Histamine downregulates CD14 expression via H2 receptors on human monocytes

Lipopolysaccharide (LPS) binds to LPS-binding protein (LBP) in plasma and is delivered to the cell surface receptor CD14 on human monocyte. LPS is transferred to the transmembrane signaling receptor toll-like receptor (TLR) 4. In the present study, the effect of histamine on the expression of CD14 on human monocytes was investigated. Histamine concentration- and time-dependently decreased the expression of cell surface CD14, whereas histamine did not decrease mRNA for CD14 nor increase soluble CD14 (sCD14). The inhibitory effects of histamine on CD14 expression were antagonized by H2-receptor antagonist, but not by H1 and H3/H4 antagonist. The effects of selective H2-receptor agonists, 4-methylhistamine and dimaprit, on CD14 expression mimicked that of histamine indicating that histamine regulated CD14 expression through the stimulation of H2-receptors. The pretreatment with histamine partially inhibited the LPS-induced TNF-alpha production in human peripheral blood mononuclear cells (PBMC). Such inhibition might be due to the down-regulation of CD14 expression on monocytes by histamine.     

Deficient IFN-γ Expression in Umbilical Cord Blood (UCB) T Cells Can Be Rescued by IFN-γ-Mediated Increase in NFATc2 Expression

Regulation of nuclear factor of activated T cells-c2 (NFATc2) gene expression is not clearly defined. We previously reported reduced NFATc2 protein expression in cord blood T lymphocytes. Here we show that NFATc2 expression in T cells is dependent in part on the presence of IFN-gamma during primary stimulation, as blocking of IFN-gamma blunted NFATc2 protein and mRNA upregulation. Conversely, addition of exogenous IFN-gamma during stimulation resulted in increased expression of NFATc2 in cord blood T lymphocytes. This correlated with rescue of deficient IFN-gamma expression by cord blood T cells. Rescue of IFN-gamma expression in cord blood T cells was dependent on the presence of antigen-presenting cells, as addition of IFN-gamma during stimulation of purified cord blood T cells did not result in an increase of IFN-gamma expression, and depletion of monocytes ablated the rescue of IFN-gamma expression. Our results point to impaired function in the antigen-presenting cell population of cord blood, playing a role in the hyporesponsiveness of T cells.     

Two cases of misinterpretation of molecular results in incontinentia pigmenti,and a PCR-based method to discriminate NEMO/IKKgamma dene deletion

Familial incontinentia pigmenti (IP) is a rare X-linked dominant disorder that affects ectodermal tissues. Over 90% of IP carrier females have a recurrent genomic deletion of exons 4-10 of the NEMO (IKBKG-IKKgamma) gene, which encodes a regulatory component of the IkB kinase complex, required to activate the NF-kB pathway. In IP, mutations in NEMOlead to the complete loss of NF-kB activation creating a susceptibility to cellular apoptosis in response to TNF-alpha. This condition is lethal for males during embryogenesis while females, who are mosaic as a result of X-inactivation, can survive. Recently, a second nonfunctional copy of the gene, DeltaNEMO, was identified, opposite in direction to NEMO in a 35.5-kb duplicated sequence tract. PCR-based detection of the NEMO deletion is diagnostic for IP disease. However, we present instances in which ex 4-10 DeltaNEMO pseudogene deletion occurs in unaffected parents of two females with clinically characteristic IP. These were missed by the currently standard PCR-based method, but can be easily discriminated by a new PCR-based test reported here that permits unambiguous molecular diagnosis and proper familial genetic counseling for IP.     

Mechanisms of inhibitory noradrenergic transmission in the rabbit facial vein

In isolated buccal segment of the rabbit facial vein, electrical responses produced by perivascular nerve stimulation and exogenously applied noradrenaline (NA) were recorded from the smooth muscle cells using microelectrode. Perivascular nerve stimulation hyperpolarized the smooth muscle cell membrane. The hyperpolarization was converted to depolarization after application of the -adrenoceptor antagonist, propranolol, and the depolarization was blocked by ₂ antagonists, yohimbine. These responses elicited by nerve stimulation were blocked by tetrodotoxin or guanethidine, but not by atropine. Exogenously applied NA mimicked the responses elicited by nerve stimulation. The amplitude of the -adrenoceptor-mediated hyperpolarization was increased in low potassium solution, decreased in high potassium solution, but unaltered by low sodium or low chloride solution, i.e., the hyperpolarization may be generated by an increase in potassium conductance of the membrane. An involvement of the apamin-sensitive (Ca-dependent) potassium channel or sodium-potassium ATPase in the hyperpolarization was ruled out.     

High energy phosphate utilization for work production and tension maintenance in frog muscle

Frog (Rana esculenta, L.) gastrocnemii (161 pairs) or sartorii (8 pairs) were stimulated by intermittent tetani at 10°C (20 Hz, supramaximal intensity, N₂ atmosphere) isometrically (IM) or isotonically (IT) for various durations (6–30 s) at different tensions (0.05–1.00 P₀=maximal IM tension at resting length,l _o). The energy expenditure (E) was measured from ATP and phosphocreatine breakdown and the high energy phosphate equivalent of lactic acid production. Both in IM and IT conditions,E was found to be a linear function of the summated tetanus duration (t):E=a+b t, whereb is the energy cost of tension maintenance. For the gastrocnemius, both in IM and IT,b was independent of the tension developed and equal to 0.45 mol P · g^–1 · s^–1, whereas for the sartoriusb was tension-dependent, varying between 0.58 and 0.28 mol P · g^–1 · s^–1 for P₀ and 0.18 P₀, respectively. The constancy of theb value in muscles with pennate structure may be tentatively attributed, at least in part, to the greater internal energy dissipation, regardless of the tension developed. The terma of the above equation is due to all time-independent processes of muscle contraction, i.e.: (1) activation energy; (2) internal work and (3) external work (in IT only). Based on the measured value ofa and on the work performed, the mechanical equivalent of P splitting was calculated as 17.7 kJ · mol^–1, a figure close to that previously obtained on frog sartorius (16.7 kJ · mol^–1) and dog gastrocnemius (19.2 kJ · mol^–1) When taking into account the energy due to the activation processes, the calculated net mechanical equivalent of P splitting amounted to 25.5 kJ · mol^–1.     

FLK1265-2493与C3d3融合基因真核表达质粒的构建及表达

目的 构建FLK1265-2493与C3d3融合基因真核表达质粒，并在体外进行表达和鉴定。方法采用PCR技术扩增FLK1265—2493片段，将该片段插入到pMDT-18载体中后，亚克隆至真核表达载体pSG．SS．C3d3．YL，构建FLD1265-2493与C3d3融合基因表达质粒。经限制性酶切鉴定和DNA序列测定结果证实后，将重组质粒pSG．SS．FLK1265—2493．C3d3．YL转染Hela细胞，检测其体外表达。结果免疫印迹和免疫细胞化学分析结果分别表明转染细胞上清液和胞浆内均有目的分子的存在。结论构建的DNA疫苗可在真核细胞内正确表达，这为进一步的动物实验打下了基础。     

Human defensins

Antimicrobial peptides are small, cationic, amphiphilic peptides of 12–50 amino acids with microbicidal activity against both bacteria and fungi. The eukaryotic antimicrobial peptides may be divided into four distinct groups according to their structural features: cysteine-free -helices, extended cysteine-free -helices with a predominance of one or two amino acids, loop structures with one intramolecular disulfide bond, and -sheet structures which are stabilised by two or three intramolecular disulfide bonds. Mammalian defensins are part of the last-mentioned group. The mammalian defensins can be subdivided into three main classes according to their structural differences: the -defensins, -defensins and the recently described -defensins. Mammalian -defensins are predominantly found in neutrophils and in small intestinal Paneth cells, whereas mammalian -defensins have been isolated from both leukocytes and epithelial cells. Recently, two novel human -defensins, human beta-defensin-3 (HBD-3), and human beta-defensin-4 (HBD-4) have been discovered. Similar to HBD-1 and HBD-2, HBD-3 has microbicidal activity towards the Gram-negative bacteria (Pseudomonas aeruginosa, Escherichia coli) and the yeasts Candida albicans and Malassezia furfur. In addition, HBD-3 kills Gram-positive bacteria such as Streptococcus pyogenes or Staphylococcus aureus, including multi-resistant S. aureus strains, and even vancomycin-resistant Enterococcus faecium. In contrast to HBD-1 and HBD-2, significant expression of HBD-3 has been demonstrated in non-epithelial tissues, such as leukocytes, heart and skeletal muscle. HBD-4 is expressed in certain epithelia and in neutrophils. Its bactericidal activity against P. aeruginosa is stronger than that of the other known -defensins. Here we present an overview of human antimicrobial peptides with some emphasis on their antifungal properties.J.J. Schneider and A. Unholzer contributed equally to this work     

Chemically modified tetracycline (CMT)-3 inhibits histamine release and cytokine production in mast cells: possible involvement of protein kinase C

Objective: To find novel inhibitors of mast cell function we have studied the effect of a potent, non-antimicrobial, chemically modified tetracycline, CMT-3 or COL-3, on key functions of mast cells.Methods and Results: In the presence of 25 μM CMT-3, the 48/80-induced histamine release from rat serosal mast cells was inhibited significantly, to 43.0 ± 7.3% of control. Similarily, the activation-induced secretion of TNF-α and IL-8 by HMC-1 cells were decreased in the presence of 25 μM CMT-3 to 13.5 ± 4.1% and 9.7 ± 1.1% of control, respectively. CMT-3 did not cause intracellular accumulation of TNF-α but instead it reduced the expression of TNF-α mRNA in HMC-1 cells. Moreover, CMT-3 was found to significantly inhibit the protein kinase C (PKC) activity with IC₅₀ value of 31 μM. CMT-3 inhibited effectively both human recombinant PKCalpha and PKCdelta isoforms. In comparison to doxycycline, CMT-3 was more effective as an inhibitor of both cytokine production and PKC activity.Conclusions: Considering the central role of PKC in mast cell activation, PKC inhibition could, at least partially, explain the observed inhibitory effects of CMT-3. The inhibition of the key proinflammatory functions of mast cells by CMT-3 suggests its potential clinical usefulness in the treatment of allergic and inflammatory disorders.Received 18 February 2005; returned for revision 7 March 2005; accepted by A. Falus 21 April 2005