Ultrafast CT and the cardiovascular system.

Ultrafast computed tomography (CT) is a new imaging technique that relies on electron beam technology. Its rapid image acquisition speeds make it ideal for evaluating the cardiovascular system. The high-resolution, flow, and cine-modes are unique and provide complimentary information about cardiovascular anatomy, function, and flow dynamics. Ultrafast CT can provide quantitative measurements of cardiac output, ejection fraction, ventricular volumes, and ventricular mass as well as evaluation of segmental cardiac function. This technique can be used to assess coronary artery bypass graft patency as well as to screen for coronary artery calcium. Intracardiac thrombus or tumor, valvular disease, and disorders of the pericardium can be evaluated and characterized using ultrafast CT. The diagnosis of congenital lesions of the heart and great vessels is facilitated by this imaging modality, which can help determine complex anatomic abnormalities and quantitate shunt lesions. Acquired lesions of the great vessels, such as aortic dissection and aneurysm, can be diagnosed by ultrafast CT, which can also be used for serial examination and conservative management.


Macrophage priming by interferon gamma: a selective process with potentially harmful effects.

The tissue-fixed macrophage is a key cellular element in the initiation and regulation of inflammation. Understanding the regulation of macrophage activation may provide valuable clues to the mechanisms involved in both beneficial and deleterious effects of inflammation. The lymphokine interferon-gamma (IFN-gamma) is capable of producing paradoxical immunoinflammatory effects. In the immunocompromised host it up-regulates a variety of immune functions and improves survival, but it is also capable of producing harmful effects by sensitizing immunocompetent animals to subclinical doses of endotoxin. These paradoxical effects suggest that the state of activation or priming of the host immune system is a key determinant of its response to endotoxemia. Because tumor necrosis factor (TNF) and procoagulant activity (PCA) elaboration by the tissue-fixed macrophage play a central role in the host response to endotoxin, we asked whether the paradoxical effects of IFN-gamma may be caused by priming of the macrophage for TNF and/or PCA production. In vitro, IFN-gamma produces a marked augmentation in TNF but does not alter PCA elaboration in response to endotoxin, demonstrating the selectivity of IFN-gamma priming of the macrophage. In vivo, IFN-gamma pretreatment followed by an established subclinical endotoxin exposure enhances toxicity while simultaneously increasing peak serum TNF levels. Exogenous priming by IFN-gamma alters the activation state of the macrophage and modifies the host response to endotoxin. Because this response is also dependent on the host's underlying immune state, IFN-gamma treatment in the immunocompetent host has the potential to produce deleterious effects by eliciting an exaggerated TNF response during endotoxemia.


The gene for human eosinophil Charcot-Leyden crystal protein directs expression of lysophospholipase activity and spontaneous crystallization in transiently transfected COS cells.

Expression of the gene encoding human eosinophil lysophospholipase, the Charcot-Leyden crystal (CLC) protein, was studied in transiently transfected COS cells. Recombinant CLC (rCLC) protein expression was demonstrated both by Western blot and radioimmunoassay inhibition analyses of transfected COS cell extracts and by immunofluorescent staining and ultrastructural immunogold analyses of intact cells. The rCLC protein was immunochemically indistinguishable from native eosinophil-derived CLC protein, and each transfected COS cell expressed approximately 11 pg of rCLC protein as determined by radioimmunoassay and assessment of transfection efficiency. Immunofluorescent microscopy and ultrastructural immunogold analyses localized rCLC protein to the nucleus, cytoplasm, and plasma membrane of COS cells. Lysates from transfected COS cells producing CLC protein expressed significant lysophospholipase activity. Furthermore, rCLC protein expressed in COS cells spontaneously formed the distinctive intracytoplasmic and intranuclear hexagonal bipyramidal crystals characteristic of the native eosinophil and basophil-derived protein. Expression of the CLC gene confirms the authenticity of the CLC cDNA, the expression of lysophospholipase activity by this unique eosinophil and basophil constituent, and will facilitate the routine purification of the active enzyme for in vitro and animal model studies of its role (or roles) in eosinophil and basophil associated allergic inflammation and eosinophil-parasite interactions.


Induction of transforming growth factor-beta and prostaglandin E2 production by ethanol in human monocytes.

To test our hypothesis that monocytes (M phi) and their mediators are major contributors to ethanol-related immunodepression, the modulating capacity of acute ethanol treatment was assessed on the production of transforming growth factor-beta (TGF beta) and prostaglandin E2 (PGE2) by human peripheral blood M phi. We demonstrate that acute in vitro treatment of adherent M phi with either 50 or 150 mM ethanol induced a significant increase in the production of TGF beta (P < 0.045 and P < 0.001, respectively). Furthermore, M phi pretreatment with both 50 and 150 mM ethanol augmented TGF beta production in response to subsequent stimulation with the synthetic bacterial analog, muramyl dipeptide (MDP) (P < 0.05 and P < 0.001, respectively). Ethanol also increased TGF beta production in interferon gamma (IFN gamma-activated M phi in response to MDP stimulus (P < 0.05). M phi TGF beta levels, however, were always lower in IFN gamma-activated than in non-IFN gamma-activated M phi after the same stimulation with ethanol plus MDP, suggesting that M phi preactivation by IFN gamma can partially counteract the TGF beta inducing potential of ethanol. Similar to its TGF beta-inducing potential, ethanol (150 mM) had the capacity to induce PGE2 production in adherent human M phi (P < 0.045). However, ethanol failed to augment M phi PGE2 production induced by the PGE2 secretagogue, MDP. TGF beta induction by ethanol was unaffected by the presence of cyclooxygenase inhibitor, suggesting that ethanol-induced M phi TGF beta production does not require M phi PGE2 production. These results indicate that ethanol is a potent inducer for inhibitory M phi mediators, TGF beta and PGE2, and also has the capacity to augment M phi TGF beta production in response to subsequent stimulation. Thus, ethanol-induced elevation of M phi TGF beta and PGE2 production might contribute to decreased T cell proliferation and abnormal M phi functions after alcohol exposure, resulting in a depressed immune response.


Eosinophil granule proteins in peripheral blood granulocytes.

Eosinophils contain four principal cationic proteins, major basic protein (MBP), eosinophil-derived neurotoxin (EDN), eosinophil cationic protein (ECP), and eosinophil peroxidase (EPO). To determine the quantities of these proteins in granulocytes and whether they are specific to eosinophils, their concentrations in lysates of human granulocytes were measured using specific radioimmunoassays. The effect of different methods for eosinophil lysis on the recovery of the proteins was also studied. Maximal recovery occurred at pH 2 for MBP and pH 5.6 for the other granule proteins. The proteins cosedimented with eosinophils and their concentrations (mean +/- SEM) in ng/10(6) eosinophils (and in nM/10(6) eosinophils) were: MBP, 8,982 +/- 611 (641.6); EDN, 3,283 +/- 116 (178.4); ECP, 5,269 +/- 283 (250.9); and EPO, 12,174 +/- 859 (171.5). Basophils from a normal person contained (in ng/10(6) cells) MBP, 2,374; EDN, 214; ECP, 77; and EPO, 17. Highly purified neutrophils contained (in ng/10(6) cells) MBP, 3 +/- 0.5; EDN, 72 +/- 9; and ECP, 50 +/- 12. Therefore we conclude that these proteins are mainly expressed in eosinophils, but that certain ones are present in basophils and neutrophils.


Killing of Legionella pneumophila by nitric oxide in gamma-interferon-activated macrophages.

The role of nitric oxide (NO) radicals in killing the intracellular bacterial pathogen Legionella pneumophila (Lp) was examined in infected macrophages. Murine (RAW 264.7) and human (HL-60) cell monolayers were treated with 100 U/ml gamma-interferon (IFN) and cocultured with Lp in the presence and absence of NGMMA, a specific inhibitor of NO production. Viable Lp in IFN-treated RAW 264.7 cells decreased from 3.8 to 0.7 +/- 0.12 log CFU/ml after 24 h incubation, whereas in IFN+NGMMA-treated RAW 264.7 cells, viable Lp persisted at 2.2 +/- 0.2 log CFU/ml after 24 h. This increased survival corresponded with an inhibition of NO production (5.65 +/- 2.99 microM with NGMMA vs. 58.6 +/- 5.36 microM without NGMMA). Viable Lp were susceptible to killing, in a dose-dependent fashion, by 0, 2.5, and 5.0 mM sodium nitroprusside, a source of NO radicals. IFN-treated RAW 264.7 cells also had significantly decreased levels of intracellular iron (below assay limit) when compared to IFN+NGMMA-treated cells (72.0 +/- 0.78% of control). Normally permissive HL-60 cells treated with IFN were bacteriostatic rather than bactericidal, and NO production was not detected above background. Thus, NO radicals play a critical role in the bactericidal activity against Lp by IFN-treated RAW 264.7 cells, but the absence of NO production limits IFN-treated HL-60 cells to bacteriostasis.


Tumor necrosis factor-alpha decreases neutrophil chemotaxis to N-formyl-1-methionyl-1-leucyl-1-phenylalanine: analysis of single cell movement.

Tumor necrosis factor-alpha (TNF-alpha), a cytokine produced by mononuclear cells in response to endotoxin, inhibits neutrophil chemotaxis. We analyzed the effects of TNF-alpha on the orientation and movement of individual neutrophils in a chemoattractant gradient. Neutrophils, treated or untreated with TNF-alpha, were observed migrating in a gradient of the chemotactic peptide N-formyl-1-methionyl-1-leucyl-1-phenylalanine (fMLP) on a specially constructed chamber (Zigmond bridge). The movement of these cells was videotaped, digitized, and then tracked using a newly designed computer algorithm. The data obtained from this algorithm were then utilized to calculate distance traveled, speed and ability to polarize and migrate in a directed manner for each individual cell. TNF-alpha-treated cells behaved like cells not exposed to fMLP in that they failed to orient in a chemotactic gradient and moved in a manner similar to randomly migrating cells. This study provides unique observations of the effect of TNF-alpha on multiple parameters of PMN migration.


Phorbol ester reduces constitutive nuclear NF kappa B and inhibits HIV-1 production in mature human monocytic cells.

NF kappa B is a potent mediator of specific gene expression in human monocytes and has been shown to play a role in transcription of the HIV-1 genome in promonocytic leukemias. There is little information available on the response of NF kappa B to cytokines in normal human monocytes. We have used a 32P-labeled oligonucleotide derived from human immunodeficiency virus (HIV-1) long terminal repeat, which contains a tandem repeat of the NF kappa B binding sequence, as a probe in a gel retardation assay to study this transcription factor. Using this assay, we have detected NF kappa B in extracts of nuclei from normal human monocytes. Treatment of normal monocytes with 12-0-tetradecanoyl phorbol-13-acetate (TPA) for 4-24 h caused the complete disappearance of NF kappa B from nuclear extracts of monocytes. A similar result was obtained with the mature monocytic leukemia cell line THP-1. The constitutive transcription factor SP1 was unaffected by addition of TPA. The disappearance of NF kappa B from the nucleus was concentration dependent between 10 and 50 ng/ml of phorbol ester. In THP-1 cells, TPA also induced a new, faster-migrating NF kappa B species not induced in monocytes. Protein kinase C inhibitor staurosporine, but not cyclic nucleotide-dependent protein kinase inhibitor HA-1004, also dramatically reduced constitutive levels of nuclear NF kappa B. Finally, TPA addition to monocytes infected with HIV-1 inhibited HIV-1 replication, as determined by reverse transcriptase assays, in a concentration-dependent manner. These results are in striking contrast to the increase in nuclear NF kappa B and HIV-1 replication induced by phorbol esters in promonocytic leukemia cells U937 and HL-60, and emphasize the importance of studying cytokine regulation of HIV-1 in normal monocytes.


Cytokines and suppressor macrophages cause tumor-bearing host CD8+ T cells to suppress recognition of allogeneic and syngeneic MHC class II molecules.

Quantitative and qualitative tumor-associated changes in T cell phenotype and function were identified in CD8+ T cells. Tumor growth changed splenic CD4+/CD8+ T cell ratios and induced the appearance of more cells with the CD8+ phenotype. In comparison to equal concentrations of normal host (NH) counterparts, tumor-bearing host (TBH) CD8+ T cells were highly suppressive to allorecognition and autorecognition. Suppression was not due to quantitative reductions in CD4+ T cells, although minor qualitative differences were observed. Suppression appeared to be mediated partly by prostaglandin E2 (PGE2). Interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) contributed to TBH CD8+ T cell-mediated suppression. Blocking studies using monoclonal antibodies (mAb) in conjunction with indomethacin suggested that cytokine networks involving IFN-gamma, IL-4, and PGE2 were disrupted during tumor growth and promoted TBH CD8+ T cell suppression. Alloresponses and autoresponses were significantly suppressed when TBH CD8+ T cells mediated these reactions simultaneously with TBH Ia- macrophages. Inhibition of PGE2 production was unable to reverse the additive suppression caused by these two cell types. These results collectively suggest that tumor-induced changes in CD8+ T cells lead to suppressed allo-recognition and autorecognition through both soluble mediator molecules and cellular interactions.


Phosphatidylinositol hydrolysis by phospholipase A2 and C activities in human peripheral blood neutrophils.

We describe here and partially characterize a Ca(2+)-independent phospholipase A2 that acts on phosphatidylinositol in normal human peripheral blood neutrophils. Neutrophils incubated with myo-[3H]inositol to form [3H]phosphatidylinositol and then stimulated with the calcium ionophore A23187 produced [3H]lysophosphatidylinositol. This deacylation was further characterized in cell sonicates by the specific release of [3H]arachidonic acid from exogenous [1-14C]stearoyl-2-[3H]arachidonyl-phosphatidylinositol. This phospholipase A2 is Ca2+ independent, retaining full activity in the presence of 10 mM EDTA, and is optimally active at alkaline pH (pH 9). A phosphatidylinositol-hydrolyzing phospholipase C activity was characterized by the production of [3H]-/[14C]-diglycerides. This phospholipase C activity is dependent on the presence of exogenous Ca2+ and is optimally active at neutral pH (pH 7.5). The lipoxygenase/cyclooxygenase inhibitors eicosatetraenoic acid and nordihydroguaiaretic acid and the calmodulin antagonist trifluoperazine were the only compounds tested that showed significant inhibition of phospholipase A2 activity. However, none of these phosphatidylinositol-hydrolyzing phospholipase A2 inhibitory compounds resulted in the accumulation of any radiolabeled diglyceride, monoglyceride, or phosphatidic acid intermediates. Following subcellular fractionation on sucrose density gradients, it was found that the plasma membrane-enriched fractions contained the highest specific activity for phospholipase A2; however, the cytosolic fraction contained a large part of the total phospholipase A2 activity. Furthermore, when neutrophils were first exposed to several agents, including lipopolysaccharide, phorbol myristate acetate, or N-formyl-methionyl-leucyl- phenylalanine, and then subfractionated, there was a significant translocation of the enzyme activity from the cytosolic fraction to the membrane-enriched fractions. These data suggest that this Ca(2+)-independent, phosphatidylinositol-hydrolyzing phospholipase A2 may play an important role in early cell activation, providing free arachidonic acid for subsequent metabolism into biologically active eicosanoids.


Liposome-encapsulated hemoglobin inhibits tumor necrosis factor release from rabbit alveolar macrophages by a posttranscriptional mechanism.

Macrophages contribute to the systemic inflammatory response that characterizes the sepsis syndrome through the production of inflammatory cytokines such as tumor necrosis factor (TNF). Liposome-encapsulated hemoglobin (LEH), a potential red cell substitute, is cleared by fixed tissue macrophages. In these studies, in vitro incubation of alveolar macrophages with stored LEH was shown to inhibit the expression of TNF induced by endotoxin (lipopolysaccharide, LPS) stimulation. This effect was dependent on LEH dose but independent of the period of exposure to the LEH. Despite inhibition of TNF expression, Northern blot analysis of total cellular RNA from LPS-stimulated macrophages revealed accumulations of TNF-specific transcripts in cells treated with or without LEH. Thus the mechanism of LEH inhibition of TNF expression appears to involve a posttranscriptional event. Although these results suggest a potential advantage of resuscitation with LEH when sepsis complicates hemorrhagic shock, immunomodulation in vivo remains to be defined.


Interleukin-1 beta (IL-1 beta), IL-1 receptor antagonist, and TNF alpha production in whole blood.

The ability of an individual to mount defense responses to infection depend in part on the capacity to produce cytokines such as interleukin 1 (IL-1) and tumor necrosis factor (TNF). The specialized equipment, labor intensity, and sterile practice required for the standard in vitro evaluation of cytokine production can make such evaluation impractical in some clinical situations. We report a method for stimulating whole blood to produce cytokines that can be implemented in laboratories without tissue culture facilities and requires minimal sample preparation. IL-1 beta and TNF alpha production in whole blood samples was stimulated with endotoxin and/or phytohemagglutinin in standard EDTA-containing vacuum collection tubes. After incubation, plasma was removed and frozen for later assay. Comparison of this whole blood method with isolated mononuclear cell cultures indicated a significant correlation for IL-1 beta production (r = 0.746, P = 0.005). This technique also produced the newly described cytokine, IL-1 receptor antagonist. We conclude that the whole blood method is an acceptable alternative to isolated cell culture methods for measuring IL-1 beta in situations that preclude the standard in vitro approach.


12 alpha-hydroxylase activity in human liver and its relation to cholesterol 7 alpha-hydroxylase activity.

Interruption of the enterohepatic circulation by cholestyramine causes a several-fold increase in bile acid synthesis, reflected in a stimulation of cholesterol 7 alpha-hydroxylase activity; the synthesis of cholic acid being stimulated to a greater extent than chenodeoxycholic acid. It is not known if this preferential increase in cholic acid is due to an increase of the 12 alpha-hydroxylase activity. The present study aimed at investigating the 12 alpha-hydroxylase activity and its relation to cholesterol 7 alpha-hydroxylase activity in liver microsomes of patients with different levels of cholesterol 7 alpha-hydroxylase activity. Liver biopsies were obtained from four gallstone-free patients, and seven untreated and two cholestyramine-treated gallstone patients undergoing cholecystectomy, and four patients with Crohn's disease undergoing intestinal resection. The combined group of cholestyramine-treated and ileum-resected patients had four times higher cholesterol 7 alpha-hydroxylase activity and two times higher 12 alpha-hydroxylase activity than the other patients. A positive correlation was obtained between cholesterol 7 alpha-hydroxylase activity and 12 alpha-hydroxylase activity (r = +0.69; n = 16). These results indicate that the increased ratio between the synthesis of cholic acid and chenodeoxycholic acid during cholestyramine treatment is due to a compensatory increase of the 12 alpha-hydroxylase activity.


Peroxisomal beta-oxidation of branched chain fatty acids in human skin fibroblasts.

Human skin fibroblasts in suspension are able to degrade [1-14C]-labeled alpha- and gamma-methyl branched chain fatty acids such as pristanic and homophytanic acid. Pristanic acid was converted to propionyl-CoA, whereas homophytanic acid was beta-oxidized to acetyl-CoA. Incubation of skin fibroblasts with [1-14C]-labeled fatty acids for longer periods produced radiolabeled carbon dioxide, presumably by further degradation of acetyl-CoA or propionyl-CoA generated by beta-oxidation. Under the same conditions similar products were produced from very long chain fatty acids, such as lignoceric acid. Inclusion of digitonin (> 10 micrograms/ml) in the incubations strongly inhibited carbon dioxide production but stimulated acetyl-CoA or propionyl-CoA production from fatty acids. ATP, Mg2+, coenzyme A, NAD+ and L-carnitine stimulated acetyl-CoA or propionyl-CoA production from [1-14C]-labeled fatty acids in skin fibroblast suspensions. Branched chain fatty acid beta-oxidation was reduced in peroxisome-deficient cells (Zellweger syndrome and infantile Refsum's disease) but they were beta-oxidized normally in cells from patients with X-linked adrenoleukodystrophy (ALD). Under the same conditions, lignoceric acid beta-oxidation was impaired in the above three peroxisomal disease states. These results provide evidence that branched chain fatty acid, as well as very long chain fatty acid, beta-oxidation occurs only in peroxisomes. As the defect in X-linked ALD is in a peroxisomal fatty acyl-CoA synthetase, which is believed to be specific for very long chain fatty acids, we postulate that different synthetases are involved in the activation of branched chain and very long chain fatty acids in peroxisomes.


Intestinal lipids and lipoproteins in the human fetus: modulation by epidermal growth factor.

The aim of the present investigation was first, to examine the ability of human fetal intestine (17-20 wk) to incorporate fatty acid into esterified lipids; and second, to study in vitro lipoprotein synthesis and secretion by fetal explants, as well as the effect of epidermal growth factor (EGF) on these processes. Cultured fetal jejunal explants were incubated in Leibovitz medium for 42 h with [14C]oleate. Both triglycerides (TG) and phospholipids (PL) were the major labeled products. Whereas TG were predominant (80%) in the culture medium, PL accounted for more than 50% of total tissue lipids. More than 60% of the radioactivity in PL was associated with phosphatidylcholine. Some labeling (< 5%) was also recovered in the cholesteryl ester fraction. Active exocytosis was demonstrated by the accumulation of newly synthesized esterified lipids in the medium and the presence of lipoproteins in the basolateral membrane region and intercellular spaces. Most of the newly synthesized lipids were found in lipoproteins of d < 0.97 g/ml (51.2%) and d < 1.21 g/ml (39.3%), whereas the rest were recovered in d < 1.006 g/ml (9.8%) and 1.063 g/ml (5.6%). A similar trend characterized the lipoprotein secretion. The synthesis of the d < 0.97 g/ml fraction (30,653 +/- 4,122 dpm/mg protein) was significantly greater than the 1.006 g/ml fraction (5,897 +/- 1,734), P < 0.005. The secretion of d < 0.97 g/ml particles into the medium was also five fold higher than that of the d < 1.006 g/ml fraction (P < 0.01). The addition of EGF to the culture medium (25, 50, and 100 ng/ml) significantly enhanced the d < 0.97 g/ml lipoprotein secretion (25-40%) and decreased the d 1.006 g/ml and 1.063 g/ml fraction output. The lipid composition of these lipoprotein fractions was never altered by the presence of EGF, suggesting that the number of lipoprotein particles, rather than size, was modified by the growth factor. The present findings provide the first evidence that the human fetal intestine has the capacity to elaborate lipoprotein fractions for the transport of newly synthesized lipids. Furthermore, our data suggest that EGF, present in significant quantity in saliva, amniotic fluid, and bile, can modulate the release of TG-rich lipoproteins by fetal intestinal explants.


Effects of dietary fat on cholesterol movement between tissues in CBA/J and C57BR/cdJ mice.

Differences in dietary fats cause differences in cholesterol metabolism in mice. CBA/J mice are resistant to diet-induced hypercholesterolemia and atherosclerosis; they adjust hepatic hydroxymethyl-glutaryl-CoA reductase activity (HMGR) to maintain homeostasis; C57BR/cdJ mice are susceptible, but young animals are thought to maintain homeostasis by changing fecal excretion of sterols. Compartmental modelling of movement of [4-14C]cholesterol was used to analyze movement of cholesterol between serum and liver, heart, and carcass in mice fed 40 en% fat, polyunsaturated to saturated fatty acid ratio (P/S) = 0.24 (US74) or 30 en% fat, P/S = 1 (MOD). Dietary effects were quite pronounced, while strain effects were more subdued. The C57/cdJ animals appear to regulate the overall cholesterol balance by reducing synthesis, as do the CBA/J animals, even though synthesis is not reduced to the same degree as in the CBA/J animals. Both diet and strain influence the whole-animal turnover rate, with slower turnover occurring for C57BR/cdJ animals and animals fed the US74 diet.


Sulfonate analogues of chenodeoxycholic acid: metabolism of sodium 3 alpha, 7 alpha-dihydroxy-25-homo-5 beta-cholane-25-sulfonate and sodium 3 alpha, 7 alpha-dihydroxy-24-nor-5 beta-cholane-23-sulfonate in the hamster.

This report describes the chemical synthesis of a new bile acid analogue, namely, sodium 3 alpha, 7 alpha-dihydroxy-25-homo-5 beta-cholane-25-sulfonate from homochenodeoxycholic acid. The structure of the new compound was assigned by proton magnetic resonance and infrared spectrometry. Its metabolism was studied in the hamster in comparison with sodium 3 alpha, 7 alpha-dihydroxy-24-nor-5 beta-cholane-23-sulfonate and sodium taurochenodeoxycholate. After intraduodenal administration of the 3H-labeled analogues into bile fistula hamsters, both sulfonates were absorbed from the intestine and nearly 80% of the radioactivity was secreted into bile within 8 h. Intra-ileal administration revealed that these compounds resembled taurochenodeoxycholate in that they were much more rapidly absorbed from the ileum than from the proximal small intestine: more than 85% of the radioactivity was recovered in bile within 1 h. After intravenous infusion the sulfonates were efficiently extracted by the liver at rates similar to that of sodium taurochenodeoxycholate. Chromatographic analysis of the bile showed that, regardless of the route of administration, most (> 95%) of the sulfonates were not biotransformed and they became major biliary bile acids. Sodium 3 alpha, 7 alpha-dihydroxy-25-homo-5 beta-cholane-25-sulfonate and, to a lesser extent, sodium 3 alpha, 7 alpha-dihydroxy-24-nor-5 beta-cholane-23-sulfonate induced cholestasis at infusion rates at which sodium taurochenodeoxycholate produced choleresis.


Transgenic mice expressing human apolipoprotein A-I have sera with modest trypanolytic activity in vitro but remain susceptible to infection by Trypanosoma brucei brucei.

Although Trypanosoma brucei brucei fatally infects livestock in much of sub-Saharan Africa, humans are innately resistant to infection, apparently because high-density lipoproteins (HDL) in human serum lyse this unicellular protozoan parasite. Recently, we demonstrated that purified human apolipoprotein (apo) A-I, the major protein (M(r) 28,016) constituent of HDL, had full trypanolytic activity in vitro whereas the apoA-I of cattle and sheep was non-lytic. In the present study, we have sought to confirm the trypanocidal capability of human apoA-I by studying four lines of transgenic mice expressing (supra)physiological serum levels of this polypeptide. Although trypanolysis in vitro by sera from transgenic mice (15.1 +/- 1.3% [mean +/- SEM], n = 30) was considerably less than by human sera (typically 60-80%), it was nevertheless significantly greater than by control sera (8.5 +/- 1.1%, n = 10; P < 0.001) and correlated with the concentration of human apoA-I (r = 0.56, P < 0.001). When trypanosomes were incubated at 37 degrees C with human serum or with human apoA-I for 30 min (i.e., within the pre-lytic period) they lost their ability to subsequently infect mice; trypanosomes incubated with transgenic mice serum remained infective. Furthermore, transgenic mice were fully susceptible to infection when inoculated with 10(3) trypanosomes; both the initial detection of trypanosomes in the blood (3-4 days) and the time to death (5-6 days) were no longer than control mice. This apparent paradox between the action of human apoA-I in human serum and in mouse serum was investigated further.(ABSTRACT TRUNCATED AT 250 WORDS)


Identification and properties of the proline664-leucine mutant LDL receptor in South Africans of Indian origin.

The incidence of familial hypercholesterolemia (FH) is high among South African Indians. The proline664-leucine low density lipoprotein (LDL)-receptor mutation was detected in four apparently unrelated Indian FH families in South Africa. This mutation was originally described in an FH subject (MM) of Indian (Gujerat province) origin (Soutar et al. 1989. Proc. Natl. Acad. Sci. 86: 4166-4170). All four South African families trace their origin to the vicinity of Surat in the Gujerat province of India. Haplotype analyses revealed that both LDL receptor genes in one of the homozygous patients are the same as those in the subject MM. The phenotype of the mutant protein was analyzed in skin fibroblasts of homozygous patients. [35S]methionine pulse-chase experiments revealed that the receptor precursors were slowly processed to mature receptors. Mature mutant receptors were degraded at faster than normal rates. This mutation, which is in the epidermal growth factor (EGF)-precursor-like domain of the LDL receptor, was previously reported to yield binding-defective receptors. Here we report that the affinity of the mutant LDL receptor for both LDL and beta-very low density lipoprotein (beta-VLDL) was normal and that the steady-state level of mutant receptors was about 20% of normal. Thus, the disease FH in these subjects is presumably due to the low steady-state level of receptor molecules that are functionally normal but exhibit accelerated turnover.


Bisphosphonates used for the treatment of bone disorders inhibit squalene synthase and cholesterol biosynthesis.

Some bisphosphonates used for the treatment of bone disorders are also potent inhibitors of squalene synthase, a critical enzyme for sterol biosynthesis. Among seven drugs tested, YM 175 (cycloheptylaminomethylene-1,1-bisphosphonic acid) was the most potent inhibitor of rat liver microsomal squalene synthase (Ki = 57 nM) and sterol biosynthesis from [14C]mevalonate in rat liver homogenate (IC50 = 17 nM). EB 1053 (3-(1-pyrolidino)-1-hydroxypropylidene-1,1-bisphosphonic acid) and PHPBP (3-(1-piperidino)-1-hydroxypropylidene-1,1-bisphosphonic acid) were less potent inhibitors in both these assays. Pamidronate and alendronate were poor inhibitors of squalene synthase (IC50 > 10 microM) but were potent inhibitors of sterol biosynthesis from mevalonate (IC50 = 420 and 168 nM, respectively), suggesting that the latter two agents may have inhibited other enzymes involved in the synthesis of farnesyl pyrophosphate from mevalonate. Etidronate and clodronate were inactive in both these assays. YM 175 also inhibited sterol biosynthesis in mouse macrophage J774 cells (IC50 = 64 microM) and in rats, when administered acutely, it inhibited cholesterol biosynthesis in the liver (ED50 = 30 mg/kg, s.c.). Structural modifications on YM 175 to enhance cell permeability may result in a new class of cholesterol-lowering agents.


Evidence for a common biliary cholesterol and VLDL cholesterol precursor pool in rat liver.

Hepatic free cholesterol levels are influenced by cholesterol synthesis and ester formation, which, in turn, might regulate cholesterol secretion into bile and plasma. We manipulated the rates of hepatic cholesterol synthesis and esterification and measured biliary and very low density lipoprotein (VLDL) cholesterol secretion, and bile acid synthesis. Mevalonate decreased HMG CoA reductase by 80%, increased acyl coenzyme A: cholesterol acyltransferase (ACAT) by 60% and increased [3H]oleate incorporation into microsomal and VLDL cholesteryl esters by 174% and 122%, respectively. Microsomal and biliary free cholesterol remained constant at the expense of increased microsomal and VLDL cholesteryl ester content. Mevalonate did not change bile acid synthesis. 25-OH cholesterol decreased HMG-CoA reductase by 39%, increased ACAT by 24%, but did not effect 7 alpha-hydroxylase. 25-OH cholesterol increased [3H]oleate in microsomal and VLDL cholesterol esters by 71% and 120%. Biliary cholesterol decreased by 40% and VLDL cholesteryl esters increased by 83%. A small and unsustained decrease in bile acid synthesis (14CO2 release) occurred after 25-OH cholesterol. After orotic acid feeding, HMG-CoA reductase increased 352%, and [3H]oleate in microsomal and VLDL cholesteryl esters decreased by 43% and 89%. Orotic acid decreased all VLDL components including free cholesterol (68%) and cholesteryl esters (55%), and increased biliary cholesterol by 160%. No change in bile acid synthesis occurred. Hepatic cholesterol synthesis and esterification appear to regulate a cholesterol pool available for both biliary and VLDL secretion. Changing cholesterol synthesis and esterification did not alter bile acid synthesis, suggesting that either this common bile/VLDL secretory pool is functionally distinct from the cholesterol pool used for bile salt synthesis, or that free cholesterol availability in this precursor pool is not a major determinant of bile acid synthesis.


Effects of apolipoprotein E, beta-very low density lipoproteins, and cholesterol on the extension of neurites by rabbit dorsal root ganglion neurons in vitro.

Previous studies suggest that during nerve regeneration apoE acts as a lipid transport protein that assists in the rapid initial extension of axons and then in their myelination. To determine whether apoE and/or apoE-containing lipoproteins can modulate axon growth, we assessed their effect on the out-growth of neurites from neurons in mixed cultures of fetal rabbit dorsal root ganglion cells in vitro. Incubation with beta-very low density lipoprotein (beta-VLDL) particles, which are rich in apoE and cholesterol, increased neurite outgrowth and branching. Unesterified cholesterol added to the cultures had a similar, but less pronounced, effect. These data suggest that cholesterol might be the component responsible for the enhanced neurite growth. In contrast, purified, lipid-free apoE added to the cultures reduced neurite branching. Neurite branching was also reduced when purified apoE was added along with beta-VLDL or cholesterol; however, the striking finding was that under these conditions the neurites extended farther from the neuronal cell body. Dorsal root ganglion cells were examined for the presence of receptors for native and apoE-enriched beta-VLDL. Immunocytochemistry, ligand blots, 45Ca2+ blots, and studies of the interaction of the cells with fluorescent lipoproteins provided evidence of two types of receptors for apoE-containing lipoproteins on neurons: the low density lipoprotein (LDL) receptor, which binds native beta-VLDL, and the LDL receptor-related protein, which binds apoE-enriched beta-VLDL. These findings indicate that apoE may play two complementary roles in neurite outgrowth. When complexed with lipoproteins, apoE stimulates neurite growth by the receptor-mediated delivery of cholesterol and perhaps other components necessary for neurite outgrowth. When apoE as a free protein is added together with apoE-containing lipoproteins, apoE decreases neurite branching and promotes neurite extension away from the cell body. These actions, which would be complementary in promoting target-directed nerve growth in vivo, provide the first direct evidence that apoE and apoE-containing lipoproteins can modulate the outgrowth of neuronal processes.


Metabolism of high density lipoprotein lipids by the rat liver: evidence for participation of hepatic lipase in the uptake of cholesteryl ester.

In order to determine the role of hepatic lipase in the hepatic uptake and metabolism of high density lipoprotein (HDL) triglycerides, cholesteryl esters, and phospholipids, isolated rat livers were perfused with a reconstituted HDL (rHDL) radiolabeled with [3H]triolein and [14C]cholesteryl oleate or palmitoyl-[14C]linoleoyl phosphatidylcholine. A bolus of radiolabeled rHDL was injected into the portal vein and livers were perfused for 5 min using a nonrecirculating perfusion system. Recovery of rHDL triolein in the liver as intact triolein was used to determine the amount of unmetabolized rHDL remaining in the liver. After correcting for the amount of unmetabolized rHDL remaining in the liver, about 30% of the rHDL triolein was hydrolyzed of which 19% was recovered in the liver and 11% in the perfusate. Moreover, about 7% of the rHDL phosphatidylcholine was hydrolyzed to lysophosphatidylcholine, all of which was recovered in the perfusate. Although there was no hydrolysis of rHDL cholesteryl oleate, about 30% of the cholesteryl oleate was taken up by the liver. Preperfusion of the liver with heparin to deplete the liver of hepatic lipase resulted in about a 70% reduction in rHDL triolein hydrolysis and about a 75% reduction in rHDL cholesteryl oleate uptake. Although hepatic lipase hydrolyzes both triglycerides and phosphatidylcholines, elimination of the triolein from rHDL had no effect on the uptake of rHDL cholesteryl oleate, but replacement of the rHDL phosphatidylcholine with a nonhydrolyzable phosphatidylcholine diether resulted in an 87% reduction in cholesteryl oleate uptake. These results indicate that hepatic lipase is necessary for the hepatic uptake of both HDL triglycerides and cholesteryl esters and that the uptake of cholesteryl esters is not dependent on the hydrolysis of HDL triglycerides but is dependent on the hydrolysis of HDL phospholipids.


Efflux of lipid from fibroblasts to apolipoproteins: dependence on elevated levels of cellular unesterified cholesterol.

Earlier work from this laboratory showed that enrichment of cells with free cholesterol enhanced the efflux of phospholipid to lipoprotein acceptors, suggesting that cellular phospholipid may contribute to high density lipoprotein (HDL) structure and the removal of sterol from cells. To test this hypothesis, we examined the efflux of [3H]cholesterol (FC) and [32P]phospholipid (PL) from control and cholesterol-enriched fibroblasts to delipidated apolipoproteins. The percentages of [3H]cholesterol and [32P]phospholipid released from control cells to human apolipoprotein A-I were 2.2 +/- 0.5%/24 h and 0.8 +/- 0.1%/24 h, respectively. When the cellular cholesterol content was doubled, efflux of both lipids increased substantially ([3H]FC efflux = 14.6 +/- 3.6%/24 h and [32P]PL efflux = 4.1 +/- 0.3%/24 h). Phosphatidylcholine accounted for 70% of the radiolabeled phospholipid released from cholesterol-enriched cells. The cholesterol to phospholipid molar ratio of the lipid released from cholesterol-enriched cells was approximately 1. This ratio remained constant throughout an incubation time of 3 to 48 h, suggesting that there was a coordinate release of both lipids. The concentrations of apoA-I, A-II, A-IV, E, and Cs that promoted half-maximal efflux of phospholipid from cholesterol-enriched fibroblasts were 53, 30, 68, 137, and 594 nM, respectively. With apoA-I and A-IV, these values for half-maximal efflux of phospholipid were identical to the concentrations that resulted in half-maximal efflux of cholesterol. Agarose gel electrophoresis of medium containing apoA-I that had been incubated with cholesterol-enriched fibroblasts revealed a particle with alpha to pre-beta mobility. We conclude that the cholesterol content of cellular membranes is an important determinant in the ability of apolipoproteins to promote lipid removal from cells. We speculate that apolipoproteins access cholesterol-phosphatidylcholine domains within the plasma membrane of cholesterol-enriched cells, whereupon HDL is generated in the extracellular compartment. The release of cellular lipid to apolipoproteins may serve as a protective mechanism against the potentially damaging effects of excess membrane cholesterol.


High sensitivity negative ion GC-MS method for detection of desaturated and chain-elongated products of deuterated linoleic and linolenic acids.

A sensitive negative chemical ionization (NCI) gas chromatography-mass spectrometry (GC-MS) method for the detection of pentafluorobenzyl (PFB) esters of deuterated fatty acids is described. Deuterated linoleic [18:2n-6 2H4-9,10,12,13] and linolenic [18:3n-3 2H5-17,17,18,18,18] acids were converted to chain-elongated and desaturated products during incubations with homogenates prepared from rat liver. The extracted fatty acids were derivatized with pentafluorobenzyl bromide and analyzed in the negative ion mode by GC-MS. The detection limit of the PFB esters in NCI using selected ion monitoring was below 10 femtograms. In general, detection of the PFB derivatives using the negative ion mode was more than three orders of magnitude more sensitive than using a positive chemical ionization (PCI) method with methyl ester derivatives. The PFB esters of the 2H4-18:2n-6 metabolites eluted with their unlabeled analogues, whereas the PFB esters of the 2H5-18:3n-3 metabolites were resolved from the unlabeled compounds on polar capillary FFAP columns. Isotope ratios of the 2H4-18:2n-6 metabolites were used to quantify the deuterated compounds from standard dilution curves generated from the ion abundances of the unlabeled fatty acids. The 2H5-18:3n-3 metabolites were quantified similarly using 18:3n-3. This method is feasible for the study of the in vivo metabolism of deuterated essential fatty acids in whole animals.


An in vitro model for essential fatty acid deficiency: HepG2 cells permanently maintained in lipid-free medium.

A stable essential fatty acid-deficient cell type, known as HepG2-EFD, was derived from the lipoprotein-producing human hepatoma cell line HepG2. These cells are particularly useful for quantitative studies involving essential fatty acids (n-6 and n-3 fatty acids) in secreted lipoproteins. Radiolabeled essential fatty acids can be delivered to these cells without altering the specific activity of the fatty acids, since the deficient cells contain no endogenous essential fatty acids. Using these cells, radioactivity data (dpm) from metabolic studies can be converted directly to mass, and masses as low as a few pmoles can be accurately measured. HepG2-EFD cell cultures were established by growing HepG2 cells in medium containing delipidated serum. After 10 days of growth in delipidated medium, HepG2 cells were completely depleted of all essential fatty acids. Compensatory increases in nonessential fatty acids (n-9 and n-7 fatty acids) including 20:3n-9 (the Mead acid), which is the hallmark fatty acid of essential fatty acid deficiency, were also observed in HepG2-EFD cells. Despite the lack of exogenous fatty acids in the medium and the lack of essential fatty acids in the cells, export of very low density lipoprotein (VLDL)-associated apolipoprotein B by HepG2-EFD was the same as observed for parent HepG2 cells. However, the activity of beta-oxidation of fatty acids in HepG2-EFD cells was much lower than in the parent cell line.(ABSTRACT TRUNCATED AT 250 WORDS)


A quantitative model for the regulation of naturally occurring cell death in the developing vertebrate nervous system.

Throughout the animal kingdom, the formation of the nervous system involves the elimination of many cells soon after their generation. This phenomenon, known as naturally occurring cell death, has precise time schedules and is observed in the vast majority of neural structures. It causes the loss of 15-85% of the neurons generated. Manipulations of the target structure can considerably affect the amount of cell death in a nervous center, but the regulation of this process is still controversial. While in some experiments cell death leads to a linear relationship between the size of the target and that of the input, other experiments show dramatic deviations from a linear prediction. It is quite possible that cell death is regulated by different mechanisms in different cases and that the search for a single explanation would be doomed to failure. However, it is shown here that if mutual trophic interactions are assumed to occur between connected structures, a general model can be developed for the regulation of histogenetic cell death in the developing nervous system of vertebrates. The model relies on few assumptions, all derived from a number of experimental studies. Cells destined to form a neural center are generated according to a program and die around a certain age unless a trophic factor is supplied that prevents their death. Target cells exert a trophic influence on input cells and vice versa. The model quantitatively describes the time course and the amount of cell death in neural structures, thereby reconciling in a unitary framework experimental findings that until now have appeared conflicting.


Neuronal activity in monkey ventral striatum related to the expectation of reward.

Projections from cortical and subcortical limbic structures to the basal ganglia are predominantly directed to the ventral striatum. The present study investigated how the expectation of external events with behavioral significance is reflected in the activity of ventral striatal neurons. A total of 420 neurons were studied in macaque monkeys performing in a delayed go-no-go task. Lights of different colors instructed the animal to do an arm-reaching movement or refrain from moving, respectively, when a trigger light was illuminated a few seconds later. Task performance was reinforced by liquid reward in both situations. A total of 60 ventral striatal neurons showed sustained increases of activity before the occurrence of individual task events. In 43 of these neurons, activations specifically preceded the delivery of reward, independent of the movement or no-movement reaction. In a series of additional tests, these activations were time locked to the subsequent reward, disappeared within a few trials when reward was omitted, and were temporally unrelated to mouth movements. Changes in the appetitive value of the reward liquid modified the magnitude of activations, suggesting a possible relationship to the hedonic properties of the expected event. Activations also occurred when reward was delivered in a predictable manner outside of any behavioral task. These data suggest that neurons in the ventral striatum are activated during states of expectation of individual environmental events that are predictable to the subject through its past experience. The prevalence of activations related to the expectation of reward suggests that ventral striatal neurons have access to central representations of reward and thereby participate in the processing of information underlying the motivational control of goal-directed behavior.


The expression of nicotinic acetylcholine receptors by PC12 cells treated with NGF.

The expression of neuronal nicotinic ACh receptors (nAChRs) and the subunits that compose these receptors by PC12 cells exposed to NGF has been studied. The analysis of total RNA reveals that the neuronal nAChR subunits alpha 3, alpha S, beta 2, beta 3, and beta 4, but not alpha 2 and alpha 4, are expressed in our PC12 cells. Within 48 hr of adding NGF to cultures, the RNA corresponding to alpha 3, alpha 5, beta 3, and beta 4 is decreased, but the beta 2 RNA increases for up to 6 d after NGF treatment. To determine the influence of NGF treatment on subunit protein expression, subunit-specific antisera were prepared. Immunocytochemistry detected antigen for alpha 3, alpha 5, beta 2, beta 3, and beta 4 (but not alpha 2 and alpha 4) in both NGF-treated and nontreated PC12 cells. The expression of nAChR subunit proteins, as measured by direct binding of antibodies to PC12 cells, does not change subsequent to 6 d of treatment with NGF. Whole-cell recording of PC12 cells shows that both the individual cell current density and response to the agonist cytisine were not altered after 5-7 d in NGF. However, the number of cells exhibiting detectable ACh-induced currents doubled. These results indicate that NGF increases the number of PC12 cells expressing ACh-sensitive nAChR currents but the activation is not the result of altering the amounts of individual nAChR subunit proteins. These data, taken together with the decrease in most nAChR subunit RNAs (except beta 2), suggest that NGF regulation of nAChRs may be through a posttranscriptional mechanism.


Lesions of the perirhinal cortex but not of the frontal, medial prefrontal, visual, or insular cortex block fear-potentiated startle using a visual conditioned stimulus.

The present study is part of an ongoing series of experiments aimed at delineation of the neural pathways that mediate fear-potentiated startle, a model of conditioned fear in which the acoustic startle reflex is enhanced when elicited in the presence of a light previously paired with shock. A number of cortical areas that might be involved in relaying information about the visual conditioned stimulus (the light) in fear-potentiated startle were investigated. One hundred thirty-five rats were given 10 light-shock pairings on each of 2 consecutive days, and 1-2 d later electrolytic or aspiration lesions in various cortical areas were performed. One week later, the magnitude of fear-potentiated startle was measured. Complete removal of the visual cortex, medial prefrontal cortex, insular cortex, or posterior perirhinal cortex had no significant effect on the magnitude of fear-potentiated startle. Lesions of the frontal cortex attenuated fear-potentiated startle by approximately 50%. However, lesions of the anterior perirhinal cortex completely eliminated fear-potentiated startle. The effective lesions included parts of the cortex both dorsal and ventral to the rhinal sulcus and extended from approximately 1.8 to 3.8 mm posterior to bregma. Lesions slightly more posterior (2.3-4.8 mm posterior to bregma) or lesions that included only the perirhinal cortex dorsal to the rhinal sulcus had no effect. The region of the perirhinal cortex in which lesions blocked fear-potentiated startle projects to the amygdala, and thus may be part of the pathway that relays the visual conditioned stimulus information to the amygdala, a structure that is also critical for fear-potentiated startle. In addition, the present findings are in agreement with numerous studies in primates suggesting that the perirhinal cortex may play a more general role in memory.


Neural regulation of N-cadherin gene expression in developing and adult skeletal muscle.

Using monoclonal antibody and cDNA probes, we have studied N-cadherin gene expression in developing and adult chick skeletal muscle. N-cadherin was expressed by developing myotubes during the period of initial nerve-muscle contact but was downregulated within days of innervation. Treatment of embryos with d-tubocurare partially reversed this downregulation. In the adult, there were muscle fiber type differences in N-cadherin expression. N-cadherin was undetectable on normally innervated twitch fibers, while multiply innervated tonic muscle fibers expressed low but readily detectable levels of N-cadherin. Denervation led to the renewed expression of N-cadherin in twitch fibers as well as a marked increase in expression in tonic fibers. Levels of N-cadherin expressed by tonic fibers could also be modulated by animal housing conditions that favored either increased or decreased levels of daily motor activity. Increased motor activity was correlated with decreased levels of N-cadherin, while decreased motor activity correlated with increased levels of muscle N-cadherin. Results of in vitro studies using the calcium channel agonist ryanodine suggest that changes in intracellular calcium may be the initial signal linking neural stimulation with changes in muscle fiber expression of N-cadherin. Together, our results indicate that neural stimulation of chick skeletal muscle fibers dynamically and reversibly downregulates the expression of N-cadherin mRNA and protein. This pattern of regulation may be functionally important in limiting and/or promoting axon growth in innervated versus denervated muscle and may serve as a molecular model system for studies of the activity-dependent regulation of gene expression.


Role of the target in synapse elimination: studies in cerebellum of developing lurcher mutants and adult chimeric mice.

As the mature nervous system is sculpted out of its embryonic anlage, regressive events are a surprisingly common feature. As one example, the establishment of adult innervation in the CNS and PNS often involves a massive withdrawal of previously formed functional synapses. In the cerebellum, the one-to-one relationship of inferior olivary climbing fibers to Purkinje cells is preceded by a transient stage in which each Purkinje cell is multiply innervated. The regulation of this regressive event is still not fully understood; previous studies suggested that some stimulus from the maturing granule cells is necessary. We have used the lurcher (Lc) mutation as a model system in which to study this phenomenon. In lurcher mice, Purkinje cells degenerate during the first few postnatal weeks, after receiving synaptic contacts from both inferior olivary neurons and granule cells. We have recorded the climbing fiber responses both in lurcher mutants at postnatal days 14-20 (P14-P20) and in adult lurcher chimeras. In the latter, experimental genetics are used to create a situation in which untreated wild-type Purkinje cells are present in an environment that ranges from 100% wild-type to nearly mutant. We found that in P14-P16 lurcher mutants, most of the cells recorded (75%) remained polyinnervated, whereas in wild-type control mice, only 10% of the Purkinje cells retained their multiple innervation. By P18-20, it was difficult to find Purkinje cells in the lurcher mutants that would withstand an intracellular electrode. Nonetheless, in those cells that were successfully impaled, most remained multiply innervated. By this age in wild-type mice, 100% of the Purkinje cells are monoinnervated.(ABSTRACT TRUNCATED AT 250 WORDS)


Development of binocular vision in the kitten's striate cortex.

Studies of the development and plasticity of the visual pathway are well documented, but a basic question remains open: what is the physiological status of the system prior to extensive visual experience? Somewhat conflicting answers have been put forward, and in a major area, binocular vision, reports have ranged from severe immaturity to well-developed maturity. This is an important question to resolve since binocular cells in the visual cortex are thought to be the neural substrate for stereoscopic depth perception. We have addressed this question by recording from single cells in the striate cortex of kittens at postnatal ages 2, 3, and 4 weeks and from adults for comparison. Gratings with sinusoidal luminance distribution are presented to left, right, or both eyes. For each cell, we determine optimal values for orientation and spatial frequency. Relative phase (retinal disparity) is then varied in a dichoptic sequence so that binocular interaction may be studied. Results are as follows. In the normal adult, we have shown in previous work that most binocular interaction in the visual cortex can be accounted for on the basis of linear summation. Results from 3 and 4 week postnatal kittens are closely similar to those from adults. All types of binocular interaction found in adults are present in kittens. This includes phase-specific and non-phase-specific suppression or facilitation. Furthermore, monocular and binocular tuning characteristics are comparable in kittens and adults. The clear changes that occur with age are optimal spatial frequencies and peak responses. In addition, at 2 weeks, there is a substantially higher proportion of monocular cells compared to other ages and correspondingly, lower relative numbers of cells that exhibit phase-specific or suppressive binocular interactions. From increases in optimal spatial frequency and interpupillary distance with age, we calculated predicted changes in binocular disparity thresholds (stereo acuity) with age. Although there are methodological limits with respect to the behavioral testing of young kittens, the predicted results are comparable to some of the values obtained. Considered together, our results show that the physiological apparatus for binocular vision is functional at an early stage in postnatal development. It is possible that the connections that underlie this function are developed rapidly during early postnatal experience. An alternative possibility is that there is an elaborate genetic organization of binocular vision, but our study does not address this issue directly. A combination of these factors may be applicable.(ABSTRACT TRUNCATED AT 400 WORDS)


The analysis of visual motion: a comparison of neuronal and psychophysical performance.

We compared the ability of psychophysical observers and single cortical neurons to discriminate weak motion signals in a stochastic visual display. All data were obtained from rhesus monkeys trained to perform a direction discrimination task near psychophysical threshold. The conditions for such a comparison were ideal in that both psychophysical and physiological data were obtained in the same animals, on the same sets of trials, and using the same visual display. In addition, the psychophysical task was tailored in each experiment to the physiological properties of the neuron under study; the visual display was matched to each neuron's preference for size, speed, and direction of motion. Under these conditions, the sensitivity of most MT neurons was very similar to the psychophysical sensitivity of the animal observers. In fact, the responses of single neurons typically provided a satisfactory account of both absolute psychophysical threshold and the shape of the psychometric function relating performance to the strength of the motion signal. Thus, psychophysical decisions in our task are likely to be based upon a relatively small number of neural signals. These signals could be carried by a small number of neurons if the responses of the pooled neurons are statistically independent. Alternatively, the signals may be carried by a much larger pool of neurons if their responses are partially intercorrelated.


Does loss of nerve growth factor receptors precede loss of cholinergic neurons in Alzheimer's disease? An autoradiographic study in the human striatum and basal forebrain.

The mechanism by which cholinergic neurons degenerate in Alzheimer's disease is not known. Some of these neurons depend, however, on trophic support from NGF via a membrane receptor. We have analyzed the state of these receptors by autoradiography, with 125I-NGF as the ligand, in the caudate nucleus, putamen, ventral striatum, nucleus basalis of Meynert, and nucleus tegmenti pedunculopontinus of six patients with Alzheimer's disease and five controls, matched for age and postmortem delay. The binding characteristics were similar in the striatum (including caudate nucleus, putamen, and ventral striatum) and basal forebrain of control subjects and patients with Alzheimer's disease (Kd = 2.5-4 x 10(-11) M). In control brains, high levels of 125I-NGF binding were observed in the basal forebrain and striatum (0.32-0.49 fmol/mg tissue equivalent), but no specific binding was detected in the nucleus tegmenti pedunculopontinus. NGF binding sites were distributed heterogeneously in the striatum with patches of low density, corresponding to AChE-poor striosomes, surrounded by a matrix in which receptor density was significantly greater. In Alzheimer's disease, the density of NGF receptors was markedly decreased in the caudate nucleus, putamen, ventral striatum, and nucleus basalis of Meynert. In contrast, AChE staining decreased less in the nucleus basalis of Meynert in all Alzheimer's disease patients, and in the ventral striatum of those most severely affected. These results indicate that if NGF receptors are located on cholinergic neurons, receptor loss and the consequent decrease in trophic support may precede cell degeneration in Alzheimer's disease. The relationship between the loss of these receptors and the pathogenesis of the disease remains to be determined.


Axo-glial interactions at the dorsal root transitional zone regulate neurofilament protein synthesis in axotomized sensory neurons.

After dorsal root crush, dramatic ultrastructural differences are observed between regenerated dorsal root axonal endings that are physically blocked at a ligation neuroma and those that are allowed to form axo-glial endings among the astrocytes at the dorsal root transitional zone (DRTZ). Physically blocked axonal endings swell immensely with membranous organelles and neurofilaments (NFs) while axo-glial endings do not, suggesting that DRTZ astrocytes stop axonal growth by activating a physiological stop pathway within those endings. Since protease-dependent NF degradation at axonal endings is a part of this pathway, this study addresses the question of whether NF subunit synthesis in the dorsal root ganglion (DRG) is regulated by the pathway. Lumbar dorsal roots were crushed and, at various postinjury times, the attached DRGs were removed and pulse-labeled in vitro with 35S-methionine for subsequent analysis of protein synthesis by electrophoresis and fluorography. Within 24 hr of axotomy, there was a down-regulation of the 68 kDa (NF-L) and 145 kDa (NF-M) NF subunits. At 14 d postcrush, a time when most of the regenerating axons have reached and been stopped by DRTZ astrocytes, NF protein synthesis returned to control levels. By contrast, when the axons were prevented from reaching the DRTZ by ligating or removing segments of the roots, NF synthesis failed to return to normal levels. These data suggest that activation of the physiological stop pathway by DRTZ astrocytes regulates NF protein synthesis in the DRG.


Microtubule-based filopodium-like protrusions form after axotomy.

The growth cone at the front of a growing neurite often has F-actin-rich structures--digitate filopodia and sheet-like veils and lamellipodia--whose protrusion advances the leading edge. Microtubules and other cytoplasmic constituents later fill the protruded area, transforming it into new neuritic length. Growth can be initiated from an axon by transecting it. We have used video-enhanced contrast-differential interference contrast microscopy to observe the early events following transection of Aplysia axons in culture. Many filopodium-like protrusions (FLPs) grew rapidly (average instantaneous velocity of 1.6 microns/sec) from the sides and end of the axon stump within minutes of transection. Some of these displayed bidirectional transport of swellings, at a rate similar to fast axonal transport. Dihydrocytochalasin B, which blocks actin polymerization, only halved the number of FLPs that formed within 10 min of transection, and actually increased the number of transporting FLPs. Nocodazole, a microtubule-specific drug, also halved the number of FLPs, but none of them displayed transport of swellings. No FLPs formed in the presence of both drugs. In transected axons that had not been exposed to either drug, removal of the plasma membrane revealed fibers in many of the FLPs; immunofluorescence showed these fibers to be microtubules. Thus, a substantial number of the FLPs that form soon after axotomy are microtubule based, rather than actin based, underscoring the potential of microtubules to drive the rapid extension of neuritic precursors.


Changes in PKC gamma immunoreactivity in mouse hippocampus induced by spatial discrimination learning.

In the present study, we examined changes in immunoreactivity (ir) for the gamma-isoform of protein kinase C (PKC gamma) in mouse hippocampus in relation to spatial memory processes employing the monoclonal antibody 36G9 raised against purified PKC gamma. Learning and memory were assessed by performance in a free-choice spatial pattern paradigm in a hole board in which the animals learned the pattern of 4 baited holes out of 16 holes. Adult male house mice were used, divided in four groups. Three control groups were formed: group N, naive (blank controls); group H, habituated (animals were for 5 consecutive days introduced to the hole board with all holes baited); and group PT, pseudotrained (animals were for 13 consecutive days introduced to the hole board with all holes baited). The T (trained) group was for 5 consecutive days introduced to the hole board with all holes baited (similar to the H and PT groups) followed by 8 successive days with only four holes baited in a fixed pattern. Behaviorally, following the first 5 d, the PT group crossed the hole board randomly, whereas the T group gradually learned to orientate in the hole board. The mice were killed 24 hr after the last performance. A shift in 36G9-ir appeared from the cell somata to the dendrites of hippocampal principal neurons when comparing the H and PT group, respectively. In contrast, the T group showed strong PKC gamma-ir in both cell somata and dendrites, which clearly exceeded that of the H and PT mice. In this way, 36G9-ir reveals the physiologically activated neurons involved in hole board learning.(ABSTRACT TRUNCATED AT 250 WORDS)


Selective vulnerability of dentate hilar neurons following traumatic brain injury: a potential mechanistic link between head trauma and disorders of the hippocampus.

Despite intensive study, the neurobiological basis of epilepsy and persistent memory impairment following traumatic head injury remains unknown. Since abnormalities of the hippocampus are known to be associated with temporal lobe seizures and memory dysfunction, we investigated the effects of experimental traumatic brain injury on hippocampal structure and function in the rat. Using a model of fluid-percussion injury, we have discovered that neurons of the dentate hilus are vulnerable to a brief, unilateral impact to the extradural surface of the brain. One week after trauma, there was a dramatic reduction in hilar neurons ipsilateral to the impact, and a milder but significant decrease in neurons on the contralateral side as well. This neuronal loss was highly selective since adjacent dentate granule and pyramidal neurons appeared relatively unaffected. Immunocytochemistry showed that the hilar cell loss included a loss of somatostatin-immunoreactive neurons, and degeneration stains provided evidence that irreversible hilar injury occurred within 4 hr of impact. To assess the functional effects of the hilar damage, dentate granule cell field potentials were measured in response to perforant path stimulation. This revealed abnormal dentate granule cell hyperexcitability at 2.0 Hz stimulation in many of the injured animals. The presence of abnormal hyperexcitability correlated with the loss of hilar neurons. Thus, a momentary impact to the surface of the brain can cause selective, bilateral hippocampal injury with associated abnormalities in dentate gyrus physiology. Furthermore, the pattern of cell loss is similar to that observed in some patients with temporal lobe epilepsy.(ABSTRACT TRUNCATED AT 250 WORDS)


Protein synthesis within neuronal growth cones.

This study investigates the capacity of neuronal growth cones to synthesize protein locally and independently of their cell body. Isolated growth cones were prepared from cultures of neurons from the snail Helisoma by transecting neurites proximal to the growth cone. The capacity for protein synthesis was tested by radiolabeling cultures with 3H-leucine and analyzing the resultant autoradiograms. Isolated growth cones displayed incorporation of 3H-leucine that was inhibited by treatment with the protein synthesis inhibitors anisomycin and pactamycin, indicating that ribosomal-dependent translation occurs in growth cones. Ultrastructural analyses of growth cones demonstrated the presence of polyribosomes, the machinery for protein synthesis. The density of polyribosomes varied between growth cones, even between different growth cones on the same neuron, suggesting that growth cones express a range of protein synthetic capabilities. That different types of growth cones possess differing capabilities for protein synthesis is suggested in autoradiograms of 3H-leucine incorporation by the growth cones of axonal and nonaxonal neurites; incorporation was radically reduced in axonal growth cones in comparison with non-axonal growth cones. Finally, growth cones that were isolated for 2 d prior to radiolabeling incorporate 3H-leucine in a eukaryotic ribosomal-dependent manner, suggesting that the capacity for translation is long-lived in growth cones. Taken together, these studies reveal a capacity for protein synthesis confined totally to the neuronal growth cone proper. The synthesis of proteins in growth cones could afford a mechanism for the alteration of growth cone structure or function. This is in accord with the view that growth cones participate autonomously, to at least some extent, in the processes of synaptogenesis and the construction of neuronal architecture.


Increased c-fos expression in spinal neurons after irritation of the lower urinary tract in the rat.

This study utilized neuronal c-fos expression to examine the spinal pathways involved in processing nociceptive and non-nociceptive afferent input from the lower urinary tract (LUT) of the urethane-anesthetized rat. C-fos protein was detected immunocytochemically in only a small number of cells (< 2 cells/L6 section) in control animals. However, chemical irritation with 1% acetic acid or mechanical stimulation of the LUT markedly increased the number of c-fos-positive neurons (56-180 cells/L6 section) in four regions of the caudal lumbosacral (L6-S1) spinal cord: medial dorsal horn (MDH), lateral dorsal horn, dorsal commissure (DCM), and sacral parasympathetic nucleus (SPN). Only small numbers of c-fos-positive cells were detected in rostral lumbar segments, a region that is thought to receive nociceptive input from the LUT via afferent pathways in sympathetic nerves. The distribution of c-fos-positive cells in the L6 spinal cord varied according to the stimulus (i.e., urethral catheter, bladder distension, or chemical irritation). Distension of the urinary bladder increased the number of c-fos-positive cells mainly in DCM and SPN regions of the cord. In contrast, irritation of the LUT increased c-fos expression largely in DCM and MDH areas. Spinal cord transection (T8 level) did not alter the c-fos expression induced by a catheter or chemical irritation, indicating that gene expression was mediated by spinal pathways. Denervation experiments showed that c-fos expression was induced by activation of afferent pathways in the pelvic and pudendal nerves. These results suggest that neurons in several regions of the spinal cord are involved in processing afferent input from different parts of the LUT. Neurons in the DCM appear to have an important role since they respond to both nociceptive and non-nociceptive inputs and to visceral (pelvic nerve) and somatic (pudendal nerve) afferent pathways. Thus, these neurons may be involved in the mechanisms of visceral-somatic referred pain.


Presynaptic localization of sodium/calcium exchangers in neuromuscular preparations.

Calcium ions play a critical role in neurotransmitter release. The cytosolic Ca2+ concentration ([Ca2+]cyt) at nerve terminals must therefore be carefully controlled. Several different mechanisms, including a plasmalemmal Na/Ca exchanger, are involved in regulating [Ca2+]cyt. We employed immunofluorescence microscopy with polyclonal antiserum raised against dog cardiac sarcolemmal Na/Ca exchanger to determine the distribution of the exchanger in vertebrate neuromuscular preparations. Our data indicate that the Na/Ca exchanger is concentrated at the neuromuscular junctions of the rat diaphragm. The exchanger is also present in the nonjunctional sarcolemma, but at a much lower concentration than in the junctional regions. Denervation markedly lowers the concentration of the exchanger in the junctional regions; this implies that the Na/Ca exchanger is concentrated in the presynaptic nerve terminals. In Xenopus laevis nerve and muscle cell cocultures, high concentrations of the exchanger are observed along the neurites as well as at the nerve terminals. The high concentrations of Na/Ca exchanger at presynaptic nerve terminals in vertebrate neuromuscular preparations suggest that the exchanger may participate in the Ca-dependent regulation of neurotransmitter release. The Na/Ca exchanger is also abundant in developing neurites and growth cones, where it may also be important for Ca2+ homeostasis.


Intracranial arterial aneurysms: a review of neurosurgical results.

The results of elective surgery for intracranial aneurysms are now excellent, with a 90% cure rate, but they do not reflect the overall results of treatment of aneurysmal pathology. Despite improvements in microsurgical techniques, these results remain disappointing (56.4% cured, 27.3% dead in the 1983 co-operative study, this is mainly due to the severity of the initial haemorrhage caused by rupture of the aneurysms, but also to the problems encountered in transferring patients, as quickly as possible, to a specialized surgical unit. Only an early care of symptomatic patients will improve these results by reducing the frequency of rebleeding and vasospasm. The most dangerous complications to which patients who survived the first haemorrhage are exposed.


Endovascular treatment of subarachnoid arterial aneurysms in the acute period. Preliminary results.

The authors report their preliminary experience concerning the endovascular treatment of ruptured subarachnoid aneurysms during the acute phase in 9 patients. The endovascular approach was only used in Hunt and Hess grade IV or V patients, as an alternative to surgical clipping and in cases considered technically difficult for surgery (extremity of the basilar artery). No particular technical difficulties were encountered, except for a case with vasospasm. Further experience is needed to evaluate the benefit of this method during the acute phase.


Traumatic aneurysms of the internal and external carotid arteries. One case and a review of the literature.

The case of a female patient with two traumatic aneurysms diagnosed by serial angiography is reported. The first aneurysms, located on the orbito-frontal branch of the left anterior cerebral artery, was removed. The second aneurysm involving the right middle meningeal artery was treated by endovascular embolization. The causes and physiopathology of such lesions are reviewed. The frequency of traumatic cerebral aneurysms has been reduced by the routine use of computed tomography. Aggressive treatment of these aneurysms is advocated because of their natural history.


Dural fistulae of the cavernous sinus treated by embolization. Ten cases.

We report ten cases of dural fistulae of the cavernous sinus treated by the endovascular route. The fistulae were fed by the internal and external carotid arteries. All patients presented with ophthalmic symptoms and/or headache and/or intracranial murmur. Embolization was decided on the ground of all in visual acuity, exophthalmos, ophthalmoplegia or sudden exacerbation of clinical symptoms. It was performed after selective catheterization of external carotid feeders with non-resorbable particles. The shunt was totally occluded in 7 cases, and local signs were improved in all patients: the intracranial murmur disappeared in 2 out of 4 cases and headache was relieved in 2 out of 3 cases. No permanent complication was observed. Among the various possible treatments of these dural fistulae, embolization of branches of the external carotid artery is a simple, effective and safe method, provided its principles and indications are respected.


MRI in neurosarcoidosis.

Nine patients with neurological manifestations of sarcoidosis were explored by MRI and, in some cases, CT. The MRI examinations were performed in T1- and T2-weighted spin-echo sequences in all patients, with gadolinium injection in seven. Several non-specific lesions were demonstrated, the most frequent of them showing on T2-weighted sequences as high-intensity signals in the periventricular white matter and the semi-oval centres. Other abnormalities, such as infiltrations of the hypothalamo-pituitary region, granulomatous masses within the brain tissue and meningeal infiltrations, were observed. These findings were in agreement with those found in the literature. Gadolinium injection seems to be important in this pathology, as it demonstrates lesions that are not visible on conventional T1- and T2-weighted spin-echo sequences.


MRI in Sturge-Weber syndrome.

Five children (3 girls, 2 boys, aged from 1 to 18 years) presenting with Sturge-Weber syndrome were explored by MRI with contrast injection in 2 cases. The respective positions of CT and MRI are discussed: CT is the method of choice to display leptomeningeal calcifications. MRI seems to be more accurate than CT to determine the location of the lesions and to demonstrate the enhancement of the angioma after contrast injection.


The aid of computerized tomography in hemifacial spasm.

Although the physiopathological mechanisms causing hemifacial spasm are not clearly understood, it is generally accepted that the causative factor is compression of the facial nerve at its root exit zone. In this paper we present seven cases of hemifacial spasm which were studied by dynamic CT scanning in the preoperative period, and treated with microvascular decompression. We suggest that CT scanning has considerable diagnostic value in disclosing the asymmetrical vascular formation causing root exit zone compression and that microvascular decompression should be the treatment of choice in this condition.


The P300 event-related potential. The effects of sleep deprivation.

We monitored 15 healthy young adults during 18 hours of sleep deprivation. Subjects were repeatedly tested on measures of the P300 (P3) event-related potential, reaction time, body temperature, and a subjective rating of fatigue. Statistically significant decreases in P3 amplitude (P < or = .01) and increases in P3 latency (P < or = .0001) were found during sleep deprivation. These changes correlated with body temperature and fatigue and were not due to circadian variation. Reaction time did not show a significant change over time. We conclude that the P3 potential is a more sensitive cognitive measure of sleep deprivation than reaction time, a measure commonly used in previous studies. We provide a review of the literature on the P3 and recommend the use of the P3 as a marker to examine sleep deprivation in health professionals.


Sleep deprivation and house staff performance. Update 1984-1991.

All English language studies since 1984 of sleep deprivation and house staff performance are reviewed. Manual and computer surveys identified 14 such studies. Our goal is to examine whether the current literature supports the hypothesis that sleep deprivation significantly impairs house staff performance. Our review finds that the current data remain inconclusive. The causes for this uncertainty include different methodologies in assessing performance, the arbitrary definitions of sleep deprived and rested states, and lack of separation of the effects of acute and chronic sleep deprivation. Our review notes striking difference in reported effects of sleep deprivation depending on medical specialty. Surgical studies most frequently show little effect of sleep deprivation. All studies since 1990 support the hypothesis that sleep deprivation significantly impairs performance. Most studies support the impairment of physician mood with increasing sleep deprivation.


A benefit-to-cost analysis of a work-site health promotion program.

An analysis of the benefit-to-cost ratio of a major health promotion program was conducted for the years 1986 through 1990, with projections to the year 2000. Program costs for personnel, capital expenses, materials, and rent were determined and compared with program benefits (discounted to account for increasing pension liability) for heath care cost savings, increased productivity, decreased absenteeism, decreased life insurance claims, and program-generated income. After adjusting future monies to net present value, a benefit-to-cost ratio of 3.4 was estimated for the program. Despite the limitations of the study design, the authors concluded that the program realizes a positive return on its investment. Several program recommendations are presented to increase the probability of achieving positive benefit-to-cost ratios in future program offerings.


A comparison of smokers and nonsmokers with respect to oncogene products and cytogenetic parameters.

Human monitoring studies can be valuable tools for assessing the adverse effects of chemicals. Cytogenetic parameters have been frequently employed but are rarely related directly to possible adverse health effects. Recently, the measurement of oncoprotein levels in plasma has been proposed as a possible and more appropriate indicator of exposure and carcinogenic risk but, unlike chromosome damage, little is known about the effects of possible confounding factors. This study compared the effect of smoking on chromosome aberrations, sister chromatid exchange, and plasma ras oncoprotein levels, in forty humans not otherwise known to be exposed to any specific chemical hazard. No effect was found on any of these end points, with the exception of a moderate, statistically nonsignificant elevation of sister chromatid exchange levels. It is concluded that smoking is unlikely to be a confounding factor in human monitoring studies using oncoprotein levels as an end point.


Training and proficiency in the medical review of job applicant drug screens.

A survey was performed to examine the proficiency of urine drug screen medical review officers (MROs) and the effect of training upon their performance. One hundred thirty-nine attendees of the ACOEM basic MRO training course, and 82 attendees of the advanced course completed both pre- and postcourse surveys. The questionnaires included 10 vignettes with positive analytical results; of which only half should be reported as positive to management. The mean number of correct responses among the 10 cases presented were: basic pre-test--4.91; basic post-test--7.45; advanced pre-test--6.68; advanced post-test--7.33. Differences between all four groups were significant (P < .001 to P = .003), except for the basic post-test versus the advanced post-test, for which there was no significant difference. It is concluded that 1) inadequate knowledge and skill in the medical review of drug screens may contribute to errors in reports to management, 2) physician's performance can be improved with training, and 3) certification of MROs in both the private and public sectors should be considered.


Sick building syndrome. Medical evaluation of two work forces.

Sick building syndrome, characterized by upper respiratory irritative and central nervous system symptoms, is poorly understood. Building ventilation problems are frequent, although causative agent(s) are unknown. Few studies have addressed clinical characterization of symptomatic building occupants. Employees from two sites underwent standardized evaluation including medical history, physical examination and screening neurologic and neuropsychologic testing while acutely symptomatic. Both symptomatic and asymptomatic individuals were evaluated when one of the sites was evacuated. Baseline evaluation results for this group were available for comparison. Symptoms of both work forces mirrored those reported in the literature. General medical examination abnormalities were few and minor, while neurologic and neuropsychologic examinations documented mental status, cerebellar, and neurobehavioral deficits. There were statistically significant changes from baseline. Abnormalities were self-limited. Controlled evaluations of symptomatic sick building occupants should be performed to verify these findings.


[Factors influencing the pitch and loudness of tinnitus].

Pitch match and loudness balance tests were given to 397 cases with tinnitus. The factors which influenced tinnitus pitch and loudness were analyzed statistically from the clinical point of view. The results obtained were as follows: 1) Onomatopoeia of tinnitus, either [Keeeen] or [Jeeeen], were observed in a majority of cases. 2) Significantly sharp sounding onomatopoeia such as [Keeeen] or [Meeeen] had high pitches, over 4kHz, and dull sounds like [Gooooh] or [Buuuun] had low pitches, below 500Hz. 3) Acute stage tinnitus, within one month of onset, had a significantly depressed pitch and walked loudness, above 6dB. 4) The pitches observed in cases with Meniere's disease and chronic otitis media were distributed evenly from low frequencies to high. In other cases, especially presbyacusis and noise deafness, high pitch tinnitus (above 4kHz) was frequently noted. The loudness of tinnitus without hearing loss was significantly greater than in other diseases. 5) As a rule the more deteriorated the hearing level was, the lower the frequency of the pitch, and the smaller the loudness in tinnitus. 6) A high pitch of tinnitus nearly corresponded with hearing type, that is, the pitch of tinnitus was also in accordance with the disturbed frequency in the hearing threshold.


[Masking in bone-conduction testing--proposal of ABC method].

A new strategic masking technique, namely the ABC method, has been developed. In performing this method of measuring thresholds of bone-conduction, the vibrator is placed at the forehead with both ears occluded by air-conduction earphones. One of the earphones is for masking noise and the other is a dummy which balances out the occlusive effect of the test ear against the nontest ear. The ABC method is based on the ABC rule that, in bone-conduction testing, the effective masking noise level necessary to block out the nontest ear can be calculated by a simple equation: right AC (A) + left AC (B)--unmasked BCu (C) under the assumption that the BCu belongs to the nontest ear. In some cases of hearing loss, the above noise level might produce overmasking, then an additive safety noise level, BCu + Interaural Attenuation, is employed. This method offers testers step by step directions which consist of indications of the noise level and a criterion for determining whether the measured bone-conduction is free from cross hearing and overmasking for the given configuration of air-conduction of both ears, BCu, and the masking noise level. Compared to the well known Plato method, in which measurements of thresholds are repeated at several masking noise levels in order to find a single bone-conduction threshold, the ABC method can essentially find the threshold at only one masking noise level. Therefore the ABC method makes it possible to save a great deal of time in performing bone conduction testing.


[The pathogenic role of Chlamydia trachomatis in otitis media with effusion].

There are conflicting views concerning middle ear infections due to Chlamydia trachomatis. To ascertain the etiological role of this agent in otitis media with effusion, middle ear effusions were cultured for C. trachomatis and other bacterial flora. A total of 102 patients with otitis media with effusion (OME) were recruited for this study. The study population included 66 patients with acute OME (AOME) and 36 patients with chronic OME (COME). As Chlamydia pneumoniae, the third species of Chlamydia, is also known to be isolated from middle ear effusion of OME, the fluorescent-antibody technique using anti-C. pneumoniae or anti-C. trachomatis antibodies was employed in order to identify the inclusion bodies isolated on HeLa 229 cells as C. trachomatis. C. trachomatis was recovered from 7 patients (10.6%) with AOME and from 8 (22.2%) patients with COME. Bacteria were cultured from 20 of 63 patients with AOME and from 13 of 28 patients with COME. Pathological bacteria were cultured from only 2 patients with C. trachomatis infection in the middle ear. Only normal skin flora, no bacterial pathogens, were isolated from the remainder. Antibodies to C. trachomatis in serum were measured by a microimmunofluorescent method in 13 patients with C. trachomatis infection in the middle ear. Antichlamydial antibody of the IgG type was detected in 84.6% (11/13) of these patients. These results suggest that C. trachomatis causes middle ear infections and plays an etiological role in the pathogenesis of otitis media with effusion.


[Immunohistological investigation of type IV collagen in the basement membrane surrounding the cancer nest (cancer nest membrane) of head and neck squamous cell carcinoma--its relation to frequency of cervical lymph node metastasis].

Lymphoducts and blood vessels exist in the stroma, while none can be detected in the cancer nest itself within cancerous tissue. This explains why metastasis of carcinoma cannot occur without the escape of tumor cells through the basement membrane surrounding the cancer nest into the stroma. Accordingly, observation of the continuity of the basement membrane, what we call the cancer nest membrane, is essential for elucidating the first step of metastasis. Since type IV collagen is the most important structure composing the basement membrane, investigation of the immunohistological localization and continuity of type IV collagen is of value in predicting the metastatic aggressiveness of squamous cell carcinoma. We therefore studied biopsy tissues from the advancing lesion of head and neck squamous cell carcinoma in 95 untreated patients. The tissues were fixed in 85% ethanol and embedded in paraffin, and 5-um thin sections prepared were then immunohistochemically stained for type IV collagen by the ABC method for observation of the continuity status of the cancer nest membrane in relation to metastasis. The basement membranes of normal mucosal epithelium and normal interstitial capillaries were utilized as positive controls, and negative controls were obtained by using PBS in place of the primary antibodies for the immunohistochemical reaction. Membrane discontinuity (breaks or absence) correlated significantly with cervical lymph node metastasis, while intact membrane was associated with a low frequency of cervical lymph node metastasis. There was no obvious relation between the clinical T category and the continuity of the membrane; pN (+) carcinomas with membrane discontinuity included even T1 supraglottic and hypopharyngeal carcinomas, as well as T2 or higher oral mucosal carcinomas and T3 or higher glottic carcinomas, suggesting variation with tumor site. Hypopharyngeal and supraglottic carcinoma was associated with membrane discontinuity and a high incidence of cervical lymph node metastasis. On the other hand, glottic and oral carcinoma more often presented with intact membranes and had a lower incidence of metastasis, although carcinomas in these sites that did present with discontinuity of the membrane were associated with a high incidence of cervical metastasis. Therefore, observation of the continuity of the cancer nest membrane by the expression of type IV collagen may be useful in selecting better specific therapies and determining the necessity of prophylactic neck dissection. A correlation between the degree of tumor differentiation and the continuity of the membrane was also found; well-differentiated tumors with discontinuity of the membrane were frequently associated with cervical lymph node metastasis.(ABSTRACT TRUNCATED AT 400 WORDS)


[Influence of environmental temperature (cold exposure) on nasal resistance].

Changes in nasal resistance were examined in 33 healthy subjects in order to investigate the physiological reactions of nasal mucosa to cold exposure. Each examinee was exposed to changes in room temperature from 25 degrees C to 15 degrees C with the same humidity in an artificial atmosphere chamber, regulated by an environmental control unit. Nasal resistance was measured by means of the active-anterior method using a rhinomanometer MPR-2200. The same experiments were repeated on different dates in all 18 subjects. The results can be summarized as follows. 1) The average value of nasal resistance significantly increased after room temperature was decreased from 25 degrees C to 15 degrees C. 2) Patterns of nasal resistance responses to cold exposure were classified into two types. One type was increased nasal resistance after cold exposure and the other was maintenance of resistance despite cold exposure. Each individual manifested a different pattern in experiments carried out on different dates. 3) The nasal resistance value in the chamber at 25 degrees C was significantly higher in winter than in summer, and the increase in nasal resistance in response to cold exposure was significantly larger in summer than in winter. This result was assumed to be due to cold adaptation of the nasal mucosa in winter. 4) Heart rate decreased significantly with cold exposure. However, changes of heart rate in response to cold exposure did not correlate with changes in nasal resistance. No correlation between changes in nasal resistance in response to cold exposure and orthostatic loading, which was carried out after the cold exposure experiment, was detected. 5) A nasal cycle was observed in 7 (14%) of the 33 cases. In 6 of these total nasal resistance was maintained despite cold exposure.


[T-cell subsets and intercellular adhesion molecule-1 in human allergic nasal mucosa].

We performed an immunohistological study of T-cell subsets and intercellular adhesion molecules in allergic and non-allergic nasal mucosa. In allergic mucosa, the number of CD4 positive cells tended to dominate that of CD8 positive cells. CD8 positive cells were detected randomly in all regions of the mucosa, whereas CD4 positive cells tended to be clustered in the superficial portion of the lamina propria. Furthermore, the number of CD4 positive cells correlated with the intensity of ICAM-1 expression, especially in the superficial portion of the lamina propria.


[Spontaneous cerebrospinal fluid otorrhea in a normal inner ear. A case report].

A 49-year-old male with spontaneous cerebrospinal fluid (CSF) otorrhea, an extremely rare condition, is presented as the third reported case in Japan. The patient visited us complaining of sudden hearing loss and fullness of the ear on the left side. A tegmen defect of the posterior cranial fossa and CSF leakage from the fistula into the mastoid air cells, but a normal inner ear, were found by means of metrizamide CT. The fistula was surgically occluded with fascia temporalis and an artificial bone with fibrin glue. The CSF otorrhea from the tegmen defect was successfully stopped by this procedure and no recurrence has been observed. We discuss this extremely rare disease in this paper.


[A study of factors regulating ciliary activity using cultured human paranasal sinus mucosa].

Whether temperature, pH, osmotic pressure, Ca2+, Mg2+, and ATP have promotive effects on ciliary activity was investigated by a photo-electric method using cultured human ethmoid sinus mucosa. The results obtained were as follows: 1. Ciliary activity was reversible between 11 and 43 degrees C. It was activated with rising temperature, and inactivated at 40 degrees C. A temperature between 36 and 40 degrees C was optimal for activation of ciliary activity. 2. In the case of brief immersion (30 min.) in solutions between pH 4.5 and 8.5 ciliary activity was reversible, and with prolonged immersion (72 h) between pH 6.5 and 7.5 it was also reversible. with brief immersion within 30 min, pH 8.5 was an activating factor. 3. Ciliary activity was reversible in solutions between 143 and 1,140mOsm/Kg for 30 min. However, the ciliary reaction was different even at the same osmotic pressure according to the substance, NaCl or glucose, used to adjust it. Inhibited beating was reversible after prolonged immersion (72 h) in an osmotic pressure between 285 and 423mOsm/Kg adjusted by NaCl, and between 285 and 570mOsm/Kg adjusted by glucose. Any osmotic pressure of 428mOsm/Kg adjusted by NaCl activated ciliary activity. 4. Neither Ca2+ nor Mg2+, between 10(-2) and 10(-6) Mol, increased ciliary activity. 5. Between 10(-3) and 10(-5) Mol of Na-ATP, given exogenously, slightly activated ciliary movement, while Mg-ATP of 10(-3) Mol also activated it but only slightly. It is important to bear in mind the above results in administering local aerosol therapy for nasal and paranasal sinus diseases.


Shifting the paradigm: toward integrative research on mothers and children.

The mother-child relationship has been examined extensively but most research has centered on maternal characteristics associated with poor childhood developmental outcomes or maternal response to stressors. The "maternal deficit" model has misdirected research and intervention. Research on maternal depression based on the maternal deficit model has resulted in research designs that do not adequately address maternal outcomes. Research must be conceptualized to address parent-child relationships and situational factors that influence them.