Carvedilol Modulates In‐Vitro Granulocyte‐Macrophage Colony‐Stimulating Factor‐Induced Interleukin‐10 Production in U937 Cells and Human Monocytes

Both granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and interleukin‐10 (IL‐10) are important mediators regulating inflammatory responses. Inflammatory processes have an important role in atherogenesis. In this paper, the effects of carvedilol on GM‐CSF‐induced IL‐10 production were examined on human monocytic cell line, U937, and purified human monocytes. First, we showed that one‐time carvedilol pretreatment at concentrations 0.3–10 μM dose‐dependently inhibited GM‐CSF‐induced IL‐10 production in U937 cells. In addition, we found carvedilol to be non‐cytotoxic at concentrations equal to or less than 10 μM. However, at concentrations higher than 10 μM, carvedilol induced programmed cell death in U937 cells. The inhibition of GM‐CSF‐induced IL‐10 production by carvedilol was also observed at the expression of mRNA. Furthermore, the inhibition of IL‐10 production was demonstrated in GM‐CSF‐activated purified human peripheral blood monocytes. Finally, long‐term carvedilol pretreatment of U937 cells up to 2 months at concentrations of 1.0 μM mildly enhanced the IL‐10 production. Our observations that carvedilol modulated GM‐CSF‐induced IL‐10 production may have some implication in understanding the broad‐spectrum effects of carvedilol in regulating inflammatory reactions.     

Long‐term exercise‐specific neuroprotection in spinal muscular atrophy‐like mice

Key points

• The real impact of physical exercise parameters, i.e. intensity, type of contraction and solicited energetic metabolism, on neuroprotection in the specific context of neurodegeneration remains poorly explored.
• In this study behavioural, biochemical and cellular analyses were conducted to compare the effects of two different long‐term exercise protocols, high intensity swimming and low intensity running, on motor units of a type 3 spinal muscular atrophy (SMA)‐like mouse model.
• Our data revealed a preferential SMA‐induced death of intermediate and fast motor neurons which was limited by the swimming protocol only, suggesting a close relationship between neuron‐specific protection and their activation levels by specific exercise.
• The exercise‐induced neuroprotection was independent of SMN protein expression and associated with specific metabolic and behavioural adaptations with notably a swimming‐induced reduction of muscle fatigability.
• Our results provide new insight into the motor units’ adaptations to different physical exercise parameters and will contribute to the design of new active physiotherapy protocols for patient care.

Abstract

Spinal muscular atrophy (SMA) is a group of autosomal recessive neurodegenerative diseases differing in their clinical outcome, characterized by the specific loss of spinal motor neurons, caused by insufficient level of expression of the protein survival of motor neuron (SMN). No cure is at present available for SMA. While physical exercise might represent a promising approach for alleviating SMA symptoms, the lack of data dealing with the effects of different exercise types on diseased motor units still precludes the use of active physiotherapy in SMA patients. In the present study, we have evaluated the efficiency of two long‐term physical exercise paradigms, based on either high intensity swimming or low intensity running, in alleviating SMA symptoms in a mild type 3 SMA‐like mouse model. We found that 10 months’ physical training induced significant benefits in terms of resistance to muscle damage, energetic metabolism, muscle fatigue and motor behaviour. Both exercise types significantly enhanced motor neuron survival, independently of SMN expression, leading to the maintenance of neuromuscular junctions and skeletal muscle phenotypes, particularly in the soleus, plantaris and tibialis of trained mice. Most importantly, both exercises significantly improved neuromuscular excitability properties. Further, all these training‐induced benefits were quantitatively and qualitatively related to the specific characteristics of each exercise, suggesting that the related neuroprotection is strongly dependent on the specific activation of some motor neuron subpopulations. Taken together, the present data show significant long‐term exercise benefits in type 3 SMA‐like mice providing important clues for designing rehabilitation programmes in patients.

Abbreviations

ChAT

choline acetyltransferase

Chodl

chondrolectin

CK

creatine kinase

CMAP

compound muscle action potential

ERRβ

oestrogen‐related receptor β

MyHC

myosin heavy chain

NMJ

neuromuscular junction

SMA

spinal muscular atrophy

SMN

survival of motor neuron

TBS

Tris‐buffered solution

     

Thrombospondin‐1‐dependent immune regulation by transforming growth factor‐β 2‐exposed antigen‐presenting cells

An important role of transforming growth factor-β (TGF-β) in the development of regulatory T cells is well established. Although integrin-mediated activation of latent TGF-β₁ is considered essential for the induction of regulatory T (Treg) cells by antigen-presenting cells (APCs), such an activation mechanism is not applicable to the TGF-β₂ isoform, which lacks an integrin-binding RGD sequence in its latency-associated peptide. Mucosal and ocular tissues harbour TGF-β₂-expressing APCs involved in Treg induction. The mechanisms that regulate TGF-β activation in such APCs remain unclear. In this study, we demonstrate that murine APCs exposed to TGF-β₂ in the environment predominantly increase expression of TGF-β₂. Such predominantly TGF-β₂-expressing APCs use thrombospondin-1 (TSP-1) as an integrin-independent mechanism to activate their newly synthesized latent TGF-β₂ to induce Foxp3⁺ Treg cells both in vitro and in vivo. Expression of Treg induction by TGF-β₂-expressing APCs is supported by a TSP-1 receptor, CD36, which facilitates activation of latent TGF-β during antigen presentation. Our results suggest that APC-derived TSP-1 is essential for the development of an adaptive regulatory immune response induced by TGF-β₂-expressing APCs similar to those located at mucosal and ocular sites. These findings introduce the integrin-independent mechanism of TGF-β activation as an integral part of peripheral immune tolerance associated with TGF-β₂-expressing tissues.     

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.     

Hyper‐responsive Toll‐like receptor 7 and 9 activation in NADPH oxidase‐deficient B lymphoblasts

Chronic granulomatous disease (CGD) is an inherited immunodeficiency linked with mutations in the multi-subunit leucocyte NADPH oxidase. Myeloid-derived phagocytic cells deficient in NADPH oxidase fail to produce sufficient levels of reactive oxygen species to clear engulfed pathogens. In this study we show that oxidase also influences B-cell functions, including responses to single-stranded RNA or unmethylated DNA by endosomal Toll-like receptors (TLRs) 7 and 9. In response to TLR7/9 ligands, B-cell lines derived from patients with CGD with mutations in either the NADPH oxidase p40^phox or p47^phox subunits produced only low levels of reactive oxygen species. Remarkably, cytokine secretion and p38 mitogen-activated protein kinase activation by these oxidase-deficient B cells was significantly increased upon TLR7/9 activation when compared with oxidase-sufficient B cells. Increased TLR responsiveness was also detected in B cells from oxidase-deficient mice. NADPH oxidase-deficient patient-derived B cells also expressed enhanced levels of TLR7 and TLR9 mRNA and protein compared with the same cells reconstituted to restore oxidase activity. These data demonstrate that the loss of oxidase function associated with CGD can significantly impact B-cell TLR signalling in response to nucleic acids with potential repercussions for auto-reactivity in patients.     

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.     

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.     

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.     

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.     

Interleukin‐23 (IL‐23), independent of IL‐17 and IL‐22, drives neutrophil recruitment and innate inflammation during Clostridium difficile colitis in mice

Our objective was to determine the role of the inflammatory cytokine interleukin‐23 (IL‐23) in promoting neutrophil recruitment, inflammatory cytokine expression and intestinal histopathology in response to Clostridium difficile infection. Wild‐type (WT) and p19^−/− (IL‐23KO) mice were pre‐treated with cefoperazone in their drinking water for 5 days, and after a 2‐day recovery period were challenged with spores from C. difficile strain VPI 10463. Interleukin‐23 deficiency was associated with significant defects in both the recruitment of CD11b^High Ly6G^High neutrophils to the colon and the expression of neutrophil chemoattractants and stabilization factors including Cxcl1, Cxcl2, Ccl3 and Csf3 within the colonic mucosa as compared with WT animals. Furthermore, the expression of inflammatory cytokines including Il33, Tnf and Il6 was significantly reduced in IL‐23‐deficient animals. There was also a trend towards less severe colonic histopathology in the absence of IL‐23. The induction of Il17a and Il22 was also significantly abrogated in IL‐23KO mice. Inflammatory cytokine expression and neutrophilic inflammation were not reduced in IL‐17a‐deficient mice or in mice treated with anti‐IL‐22 depleting monoclonal antibody. However, induction of RegIIIg was significantly reduced in animals treated with anti‐IL‐22 antibody. Taken together, these data indicate that IL‐23, but not IL‐17a or IL‐22, promotes neutrophil recruitment and inflammatory cytokine and chemokine expression in the colon in response to C. difficile infection.     

The Effect of l‐Arginine on Porphyromonas gingivalis‐Induced Phagocytosis of a Murine Macrophage‐like Raw 264.7 Cell Line

The aim of this study was to determine the effect of l‐arginine on Porphyromonas gingivalis‐induced phagocytosis by RAW 264.7 cells. The cells were pretreated with l‐arginine or d‐arginine prior to incubation with either unopsonized or opsonized P. gingivalis. In other experiments, the cells were pretreated with l‐arginine and various concentrations of NMLA (N^G‐monomethyl‐l‐arginine) prior to incubation with the bacteria. The phagocytosis was microscopically assessed and determined by the phagocytic index. The results showed that l‐arginine, but not d‐arginine enhances the ability of RAW264.7 cells to engulf the bacteria. The upregulatory effect of l‐arginine on P. gingivalis‐induced phagocytosis was abolished by NMLA. The results of the present study suggest that l‐arginine may upregulate the P. gingivalis‐induced phagocytic activity of RAW264.7 cells, perhaps, via modulation of nitric oxide synthase.     

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.     

Open‐ and closed‐loop smooth‐pursuit eye movements in normal children: An analysis of a step‐ramp task

We previously reported that the visual ability to track a moving target (smooth‐pursuit tracking) improves as children age from 8 to 15 years old. This study used infrared oculography during step‐ramp tasks to determine whether the age‐related improvement in smooth‐pursuit tracking is due to developmental changes in the ability to perceive and match eye velocity to target velocity (open‐loop tracking). Infrared oculography was used to assess the ability to track a moving stimulus (smooth‐pursuit tracking) during step‐ramp tasks in 51 normal children between 8 and 15 years old. The first 100 msec of tracking (initial pursuit) occurs before any visual feedback (open‐loop tracking) and represents sensorimotor transformation. Ongoing pursuit (measured by smooth‐pursuit gain) includes feedback information as to the success of pursuit (closed‐loop pursuit) and depends on sensorimotor transformation as well as higher order abilities, including the ability to sustain focused attention. Open‐loop pursuit is not affected by age of the subject. In contrast, during closed‐loop pursuit, when target step and target motion are in opposite directions, age is significantly correlated with closed‐loop pursuit gain, Spearman's R = 0.40, p < .003. The ability to perceive and match eye velocity to target velocity is fully developed by 8 years of age.     

A shift in paradigm towards human biology‐based systems for cholestatic‐liver diseases

IntroductionLiver and gall bladder diseases are very common all over the world, posing a significant health burden worldwide (Shaheen et al. 2006; Williams, 2006). It is now known that diseases related to modern lifestyle such as obesity, diabetes, non‐alcoholic fatty liver disease and other nutritional/metabolic disorders are either the cause or the consequences of liver and gall bladder diseases (Fig. 1). Liver is a very complex vital organ performing a diverse range of metabolic functions including the regulation of carbohydrate metabolism; lipid synthesis and secretion of plasma lipoproteins; cholesterol metabolism; synthesis and secretion of bile salts; digestion; storage of nutrients, vitamins and minerals; synthesis and secretion of serum albumin, clotting factors, enzymes and other proteins; ammonia detoxification through urea and glutamine formation; and biotransformation/detoxification of drugs and other xenobiotics. Liver disorders can result from various insults such as infections, drugs, toxins, ischaemia and autoimmune disorders. Persisting disturbances in liver functions due to resulting hepatocellular injury lead to chronic liver disease(s). The diverse functions of liver are performed by parenchymal (hepatocytes) and non‐parenchymal cells (mainly Kupffer cells, stellate cells, sinusoidal endothelial cells and biliary epithelial cells) communicating and working together. The liver parenchyma accounts for approximately 60% of total liver mass with non‐parenchymal cells making up the rest.Open in a separate windowFigure 1 Diseases of civilization are usually a cause or consequence of liver and gall bladder diseases Biliary epithelial cells or cholangiocytes constitute around 5% of liver cells (Sirica et al. 2008). These form an intricate network of tiny channels (bile canaliculi) that merge to form the bile ducts channeling and collecting bile from liver lobules for storage in the gall bladder and subsequent intestinal secretion. Cholangiocytes that line the bile ducts have secretory functions and those that line the smaller bile ducts and canaliculi play roles in inflammatory and proliferative responses.Bile mainly comprises the bile acids (BAs), which are the end products of cholesterol metabolism. From cholesterol to BAs, there are 17 energy consuming enzymatic reactions (Russell, 2009); and hence a very efficient and controlled recycling system for BAs exists in humans. About 95% of BAs are reabsorbed through the enterohepatic circulation. The functional BA pool is maintained by an extended system of transporters (Thomas et al. 2008) as shown in Fig. 2. As hepatocytes in the liver are organized over the sinusoids, specific hepatic transporters are expressed at the polarized membranes (Table 1). These transporters are involved in adaptive response to BAs overload and accumulation e.g. in disease conditions such as cholestasis. BAs, having hormonal functions; exert effects via the nuclear receptors (Fig. 3) in the regulation of a variety of metabolic effects – including glucose, lipid and energy metabolism (Watanabe et al. 2006; Lefebvre et al. 2009; Wei et al. 2009; Torres et al. 2012; Li & Chiang, 2015); cholesterol uptake, metabolism and secretion (De Fabiani et al. 2003); xenobiotic metabolism (Hofmann & Hagey, 2008; Zollner & Trauner, 2009); endocrine (Houten et al. 2006; Keitel et al. 2008) and immunological signalling (Ishizawa et al. 2008; Makishima et al. 2002) – and have antimicrobial effects in the digestive tract (Begley et al. 2005; Kurdi et al. 2006).ConclusionCLDs are complex and although manifest as cholestasis resulting from perturbed bile acid homeostasis, they are intertwined with glucose, lipid and energy metabolism as well as the immune response of the patient. Such complex pathogenesis requires a systems understanding leaning on new technologies. Although animal studies have advanced our knowledge of CLDs, there has not been significant clinical translation of that knowledge in the treatment or prevention of these diseases. There is a huge amount of clinical and animal data available on cholestasis. Modern in vitro methods based on human cells (and co‐cultures) maintained in in vivo‐like conditions provide an invaluable tool for the investigation and validation of human‐relevant mechanisms involved in the development of cholestasis and its progression. Omics technologies and computational modelling will enhance the knowledge and allow prediction. The shift in paradigm towards a human‐relevant systems approach to the understanding of cholestasis seems essential to bring a breakthrough that will pave the way for new therapeutic options for CLDs and eventually personalized therapy.     

Self‐recognition in everyday life

Introduction: A sample of everyday difficulties was collected, encompassing errors and unusual experiences participants had encountered when recognising their own faces in everyday life, with the aim of characterising similarities and differences between the reported difficulties and the major forms of self‐recognition impairments described in the neuropsychological and neuropsychiatric literatures (prosopagnosia, mirrored‐self misidentification, and Capgras delusion).

Method. A total of 70 participants recalled experiences from memory. Incidents (n = 51) were recorded on questionnaire sheets that were filled out at home. Reports of three categories of incidents were analysed: misidentifications (the participant misidentified her/his own face as being that of another familiar person; n = 5), recognition failures (the participant judged that his/her own face was that of an unfamiliar person; n = 20) and perception of unusual aspects (the participant confidently recognised his/her own face but found that the seen face did not fit well the representation she/he had of his/her own face; n = 26).

Results and discussion. In the reported incidents, experiences showing some similarities to those of patients with prosopagnosia, Capgras delusion or mirrored‐self misidentification were noted. However, across the whole study, no incident involved a failure of reality testing; in contrast to pathological forms of error, in all of the reported incidents from our study the participant realised that a mistake had been made. The importance of decision processes in pathological forms of own‐face misrecognition is discussed.