Truncated trkB receptors on nonneuronal cells inhibit BDNF-induced neurite outgrowth in vitro

The function of truncated trkB receptors during nervous system plasticity and regeneration is currently unknown. The extensive nonneuronal localization of truncated trkB-T1 receptors, coupled with their up-regulation by CNS glial cells in response to injury, has led to the speculation that these receptors may sequester BDNF and NT-4/5 to reduce their local availability and, thus, limit axonal sprouting. Conversely, trkB-T1 receptors could bind and present neurotrophins to injured axons and facilitate their regeneration in a manor analogous to that proposed for p75(NTR) receptors on Schwann cells. To address this issue, we used an in vitro coculture paradigm in which wild-type 3T3 NIH fibroblasts or two different 3T3 cell clones stably expressing trkB-T1 receptors served as monolayer substrates upon which to evaluate the effect of trkB-T1 receptors on nonneuronal cells to influence neurotrophin (NGF, BDNF, NT-3, and NT-4/5)-induced neurite outgrowth from retinoic acid (RA)-treated SY5Y neuroblastoma cells. In these experiments, BDNF and NT-4/5 produce a strong phosphorylation of trk receptors on the RA-SY5Y cells and induce differentiation of the SY5Y cells (as measured by the development of neurofilament-positive neuritic processes). This ability of the trkB ligands to stimulate neurite outgrowth is dose dependent since increasing concentrations of BDNF (5, 25, and 100 ng/ml) result in an increased percentage of SY5Y cells developing neurites and in progressively longer neurites from SY5Y cells on the control 3T3 monolayers. In these experiments, BDNF and NT-4/5 induce the strongest neurite outgrowth, followed by NT-3 and then NGF. When trkB-T1 receptors are present on the 3T3 cell substratum both BDNF- and NT-4/5-induced neurite extension from the SY5Y cells are strongly inhibited. In contrast, NGF-induced neurite growth is unaffected and NT-3-associated growth is somewhat reduced. These results suggest that the inhibitory effect of the trkB-T1 receptors on the nonneuronal cell substrates is selective for neurite outgrowth that is mediated via the trkB-kinase receptors on the neuroblastoma cells. This ability of trkB-T1 receptors on the nonneuronal substratum to inhibit BDNF-induced neurite outgrowth can be overcome by the addition of high concentrations of BDNF (1 microg/ml). Binding assays using 125I-BDNF suggest that this inhibitory effect could be mediated via binding and internalization of BDNF by the trkB-T1 receptors on the 3T3 cells. These results provide strong support for the hypothesis that the up-regulation of trkB-T1 receptors on astrocytes following CNS lesions enhances the sequestration of the trkB ligands, BDNF and NT- 4/5, at the site of reactive gliosis and, thus, contributes to the inhibition of CNS axonal regeneration from neurons expressing trkB-kinase receptors by removing their ligands from the extracellular environment.     

Induction of biologically active IL-1 beta-converting enzyme and mature IL-1 beta in human keratinocytes by inflammatory and immunologic stimuli

IL-1beta, a major mediator of inflammatory and immunologic skin disease, undergoes post-translational site-specific cleavage by a novel cysteine protease termed IL-1beta-converting enzyme (ICE). Although in human skin keratinocytes produce significant amounts of the 31-kDa IL-1beta precursor protein, they fail under nonpathologic conditions to convert it to the 17.5-kDa bioactive form. In this study, we examined whether haptens and inflammatory agents might serve as stimuli for ICE activity in human keratinocytes, and, if so, whether ICE activity might precipitate enzymatic processing of IL-1beta to its 17.5-kDa form. Baseline levels of ICE mRNA were detected in keratinocyte cultures devoid of Langerhans cells and were up-regulated by nontoxic concentrations of the reactive hapten urushiol and by the irritant chemicals sodium lauryl sulfate and PMA. Although untreated keratinocytes expressed the 31-kDa form of the protein, 17.5-kDa IL-1beta was easily detected in keratinocytes and keratinocyte supernatants treated with either urushiol or the irritant chemicals. Enzymatic conversion from the 31-kDa to the 17.5-kDa form of IL-1beta was blocked by addition of a highly specific aldehyde inhibitor that contained a tetrapeptide recognition sequence specific for ICE, but not by an aldehyde inhibitor of a related ICE-like cysteine protease. Induction of IL-1beta-converting enzyme by immunologic and inflammatory stimuli may be one of the key regulatory elements in the pathogenesis of allergic and irritant contact hypersensitivity.     

A prospective study of trephined bone grafts of the tibial shaft and iliac crest

BACKGROUND: Our purpose was to analyze the expression of Fas antigen on CD4+ lymphocytes in the aqueous humor (AH) and cerebrospinal fluid (CSF) of patients with Vogt-Koyanagi-Harada disease (VKH). METHODS: Using three-color flow cytometry, we assessed T-lymphocyte subsets stained with fluorescence-conjugated anti-CD3, CD4, CD8, CD29, CD45RA, CD45RO, HLA-DR, and Fas monoclonal antibodies in AH, CSF and peripheral blood (PB) from 8 patients with active VKH. RESULTS: CD3+ T cells constituted the majority of lymphocytes in AH and CSF, in contrast to with PB. The percentages of CD4+ lymphocytes in uveitic AH and CSF were significantly higher than that in PB (P < 0.01). Activated CD4+ and CD8+ cells were significantly more frequent in AH than in CSF and PB (P < 0.01). Although the percentages of CD45RA+ cells within CD4+ cells in AH and CSF were extremely low compared with those in PB, the proportions of CD29+ and CD45RO+ (memory) cells within CD4+ were much higher than those in PB (P < 0.01). Fas antigen was also highly expressed on such CD4+ cells in AH, as in other uveitis patients and on such cells in CSF. Moreover, the percentages of Fas+ and memory cells in AH were significantly higher than those in CSF. CONCLUSIONS: The majority of CD4+ lymphocytes in AH and CSF from patients with active VKH were activated memory cells, on which Fas antigen was also highly expressed. Although this Fas expression may not be an apoptosis-related phenomenon, accumulation of Fas+ memory T lymphocytes in AH and CSF probably reflects the immunopathologic mechanism of VKH.     

The effects of knee reconstruction on combined anterior cruciate ligament and anterolateral capsular deficiencies

We tested the effect of intraarticular reconstructions of the anterior cruciate ligament alone and in combination with extraarticular reconstructions in 10 cadaveric knees. These knees had anterior cruciate ligament deficiency alone or in combination with anterolateral capsuloligamentous deficiencies. In the knees with combined injury, intraarticular reconstruction returned anterior stability to levels not significantly different from levels found for the knees deficient in the anterior cruciate ligament alone and treated with this procedure. After intraarticular reconstruction, rotational stability of the knee with combined injuries failed to return to the levels seen in the knee with isolated anterior cruciate ligament deficiencies that underwent the same treatment. When a tenodesis with either 0 N or 22 N of tension was added to the intraarticular reconstruction in the knee with combined injuries, we found that excessive internal rotation significantly decreased at all angles of flexion, except at full extension with 0 N of tension. In addition, the extraarticular reconstruction with 22 N of tension in the tenodesis overconstrained the knee in internal rotation between 30 degrees and 90 degrees of knee flexion. The tenodesis with 0 N of tension overconstrained the knee at only 60 degrees and 90 degrees of flexion. These results suggest extraarticular reconstruction as an adjunct to the intraarticular operation for the knee with anterior cruciate ligament and anterolateral structural injuries. The results also suggest that the surgeon can affect anterior and rotational laxity by adjusting the tension in the tenodesis.     

Intimal hyperplasia thickness at follow-up is independent of stent size: a serial intravascular ultrasound study

The purpose of this study was to determine whether the thickness of the intimal hyperplasia (IH) layer that accumulates within Palmaz-Schatz stents is dependent on stent size. Intravascular ultrasound (IVUS) and quantitative angiographic (QCA) studies were performed after stent implantation and at follow-up (5.4 +/- 3.8 months) in 161 patients with 177 lesions treated with 221 Palmaz-Schatz stents. Stent and lumen cross-sectional area (CSA) were measured. IH CSA and thickness at follow-up were calculated and compared with stent CSA and circumference. Maximum IH CSA and thickness were measured at the smallest follow-up lumen CSA; mean IH CSA and thickness was averaged over the length of the stent. Maximum IH CSA measured 4.8 +/- 2.4 mm2, and mean IH CSA measured 2.8 +/- 2.2 mm2. Maximum IH thickness (at the smallest follow-up lumen CSA) measured 0.60 +/- 0.36 mm, and mean IH thickness (over the length of the stent) measured 0.30 +/- 0.19 mm. There was a weak, but significant correlation between mean and maximum IH CSA versus stent CSA (r = 0.215, p <0.0001 and r = 0.355, p <0.0001, respectively). However, there was no correlation between mean or maximum IH thickness versus stent CSA (r = 0.018, p = 0.643 and r = 0.056, p = 0.463, respectively) or stent circumference (r = 0.002, p = 0.956 and r = 0.069, p = 0.361, respectively). IH thickness was found to be independent of the stent size. This explains the known higher frequency of restenosis in smaller stents compared with larger stents.     

Immunization with synthetic peptide segments of a sperm protein impair fertility in rats

The nucleotide sequence of the cDNA encoding a sperm protein (rSMP-B) was determined in a previous study. Two peptide segments corresponding to the extracellular domain of the deduced sperm polypeptide were synthesized as multiple antigen peptide (MAP) and designated as rSMP-229 and rSMP-230. Polyclonal antibodies were raised against the two MAPs. Sera obtained from rabbits immunized with rSMP-230 interacted with human and rabbit sperm membrane proteins with estimated molecular sizes of 72 and 20.1 kD, respectively. Adult female and male rats were immunized with the MAPs and their fertilities determined. Immunization of female rats with rSMP-229 and rSMP-230 induced infertility in 25% and 83% of the treated animals, respectively. All male rats immunized with rSMP-229 remained fertile; whereas animals immunized with rSMP-230 did not mate with normal cycling female rats. Three impotent male rats were found to regain their mating potency 45 days after the last booster injection. These findings demonstrated that immunization with rSMP-230 induced a reversible impotency in male rats. Serum testosterone and LH levels were reduced in rSMP-230-immunized male rats and were elevated in rSMP-229-immunized animals. Histopathological examination of sections of testes from male rats immunized with rSMP-230 showed impairment of spermatogenesis and sloughing of germ cells into the lumen of the seminiferous tubules. The testes of male rats immunized with rSMP-229 showed normal morphology and active spermatogenesis with scattered foci of nodular hyperplasia of Leydig cells in the interstitial areas. In conclusion, immunization with synthetic peptide segments corresponding to different domains of a deduced sperm protein induced infertility in a significant number of female rats and transient impotency in male rats.     

Genetic relationship between soxRS and mar loci in promoting multiple antibiotic resistance in Escherichia coli

Multiple antibiotic resistance in Escherichia coli has typically been associated with mutations at the mar locus, located at 34 min on the E. coli chromosome. A new mutant, marC, isolated on the basis of a Mar phenotype but which maps to the soxRS (encoding the regulators of the superoxide stress response) locus located at 92 min, is described here. This mutant shares several features with a known constitutive allele of the soxRS gene, prompting the conclusion that it is a highly active allele of this gene. The marC mutation has thus been given the designation soxR201. This new mutant was used to examine the relationship between the mar and sox loci in promoting antibiotic resistance. The results of these studies indicate that full antibiotic resistance resulting from the soxR201 mutation is partially dependent on an intact mar locus and is associated with an increase in the steady-state level of mar-specific mRNA. In addition, paraquat treatment of wild-type cells is shown to increase the level of antibiotic resistance in a dose-dependent manner that requires an intact soxRS locus. Conversely, overexpression of MarA from a multicopy plasmid results in weak activation of a superoxide stress response target gene. These findings are consistent with a model in which the regulatory factors encoded by the marA and soxS genes control the expression of overlapping sets of target genes, with MarA preferentially acting on targets involved with antibiotic resistance and SoxS directed primarily towards components of the superoxide stress response. Furthermore, compounds frequently used to induce the superoxide stress response, including paraquat, menadione, and phenazine methosulfate, differ with respect to the amount of protection provided against them by the antibiotic resistance response.