Interleukin-4 (IL-4) plays a major role in immunoglobulin E (IgE) production. Its signal is conferred to effector cells through binding to the alpha chain of the IL-4 receptor (IL-4Ralpha). We present further evidence for polymorphisms in the IL-4Ralpha gene having an effect on IgE regulation. For two of four common polymorphisms, S503P and Q576R, we found an association with lowered total IgE concentrations (P=0.0008 if occurring together). The polymorphism S503P has not yet been described and is located within the I4R motif of the receptor. In vitro analyses using synthetic peptides of this region showed that the tyrosine kinase Janus kinase 1 (JAK1), as well as IRS-1 and IRS-2 bind to the I4R motif irrespective of the polymorphism or a tyrosine phosphorylation. In vivo immunoassays using T cells of four different groups of individuals (S503/Q576; P503/Q576; S503/R576; P503/R576) revealed that only in case of both polymorphisms the phosphorylation of IRS-1 and IRS-2, but not JAK1 was increased. We found no binding of STAT6 to the I4R synthetic peptides; however, the phosphorylation was reduced in the presence of any of the two polymorphisms, including P503 alone. We discuss possible conformational changes of the receptor leading to the observed effects on the phosphorylation status of IRS-1, IRS-2 and STAT6, in addition to previous findings that Q576R alters STAT6 binding. We conclude that P503 and R576 influence the signal transduction pathways through the IL-4Ralpha, an effect that is magnified by the presence of both polymorphisms. This could explain the observed association effects with lowered total IgE concentrations.

The mammalian intestine is colonized by a dense microbial community, the microbiota. Homeostatic and symbiotic interactions facilitate the peaceful co-existence between the microbiota and the host, and inhibit colonization by most incoming pathogens ('colonization resistance'). However, if pathogenic intruders overcome colonization resistance, a fierce, innate inflammatory defense can be mounted within hours, the adaptive arm of the immune system is initiated, and the pathogen is fought back. The molecular nature of the homeostatic interactions, the pathogen's ability to overcome colonization resistance, and the triggering of native and adaptive mucosal immune responses are still poorly understood. To study these mechanisms, the streptomycin mouse model for Salmonella diarrhea is of great value. Here, we review how S. Typhimurium triggers mucosal immune responses by active (virulence factor elicited) and passive (MyD88-dependent) mechanisms and introduce the S. Typhimurium mutants available for focusing on either response. Interestingly, mucosal defense turns out to be a double-edged sword, limiting pathogen burdens in the gut tissue but enhancing pathogen growth in the gut lumen. This model allows not only studying the molecular pathogenesis of Salmonella diarrhea but also is ideally suited for analyzing innate defenses, microbe handling by mucosal phagocytes, adaptive secretory immunoglobulin A responses, probing microbiota function, and homeostatic microbiota-host interactions. Finally, we discuss the general need for defined assay conditions when using animal models for enteric infections and the central importance of littermate controls.

Gingipains are cysteine proteinases acting as key virulence factors of the bacterium Porphyromonas gingivalis, the major pathogen in periodontal disease. The 1.5 and 2.0 A crystal structures of free and D-Phe-Phe-Arg-chloromethylketone-inhibited gingipain R reveal a 435-residue, single-polypeptide chain organized into a catalytic and an immunoglobulin-like domain. The catalytic domain is subdivided into two subdomains comprising four- and six-stranded beta-sheets sandwiched by alpha-helices. Each subdomain bears topological similarities to the p20-p10 heterodimer of caspase-1. The second subdomain harbours the Cys-His catalytic diad and a nearby Glu arranged around the S1 specificity pocket, which carries an Asp residue to enforce preference for Arg-P1 residues. This gingipain R structure is an excellent template for the rational design of drugs with a potential to cure and prevent periodontitis. Here we show the binding mode of an arginine-containing inhibitor in the active-site, thus identifying major interaction sites defining a suitable pharmacophor.

Voltage-gated Na(+) channels (VGSCs) exist as macromolecular complexes containing a pore-forming alpha subunit and one or more beta subunits. The VGSC alpha subunit gene family consists of 10 members, which have distinct tissue-specific and developmental expression profiles. So far, four beta subunits (beta1-beta4) and one splice variant of beta1 (beta1A, also called beta1B) have been identified. VGSC beta subunits are multifunctional, serving as modulators of channel activity, regulators of channel cell surface expression, and as members of the immunoglobulin superfamily, cell adhesion molecules (CAMs). beta subunits are substrates of beta-amyloid precursor protein-cleaving enzyme (BACE1) and gamma-secretase, yielding intracellular domains (ICDs) that may further modulate cellular activity via transcription. Recent evidence shows that beta1 regulates migration and pathfinding in the developing postnatal CNS in vivo. The alpha and beta subunits, together with other components of the VGSC signaling complex, may have dynamic interactive roles depending on cell/tissue type, developmental stage, and pathophysiology. In addition to excitable cells like nerve and muscle, VGSC alpha and beta subunits are functionally expressed in cells that are traditionally considered nonexcitable, including glia, vascular endothelial cells, and cancer cells. In particular, the alpha subunits are up-regulated in line with metastatic potential and are proposed to enhance cellular migration and invasion. In contrast to the alpha subunits, beta1 is more highly expressed in weakly metastatic cancer cells, and evidence suggests that its expression enhances cellular adhesion. Thus, novel roles are emerging for VGSC alpha and beta subunits in regulating migration during normal postnatal development of the CNS as well as during cancer metastasis.

OBJECTIVE

To investigate the safety, tolerability, and pharmacokinetics of the anti-tumor necrosis factor-alpha monoclonal antibody infliximab in subjects with intravenous immunoglobulin (IVIG)-resistant Kawasaki disease (KD).

METHODS

We conducted a multicenter, randomized, prospective trial of second IVIG infusion (2 g/kg) versus infliximab (5 mg/kg) in 24 children with acute KD and fever after initial treatment with IVIG. Primary outcome measures were the safety, tolerability, and pharmacokinetics of infliximab. Secondary outcome measures were duration of fever and changes in markers of inflammation.

RESULTS

Study drug infusions were associated with cessation of fever within 24 hours in 11 of 12 subjects treated with infliximab and in 8 of 12 subjects retreated with IVIG. No infusion reactions or serious adverse events were attributed to either study drug. No significant differences were observed between treatment groups in the change from baseline for laboratory variables, fever, or echocardiographic assessment of coronary arteries.

CONCLUSIONS

Both infliximab and a second IVIG infusion were safe and well tolerated in the subjects with KD who were resistant to standard IVIG treatment. The optimal management of patients resistant to IVIG remains to be determined.

The striated muscle sarcomeres are highly organized structures composed of actin (thin) and myosin (thick) filaments that slide past each other during contraction. The integrity of sarcomeres is controlled by a set of structural proteins, among which are titin, a giant molecule that contains several immunoglobulin (Ig)-like domains and associates with thin and thick filaments, and [alpha]-actinin, an actin cross-linking protein. Mutations in several sarcomeric and sarcolemmal proteins have been shown to result in muscular dystrophy and cardiomyopathy. On the other hand, the disease genes underlying several disease forms remain to be identified. Here we describe a novel 57 kDa cytoskeletal protein, myotilin. Its N-terminal sequence is unique, but the C-terminal half contains two Ig-like domains homologous to titin. Myotilin is expressed in skeletal and cardiac muscle, it co-localizes with [alpha]-actinin in the sarcomeric I--bands and directly interacts with [alpha]-actinin. The human myotilin gene maps to chromosome 5q31 between markers AFM350yB1 and D5S500. The locus of a dominantly inherited limb-girdle muscular dystrophy (LGMD1A) resides in an overlapping narrow segment, and a new type of distal myopathy with vocal cord and pharyngeal weakness (VCPMD) has been mapped to the same locus. The muscle specificity and apparent role as a sarcomeric structural protein raise the possibility that defects in the myotilin gene may cause muscular dystrophy.

Interleukin-4 (IL-4) stimulation leads to the activation of the signal transducer and activator of transcription 6 (Stat6). In this study, we present data relating to the functional properties of Stat6. Human embryonic kidney 293 cells were shown to be deficient of Stat6 yet express all other components of the IL-4 signaling cascade. This cell line was used for transient-transfection studies of wild-type and mutant Stat6 proteins. The wild-type protein was shown to activate a reporter construct carrying multiple copies of the IL-4 response element derived from the human immunoglobulin heavy-chain germ line epsilon promoter. Similarly, a truncated protein lacking 41 amino acids of the N terminus was fully active. However, removal of the C-terminal 186 amino acids completely abolished transcription activation. Amino acid substitutions were introduced into the putative DNA binding domain (VVI at positions 411 to 413), the SH2 domain (R-562), or the tyrosine (Y-641) which presumably becomes phosphorylated upon activation. All three of these Stat6 mutants were unable to activate transcription in 293 cells. Wild-type and mutant Stat6 derivatives were also expressed in insect cells, and purified proteins were analyzed in vitro for the ability to interact with both DNA and tyrosine-phosphorylated peptides derived from the IL-4 receptor alpha chain. Mutations within the DNA binding domain, the SH2 domain, or tyrosine 641 completely abolished DNA binding. In contrast, only the SH2 mutant failed to interact with tyrosine-phosphorylated peptides. The transdominant effects of all Stat6 derivatives were analyzed by using HepG2 cells, which express endogenous Stat6 protein. Differential effects were observed with various mutants, supporting the current model of the Jak/STAT activation cycle.

Immune homeostasis is regulated by a finely tuned network of positive-negative regulatory mechanisms that guarantees proper surveillance avoiding hyperactivity that would lead to autoimmunity and inflammatory diseases. Immune responses involve the activation of immunoreceptors that contain tyrosine-based activation motifs (ITAMs). One arm of control involves immunoreceptor tyrosine-based inhibitory motif (ITIM)-bearing receptors, which upon co-aggregation initiate an inhibitory signal through recruitment of signal-aborting phosphatases. Recently, a new immunoregulatory function has been ascribed to ITAMs, which represent in fact dual function modules that, under specific configurations termed inhibitory ITAM (ITAMi), can propagate inhibitory signals. One paradigm is the immunoglobulin A (IgA) Fc receptor (FcalphaRI), which, upon interaction with IgA monomers in the absence of antigen, initiates a powerful inhibitory signal involving Src homology 2 domain-containing phosphatase 1 (SHP-1) recruitment that suppresses cell activation launched by a whole variety of heterologous receptors without co-aggregation. This explains the long known function of IgA as an anti-inflammatory isotype. The importance of this control mechanism in immune homeostasis is underlined by the high incidence of autoimmune and allergic diseases in IgA-deficient patients. ITAMi is now described for an increasing number of immunoreceptors with multiple roles in immunity. ITAMi signaling is also exploited by Escherichia coli to achieve immune evasion during sepsis. Here, we review our current understanding of ITAMi regulatory mechanisms in immune responses and discuss its role in immune homeostasis.

This paper describes several salivary components and their distribution in other mucosal secretions. Histatins are polypeptides which possess exceptional anti-fungal and anti-bacterial activities, but are nevertheless present only in saliva. Proline-rich proteins (PRPs) are members of a closely related family, of which the acidic PRPs are found solely in saliva, whereas the basic PRPs are also found in other secretions. Mucins are a group of glycoproteins that contribute to the visco-elastic character of the mucosal secretions. Despite the similarities in their structure and behavior, mucins have distinct tissue distributions and amino acid sequences. Other salivary proteins are present in one or more mucosal secretions. Lysozyme is an example of a component belonging to an ancient self-defense system, whereas secretory immunoglobulin A (sIgA) is the secreted part of a sophisticated adaptive immune system. Cystatins are closely related proteins which belong to a multigene family. Alpha-Amylase is a component that is believed to play a specific role in digestion, but is nevertheless present in several body fluids. Kallikrein and albumin are components of blood plasma. But whereas albumin diffuses into the different mucosal secretions, kallikrein is secreted specifically by the mucosal glands. The presence of these proteins specifically in saliva, or their distribution in other mucosal secretions as well, may provide important clues with respect to the physiology of those proteins in the oral cavity.

Exercise has a profound effect on the normal functioning of the immune system. It is generally accepted that prolonged periods of intensive exercise training can depress immunity, while regular moderate intensity exercise is beneficial. Single bouts of exercise evoke a striking leukocytosis and a redistribution of effector cells between the blood compartment and the lymphoid and peripheral tissues, a response that is mediated by increased hemodynamics and the release of catecholamines and glucocorticoids following the activation of the sympathetic nervous system and the hypothalamic-pituitary-adrenal axis. Single bouts of prolonged exercise may impair T-cell, NK-cell, and neutrophil function, alter the Type I and Type II cytokine balance, and blunt immune responses to primary and recall antigens in vivo. Elite athletes frequently report symptoms associated with upper respiratory tract infections (URTI) during periods of heavy training and competition that may be due to alterations in mucosal immunity, particularly reductions in secretory immunoglobulin A. In contrast, single bouts of moderate intensity exercise are "immuno-enhancing" and have been used to effectively increase vaccine responses in "at-risk" patients. Improvements in immunity due to regular exercise of moderate intensity may be due to reductions in inflammation, maintenance of thymic mass, alterations in the composition of "older" and "younger" immune cells, enhanced immunosurveillance, and/or the amelioration of psychological stress. Indeed, exercise is a powerful behavioral intervention that has the potential to improve immune and health outcomes in the elderly, the obese, and patients living with cancer and chronic viral infections such as HIV.

Biliary transport of rat immunoglobulin was studied by perfusion of isolated rat liver with blood containing radiolabeled immunoglobulin. Transport to bile was selective for polymeric IgA. Between 15 and 27% of polymeric IgA was transported from blood to bile during a 210-min perfusion period, and approximately 60% of the IgA transported to bile bore secretory component. Small quantities of IgM (0.12%) were transported; transport of IgG2 alpha, IgE, or monomeric IgA was not detected. Purification of radiolabeled polymeric IgA by affinity chromatography on human secretory component-Sepharose yielded a fraction that was transported more efficiently (i.e., up to 40% transported). In contrast, secretory IgA (colostral or biliary) was transported 1/25th to 1/12th as well as polymeric IgA myeloma protein. Complexes of 125I-labeled secretory component and polymeric IgA formed in vitro were transported poorly (0.1%) compared to polymeric IgA (26%). It was concluded that biliary transport of polymeric IgA requires combination of it with secretory component in the liver. In support of this hypothesis, rabbit IgG anti-rat secretory component antibodies were also transported to bile but normal rabbit IgG was not.

The bronchus-associated lymphoid tissue (BALT) and the nasal-associated lymphoid tissue (NALT) constitute organized lymphoid aggregates that are capable of T- and B-cell responses to inhaled antigens. BALT, located mostly at bifurcations of the bronchus in animals and humans, is present in the fetus and develops rapidly following birth, especially in the presence of antigens. Humoral immune responses elicited by BALT are primarily immunoglobulin A secretion both locally and by BALT-derived B cells that have trafficked to distant mucosal sites. Similarly located T-cell responses have been noted. On the basis of these findings, the BALT can be thought of as functionally analogous to mucosal lymphoid aggregates in the intestine and is deemed a member of the common mucosal immunologic system. NALT has been described principally in the rodent nasal passage as two separate lymphoid aggregates. It develops after birth, likely in response to antigen, and B- and T-cell responses parallel those that occur in BALT. It is not known whether NALT cells traffic to distant mucosal sites, although mucosal responses have been detected after nasal immunization. NALT appears from many studies to be a functionally distinct lymphoid aggregate when compared with BALT and Peyer's patches. It may exist, however, in humans as a diffuse collection of isolated lymphoid follicles.

The possibility that antibody responses in serum, saliva, or breast milk samples to oral vaccines or enteric infections may reflect the intestinal immune response was evaluated in Bangladeshi volunteers orally immunized with a cholera B subunit-whole-cell vaccine (B + WCV) and in patients convalescing from enterotoxin-induced diarrheal disease. Two peroral doses of B + WCV induced antitoxin and antibacterial antibody responses in the intestinal fluids of 76 and 92%, respectively, of the volunteers and in serum samples in 90 and 69% of those tested. These responses were comparable to those obtained after cholera or enterotoxigenic Escherichia coli disease. Whereas immunoglobulin A (IgA) antitoxin titer increases in saliva (44%) and breast milk (29%) specimens after vaccination were less frequent than in intestinal fluid (76%), antitoxin responses in saliva and breast milk occurred in 80 to 90% of the patients after disease. Also, antilipopolysaccharide (anti-LPS) titer increases in extraintestinal body fluids were found more frequently after disease than after vaccination. A comparison of the frequency and magnitude of antibody response in different body fluids with those in intestinal lavage fluid revealed no extraintestinal antibody that directly reflected the intestinal immunity. However, comparison of vibriocidal and IgG antitoxin antibodies in serum specimens with antitoxin and anti-LPS IgA responses in intestinal fluids after the vaccination of volunteers showed a sensitivity of 70 to 90% and a predictive accuracy of about 80% for the serum analyses reflecting the intestinal immune responses. Furthermore, antitoxin and anti-LPS antibody responses in saliva and breast milk samples seemed to be useful proxy indicators of a gut mucosal response of these antibodies after enterotoxin-induced diarrheal disease showing sensitivity vales of 70 to 90% and predictive accuracy vales of 70 to 100%.

Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease with protean clinical manifestations. The major route of infection is thought to be through subcutaneous inoculation of contaminated soil and water, although ingestion and inhalation of contaminated aerosols are also possible. This study examines infection through the intranasal route in a murine model to mimic infection through inhalation. Two strains of mice, C57BL/6 and BALB/c, exhibit differential susceptibilities to the infection, with the C57BL/6 mice being considerably more resistant. To examine host factors that could contribute to this difference, bacterial loads and cytokine profiles in the two strains of mice were compared. We found that infected BALB/c mice exhibited higher bacterial loads in the lung and spleen and that they produced significantly higher levels of gamma interferon (IFN-gamma) in the serum than C57BL/6 mice. Although tumor necrosis factor alpha and interleukin-1 could be detected in the nasal washes and sera of both strains of mice, the production in serum was transient and much lower than that of IFN-gamma. C57BL/6 mice also exhibited memory responses to bacteria upon reinfection, with the production of serum immunoglobulin G (IgG) and mucosal IgA antibodies. Thus, it is possible that the production of systemic and mucosal antibodies is important for protection against disease in C57BL/6 mice.

We used molecular cloning and functional analyses to extend the family of Neu differentiation factors (NDFs) and to explore the biochemical activity of different NDF isoforms. Exhaustive cloning revealed the existence of six distinct fibroblastic pro-NDFs, whose basic transmembrane structure includes an immunoglobulin-like motif and an epidermal growth factor (EGF)-like domain. Structural variation is confined to three domains: the C-terminal portion of the EGF-like domain (isoforms alpha and beta), the adjacent juxtamembrane stretch (isoforms 1 to 4), and the variable-length cytoplasmic domain (isoforms a, b, and c). Only certain combinations of the variable domains exist, and they display partial tissue specificity in their expression: pro-NDF-alpha 2 is the predominant form in mesenchymal cells, whereas pro-NDF-beta 1 is the major neuronal isoform. Only the transmembrane isoforms were glycosylated and secreted as biologically active 44-kDa glycoproteins, implying that the transmembrane domain functions as an internal signal peptide. Extensive glycosylation precedes proteolytic cleavage of pro-NDF but has no effect on receptor binding. By contrast, the EGF-like domain fully retains receptor binding activity when expressed separately, but its beta-type C terminus displays higher affinity than alpha-type NDFs. Likewise, structural heterogeneity of the cytoplasmic tails may determine isoform-specific rate of pro-NDF processing. Taken together, these results suggest that different NDF isoforms are generated by alternative splicing and perform distinct tissue-specific functions.

Triggering of mast cells and basophils by immunoglobulin E (IgE) and antigen induces various biochemical signals, including tyrosine kinase activation, which lead to cell degranulation and the release of mediators of the allergic reaction. The high-affinity receptor for IgE (Fc epsilon RI) responsible for initiating these events is a complex structure composed of an IgE-binding alpha-chain, a beta-chain and a homodimer of gamma-chains. It has been assumed that beta and gamma, which have extensive cytoplasmic domains, play an important but undefined role in coupling Fc epsilon RI to signal transduction mechanisms. Here we show that Fc epsilon RI engagement induces immediate in vivo phosphorylation on beta (tyrosine and serine) and gamma (tyrosine and threonine) by at least two different non-receptor kinases. We take advantage of unique features of this receptor system to demonstrate that the phosphorylation signal is restricted to activated receptors and is immediately reversible upon receptor disengagement by undefined phosphatases. Rapid phosphorylation and dephosphorylation may be a general mechanism to couple and uncouple activated receptors to other effector molecules. This could be particularly relevant to other multimeric receptors containing Fc epsilon RI gamma-chains or the related zeta and eta chains such as the T-cell antigen receptor (TCR) and the low-affinity receptor for immunoglobulin G (Fc gamma RIII, CD16).

Allergens and infections with parasitic helminths preferentially induced Th2 immune responses associated with elevated levels of serum immunoglobulin E (IgE) and expansion of eosinophils and mast cells. Interleukin-4 (IL-4) is a key cytokine in the differentiation of naive CD4+ T cells into Th2 cells, which produce a panel of cytokines including IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13 [1] and have been shown to trigger recovery from gastrointestinal nematodes [2]. Nonetheless, mice deficient for IL-4 have been shown to develop residual Th2 responses [3-5] and can expel the nematode Nippostrongylus brasiliensis [6], suggesting that there is a functional equivalent of IL-4 in these processes. IL-13 is a cytokine that shares some, but not all, biological activities with IL-4 [7,8]. There is now compelling evidence that IL-4 and IL-13 share receptor components, including IL-4R alpha and IL-13R alpha 1 [9]. In order to dissect the roles of IL-4 and IL-13 in the regulation of Th2 cells and in the response to nematode infections, we looked for differences between mice deficient for either the IL-4 gene or the IL-4R alpha gene. Unlike IL-4, IL-4R alpha was required for control of N. brasiliensis, and Th2 development during infection--as characterized by cytokine production, GATA-3 and surface CD30 expression--was more severely affected in IL-4R alpha-/- mice than in IL-4-/- mice. Injection of recombinant IL-13 induced worm expulsion in otherwise incompetent RAG2-/- mice. Our results suggest that IL-13 regulates Th2 responses to nematode infection and requires IL-4R alpha.

Immunoglobulin heavy chain binding protein (BiP, GRP 78) coprecipitates with soluble and membrane-associated variants of the T-cell antigen receptor alpha chain (TCR-alpha) which are stably retained within the ER. Chelation of Ca2+ during solubilization of cells leads to the dissociation of BiP from the TCR-alpha variants, which is dependent upon the availability of Mg2+ and hydrolyzable ATP; this suggests that Ca2+ levels can serve to modulate the association/dissociation of these proteins with BiP. In vivo treatment of cells expressing either the soluble or membrane-anchored TCR-alpha variants with the Ca2+ ionophore, A23187, or an inhibitor of an ER Ca(2+)-ATPase, thapsigargin, or the membrane-permeant Ca2+ chelator BAPTA-AM, results in the redistribution of these proteins out of the ER and their subsequent secretion or cell surface expression. Under the same assay conditions, no movement of BiP out of the ER is observed. Taken together, these observations indicate that decreased Ca2+ levels result in the dissociation of a protein bound to BiP, leading to its release from ER retention. These data suggest that the intracellular fate of newly synthesized proteins stably associated with BiP can be regulated by Ca2+ levels in the ER.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer mortality worldwide and ranks second in China. The prognosis of HCC remains dismal mainly because of its late diagnosis, especially in patients with coexisting chronic liver diseases. To identify serum biomarkers for HCC, sera from 20 healthy volunteers, 20 hepatitis B virus (HBV) infected patients and 20 HCC patients were selected for screening study and same number of sera into the same three groups were used for validation study. A strategy including sonication, albumin and <em>immunoglobulin</em> G (IgG) depletion and desalting was optimized for screening differentially expressed proteins of low abundance in serum. By 2-DE image analysis and MALDI-TOF-MS/MS identification, eight proteins including heat-shock protein 27 (HSP27), <em>alpha</em>-fetoprotein (AFP), <em>alpha</em>-1 antitrypsin, clusterin, caeruloplasmin, haptoglobin <em>alpha</em>2 chain, tranferrin and transthyretin were found significantly changed among the healthy, HBV and HCC groups. Further validation study by Western blot showed the detection of HSP27 in 90% HCC sera and two HBV sera, but in none of normal sera. Thus, 2-DE based serum proteome analysis can be useful in the screening of serum biomarkers for HCC and HSP27 could aid in the diagnosis of HCC though further validation is needed.

The physiological effects of "Shinrin-yoku" (taking in the atmosphere of the forest) were examined by investigating blood pressure, pulse rate, heart rate variability (HRV), salivary cortisol concentration, and immunoglobulin A concentration in saliva. Subjective feelings of being "comfortable", "calm", and "refreshed" were also assessed by questionnaire. The subjects were 12 male university students aged from 21 to 23 (mean+/-SD: 22.0+/-1.0). The physiological measurements were conducted six times, i.e., in the morning and evening before meals at the place of accommodation, before and after the subjects walked a predetermined course in the forest and city areas for 15 minutes, and before and after they sat still on a chair watching the scenery in the respective areas for 15 minutes. The findings were as follows. In the forest area compared to the city area, 1) blood pressure and pulse rate were significantly lower, and 2) the power of the HF component of the HRV tended to be higher and LF/(LF+HF) tended to be lower. Also, 3) salivary cortisol concentration was significantly lower in the forest area. These physiological responses suggest that sympathetic nervous activity was suppressed and parasympathetic nervous activity was enhanced in the forest area, and that "Shinrin-yoku" reduced stress levels. In the subjective evaluation, 4) "comfortable", "calm", and "refreshed" feelings were significantly higher in the forest area. The present study has, by conducting physiological investigations with subjective evaluations as supporting evidence, demonstrated the relaxing and stress-relieving effects of "Shinrin-yoku".

To compare the function of the tumor necrosis factor (TNF) and lymphotoxin (LT)alpha/beta systems in the mature immune system, these two pathways were blocked with soluble receptor-immunoglobulin (R-Ig) fusion proteins in normal adult mice. Inhibition of LT alpha/beta signaling using LT betaR-Ig or a blocking monoclonal antibody against murine LT beta had profound effects. The spleen lacked discrete B cell follicles and the marginal zone was altered. Less marked changes were detected in lymph nodes. LT alpha/beta inhibition also prevented germinal center formation in the spleen and impaired Ig production in response to sheep red blood cells (SRBC) immunization. These results show that the LT alpha/beta system is required for the maintenance of splenic architecture and normal immune responses, and not simply for the development of peripheral immune organs during ontogeny. In contrast, inhibition of the TNF/LT alpha pathway with TNF-R55-Ig did not affect the splenic architecture or the anti-SRBC response. Splenic defects and impaired antibody responses are seen in TNF-deficient mice, suggesting that TNF is important during development. Therefore relative to TNF, the LT system has the dominant influence on splenic organization and anti-SRBC Ig formation in the adult mouse.

BACKGROUND

Immunoglobulin A nephropathy (IgAN) is the most common cause of chronic renal failure among primary glomerulonephritis patients. The best treatment for IgAN remains poorly defined. We planned a long-term, prospective, open-label, multicentre, centrally randomized controlled trial to assess whether the combination of prednisone and ramipril was more effective than ramipril alone in patients with proteinuric IgAN.

METHODS

Ninety-seven biopsy-proven IgAN patients with moderate histologic lesions, 24-h proteinuria>> or =1.0 g and estimated glomerular filtration rate (eGFR)>> or = 50 ml/min/ 1.73 m(2) were randomly allocated to receive a 6-month course of oral prednisone plus ramipril (combination therapy group) or ramipril alone (monotherapy group) for the total duration of follow-up. The primary outcome was the progression of renal disease defined as the combination of doubling of baseline serum creatinine or end-stage kidney disease (ESKD). The secondary outcomes were the rate of renal function decline defined as the eGFR slope over time, and the reduction of 24-h proteinuria.

RESULTS

After a follow-up of up to 96 months, 13/49 (26.5%) patients in the monotherapy group reached the primary outcome compared with 2/48 (4.2%) in the combination therapy group. The Kaplan-Meier analysis showed a significantly higher probability of not reaching the combined outcome in the combination therapy group than in the monotherapy group (85.2% versus 52.1%; log-rank test P = 0.003). In the multivariate analysis, baseline serum creatinine and 24-h proteinuria were independent predictors of the risk of primary outcome; treatment with prednisone plus ramipril significantly reduced the risk of renal disease progression (hazard ratio 0.13; 95% confidence interval 0.03-0.61; P = 0.01). The mean rate of eGFR decline was higher in the monotherapy group than in the combination therapy group (-6.17 +/- 13.3 versus -0.56 +/- 7.62 ml/min/ 1.73 m(2)/year; P = 0.013). Moreover, the combined treatment reduced 24-h proteinuria more than ramipril alone during the first 2 years.

CONCLUSIONS

Our results suggest that the combination of corticosteroids and ramipril may provide additional benefits compared with ramipril alone in preventing the progression of renal disease in proteinuric IgAN patients in the long-term follow-up.

Flagellin, the structural component of flagellar filament in various locomotive bacteria, is the ligand for Toll-like receptor 5 (TLR5) of host cells. TLR stimulation by various pathogen-associated molecular patterns leads to activation of innate and subsequent adaptive immune responses. Therefore, TLR ligands are considered attractive adjuvant candidates in vaccine development. In this study, we show the highly potent mucosal adjuvant activity of a Vibrio vulnificus major flagellin (FlaB). Using an intranasal immunization mouse model, we observed that coadministration of the flagellin with tetanus toxoid (TT) induced significantly enhanced TT-specific immunoglobulin A (IgA) responses in both mucosal and systemic compartments and IgG responses in the systemic compartment. The mice immunized with TT plus FlaB were completely protected from systemic challenge with a 200x minimum lethal dose of tetanus toxin. Radiolabeled FlaB administered into the nasal cavity readily reached the cervical lymph nodes and systemic circulation. FlaB bound directly to human TLR5 expressed on cultured epithelial cells and consequently induced NF-kappaB and interleukin-8 activation. Intranasally administered FlaB colocalized with CD11c as patches in putative dendritic cells and caused an increase in the number of TLR5-expressing cells in cervical lymph nodes. These results indicate that flagellin would serve as an efficacious mucosal adjuvant inducing protective immune responses through TLR5 activation.