Hemoglobin stimulates mononuclear leukocytes to release interleukin-8 and tumor necrosis factor alpha

Incubation of human mononuclear leukocytes (MNL) with human stroma-free hemolysate (SFH), purified adult hemoglobin Ao (HbAo), and oxidized HbAo (METHb) caused MNL to release compounds into the supernate that mediated neutrophil (polymorphonuclear leukocytes, PMN) chemotaxis and PMN adherence to human umbilical vein endothelial cells (HUVEC). Chemotaxis and PMN adherence to HUVEC were reduced significantly when supernates were preincubated with neutralizing antibodies to interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-alpha), respectively, suggesting that IL-8 and TNF-alpha played significant roles in mediating these activities. Greatest chemotactic activity was observed in supernates of MNL treated with HbAo; while greatest PMN/endothelial cell (EC) adherence activity was observed in supernates of MNL treated with METHb. Furthermore, PMN/EC adherence activity was a function of METHb content in each hemoglobin solution. PMN chemotaxis, PMN adherence to HUVEC, and cytokine release increased as a function of increasing incubation time. Chemotactic activity was detected in HbAo- treated and METHb-treated MNL supernates after incubation for 6 hours and was maximal by 10 hours. IL-8 was detected in both HbAo and METHb- MNL supernates by 4 hours. PMN/EC adherence activity was detected in HbAo-MNL supernates at 10 hours and in METHb-MNL supernates at 4 hours. TNF-alpha was detected in METHb and HbAo-MNL supernates at 4 and 12 hours, respectively. These results suggest that hemoglobin solutions stimulate MNL to release IL-8 and TNF-alpha in quantities sufficient to induce PMN chemotaxis and PMN adherence to HUVEC. This is a US government work. There are no restrictions on its use.     

Prognostic implications of cytogenetic studies in an intensively treated group of children with acute lymphoblastic leukemia

We assessed the prognostic significance of leukemia cell cytogenetics by analyzing bone marrow aspirates obtained at time of diagnosis in 165 children on a single protocol for acute lymphoblastic leukemia (ALL). These children were assigned to six mutually exclusive cytogenetic categories as follows: (1) hyperdiploid, with 50 or more chromosomes (n = 35); (2) hyperdiploid, with 47 to 49 chromosomes (n = 11); (3) diploid (n = 42); (4) pseudodiploid (n = 34); (5) hypodiploid (n = 9); and (6) insufficient data (n = 34). At a median follow-up of 5 years, there were no statistically significant differences between any of these cytogenetic categories in either event-free or overall survival. Those children with chromosomal translocations (n = 26) appeared to fare the same as those lacking translocations (n = 105). The absence of karyotypic prognostic significance was observed not only within the overall group, but also when the results were stratified by standard- risk and high-risk status. Of the specific structural chromosome changes that we studied, only the Philadelphia chromosome (Ph) appeared to confer a poor prognosis, although there were too few such cases to achieve statistical significance. Although we did not detect the event- free survival differences that have been described previously in hyperdiploid, hypodiploid, and pseudodiploid childhood ALL, our findings must be viewed as preliminary given the small number of children in some of the cytogenetic categories. We think that the prognostic implications of these cytogenetic features might have been nullified by improvements in therapy.     

The sequential analysis of T lymphocyte subsets and interleukin-6 in polymyalgia rheumatica patients as predictors of disease remission and steroid withdrawal

CD4 and CD8 T lymphocyte subsets, the late T cell activation marker, HLA-DR, and serum interleukin-6 (IL-6) levels of 57 polymyalgia rheumatica (PMR) patients were followed over 2 yr to investigate whether they could be used to predict the safe withdrawal of steroid therapy. Cell phenotypes were studied by flow cytometry and IL-6 levels by ELISA. %CD8 cells were reduced below the normal range in PMR patients prior to steroid therapy. In 56% of patients, the %CD8 T lymphocytes failed to return to normal levels when quiescent disease allowed cessation of steroid therapy. Activated CD8 T cells, as detected by HLA-DR positivity, were above the normal range at the initiation of therapy and showed a negative correlation with %CD8 T cells. The serum concentration of IL-6 fluctuated over 24 months, and the correlation between IL-6 and erythrocyte sedimentation rate (ESR) seen prior to treatment was not seen at later intervals. The %CD8 T cell and serum IL-6 levels are not a good indicator of disease activity in PMR and are, therefore, unable to predict the safe withdrawal of steroids.      

INADEQUACY OF ACTIVE IMMUNIZATION ALONE IN PREVENTION OF RABIES (A Case Report)

A case of dog bite where prompt immunization with antirabies vaccine alone proved inadequate in prevention of fatal rabies is reported to emphasize upon the need of concomitant passive immunization with either human rabies immune globulin or equine antiserum.KEY WORDS: Rabies, Active immunization, Passive immunization     

Neurotrophin-3 down-regulates trkA mRNA, NGF high-affinity binding sites, and associated phenotype in adult DRG neurons

Neurotrophin-3 (NT-3) binds to multiple trks, not only its initially identified receptor trkC. Recent studies in our laboratory show that NT-3 negatively regulates nociceptive phenotype associated with the trkA subpopulation. Due to the extensive overlap in trkA and trkC expression it is uncertain whether there is a direct influence of NT-3 on trkA in adult sensory neurons. Thus, the aim of this study was to examine whether NT-3 might alter trkA and associated neuronal phenotype outside of the trkC subpopulation. The effect of a seven-day intrathecal infusion of NT-3 on intact, uninjured adult rat dorsal root ganglion neurons was investigated. Serial sections were processed for receptor radioautography or in situ hybridization to identify and colocalize neurons expressing high-affinity nerve growth factor (NGF) binding sites, substance P (SP), trkC, or trkA mRNAs and to examine the influence of NT-3 on these populations. NT-3 does not appear to alter trkC expression, but evokes a notable reduction in trkA, high-affinity NGF binding sites, and SP levels. It is unlikely that signalling by trkC greatly influences this response because the down-regulation of SP occurs most notably in trkA neurons that lack trkC. Moreover, we have shown here that message levels of two trkA isoforms are differentially modulated by NT-3; infusion results in greater down-regulation of the noninsert containing isoform. These findings suggest a clinically relevant role for NT-3 as an antagonist to NGF, but also raise the caution that not just trkC-positive neurons are influenced following exposure to the neurotrophin.     

Effect of axial loading on neural foramina and nerve roots in the lumbar spine

The hypothesis that the neural foramina in some patients are critically narrowed by axial compression of the spine has not been studied with direct imaging techniques. Frozen cadaveric motion segments of the lumbar spine (intervertebral disk and contiguous vertebrae) were imaged with computed tomography (CT). The segments were thawed and compressed in a hydrostatic press to simulate axial loading, and then the segments were frozen and imaged again. The motion segments were subsequently sectioned with a cryomicrotome, and the chronic degenerative changes present in the disks were classified. Pre- and post-compression CT images were compared, and anatomic relationships were studied. In 41 randomly selected segments (some with preexisting radial, transverse, and concentric annular tears), compression diminished the diameters and cross-sectional areas of the spinal canal and neural foramina. In no cases were nerve roots displaced, distorted, or compressed by axial loading. This study suggests that axial loading, such as that produced by ordinary weight bearing, does not critically compromise the neural foramina even in the presence of chronic degenerative disk changes.     

Ex vivo and in situ PLGA microspheres uptake by pig ileal Peyer's patch segment

We investigated the ability of pig ileal Peyer's patch segments to transport intestinal poly (D,L-lactide-co-glycolide) microspheres (PLGA MS) from intestinal lumen across the mucosae using in situ and ex vivo segments with confocal laser scanning microscopy (CLSM) and transmission electronic microscopy (TEM). From a global aspect, CLSM suggested that PLGA MS were translocated by M cells labelled with a FITC-conjugated anti-cytokeratin peptide 18, and transported through the follicle-associated epithelium (FAE) in the dome area in both types of experiments. At the ultrastructural level, TEM showed the traffic of PLGA MS throughout M cells, their transport into the basolateral invaginations of the M cells and their subsequent migration into the dome area and the follicular area in contact with macrophages and lymphatic vessels. Although in situ experiments allowed following the migration of PLGA MS until mesenteric lymph nodes, an ex vivo model could be used as a useful tool to study the targeting ability of PLGA MS formulations to the gut-associated lymphoid tissue (GALT).     

Role of the GLT-1 subtype of glutamate transporter in glutamate homeostasis: the GLT-1-preferring inhibitor WAY-855 produces marginal neurotoxicity in the rat hippocampus

Glutamate is the major excitatory neurotransmitter in the central nervous system and is tightly regulated by cell surface transporters to avoid increases in concentration and associated neurotoxicity. Selective blockers of glutamate transporter subtypes are sparse and so knock-out animals and antisense techniques have been used to study their specific roles. Here we used WAY-855, a GLT-1-preferring blocker, to assess the role of GLT-1 in rat hippocampus. GLT-1 was the most abundant transporter in the hippocampus at the mRNA level. According to [(3)H]-l-glutamate uptake data, GLT-1 was responsible for approximately 80% of the GLAST-, GLT-1-, and EAAC1-mediated uptake that occurs within dissociated hippocampal tissue, yet when this transporter was preferentially blocked for 120 h with WAY-855 (100 microm), no significant neurotoxicity was observed in hippocampal slices. This is in stark contrast to results obtained with TBOA, a broad-spectrum transport blocker, which, at concentrations that caused a similar inhibition of glutamate uptake (10 and 30 microm), caused substantial neuronal death when exposed to the slices for 24 h or longer. Likewise, WAY-855, did not significantly exacerbate neurotoxicity associated with simulated ischemia, whereas TBOA did. Finally, intrahippocampal microinjection of WAY-855 (200 and 300 nmol) in vivo resulted in marginal damage compared with TBOA (20 and 200 nmol), which killed the majority of both CA1-4 pyramidal cells and dentate gyrus granule cells. These results indicate that selective inhibition of GLT-1 is insufficient to provoke glutamate build-up, leading to NMDA receptor-mediated neurotoxic effects, and suggest a prominent role of GLAST and/or EAAC1 in extracellular glutamate maintenance.     

Developmental down-regulation of GAP-43 expression and timing of target contact in rat corticospinal neurons

During early nervous system development axons grow toward the target tissue that they will innervate. As axons invade target tissue, growth slows and ceases. Neurons express high levels of the growth-associated protein GAP-43 during developmental axon growth, declining with maturation. It has been suggested that target contact provides a signal which down-regulates GAP-43 expression. To study this issue in more detail, we used in situ hybridization to quantify relative changes in GAP-43 mRNA in corticospinal tract neurons identified by Fast Blue retrograde labeling. We also used anterograde transport of biotinylated dextran amine to study the invasion of target by corticospinal axons. We find that GAP-43 mRNA is high during the first postnatal week and then declines in two phases. Approximately half of the initial level of GAP-43 expression in corticospinal neurons is lost by P12; then expression remains at a plateau until P21. Between P21 and P28, GAP-43 expression again declines by half and then remains steady at the adult level (one fourth of initial level). Corticospinal axons initially invade spinal gray matter during the first 2 postnatal weeks, in a rostrocaudal gradient. Varicosities suggestive of terminal boutons become numerous during the third and fourth week, and the morphology of corticospinal axon terminals achieves the mature form at the end of the fourth week. These data suggest that the first phase of down-regulation of GAP-43 in corticospinal neurons is coincident with initial target contact and that the second phase is coincident with final maturation of terminal arborization.