Effects of Immune Complexes with Different Molecular Weights on Functional Activity and Intracellular pH of Neutrophils Exposed or Not Exposed to UV Light

Loading of neutrophils with immune complexes changed their functional activity and intracellular pH. These changes depended on the size of immune complexes. UV irradiation of neutrophils loaded with immune complexes led to a molecular weight-dependent decrease in intracellular pH. After irradiation the chemiluminescence response to latex increased only in neutrophils loaded with non-phagocytized immune complexes with low (less than 10 units IgG) and high molecular weights (more than 25 units IgG).     

Doxycycline vs. macrolides in combination therapy for treatment of community-acquired pneumonia

We assessed the comparative efficacy of empirical therapy with beta-lactam plus macrolide vs. beta-lactam plus doxycycline for the treatment of community-acquired pneumonia (CAP) among patients in the Australian Community-Acquired Pneumonia Study. Both regimens demonstrated similar outcomes against CAP due to either ‘atypical’ (Chlamydophila, Legionella or Mycoplasma spp.) or typical bacterial pathogens.     

Successful salvage treatment of steroid-refractory bronchiolar COP with low-dose macrolides

Cryptogenic organizing pneumonia (COP) is an idiopathic interstitial pneumonia characterized by fibroblastic tissues that occupy the lumina of alveoli and alveolar ducts or respiratory bronchioles. Although adequate doses and durations of glucocorticoids can improve its condition, COP is sometimes resistant to glucocorticoid therapy and is often lethal.Herein, a very rare case of 'bronchiolar COP' that was confined to the respiratory bronchioles is reported. This case indicates that macrolides may act as anti-inflammatory agents in patients with COP. Timely and precise pathological diagnosis may corroborate clinician diagnoses and eventually improve chances to overcome the disease.     

Antiangiogenic and antitumor effects of 14-membered ring macrolides on mouse B16 melanoma cells

We examined the effects of macrolide antibiotics on tumor angiogenesis, tumor growth and metastasis in the B16BL6 mouse melanoma and C57BL mouse system. Two 14-membered ring macrolide antibiotics, roxithromycin and clarithromycin, significantly reduced the dense capillary network area in a mouse dorsal air sac angiogenesis model, whereas a 15-membered ring macrolide, azithromycin, and a 16-membered ring macrolide, josamycin, did not show any inhibitory effect on angiogenesis at the same dose. Intraperitoneal administration of roxithromycin and clarithromycin at 50 mg/kg/day reduced the tumor size of B16BL6 melanoma to about 41% and 56%, respectively, of that of the control, and significantly suppressed pulmonary metastasis of B16BL6 cells in a spontaneous system. Azithromycin and josamycin, on the other hand, did not inhibit tumor growth or pulmonary metastasis of B16BL6 cells. Immunohistochemistry revealed that roxithromycin and clarithromycin reduced the tumor vascularity and increased apoptosis of the tumor cells in vivo. These results suggest that 14-membered ring macrolides have antiangiogenic and antitumor effects and might have possible therapeutic applications.     

Increase of resistance to macrolides in invasive Streptococcus pneumoniae in Spain (2000–2001)

This study examined the antimicrobial resistance of 1,278 invasive Streptococcus pneumoniae isolates from 41 Spanish laboratories participating in the European Antimicrobial Resistance Surveillance System (EARSS) during 2000 and 2001. Twenty-nine laboratories participated during both years and provided 950 of the isolates. Each laboratory used its own susceptibility testing methods. External quality assessment was performed annually by each participating laboratory. Significant increases in penicillin and erythromycin resistance were observed between 2000 and 2001. This increase was particularly noticeable in isolates from the laboratories participating during both years and in isolates from children and elderly patients.     

Elicitation of peritoneal polymorphonuclear neutrophils from mice

Although the mouse has been used extensively as a model for the study of host-parasite relationships, murine neutrophils have not been used nearly as often as PMNs from other species for in vitro functional assays due to lack of a commonly used procedure for murine neutrophil collection. These studies compared two eliciting agents and characterized the phagocytic and bactericidal activity of murine polymorphonuclear neutrophils elicited from the peritoneal cavity. We examined the effects of mouse strain (BALB/c, C57BL/6 and DBA/2) and sex, eliciting agent (0.2% glycogen vs. 3% fluid thioglycolate medium) and donor sacrifice method (ether vs. cervical dislocation) on the number of neutrophils recovered in peritoneal exudate. The greatest number of neutrophils was harvested when mice were sacrificed 5 h after intraperitoneal injection of 2.5 ml of 3% thioglycolate medium. This method as described allows reproducible collection of adequate numbers of neutrophils for use in in vitro assays of neutrophil phagocytic and bactericidal function.     

Activity of five quinolones, three macrolides and telithromycin against 12 Haemophilus influenzae strains with different resistance phenotypes

Gemifloxacin MICs for 12 Haemophilus influenzae strains with different resistance phenotypes were 0.001-0.015 mg/L. Gemifloxacin was bactericidal against all 12 strains after 24 h at 2 x MIC. Ciprofloxacin, levofloxacin, gatifloxacin and moxifloxacin had MICs of 0.008-0.03 mg/L and similar kill kinetics. Macrolides and telithromycin had unimodal MICs (1.0-8.0 mg/L), except for two strains without efflux systems (0.0125-0.5 mg/L) and two with efflux systems and ribosomal protein mutations (> 64.0 mg/L), and were bactericidal against eight to ten strains tested at 2 x MIC after 24 h.     

Expression of the CXCR6 on polymorphonuclear neutrophils in pancreatic carcinoma and in acute, localized bacterial infections

The chemokine receptor CXCR6 has been described on lymphoid cells and is thought to participate in the homing of activated T-cells to non-lymphoid tissue. We now provide evidence that the chemokine receptor CXCR6 is also expressed by activated polymorphonuclear neutrophils (PMN) in vivo: Examination of biopsies derived from patients with pancreatic carcinoma by confocal laser scan microscopy revealed a massive infiltration of PMN that expressed CXCR6, while PMN of the peripheral blood of these patients did not. To answer the question whether CXCR6 expression is a property of infiltrated and activated PMN, leucocytes were collected from patients with localized soft tissue infections in the course of the wound debridement. By cytofluorometry, the majority of these cells were identified as PMN. Up to 50% of these PMN were also positive for CXCR6. Again, PMN from the peripheral blood of these patients were nearly negative for CXCR6, as were PMN of healthy donors. In a series of in vitro experiments, up-regulation of CXCR6 on PMN of healthy donors by a variety of cytokines was tested. So far, a minor, although reproducible, effect of tumour necrosis factor (TNFalpha) was seen: brief exposure with low-dose TNFalpha induced expression of CXCR6 on the surface of PMN. Furthermore, we could show an increased migration of PMN induced by the axis CXCL16 and CXCR6. In summary, our data provide evidence that CXCR6 is not constitutively expressed on PMN, but is up-regulated under inflammatory conditions and mediates migration of CXCR6-positive PMN.     

Mononuclear cells from HIV-infected patients produce factors which enhance functional activity of polymorphonuclear neutrophils from healthy subjects.

The influence of mononuclear cell supernatants (MNCS) from nine healthy donors and 35 HIV-infected patients (17 with lymphoadenopathy syndrome (LAS), 15 with ARC and three with AIDS) on functional activity of polymorphonuclear neutrophils (PMN) from healthy donors was investigated. MNC after short-term cultivation (24 h) produced factors which enhanced chemiluminescence (CL) and chemotaxis of PMN. This augmentation did not depend on stimulation of MNC by mitogens (lipopolysaccharide Escherichia coli (LPS) and concanavalin A (Con A)) or on activation of PMN by FMLP. After 48 h of cultivation only MNC stimulated by LPS produced these factors. MNCS from HIV-infected patients provoked a more pronounced augmentation of PMN CL compared with MNCS from healthy subjects. This enhancement was observed in patients at all stages of infection, but was more pronounced in patients with LAS. MNCS impact on PMN CL was not connected with proliferative activity of MNC but was correlated with the level of CD4 cells. It was shown that removal of adherent cells from MNC fraction resulted in decreased MNCS impact. Treatment of MNCS by antibody to IL-1 beta, IL-8, interferon-alpha (IFN-alpha) and tumour necrosis factor-alpha (TNF-alpha) did not decrease MNCS impact on PMN CL.     

Intracellular regulation of neuronal nicotinic cholinorceptors

Experiments on isolated superior cervical ganglia from rats were used to study the effects of substances affecting intracellular second messengers on membrane currents evoked by iontophoretic application of acetylcholine (ACh currents) and on excitatory postsynaptic currents (EPSC) induced by single discharges of preganglionic nerve fibers. These studies showed that the adenylate cyclase activator forskolin, the phosphodiesterase inhibitor isobutylmethylxanthine (IMBX), and the protein kinase C activator phorbol ester decreased the amplitude of the ACh current. Neither IMBX nor phorbol ester had any effect on the amplitude or decay time constant of EPSC, while forskolin increased the amplitude of EPSC without altering its decay time constant. Thapsigargin, which liberates intracellular calcium, not only decreased the amplitude of the ACh current, but also decreased EPSC amplitude without affecting its decay time constant. These results suggest that intracellular signaling via protein kinases A and C may affect neuronal nicotinic cholinoceptors (nAChR) only by altering receptor desensitization and not affecting receptor sensitivity to transmitters released from nerves or the kinetics of receptor ion channels. At the same time, neuronal nAChR are influenced by intracellular calcium, which decreases their ability to be activated by exogenous (perhaps acting via desensitization) and nerve-released acetylcholine without affecting the kinetics of ion channel function. Translated from Rossiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 84, No. 10, pp. 985–993, October, 1998.     

截短型人骨保护素在CHO-DHFR-细胞中的表达及活性测定

目的:构建截短型人骨保护素(hTOPG)哺乳动物表达载体pcDNA3.1/DHFR-hTOPG, 实现其在CHO-DHFR-细胞中的高效表达, 获取生物活性较高的重组蛋白.方法:利用基因重组技术构建重组表达载体pcDNA3.1/DHFR-TOPG, 按LipofectamineTM 2000试剂盒说明书转染CHO-DHFR-细胞, 以含50 mL/L透析血清的IMDM培养基培养, 氨甲喋呤(MTX)加压筛选高表达细胞株, ELISA法和RT-PCR法测定重组蛋白和基因的表达, 并采用破骨细胞样细胞(OLC)诱导分化抑制实验测定重组蛋白的体外活性.结果:重组蛋白的表达量最高可达6 mg/L·72 h , 且能够明显抑制OLC生成(P<0.05).结论:截短型人OPG在CHO-DHFR-细胞中成功高效表达, 并具有良好的生物学活性, 为进一步的实验研究和临床应用提供了基础.     

Intracellular signalling involved in modulating human endothelial barrier function

The endothelium dynamically regulates the extravasation of hormones, macromolecules and other solutes. In pathological conditions, endothelial hyperpermeability can be induced by vasoactive agents, which induce tiny leakage sites between the cells, and by cytokines, in particular vascular endothelial growth factor, which increase the exchange of plasma proteins by vesicles and intracellular pores. It is generally believed that the interaction of actin and non-muscle myosin in the periphery of the endothelial cell, and the destabilization of endothelial junctions, are required for endothelial hyperpermeability induced by vasoactive agents. Transient short-term hyperpermeability induced by histamine involves Ca2+/calmodulin-dependent activation of the myosin light chain (MLC) kinase. Prolonged elevated permeability induced by thrombin in addition involves activation of the small GTPase RhoA and Rho kinase, which inhibits dephosphorylation of MLC. It also involves the action of other protein kinases. Several mechanisms can increase endothelial barrier function, depending on the tissue affected and the cause of hyperpermeability. They include blockage of specific receptors, and elevation of cyclic AMP by agents such as beta2-adrenergic agents. Depending on the vascular bed, nitric oxide and cyclic GMP can counteract or aggravate endothelial hyperpermeability. Finally, inhibitors of RhoA activation and Rho kinase represent a potentially valuable group of agents with endothelial hyperpermeability-reducing properties.     

Activated neutrophils express proteinase 3 on their plasma membrane in vitro and in vivo.

Apart from the diagnostic value of anti-neutrophil cytoplasmic antibodies (ANCA), their detailed characterization and that of their corresponding antigens have opened new ways for the exploration of the pathogenesis of primary systemic vasculitis. ANCA are now thought to play an important functional role via activation of phagocytic cells (e.g. polymorphonuclear neutrophils (PMN)). In this study we examined the mechanisms by which ANCA could gain access to proteinase 3 (PR3) in intact PMN, at two levels: ex vivo by analysing the presence of PR3 on the plasma membrane of PMN from patients with ANCA-associated vasculitis, and in vitro by stimulation of PMN using different cytokines, including recombinant tumour necrosis factor-alpha (rhTNF-alpha) and two forms of IL-8 (produced by monocytic and endothelial cells). Using immunocytochemical staining techniques (FACS and immunoelectronmicroscopy) PR3 has been detected on the plasma membrane of PMN from patients with active ANCA-associated vasculitis. However, this phenomenon is also seen in patients with sepsis who do not have ANCA. In addition, TNF-alpha and both forms of IL-8 act synergistically and induce a translocation of PR3 from the intragranular loci to the cell surface of PMN. These results provide strong evidence for the hypothesis that ANCA are directly pathogenic by binding to PR3 which is expressed on the cell surface of primed/activated PMN.     

人白细胞介素32基因的克隆表达及其生物学活性的研究

目的:克隆人白细胞介素32(hIL-32)基因,构建高效稳定的hIL-32大肠杆菌表达菌株,并对纯化的目的蛋白进行生物学活性的初步测定.方法:将人外周血单个核细胞( PBMC)经刀豆素A( Con A)刺激60 h后提取细胞的总RNA,通过逆转录聚合酶链式反应( RT-PCR)从刺激的PBMC中扩增出hIL-32的基因,通过基因克隆技术,构建hIL-32在pET-30 a(+)中的重组质粒pET30a-hIL32,用IPTG进行诱导,得到hIL-32的原核表达蛋白;采用Ni2+ -NTA亲和层析方法纯化目的蛋白;应用ELISA方法检测纯化蛋白诱导人PBMC产生IL-6的情况.结果:序列测定表明,hIL-32基因核苷酸长度为567 bp,编码189个氨基酸.重组质粒pET30 -hIL32转化至大肠杆菌BL21( DE3),重组菌菌体裂解物SDS-PAGE可检测到相对分子质量(Mr)为28 000的重组蛋白,表达的重组蛋白IL-32可促进人PBMC产生IL-6,浓度达到(127±4.8)ng/L,而对照组IL-6的浓度仅为(25±2.3)ng/L.结论:成功克隆了hIL-32基因并构建了高效且稳定表达hIL-32基因的大肠杆菌菌株,且表达的目的蛋白能诱导IL-6细胞因子的产生.     

Intracellular bacteria and adverse pregnancy outcomes

This review considers the role of intracellular bacteria in adverse pregnancy outcomes, such as miscarriage, stillbirths, and preterm labour. The cause of miscarriage, stillbirth and preterm labour often remains unexplained. Intracellular bacteria that grow either poorly or not at all on media used routinely to detect human pathogens could be the aetiological agents of these obstetric conditions. For example, Listeria monocytogenes and Coxiella burnetti are intracellular bacteria that have a predilection for the fetomaternal unit and may induce fatal disease in the mother and/or fetus. Both are important foodborne or zoonotic pathogens in pregnancy. Preventive measures, diagnostic tools and treatment will be reviewed. Moreover, we will also address the importance in adverse pregnancy outcomes of other intracellular bacteria, including Brucella abortus and various members of the order Chlamydiales. Indeed, there is growing evidence that Chlamydia trachomatis, Chlamydia abortus and Chlamydia pneumoniae infections may also result in adverse pregnancy outcomes in humans and/or animals. Moreover, newly discovered Chlamydia-like organisms have recently emerged as new pathogens of both animals and humans. For example, Waddlia chondrophila, a Chlamydia-related bacterium isolated from aborted bovine fetuses, has also been implicated in human miscarriages. Future research should help us to better understand the pathophysiology of adverse pregnancy outcomes caused by intracellular bacteria and to determine the precise mode of transmission of newly identified bacteria, such as Waddlia and Parachlamydia. These emerging pathogens may represent the tip of the iceberg of a large number of as yet unknown intracellular pathogenic agents.     

Intracellular localization of natural and modified oligonucleotides in primary human endothelial cells

Intracellular localization of natural and fluorescent-labeled oligonucleotides in primary human endothelial cells was studied by means of fluorescence microscopy and radioisotope analysis. Transport and distribution of oligonucleotides in endotheliocytes depended on their structure and resistance to hydrolysis under the effect of cell nucleases. Modification of 5′-terminal phosphate and 3′-terminal oligonucleotide increased the stability and ensures nuclear localization of oligonucleotides in cells. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 143, No. 2, pp. 163–165, February, 2007     

Intracellular signal transduction mediated by ligation of MHC class I molecules

Abstract: A great deal of knowledge has accumulated regarding signal transduction after ligation of MHC class I (MHC-I) molecules. In recent years focus has been given to delineation of the intracellular signal pathways activated after MHC-I ligation. Activation of tyrosine kinases leading to a rise in the intracellular free calcium concentration ([Ca²⁺]_i) is the major initial event occurring after MHC-I ligation of T cells. Curiously, the MHC-I-induced signaling is not dependent upon the cytoplasmic tail of the MHC-I molecule, suggesting that the MHC-I molecule induces intracellular signaling through association with other membrane-embedded molecules. More distal signaling events after MHC-I ligation includes activation of the Jak/Stat pathway leading to Stat-3 activation, and activation of the PI3-kinase leading to JNK activation and apoptosis. This review will sum up what is currently known about signaling induced by ligation of MHC-L