Effects of peripheral κ opioid receptor activation on inflammatory mechanical hyperalgesia in male and female rats

Activation of peripheral κ opioid receptors (KOR) effectively relieves pain and hyperalgesia in preclinical and clinical models of pain. Although centrally located KOR activation results in sexually dimorphic effects, it is unclear whether peripheral KOR also produces sex dependent effects in persistent inflammatory pain conditions. In this study, we investigated whether local administration of a specific KOR agonist, U50, 488 relieve mechanical hyperalgesia induced by the injection of complete Freund's adjuvant (CFA) in the rat hindpaw, and whether there are sex differences. The effects of U50, 488 were assessed three days after the induction of CFA-induced inflammation, a time point at which mechanical hyperalgesia was most prominent. There were no sex differences in baseline and CFA-induced changes in mechanical thresholds between male and female rats. Local treatment of U50, 488 produced moderate, but significant, anti-hyperalgesia in both male and female rats. However, U50, 488 was significantly more effective in male rats at the highest dose of U50, 488. We confirmed that the highest dose of U50, 488 used in this study did not produce systemic effects, and that the drug effect is receptor specific. On the basis of these results, we suggest that local KOR agonists are effective in mitigating mechanical hyperalgesia under a persistent inflammatory pain condition and that sex differences in anti-hyperalgesic effects become more evident at high doses.     

Assessing the role of Asp 194 in the transmembrane domains of the α-chain of the high-affinity receptor complex for immunoglobulin E in signal transduction

The high-affinity receptor complex for IgE plays a pivotal role in allergic responses since cross-linking of the high-affinity IgE receptor (Fc?RI) on target cells initiates a signaling cascade facilitating release of inflammatory mediators causing allergic responses. The transmembrane regions of the ligand binding domains of the high-affinity IgE and low-affinity IgG receptors share an invariant motif (LFAVDTGL) containing a polar aspartate within a predominantly non-polar setting. The functional importance of this aspartate residue (D194) in Fc?RI-mediated receptor signaling was assessed by site-directed mutagenesis. Rat basophilic leukemia cells (RBL-2H3) transfected with the human IgE binding subunit (Fc?RIα) incorporating polar substitutions like asparagine (D194N) or threonine (D194T) resulted in the formation of a functional rat/human chimeric receptor complex. When activated via huIgE and antigen, cells transfected with these variant receptor subunits supported mediator release, intracellular calcium mobilisation and tyrosine phosphorylation of γ-chain and Syk kinase while a non-polar substitution (D194L) gave rise to cell surface expression of the mutated receptor subunit but failed to initiate downstream signaling. No cell surface expression of huFc?RIα gene constructs was observed when D194 was replaced with the non-polar Ile (D194I) residue of similar size, the larger positively charged Arg (D194R) or lysine (D194K) residues, or the negatively charged glutamate (D194E) and smaller polar Ser (D194S) non-polar Ala (D194A) and V (D194V). These observations highlight importance of the size and charge of amino acid residue at position 194 in determining IgE receptor subunit interactions, cell surface localization, and initiation of downstream signaling events.     

Early mother–child attachment and behavior problems in middle childhood: the role of the subsequent caregiving environment

The current study investigated associations between early mother–child attachment, as well as mother–child and teacher–child relationships, and internalizing and externalizing behaviors in middle childhood. Data from the NICHD Study of Early Child Care and Youth Development were used. Findings from a series of individual growth curve analyses revealed that attachment security was negatively related to internalizing and externalizing behaviors, while insecure/other and avoidant attachment were positively related to internalizing behaviors. In addition, longitudinal associations were found between mother–child and teacher–child relationships and internalizing and externalizing behaviors across middle childhood. Implications for attachment theory are discussed.     

Early activation of γδ T lymphocytes in the elderly

T cell function is altered in vivo and in vitro in elderly compared with young subjects, and this alteration is believed to contribute to morbidity and mortality in man due to the greater incidence of infection, as well as autoimmunity and cancer in elderly. The majority of T cells express TCRαβ whereas TCRγδ is expressed on a minority of T cells. Moreover, it is known that γδ T lymphocytes display major histocompatibility complex (MHC)- unrestricted cytotoxicity that is reminiscent of natural killer (NK) activity. In view of earlier findings on both T cells and NK cells in the elderly, we hypothesised a different behaviour of γδ T lymphocytes from old subjects when compared with γδ T lymphocytes obtained from young people. Therefore, to gain further insight into mechanisms of immunosenescence in this little-studied population, we studied immunofluorescence analysis γδ T cells from the elderly. Our preliminary results show that the percentage of blood γδ T cells in lymphocytes from old subjects is decreased when compared with the young. Interestingly, these cells are more activated in the elderly than in young subjects; expression of CD69, an early activation marker, is increased in γδ T lymphocytes from old subjects after three hours of in vitro culture both with and without lipopolysaccharide stimulation. Thus, our findings, which need confirmation, strongly suggest that, in humans, γδ T cells are early responders when compared with αβ T cells. They may act as ‘first aid’ cells to replace the described deficit of the specific and aspecific immunity in elderly. In this view, the proinflammatory status, observable in the elderly, renders them ready to be stimulated by exogenous agents.     

B lymphocyte proliferation is suppressed by the opioid growth factor–opioid growth factor receptor axis: Implication for the treatment of autoimmune diseases

Endogenous opioids are known to repress the incidence and progression of autoimmune diseases. One native opioid peptide, [Met⁵]-enkephalin, termed the opioid gowth factor (OGF), interacts with the OGF receptor (OGFr) to suppress the expression of experimental autoimmune encephalomyelitis. The present study examined the role of the OGF-OGFr axis in the regulation of B lymphocyte proliferation. Murine B lymphocytes were stimulated with lipopolysaccharide. Both OGF and OGFr were present in all B lymphocytes. OGF had a dose-dependent effect on growth, with cell number inhibited by up to 43% at 72 h; no other synthetic or native opioid altered cell proliferation. Exogenous OGF depressed cell number in cultures treated with siRNAs for the classical opioid receptors, MOR (μ), DOR (δ), and KOR (κ), however this peptide had no effect in preparations exposed to siRNA for OGFr. The decrease in cell number by exogenous OGF was dependent on p16 or p21 cyclin-dependent inhibitory kinase pathways. Exposure to the opioid antagonist, naltrexone, did not change cell number from control levels. These results suggest that the OGF-OGFr axis is present and functional in B lymphocytes, but this system is not an autocrine regulator of cell proliferation. Thus, at least exogenous OGF and perhaps endogenous OGF by paracrine/endocrine sources, can be an immunosuppressant. Modulation of the OGF-OGFr axis may be a novel paradigm for the treatment of autoimmune diseases.     

Effect of nicotine on mRNA levels encoding opioid peptides,vasopressin and α3 nicotinic receptor subunit in the rat

Summary The effect of acute and chronic nicotine treatment of rats on the mRNA levels coding for the three opioid peptide precursors, for provasopressin and for the a3 subunit of nicotinic receptors in brain, pituitary and/or adrenal medulla of rats was investigated. Nicotine was found to increase the levels of proenkephalin mRNA in the adrenal medulla, but did not affect the levels of PENK mRNA in striatum, hypothalamus and hippocampus. The mRNA levels of prodynorphin were increased together with that of provasopressin in the hypothalamus after nicotine, whereas the prodynorphin mRNA levels in the hippocampus and the striatum remained unchanged. Nicotine treatment resulted in an increase in the pro-opiomelanocortin mRNA levels in the anterior pituitary and in a decrease in the intermediate pituitary, but did not change the levels of pro-opiomelanocortin mRNA in the hypothalamus. The levels of mRNA coding for the ₃ subunit of nicotinic receptors in the hypothalamus and the adrenal medulla remained unchanged. The increase in the prodynorphin and provasopressin mRNA levels in the hypothalamus was most pronounced 1 day after s.c. application of two doses of 0.4 mg/kg nicotine (about 100% above control). A smaller increase in mRNA concentrations (about 30%) was found after tonic infusion of the drug for 4 days (4 mg/kg per day), whereas no change was observed after tonic infusion of nicotine for 7 and 14 days indicating the development of complete tolerance. The increase in proenkephalin mRNA levels in the adrenal medulla was highest after the short-term application of nicotine (about 150% above control). Less, but still significant increases in the mRNA levels (about 40%) were also seen after 7 and 14 days of tonic nicotine administration suggesting that no complete desensitization is developing to the effect of nicotine. This desensitization appeared to be less pronounced when the drug was applied in a pulsatile manner using minipumps, since a high increase in the PENK mRNA levels (100% above control levels) was observed after intermittent infusion of nicotine (six boli per day of 1 mg/kg for 7 days). These findings demonstrate that nicotine can alter gene expression of opioid peptides and vasopressin in certain rat tissues, and that the mode of drug administration plays an important role in this effect of nicotine. The possibility that the pulsatile administration of nicotine by cigarette smokers may also result in an altered expression of opioid peptide genes in humans is discussed.Abbreviations PENK proenkephalin - POMC pro-opiomelanocortin - MSH melanocyte stimulating hormone - PDYN prodynorphin - CRF corticotropin releasing factor     

The important role for βVLDLs binding at the fourth cysteine of first ligand-binding domain in the low-density lipoprotein receptor

The low-density lipoprotein (LDL) receptor (LDLR) is a crucial role for binding and uptaking apolipoprotein (apo) B-containing lipoproteins, such as very-low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), and LDL. The defect function of the LDLR causes familial hypercholesterolemia (FH), the phenotype of which is elevated plasma cholesterol and premature coronary heart disease (CHD). In the present study, we characterize the role of the cysteine residue of the ligand-binding domain of the LDLR. The mutant LDLR protein of cysteine for serine at codon 25 (25S-LDLR) was expressed in Chinese hamster ovary (CHO) cell line, ldl-A7. By Western blot analysis, the 25S-LDLR was detected with monoclonal antibody IgG-12D10, which reacts with the linker site of the LDLR but not with IgG-C7, which reacts with the NH₂ terminus of the receptor. The 25S-LDLR bound LDL similarly to the wild-type LDLR, but the rate of uptake of LDL by the mutant receptor was only about half of that by the wild-type receptor. In contrast, the 25S-LDLR bound and internalized VLDL more avidly than LDL. These results suggest that the fourth cysteine residue of the first ligand-binding domain of the LDLR might be important for the internalization of atherogenic lipoproteins by vascular cells despite reduced LDL uptake, leading to atherosclerosis and premature cardiovascular disease.     

What is the role of tropomyosin in the regulation of muscle contraction?

Journal of Muscle Research and Cell Motility -     

Physiology and pathophysiology of excitation–contraction coupling: the functional role of ryanodine receptor

Calcium (Ca²⁺) release from intracellular stores plays a key role in the regulation of skeletal muscle contraction. The type 1 ryanodine receptors (RyR1) is the major Ca²⁺ release channel on the sarcoplasmic reticulum (SR) of myocytes in skeletal muscle and is required for excitation–contraction (E–C) coupling. This article explores the role of RyR1 in skeletal muscle physiology and pathophysiology.     

The role of γδ T cells in the airways

Based on their T cell receptor expression two distinct T cell populations have been identified and designated as alphabeta and gammadelta T cells. While the specific role of alphabeta T cells is well understood, the specific function of gammabeta T cells remains elusive. Despite the limited knowledge on what gammadelta T cells exactly recognize as their antigen or antigens, several studies have clearly demonstrated that gammadelta T cells are important regulators of immune responses. Studies on gammadelta T cells in the lung have shown that gammadelta T cells influence B and T cell responses. For instance, gammadelta T cells modulate CD4+alphabeta T cells in tuberculosis, and in allergic inflammation they support IgE production. Furthermore, a recent study on their role as part of the innate immune response has demonstrated that gammadelta T cells regulate airway function independently of alphabeta T cells. This novel finding provides an opportunity to review previous studies on gammadelta T cells in the lung of mice and humans. The examination of these data demonstrates that understanding the role of the various subsets of gammadelta T cells will be critical in elucidating their function as "immunosurveillance cells" of the lung.     

What is the role of rituximab in the treatment of rheumatoid arthritis?

Rituximab is a monoclonal antibody against CD20 that was developed for the treatment of relapsed or refractory B-cell non-Hodgkin's lymphoma (NHL). Recent controlled trials have shown that B cell-targeted therapy with rituximab is effective in RA (which suggest that B lymphocytes may be critical in its pathogenesis of RA) and early exposure data suggest that the tolerability and safety profile of rituximab may be even better in RA than in NHL patients. Rituximab is generally well tolerated, with a low incidence of serious adverse events, including serious infections. Available evidence suggests that its clinical benefits depend on effective B cell depletion, and the fact that its novel mode of action leads to the depletion of B cells makes it distinct from other biological therapies for RA that target T cells and their related cytokines. Although complete peripheral B cell depletion is regularly seen in RA and other autoimmune diseases, especially systemic lupus erythematosus (SLE), incomplete depletion has been reported in a subset of patients, even after full dosing with rituximab.     

Sensory thresholds and the antinociceptive effects of GABA receptor agonists in mice lacking the β3 subunit of the GABAA receptor

A line of mice was recently created in which the gabrb3 gene, which encodes the β₃ subunit of the GABA_A receptor, was inactivated by gene-targeting. The existence of mice with a significantly reduced population of GABA_A receptors in the CNS enabled an investigation of the role of GABA and GABA_A receptors in nociception. The present study examined the sensory thresholds of these mice, as well as the antinociceptive effects of subcutaneously or intrathecally administered GABA_A and GABA_B receptor agonists. Homozygous null (β₃^−/−) mice displayed enhanced responsiveness to low-intensity thermal stimuli in the tail-flick and hot-plate test compared to C57BL/6J and 129/SvJ progenitor strain mice, and their wild-type (β₃^+/+) and heterozygous (β₃^+/−) littermates. The β₃^−/− mice also exhibited enhanced responsiveness to innocuous tactile stimuli compared to C57BL/6J, 129/SvJ and to their β₃^+/+ littermates as assessed by von Frey filaments. The presence of thermal hyperalgesia and tactile allodynia in β₃^−/− mice is consistent with a loss of inhibition mediated by presynaptic and postsynaptic GABA_A receptors in the spinal cord. As expected, subcutaneous administration of the GABA_A receptor agonist 4,5,6,7-tetrahydroisoxazolo-(5,4-c)pyridin-3-ol did not produce antinociception in β₃^−/− mice, whereas it produced a dose-dependent increase in hot-plate latency in C57BL/6J, 129/SvJ, β₃^+/+ and β₃^+/− mice. However, the antinociceptive effect of the GABA_B receptor agonist baclofen in the tail-flick and hot-plate tests was also reduced in β₃^−/− mice compared to the progenitor strains, β₃^+/+ or β₃^+/− mice after either subcutaneous or intrathecal administration. This finding was unexpected and suggests that a reduction in GABA_A receptors can affect the production of antinociception by other analgesic drugs as well.     

Bosentan, an endothelin receptor antagonist, ameliorates collagen-induced arthritis: the role of TNF-α in the induction of endothelin system genes

Objective

Endothelins (ETs) are involved in several inflammatory events. The present study investigated the efficacy of bosentan, a dual ETA/ETB receptor antagonist, in collagen-induced arthritis (CIA) in mice.

Treatment

CIA was induced in DBA/1J mice. Arthritic mice were treated with bosentan (100?mg/kg) once a day, starting from the day when arthritis was clinically detectable.

Methods

CIA progression was assessed by measurements of visual clinical score, paw swelling and hypernociception. Histological changes, neutrophil infiltration and pro-inflammatory cytokines were evaluated in the joints. Gene expression in the lymph nodes of arthritic mice was evaluated by microarray technology. PreproET-1 mRNA expression in the lymph nodes of mice and in peripheral blood mononuclear cells (PBMCs) was evaluated by real-time PCR. The differences were evaluated by one-way ANOVA or Student’s t test.

Results

Oral treatment with bosentan markedly ameliorated the clinical aspects of CIA (visual clinical score, paw swelling and hyperalgesia). Bosentan treatment also reduced joint damage, leukocyte infiltration and pro-inflammatory cytokine levels (IL-1β, TNFα and IL-17) in the joint tissues. Changes in gene expression in the lymph nodes of arthritic mice returned to the levels of the control mice after bosentan treatment. PreproET mRNA expression increased in PBMCs from rheumatoid arthritis (RA) patients but returned to basal level in PBMCs from patients under anti-TNF therapy. In-vitro treatment of PBMCs with TNFα upregulated ET system genes.

Conclusion

These findings indicate that ET receptor antagonists, such as bosentan, might be useful in controlling RA. Moreover, it seems that ET mediation of arthritis is triggered by TNFα.     

The role of T-cell receptor α and β genes in MHC-restricted antigen recognition

T cells use α- and β-chain genes, which are organized and diversified by somatic DNA rearrangements much like the immunoglobulin genes, to encode clonally distributed receptor molecules which confer specificity for MHC and antigen. Here Zlatko Dembic and his colleagues summarize recent mutagenesis and gene transfection experiments indicating that α- and β-chain genes are necessary and sufficient for T-cell specificity.     

What is the role of infant banging in the development of tool use?

Throughout the first year, infants are known to engage in repetitive motor behaviors. The current study examines the changes in the hand trajectory of one such behavior, banging, during the second half-year and the implications of these changes for tool use development. Fourteen (7- to 14-month-old) infants were seated at a table and presented with a small wooden cube. Kinematic measurements of their banging movements were recorded at 240 Hz. Analyses revealed stable temporal characteristics of the hand trajectories within and across infants. Results further indicated that as infants became older, their hands moved more efficiently in straighter up-and-down trajectories, with developmental changes especially pronounced for upward excursions of the hand: Younger infants’ arm movements were less straight on the way up than down, but there was no difference in the straightness of the two movement phases for older infants. These changes with age may reflect improvements in overcoming constraints associated with gravity and/or in motor planning. Additionally, the angle at which infants hit the table became more perpendicular with age. Collectively, the reported changes lead to more efficient movements, better aim and improved force delivery, enabling spontaneous banging movements to become well suited for instrumental hammering and tool use, more generally, later in childhood.