The role of interleukin-1beta in direct and toll-like receptor 4-mediated neutrophil activation and survival

The regulation of systemic and local neutrophil activation is crucial to the clearance of infections and the successful resolution of inflammation without progress to tissue damage or disseminated inflammatory reactions. Using purified lipopolysaccharide (pLPS) and highly purified neutrophils, we have previously shown that Toll-like receptor 4 signaling is a potent neutrophil activator, but a poor stimulator of survival. In the presence of peripheral blood mononuclear cells (PBMCs), however, pLPS becomes a potent neutrophil survival factor. Interleukin (IL)-1beta has been identified as an important neutrophil activator and prosurvival cytokine, and is produced in abundance by LPS-stimulated PBMCs. We now show that IL-1beta fails to activate highly purified neutrophils or enhance their survival, but in the presence of PBMCs, IL-1beta induces neutrophil survival. We hypothesized that LPS-primed neutrophils might become responsive to IL-1beta, but were unable to demonstrate this. Moreover, IL-1ra failed to prevent pLPS + PBMC-dependent neutrophil survival. In studies of IL-1R1(-/-) mice, we found that LPS was still able to mediate neutrophil survival, and neutrophil survival was enhanced by the addition of monocytic cells. Thus an important paradigm of neutrophil regulation needs to be viewed in the context of a cellular network in which actions of IL-1beta on neutrophils are indirect and mediated by other cells.     

Physical and chemical properties of a bacterial virus as related to its inhibition by streptomycin

Streptomycin specifically and reversibly inhibits the injection mechanism of a bacterial virus (P9) which attacks Streptococcus faecium. Dihydrostreptomycin does not inhibit injection, but reverses the inhibition brought about by streptomycin. Another virus of this same host (P3) is resistant to inhibition, and it is also possible to isolate mutants of P9 which are partially resistant. Streptomycin, but not dihydrostreptomycin, is able to cross-link and precipitate polyanions, and it is likely that the cross-linking function of the antibiotic is involved in the inhibitory process. Evidence is presented that there is no marked difference in the DNA characteristics of the streptomycin-sensitive and streptomycin-resistant viruses. Indirect evidence from methylene blue-mediated photodynamic inactivation studies suggests that neither SM or DHSM penetrates to the DNA of the virus. Evidence from dark inactivation of the viruses by dyes or protamine sulfate, and its reversal by the antibiotics, suggests that the antibiotics combine in some way with the protein coat of both P3 and P9. Therefore resistance to SM⁴ is not a consequence of inability to bind antibiotic. SM precipitates P3, probably by cross-linking between adjacent phage particles. SM does not precipitate P9, showing that the antiviral effect is not a consequence of interparticle precipitation. However, SM, but not DHSM, causes a marked increase in the sedimentation constant of P9 under conditions in which SM is biologically active. Under the same conditions SM, but not DHSM, causes a shrinkage of the head and tail of the phage particle as viewed under the electron microscope. It is postulated that SM causes a shrinkage of the phage particle by cross-linking between adjacent protein subunits of a single phage particle, and thus pulling the protein subunits closer together. Since SM, but not DHSM, causes both the shrinkage of the phage particle and the inhibition of injection, it is postulated that the inhibition of injection is a consequence of the ability of the antibiotic to cross-link the protein subunits of the virus particle. Neomycin, which is structurally unrelated to SM, but is an effective cross-linking agent, behaves in a manner similar to SM.     

Adhesion of human neutrophils to and activation by type-I collagen involving a beta 2 integrin.

We previously demonstrated that the alpha 1(I) polypeptide chain of collagen can bind and activate polymorphonuclear neutrophils (PMN). In the present experiments, performed in culture grade 96-well plastic plates coated with collagen, fibronectin, or other proteins, adhesion was assessed by staining the adhering cells after 30 min with crystal violet and measuring absorbance at 560 nm, and activation of PMNs was assessed by measuring the amount of O2-formed. Adhesion occurred at 17 and 37 degrees C but activation at 37 degrees C only. Monoclonal antibody anti-CD 18 inhibited adhesion, showing that the receptor of collagen I on PMNs is a beta 2 integrin. On the other hand, adhesion of PMNs to fibronectin was inhibited by monoclonal antibodies to CD18 and to CD11b.     

玉米醇溶蛋白的物理化学改性

背景：玉米醇溶蛋白作为缓控释材料和生物可降解药用辅料在药剂学领域有着广泛的应用。 目的：对玉米醇溶蛋白的物理化学改性方法进行综合分析。 方法：应用计算机检索CNKI和PubMed数据库中1998-01/2010-10关于玉米醇溶蛋白的文章,在标题和摘要中以“玉米醇溶蛋白,玉米朊,改性,接枝”或“ZEIN,Modification,Graft”为检索词进行检索。选择文章内容与玉米醇溶蛋白改性研究相关,同一领域文献则选择近期发表或发表在权威杂志文章。 结果与结论：对玉米醇溶蛋白的改性研究进行总结有利于指导玉米醇溶蛋白的改性研究,从而增加玉米醇溶蛋白在药物制剂,生物医学工程方面的利用价值。     

中子活化技术在人体组份研究中的应用

综述了人体组份无创伤测量的新进展。中子诱发的瞬发γ射线,缓发γ射线测量和体内天然放射性＾４０Ｋ测量成功对用于人体内化学元素的和人体组份,获得的关于处于正常生理状态的体内分水、蛋白质、骨矿物、脂肪等信息对于研究人体的成长、衰老、骨质疏松、营养失调以及与人体组份变化以腾的疾病非常有价值。讨论了相关的测量技术。     

Monitoring the excitability of neocortical efferent neurons to direct activation by extracellular current pulses.

1. Extracellular action potentials were recorded from antidromically activated efferent neurons in visual, somatosensory, and motor cortex of the awake rabbit using low-impedance metal microelectrodes. Efferent neurons were also activated by current pulses delivered near the soma [juxtasomal current pulses (JSCPs)] through the recording microelectrode. Action potentials generated by JSCPs were not directly observed (because of the stimulus artifact), but were inferred with the use of a collision paradigm. Efferent populations studied include callosal neurons [CC (n = 80)], ipsilateral corticocortical neurons [C-IC (n = 21)], corticothalamic neurons of layer 6 [CF-6 (n = 57)], and descending corticofugal neurons of layer 5 [CF-5, corticotectal neurons of the visual cortex (n = 48)]. 2. Most CC neurons (45/46) and all C-IC (8/8) and CF-6 neurons (39/39) were directly activated by JSCPs at near-threshold intensities. Some CF-5 neurons (9/38), however, showed evidence of indirect activation. All efferent classes had similar current thresholds (means 1.85-2.10 microA) to direct activation by JSCPs, and thresholds were inversely related to extracellular spike amplitude. For each neuron, the range of JSCP intensities that generated response probabilities of between 0.2 and 0.8 was measured, and this "range of uncertainty" was significantly greater in CF-5 neurons (mean 32.7% of threshold) than in CC (mean 19.0%) or CF-6 (mean 20.4%) neurons. 3. Several factors indicate that the threshold of efferent neurons to JSCPs is very sensitive to excitatory and inhibitory synaptic inputs. Iontophoretic applications of gamma-aminobutyric acid (GABA) increased the threshold to JSCPs, and glutamate reduced the threshold. Electrical stimulation of afferent pathways at intensities just below threshold for eliciting action potentials resulted in a dramatic decrease in JSCP threshold. This initial short-latency threshold decrease was specific to stimulation of particular afferent pathways and is thought to reflect excitability changes associated with EPSPs. Examination of such subliminal responses revealed subthreshold synaptic inputs that were not revealed by examination of all-or-none action potentials. In contrast to the specificity of the short-latency threshold decrease, a long-lasting increase in JSCP threshold was seen in virtually all neurons after stimulation of each of the afferent pathways tested. This increase in threshold usually began 20-40 ms after stimulation, lasted for 100-200 ms, and is thought to reflect excitability changes associated with a long-lasting inhibitory postsynaptic potential (IPSP) seen in many cortical neurons. 4. Many neurons in primary somatosensory cortex of rat, cat, and rabbit have no demonstrable receptive fields.(ABSTRACT TRUNCATED AT 400 WORDS)     

Physical limitations of the TOBEC method: accuracy and long-term stability

While measurement of electrical conductivity provides one way of estimating body composition in live animals, the accuracy of methods based on this principle requires further study. In this work, we evaluate the effect of the ambient conditions and sample geometry on the response given by an SA-2 model (EM-SCAN) apparatus as well as the reproducibility of the measurements over time. A 2 degrees C variation of the sample temperature (a normal range for living animals) can result in a 6-10% variation in the response. When the conductive mass of a sample is increased in length, the response of the apparatus does not increase once the sample length reaches half the length of the measurement chamber. Samples having the same conductive mass but different shapes can show up to a 17-fold variation in signal. The geometry of the sample itself appears to be of prime importance for determining the strength of the response. We find that the reference phantom provided by the manufacturer is inadequate for calibration and is unable to detect the 10% variation over time of the signal of the apparatus. Until these problems are resolved, the usefulness of the EM-SCAN SA-2 to investigate body composition accurately is questionable.     

Monosynaptic activation of a direct reticulo-spinal pathway by the dentate nucleus

Experiments were performed to test the hypothesis that the output of the dentate nucleus can affect the excitability of spinal neurons via the reticular formation. In the first group of studies, the response of neurons in the medial reticular formation to stimulation of the dentate nucleus was investigated. In the second set of experiments, stimuli were applied in the same region of the medial reticular formation in order to determine whether neurons in the dentate nucleus could be antidromically activated from this part of the brainstem. The results indicate that the output from the dentate nucleus monosynaptically activates medial reticular neurons which project to the spinal cord. This finding, together with the observation that stimulation of the medial reticular formation can antidromically activate neurons in the dentate nucleus, demonstrates that there is an anatomical substrate by which the dentate nucleus can affect the excitability of spinal neurons via a rapidly conducting reticulospinal pathway.     

Assessment of cortical and trabecular bone distribution in the beagle skeleton by neutron activation analysis

The distribution of bone calcium between morphologically identifiable cortical and trabecular bone obtained by dissection and quantitated by neutron activation analysis (NAA) is described. The skeleton of a female beagle dog was dissected into approximately 400 pieces and assayed for ⁴⁹Ca produced in the University of California, Irvine TRIGA reactor. For each of the skeletal sections, we give the initial weight of the alcohol-fixed tissue, which includes cortical bone, trabecular bone, marrow, and cartilage, and a final tissue weight after the marrow and trabecular bone have been dissected away; total section and cortical section calcium weights are reported. The level of detail is represented, for example, by the vertebrae, which were divided into three parts (body, spine, and transverse processes) and by the long bones, which were divided into 10–12 parts such that characterization of the epiphysis, metaphysis, and diaphysis was accomplished. The nedian percentage cortical calcium values for cervical, thoracic, and lumbar vertebrae were 82%, 56%, and 66%, respectively; however, variation within these groups and among individual vertebral sections was about a factor of 2. For long bones, the median percentage cortical calcium varied from 90–100% in the midshaft to below 50% in the proximal and distal sections. The final calculated cortical tissue-to-calcium mass ratio (TCR) varied from about 4.5 for midshafts of the long bones to about 9 for thoracic vertebral bodies and indicated that the mineral fraction of cortical bone is not constant throughout the skeleton. The ratio of cortical to trabecular calcium in the skeleton was 79.6:20.4.     

Measurement of total body chlorine by prompt gamma in vivo neutron activation analysis

A method of measuring total body chlorine (TBCl) by prompt gamma in vivo neutron activation analysis is described which depends on the same NaI(Tl) spectra used for determinations of total body nitrogen. From these spectra counts ratios of chlorine to hydrogen are derived and TBCl is determined using a model of body composition which depends on measured body weight, total body water (by tritium dilution) and protein (6.25 X nitrogen) as well as estimated body minerals and glycogen. The precision of the method based on scanning an anthropomorphic phantom is at present only approximately 9% (SD), for a patient dose equivalent of less than 0.30 mSv. Spectra collected from 67 normal volunteers (32 male, 35 female) yielded mean values of TBCl of 72 +/- 19 (SD) g in males and 53.6 +/- 15 g in females, in broad agreement with values reported by workers using delayed gamma methods. Results are also presented for two human cadavers analysed both by neutron activation and by conventional chemical analysis; the ratios of TBCl (neutron activation) to TBCl (chemical) were 0.980 +/- 0.028 (SEM) and 0.91 +/- 0.09. Finally, it is suggested that an improvement in precision will be achieved by increasing the scanning time (thereby increasing the radiation dose equivalent) and by adding two more detectors.     

Development of apparatus to measure calcium changes in the forearm and spine by neutron activation analysis using californium-252.

Techniques were developed to measure small changes of calcium in the forearm and spine in vivo by neutron activation analysis using two sources of 252Cf in a hospital environment. Using purpose-built part-body counters and bilateral irradiation with 7.5 cm premoderation between the sources and the bone, peripheral bone was measured with a total source strength eventually as low as 50 mCi. Two methods of spectral analysis were used and compared. Patient studies of the forearm were successfully undertaken, with a precision of 2.6% which included patient movement, and an annual bone dose of less than 10 rem and skin dose of 35 rem from six measurements. Two 100 mCi sources were used for measurements of the lumbar spine. Care was taken to minimise the problems of non-uniformity of activation which are present using unilateral irradiation. Emphasis was placed on measuring the bodies of the vertebrae with adequate sensitivity and uniformity, and the spinous processes and arches with low sensitivity. A whole body counter was used for the bilateral detection of the induced activity. The precision of the method was 3.0% with an annual peak bone dose of 2.1 rem and skin dose of 18 rem from three measurements.     

Usefulness and limitations of an in-house direct radioimmunoassay for 17-hydroxyprogesterone in serum

Since conventional radioimmunoassays (RIA) for measurement of 17-hydroxyprogesterone (17-OHP) in serum samples require a laborious solvent extraction step, a direct and rapid in-house RIA was developed for early diagnosis and management of congenital adrenal hyperplasia (CAH). In-house rabbit anti-17-OHP antiserum, tritium labelled 17-OHP and dextran-coated charcoal were used in assay buffer with low pH 5.1 and preheated serum samples. Both inter- and intra-assay CVs were < 10% and the sensitivity was 1.2 nmol/l or 12 fmol/tube. Results from the direct assay correlated well with values from an extraction assay, r = 0.88 in samples from CAH patients, r = 0.85 in adults and children, 0.69 and 0.40 in term and preterm neonates respectively, 0.66 and 0.63 in luteal phase and third trimester pregnancy; p < 0.001 in all groups except p < 0.05 in preterm neonates. However, results from the direct assay were two to three times higher in serum samples from CAH patients, normal adults and children, but were five to seven times higher in pregnancy and term neonates and thirty times higher in preterm neonates. The markedly elevated levels measured by the direct assay are probably due to cross-reactivities with water-soluble steroid metabolites such as 17-hydroxypregnenolone sulphate and dehydroepiandrosterone sulphate (DHEAS). Although the direct assay is only useful as a screening test for preterm babies, it can be used for both diagnosis and monitoring of treatment of CAH in all other age groups.     

Improved detection of viruses by electron microscopy after direct ultracentrifuge preparation of specimens

We have adapted the Beckman Airfuge air turbine ultracentrifuge and the new EM-90 particle-counting rotor to improve detection by electron microscopy of viruses in clinical specimens. Samples were clarified by centrifugation, pelleted in the EM-90 rotor directly to Formvar-coated copper grids, and strained with 1.5% sodium phosphotungstate. Virus counts and endpoint titrations of serial dilutions of partially purified preparations of poliovirus, SA11 rotavirus, herpes simplex virus, and vaccinia virus showed an increase of ca. 1.5 log10 to 3.0 log10 over the virus titers of unconcentrated preparations of the same material. An increased yield of 14% more positive specimens for rotavirus was obtained after preparation of clinical samples by direct ultracentrifugation versus a method without virus concentration (82 versus 72). A prospective study showed that detection of adenoviruses, herpesviruses, and enteroviruses increased when specimens were prepared by direct ultracentrifugation. Direct ultracentrifugation with the EM-90 rotor in the Airfuge ultracentrifuge is a rapid concentration method which enhances the rate and yield of virus detection from clinical specimens by electron microscopy and is easily adaptable to a diagnostic virology laboratory.     

A method for preparation of immunosorbents by direct coupling of dissociated immune complexes

A simple and rapid method of preparing immunosorbents by use of isolated immune complexes is described. Dissociated immune complexes may be directly coupled to activated Sepharose and an immunosorbent gel with complementary molecular populations of immunospecifically purified ligands is obtained. The antigen binding capacity of insolubilized antibodies was about 50% of the antigen bound at equivalence using liquid phase precipitation. The batch technique described may be scaled up and the only limitation is the availability of the ligands. The usefulness of the technique is illustrated in two model systems.     

Characteristics of human synovial fibroblast activation by IL-1 beta and TNF alpha.

Human synovial fibroblast cell lines (HSN), established from tissues obtained from the knee joints of arthritis patients undergoing arthoplasty, were used to investigate the effects of human interleukin-1 (IL-1) beta and tumour necrosis factor (TNF)alpha on proliferation and prostaglandin E2 (PGE2) secretion. IL-1 beta and TNF alpha were equipotent stimulators of HSN proliferation. Classical non-steroidal anti-inflammatory drugs and glucocorticoids significantly augmented this effect. In addition, IL-1 beta and TNF alpha were potent inducers of PGE2 production while exogenous PGE2 was growth inhibiting. These data suggest that the secretion of PGE2 by monokine-stimulated HSN exerts a negative feedback signal. Further examination of IL-1 beta- and TNF alpha-induced PGE2 secretion revealed IL-1 beta to be a more potent stimulator; however, this observation may be due, in part, to differences in the kinetics of induction. Rabbit anti-IL-1 beta and anti-TNF alpha specifically neutralized both proliferation and PGE2 production induced by these monokines, but anti-IL-1 beta (or anti-IL-1 alpha) did not block TNF alpha activity. It is unclear whether TNF alpha stimulates HSN to produce IL-1, but the antibody data suggest that extracellular IL-1 is not responsible for TNF alpha in vitro activity.     

Integrin beta6 mediates phospholipid and collectin homeostasis by activation of latent TGF-beta1

Surfactant lines the alveolar surface and prevents alveolar collapse. Derangements of surfactant cause respiratory failure and interstitial lung diseases. The collectins, surfactant proteins A and D, are also important in innate host defense. However, surfactant regulation in the postnatal lung is poorly understood. We found that the epithelial integrin, alphavbeta6, regulates surfactant homeostasis in vivo by activating latent transforming growth factor (TGF)-beta. Adult mice lacking the beta-subunit of alphavbeta6 (Itgb6-/-) developed increased bronchoalveolar lavage phospholipids and surfactant proteins A and D, and demonstrated abnormal-appearing alveolar macrophages, reminiscent of the human disease pulmonary alveolar proteinosis. Using lung-specific expression of constitutively active TGF-beta1 in Itgb6-/- mice, we found that TGF-beta1 was sufficient to normalize these abnormalities. Tgfbeta1-deficient mice also demonstrated increased phospholipids and surfactant proteins A and D, but mice lacking the key TGF-beta signaling molecule, SMAD3, did not. Therefore, integrin-mediated activation of latent TGF-beta1 regulates surfactant constituents independent of intracellular SMAD3. In vivo increases in surfactant protein A and D were not associated with increases in mRNA for these proteins in alveolar tissue from Itgb6-/- mice. On the other hand, isolated alveolar macrophages from Itgb6-/- mice were defective in processing phospholipids in vitro, suggesting that reduced surfactant clearance contributes to altered surfactant homeostasis in these mice in vivo. These findings show that alphavbeta6 and TGF-beta1 regulate homeostasis of phospholipids and collectins in adult mouse lungs and may have implications for anti-fibrotic therapeutics that inhibit active TGF-beta in the lung.