N‐Palmitoylation of the Radioprotective Domain of Interleukin‐1 Affords Inhibition of LPS‐Induced Nitric Oxide Generation

Interleukin‐1β (IL‐1β), a cytokine involved in homeostatic processes such as the immune system and inflammatory reactions, is a potent inducer of nitric oxide. The nonapeptide of human IL‐1β (VQGEESNDK, position 163–171, specific radioprotective domain–SRD) has been shown to retain radioprotective, immunostimulatory, and adjuvant activities of the native molecule without any inflammatory and pyrogenic properties. Unlike the parent IL‐1, SRD did not induce nitric oxide (NO) in control or irradiated RAW 264.7 cells nor did it affect inducible nitric oxide synthase (iNOS) as shown by ELISA based mRNA assay (Quantikine). A lipophillic derivative of the SRD (a palmitoyl residue at the amino terminus of the SRD) was synthesized (palmitoyl specific radioprotective domain, P‐SRD) to find out if this structural derivatization would restore the NO‐inducing ability of IL‐1. Surprisingly, P‐SRD not only did not induce NO, but significantly inhibited lipopolysaccharide (LPS) stimulated nitric oxide (NO) production. Quantikine studies indicated that P‐SRD also inhibited iNOS in LPS stimulated macrophage cells, suggesting that decrease in NO production in the presence of P‐SRD was the result of iNOS mRNA inhibition. These results indicate that N‐palmitoylation of SRD may effectively ameliorate potentially fatal symptoms of LPS‐induced endotoxemic hypotensive shock associated with IL‐1 without inflammatory and pyrogenic toxic side effects.     

Fc‐Dependent Monoclonal IgG1‐Mediated Suppression of Antibody Response

It has been known for a long time that passively administered antibodies (Abs) or immune complexes regulate the immune response to their specific antigen (Ag). IgG may sometimes suppress the humoral immune response against soluble antigens. The exact mechanism behind this phenomenon has not been understood yet and the requirement for the Fc part is still a matter of controversy. The present study was undertaken to clarify whether there is a true IgG‐mediated Fc‐dependent suppression of the immune response. Antigen and monoclonal antibody (mAb) used in this study were recombinant human interferon gamma (r‐hIFN‐γ) and mouse monoclonal antibodies specific for human IFN‐γ [anti‐hIFN‐γ mAb (CAy‐IFNγ38)] respectively. An intact IgG‐free preparation of Fab plus various Fc fragments was prepared from papain‐digested CAy‐IFNγ38. Ag/IgG and Ag/Fab complexes were prepared at various molar ratios. Keeping the Ag doses constant, mice were immunized either with Ag, Ag/IgG or Ag/Fab complexes. Primary immunization and the boosting were performed with the samples in complete and incomplete Freund's adjuvants respectively. Specific antibody levels were measured by an ELISA. Immunization performed with Ag/Fab complexes even at a molar ratio of 1:1.36 did not result in marked suppression of the response when compared to that of Ag only‐immunization. In contrast, Ag/IgG complexes resulted in nearly 90% suppression of the antibody response. Our observations suggest that Fc part of IgG molecule plays a crucial role in suppression of the in vivo antibody response against the Ag when complexed with intact IgG.     

Electrophysiological correlates of word meanings in 14‐month‐old human infants

Two studies were conducted in a preliminary attempt to determine whether electrophysiological procedures could be used to study early word learning in young infants. In Experiment 1, auditory evoked responses (AERs) were recorded from the frontal, temporal, and parietal scalp regions of a group of 14‐month‐old infants while they listened to a series of words. The brain responses reliably discriminated between words the infants were thought (by their parents and two independent raters) to understand versus those that they did not appear to know. A second experiment was conducted with a different group of infants to determine whether familiarity with the sounds alone could produce similar brainwave differences. This study, although showing that the brain‐wave patterns could discriminate familiar from novel speech‐sound sequences, did not demonstrate findings identical to those reported for differences in word understanding. These data are the first indication that AERs can be used to detect differences in word meanings in infants.     

Detection of viral antigen following exposure of one‐day‐old chickens to the Holland 52 strain of infectious bronchitis virus

One‐day‐old specific‐pathogen‐free single comb White Leghorn chickens were inoculated by eyedrop with either 0.1 ml of phosphate buffered saline containing 10^4.3 EID₅₀ of the Holland 52 strain of infectious bronchitis virus or normal allantoic fluid. Trachea, caecal tonsils and kidneys were removed from randomly selected birds at 0, 3, 5, 7 and 10 days post‐inoculation (pi) and the presence or absence of viral antigen was detected utilizing virus isolation (VI), an indirect fluorescent antibody technique (IFA), or a streptavidin‐biotin immunohistochemical (IH) technique. The presence of viral antigen as detected by the IH technique was also compared to histopathological changes in serial sections stained with haematoxylin and eosin.

Detection of viral antigen occurred more frequently with VI than with IFA or IH. The IFA and IH techniques detected viral antigen with about the same frequency.

Viral antigen was detected in the mucosa and submucosa of the trachea as early as 3 days pi, reached maximum levels at 5 days pi, and could still be detected in the mucosa at 10 days pi. In the kidney, viral antigen was not detectable by IFA or IH at 3 days pi, but could be visualized in distal convoluted tubules and collecting tubules at 5 days pi. At 7 days pi, antigen was detectable in the proximal convoluted tubules also. The presence of antigen in the caecal tonsils was sporadic, but it was detected in histiocytic cells and, occasionally, lymphoid cells of that organ.     

Determination of (+)‐3,3′,5‐Triiodo‐L‐thyronine (L‐T3) from Serum Using a Sequential Injection Analysis/Immunosensor System

Abstract

A sequential injection analysis/immunosensor system is proposed for the analysis of T₃ in serum with a rate of 75 samples/hr. The immunosensor design is based on the physical immobilization of anti‐T₃ in carbon paste. The working concentration range of the immunosensor in a sequential injection analysis system is between 3.4 and 340 ng/mL with a limit of detection of 2.19 ng/mL. The system is very reliable and very easy to design and operate.     

Effect of L‐Arginine on Age‐Related Changes in Macrophage Phagocytic Activity

Aging is associated with decline in the functioning of immune cells and reductions in serum L‐arginine and excretion of nitric oxide metabolites. Studies have shown that L‐arginine plays an important role in many physiological, biological and immunological processes. The present study was performed to determine if treatment with L‐arginine could prevent age‐related changes in phagocytic function of peritoneal macrophages. The effects of L‐arginine on phagocytic activity of peritoneal macrophages were compared between young and middle‐aged rats. Studies were performed in four groups of rats for 8 weeks: group 1 (3 month‐old) received physiological saline; group 2 (3 month‐old) received L‐arginine (160 mg/kg/day); group 3 (12 month‐old) received physiological saline; group 4 (12 month‐old) received L‐arginine (160 mg/kg/day). There were no significant differences in percentage of cells which were phagocytized. However, the phagocytosis of activated charcoal by peritoneal macrophages reduced with age. Thus, the phagocytic index was lower in macrophages of middle‐aged rats. L‐arginine treatment increased phagocytosis by peritoneal macrophages of both young and middle‐aged rats. L‐arginine‐induced augmentation in phagocytosis by macrophages were much higher in the middle‐aged rats compared with young rats. In summary, we found that L‐arginine prevented the age‐related reduction in phagocytic capability of peritoneal macrophages.     

Inhibitory Effect of Water‐Soluble Chitosan on TNF‐α and IL‐8 Secretion from HMC‐1 Cells

Mast cells are known to play an active role as effector cells in allergic inflammation and in diverse immunological and pathological processes. Activated mast cell‐derived pro‐inflammatory cytokines are important pathologic factors of progression of allergic inflammation. In this study, we investigated whether pro‐inflammatory cytokines (TNF‐α and IL‐8) can be induced by calcium stimulation in HMC‐1 cells, and high molecular weight water‐soluble chitosan (WSC) can inhibit the production of these cytokines. We provided evidence that the secretion of TNF‐α and IL‐8 from HMC‐1 cells was induced by Ca^{2 +}‐ionophore A23187 or Ca^{2 +}‐ATPase inhibitor TSG. Treatment of WSC (10 µg/ml) prior to stimulation with calcium agonists significantly blocked the secretion of TNF‐α by 65.1% for A23187 and 87.7% for TSG. IL‐8 secretion in response to A23187 or TSG was inhibited by 49.2% for A23187 and 34.1% for TSG, respectively, compared to absence of WSC. These results suggest that WSC has potential regulatory effects on allergic inflammatory diseases by down‐modulating Ca^{2 +}‐induced mast cell activation.     

Detection by immunofluorescent assay of serotype‐specific and group‐specific antigens of infectious bronchitis virus in tracheas of broilers with respiratory problems

The performances of seven immunofluorescent assays (IFAs) for infectious bronchitis virus (IBV) were examined on 115 trachea samples collected from 60 broiler flocks with clinical respiratory distress. Whether IBV strains could be serotyped directly on trachea sections by IFAs was examined using four different serotype‐specific monoclonal antibodies (MAbs). Two group‐specific IFAs using two different group‐specific MAbs, were compared with a conventional IFA using a chicken hyperimmune anti‐IBV serum. The use of the six MAbs in the IFA showed, in contrast to the use of the hyperimmune serum, no or only faint non‐specific staining. Although the sensitivities of the two group‐specific IFAs using MAbs were not higher (P> 0.05, power 80%) than the sensitivity of the IFA using hyperimmune serum, the interpretation of the staining of the first two IFAs was easier.

Seventeen of the 41 isolated IBV strains could be typed by IFA using the serotype‐specific MAbs. Serotyping by IFA was possible in about 70% of the tracheas that stained positive with group‐specific MAbs or hyperimmune serum and from which IBV was isolated. Use of serotype‐specific IFAs is a new and very fast way of diagnosing IBV infections including serotyping, providing enough time to adjust the vaccination programme for the next broiler flock.     

Thymic stromal lymphopoietin‐induced interleukin‐17A is involved in the development of IgE‐mediated atopic dermatitis‐like skin lesions in mice

Atopic dermatitis (AD) is a chronic inflammatory skin disease associated with elevated levels of allergen-specific IgE. Although thymic stromal lymphopoietin (TSLP) and interleukin-17A (IL-17A) have been considered as important factors in allergic diseases, their relationships in AD have not been fully defined. Here, we show the contribution of TSLP-induced IL-17A responses to IgE-mediated AD-like skin lesions. BALB/c mice passively sensitized by intraperitoneal injections of ovalbumin (OVA)-specific IgE monoclonal antibody (mAb) were challenged with OVA applied to the skin six times. Treatment with anti-TSLP mAb during the second to sixth challenges inhibited IgE-mediated AD-like skin lesions and IL-17A production in lymph nodes. Furthermore, the increased number of IL-17A-producing CD4⁺ and γδ T cells in lymph nodes and neutrophilic inflammation in the skin were reduced by anti-TSLP mAb. These findings prompted us to examine the roles of IL-17A. Treatment with anti-IL-17A mAb suppressed the AD-like skin lesions and neutrophilic inflammation; anti-Gr-1 mAb also inhibited them. Furthermore, treatment with CXCR2 antagonist reduced the AD-like skin lesions and neutrophilic inflammation accompanied by the reduction of IL-17A production; the increased CXCR2 expression in the epidermal cells was suppressed by anti-TSLP mAb. Meanwhile, these treatments, except for anti-Gr-1 mAb, inhibited the increased mast cell accumulation in the skin. Collectively, the mechanism of IgE mediating IL-17A-producing CD4⁺ and γδ T cells through TSLP by repeated antigen challenges is involved in AD-like skin lesions associated with skin inflammation, such as neutrophil and mast cell accumulation; TSLP may regulate CXCR2 signalling-induced IL-17A production.     

Effect of Bojungikki‐tang on Lipopolysaccharide‐Induced Cytokine Production from Peripheral Blood Mononuclear Cells of Chronic Fatigue Syndrome Patients

Abstract

Bojungikki‐tang (BIT) has been widely used to treat patients suffering from chronic fatigue syndrome (CFS). However, its effect has not been yet investigated experimentally. Based upon the clinical presentation of CFS, we hypothesized that cytokines may play a role in the pathogenesis of the disease. We studied the effect of BIT on lipopolysaccharide (LPS)‐induced various cytokines production in peripheral blood mononuclear cells (PBMC) of CFS patients. Bojungikki‐tang (1 mg/mL) significantly inhibited LPS‐induced tumor necrosis factor (TNF)‐α, interleukin (IL)‐6, IL‐10, transforming growth factor (TGF)‐β1 production by 63.55% ± 0.19%, 55.06% ± 0.27%, 48.23% ± 0.48%, 54.09% ± 0.76%, respectively (P < 0.05). Bojungikki‐tang showed a slightly lower inhibitory effect of LPS‐induced Interferon (IFN)‐γ production. These results suggest that BIT may be useful in treating fatigue associated with chronic diseases.     

Development of a PAN‐Specific,Affinity‐Purified Anti‐acetylated Lysine Antibody for Detection,Identification, Isolation,and Intracellular Localization of Acetylated Protein

Abstract

Acetylation on the lysine residue is an important event of posttranslational modification of proteins. In this study, we developed a simple method to produce and to affinity purify the specific anti‐acetylated lysine polyclonal antibody, which is useful for the detection, identification, isolation, and intracellular localization of acetylated proteins on the lysine residues. We utilized the chemically acetylated hemocyanin of keyhole limpets (KLH) as an immunogen to raise the immune serum and to isolate the population of the acetylated lysine specific antibody using the immobilized acetylated lysine as immunoaffinity‐ligand. The isolated antibody was tested to be useful for ELISA, immunoblotting detection, immunofluorescent localization, and affinity isolation of the acetylated proteins.     

Selective Isolation and Identification of HLA‐DR‐Associated Naturally Processed and Presented Epitope Peptides

Activation of CD4 helper T‐cells is mediated by the presentation of antigenic peptides in context of self‐MHC class II molecules. So far, the rules after which antigen‐presenting cells (APC) select a particular epitope within a given protein antigen have been not fully elucidated. Nevertheless, immunoaffinity purification of APC‐derived MHC class II molecules and the subsequent elutions of their with associated naturally processed and presented peptide epitopes (NPPE) have helped tremendously in understanding the nature of this rather complex process. In the present study, a novel approach for identifying such NPPEs is introduced, which is based on the culture of APCs in a completely protein‐free medium during the antigen presenting process. These APCs do still express a high level of MHC class II as determined by HLA‐DR cell surface staining, but the repertoire of the associated NPPEs is drastically reduced when compared to peptides eluted from cells maintained under normal culture condition. Actually, reverse phase‐high pressure liquid chromatography (RP‐HPLC) revealed that the entire NPPE repertoire consisted of less than ten major peaks, which is more than a 100‐fold reduction of background peptide peaks as seen in cells from serum‐containing culture conditions. Feeding APCs with exogenous antigens further confirmed the advantage of this novel system. While exogenous antigen‐derived peptide peaks in an NPPE‐eluate from RP‐HPLC are hardly to detect by conventional procedures, the very low background of serum‐ and protein‐free cultured APCs immensely facilitated this process, providing an improved tool for the identification and characterization of NPPEs.     

Impact of sympathetic nervous system activity on post‐exercise flow‐mediated dilatation in humans

AbstractTransient reduction in vascular function following systemic large muscle group exercise has previously been reported in humans. The mechanisms responsible are currently unknown. We hypothesised that sympathetic nervous system activation, induced by cycle ergometer exercise, would contribute to post‐exercise reductions in flow‐mediated dilatation (FMD). Ten healthy male subjects (28 ± 5 years) undertook two 30 min sessions of cycle exercise at 75% HR_max. Prior to exercise, individuals ingested either a placebo or an α₁‐adrenoreceptor blocker (prazosin; 0.05 mg kg⁻¹). Central haemodynamics, brachial artery shear rate (SR) and blood flow profiles were assessed throughout each exercise bout and in response to brachial artery FMD, measured prior to, immediately after and 60 min after exercise. Cycle exercise increased both mean and antegrade SR (P < 0.001) with retrograde SR also elevated under both conditions (P < 0.001). Pre‐exercise FMD was similar on both occasions, and was significantly reduced (27%) immediately following exercise in the placebo condition (t‐test, P = 0.03). In contrast, FMD increased (37%) immediately following exercise in the prazosin condition (t‐test, P = 0.004, interaction effect P = 0.01). Post‐exercise FMD remained different between conditions after correction for baseline diameters preceding cuff deflation and also post‐deflation SR. No differences in FMD or other variables were evident 60 min following recovery. Our results indicate that sympathetic vasoconstriction competes with endothelium‐dependent dilator activity to determine post‐exercise arterial function. These findings have implications for understanding the chronic impacts of interventions, such as exercise training, which affect both sympathetic activity and arterial shear stress.

Abbreviations

BF

blood flow

CO

cardiac output

FMD

flow‐mediated dilatation

HR

heart rate

LBNP

lower body negative pressure

MAP

mean arterial pressure

MSNA

muscle sympathetic nerve activity

SNS

sympathetic nervous system

SR

shear rate

SR_AUC

shear rate area under curve

SV

stroke volume

TPRi

total peripheral resistance index