatory vasodilatation. At the other end of the spectrum, inflammatory cytokines can impair endothelium-dependent dilatation and the endothelium may lose its ability to respond to circulating hormones or autacoids. This effect may case a predisposition to vessel spasm, thrombosis or atherogenesis. Studies in human vessels suggest that interleukin-1 is particularly important as a mediator of inflammatory dilatation; the underlying mechanisms include induction of the inducible isoform of nitric oxide synthase in vascular smooth muscle, or over-production of nitric oxide from the endothelial isoform of nitric oxide synthase. Induction of the enzyme GTP cyclohydrolase 1 and consequent production of tetrahydrobiopterin contributes to the increase in the activity of endothelial nitric oxide synthase. In contrast, tumour necrosis factor-alpha considerably impairs endothelium-dependent relaxation. The mechanisms of these effects are not yet fully understood, but tumour necrosis factor can induce endothelial dysfunction in human endothelial cells in culture, and human blood vessels in vitro and in vivo. The implications of these observations for cardiovascular disease are discussed.     

Antagonists of the platelet P2T receptor: a novel approach to antithrombotic therapy

The platelet P2T receptor plays a major role in platelet aggregation, and its antagonists are predicted to have significant therapeutic potential as antithrombotic agents. We have explored analogues of adenosine triphosphate (ATP), which is a weak, nonselective but competitive P2T receptor antagonist. Modification of the polyphosphate side chain to prevent breakdown to the agonist adenosine diphosphate (ADP) and substitution of the adenine moiety to enhance affinity and selectivity for the P2T receptor led to the identification of 10e (AR-C67085MX), having an IC50 of 2.5 nM against ADP-induced aggregation of human platelets. Compound 10e was the first very potent antagonist of the P2T receptor, with a selectivity for that subtype of the P2 receptor family of >1000-fold. Further modification of the structure produced compound 10l (AR-C69931MX) having an IC50 of 0.4 nM. In vivo, at maximally effective antithrombotic doses, there is little prolongation of bleeding time (1.4-fold), which is in marked contrast to the 5-6-fold found with GPIIb/IIIa antagonists.     

Identification of a homozygous deletion at 8p12-21 in a human prostate cancer xenograft

Endothelial-monocyte activating polypeptide II (EMAP II) and allograft-inflammatory factor-1 (AIF-1) are two proteins produced by activated monocytes and microglial cells. We now report expression of these factors during experimental therapy of rat neuroautoimmune diseases. Comparative analysis of two therapeutic strategies, treatment with high doses of recombinant autoantigens or with dexamethasone, revealed unexpected differences. High doses of autoantigen were most effective in experimental autoimmune encephalomyelitis and neuritis (EAE and EAN), but less effective in experimental autoimmune uveitis (EAU). Low and high doses of dexamethasone treatment greatly reduced the severity of EAE, EAN and EAU at day 11, but a relapse was observed between days 21 and 26. Only rather limited expression of EMAP II and AIF-1 is seen in the normal central nervous system (CNS). This constitutive expression is not abolished by dexamethasone treatment. In inflammatory autoimmune lesions of the rat CNS, prominent AIF-1 and EMAP II staining was seen with macrophages and monocytes. In particular, parenchymal microglial cells were now activated to express AIF-1 and EMAP II. In accordance with prevention of neurological signs, histological observations revealed that accumulation of activated monocytes expressing EMAP II and AIF-1 in the CNS or peripheral nervous system and the massive expression of these factors by parenchymal microglial cells is inhibited by high doses of autoantigen. Dexamethasone prevented or abolished local expression of EMAP II and AIF-1 at days 10-16. However, an acute and severe relapse occurred in encephalomyelitis between days 20-26. In these cases, a smoldering expression of EMAP II and AIF-1 persisting long after cessation of neurological signs was observed. Thus, expression of EMAP II and AIF-1 by infiltrating activated macrophages is a marker of disease activity and expression of these factors could be used to demonstrate 'silent' lesions in the CNS and prolonged microglial cell activation. Apparently, AIF-1 and EMAP II immunoreactivity are tools to stage activation of monocytes and microglial cells in inflammatory lesions.     

Epidemiology training for primary health care: the use of computer-assisted distance learning

The Liverpool Epidemiology Programme, based in the Liverpool School of Tropical Medicine, has designed a series of computer-based modules for use in distance learning. The purpose of this paper is to highlight the role of computers in training health workers in epidemiology in developing countries. The aim of the modules is to provide health workers with solutions to problems which they face in their everyday work. The modules are written in hypertext software for IBM compatible machines and interact with the epidemiological software Epi Info. Four modules are described: LEP-Nut which deals with nutritional surveillance, LEP-Ref which looks at the role of epidemiology within a refugee health care programme, LEP-Surv dealing with health surveillance and LEP-Rap which introduces the concept of rapid appraisal. They are also easily distributed, particularly with the development of the Internet. The modules are carefully evaluated before and after distribution. Issues related to their evaluation and subsequent revision are discussed, in particular is the content important, adequate, communicated and useful? A major advantage of computer-based learning materials is that they can be easily updated with new advancements of knowledge and experience from the field.     

Characterization of a novel simian immunodeficiency virus (SIV) from L'Hoest monkeys (Cercopithecus l'hoesti): implications for the origins of SIVmnd and other primate lentiviruses

The human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) appear to have originated by cross-species transmission of simian immunodeficiency virus (SIV) from asymptomatically infected African primates. Few of the SIVs characterized to date efficiently infect human primary lymphocytes. Interesting, two of the three identified to infect such cultures (SIVsm and SIVcpz) have appeared in human populations as genetically related HIVs. In the present study, we characterized a novel SIV isolate from an East African monkey of the Cercopithecus genus, the l'hoest monkey (C. l'hoesti), which we designated SIVlhoest. This SIV isolate efficiently infected both human and macaque lymphocytes and resulted in a persistent infection of macaques, characterized by high primary virus load and a progressive decline in circulating CD4 lymphocytes, consistent with progression to AIDS. Phylogenetic analyses showed that SIVlhoest is genetically distinct from other previously characterized primate lentiviruses but clusters in the same major lineage as SIV from mandrills (SIVmnd), a West African primate species. Given the geographic distance between the ranges of l'hoest monkeys and mandrills, this may indicate that SIVmnd arose through cross-species transmission from close relatives of l'hoest monkeys that are sympatric with mandrills. These observations lend support to the hypothesis that the primate lentiviruses originated and coevolved within monkeys of the Cercopithecus genus. Regarded in this light, lentivirus infections of primates not belonging to the Cercopithecus genus may have resulted from cross-species transmission in the not-too-distant past.