Amelioration of Compound 48/80-Mediated Itch and LL-37-Induced Inflammation by a Single-Stranded Oligonucleotide.

Numerous inflammatory skin disorders display a high prevalence of itch. The Mas-related G protein coupled receptor X2 (MRGPRX2) has been shown to modulate itch by inducing non-IgE-mediated mast cell degranulation and the release of endogenous inducers of pruritus. Various substances collectively known as basic secretagogues, which include inflammatory peptides and certain drugs, can trigger MRGPRX2 and thereby induce pseudo-allergic reactions characterized by histamine and protease release as well as inflammation. Here, we investigated the capacity of an immunomodulatory single-stranded oligonucleotide (ssON) to modulate IgE-independent mast cell degranulation and, more specifically, its ability to inhibit the basic secretagogues compound 48/80 (C48/80)-and LL-37 in vitro and in vivo. We examined the effect of ssON on MRGPRX2 activation in vitro by measuring degranulation in a human mast cell line (LAD2) and calcium influx in MRGPRX2-transfected HEK293 cells. To determine the effect of ssON on itch, we performed behavioral studies in established mouse models and collected skin biopsies for histological analysis. Additionally, with the use of a rosacea mouse model and RT-qPCR, we investigated the effect on ssON on LL-37-induced inflammation. We reveal that both mast cell degranulation and calcium influx in MRGPRX2 transfected HEK293 cells, induced by the antimicrobial peptide LL-37 and the basic secretagogue C48/80, are effectively inhibited by ssON in a dose-dependent manner. Further, ssON demonstrates a capability to inhibit LL-37 and C48/80 activation in vivo in two mouse models. We show that intradermal injection of ssON in mice is able to block itch induced via C48/80 in a dose-dependent manner. Histological staining revealed that ssON inhibits acute mast cell degranulation in murine skin treated with C48/80. Lastly, we show that ssON treatment ameliorates LL-37-induced inflammation in a rosacea mouse model. Since there is a need for new therapeutics targeting non-IgE-mediated activation of mast cells, ssON could be used as a prospective drug candidate to resolve itch and inflammation in certain dermatoses.

Mouse connective tissue mast cell proteases tryptase and carboxypeptidase A3 play protective roles in itch induced by endothelin-1.

BACKGROUND: Itch is an unpleasant sensation that can be debilitating, especially if it is chronic and of non-histaminergic origin, as treatment options are limited. Endothelin-1 (ET-1) is a potent endogenous vasoconstrictor that also has the ability to induce a burning, non-histaminergic pruritus when exogenously administered, by activating the endothelin A receptor (ETAR) on primary afferents. ET-1 is released endogenously by several cell-types found in the skin, including macrophages and keratinocytes. Mast cells express ETARs and can thereby be degranulated by ET-1, and mast cell proteases chymase and carboxypeptidase A3 (CPA3) are known to either generate or degrade ET-1, respectively, suggesting a role for mast cell proteases in the regulation of ET-1-induced itch. The mouse mast cell proteases (mMCPs) mMCP4 (chymase), mMCP6 (tryptase), and CPA3 are found in connective tissue type mast cells and are the closest functional homologs to human mast cell proteases, but little is known about their role in endothelin-induced itch. METHODS: In this study, we evaluated the effects of mast cell protease deficiency on scratching behavior induced by ET-1. To investigate this, mMCP knock-out and transgenic mice were injected intradermally with ET-1 and their scratching behavior was recorded and analyzed. RESULTS: CPA3-deficient mice and mice lacking all three proteases demonstrated highly elevated levels of scratching behavior compared with wild-type controls. A modest increase in the number of scratching bouts was also seen in mMCP6-deficient mice, while mMCP4-deficiency did not have any effect. CONCLUSION: Altogether, these findings identify a prominent role for the mast cell proteases, in particular CPA3, in the protection against itch induced by ET-1.

Mouse mast cells and mast cell proteases do not play a significant role in acute tissue injury pain induced by formalin.

Subcutaneous formalin injections are used as a model for tissue injury-induced pain where formalin induces pain and inflammation indirectly by crosslinking proteins and directly through activation of the transient receptor potential A1 receptor on primary afferents. Activation of primary afferents leads to both central and peripheral release of neurotransmitters. Mast cells are found in close proximity to peripheral sensory nerve endings and express receptors for neurotransmitters released by the primary afferents, contributing to the neuro/immune interface. Mast cell proteases are found in large quantities within mast cell granules and are released continuously in small amounts and upon mast cell activation. They have a wide repertoire of proposed substrates, including Substance P and calcitonin gene-related peptide, but knowledge of their in vivo function is limited. We evaluated the role of mouse mast cell proteases (mMCPs) in tissue injury pain responses induced by formalin, using transgenic mice lacking either mMCP4, mMCP6, or carboxypeptidase A3 (CPA3), or mast cells in their entirety. Further, we investigated the role of mast cells in heat hypersensitivity following a nerve growth factor injection. No statistical difference was observed between the respective mast cell protease knockout lines and wild-type controls in the formalin test. Mast cell deficiency did not have an effect on formalin-induced nociceptive responses nor nerve growth factor-induced heat hypersensitivity. Our data thus show that mMCP4, mMCP6, and CPA3 as well as mast cells as a whole, do not play a significant role in the pain responses associated with acute tissue injury and inflammation in the formalin test. Our data also indicate that mast cells are not essential to heat hypersensitivity induced by nerve growth factor.

CGRPα within the Trpv1-Cre population contributes to visceral nociception.

The role of calcitonin gene-related peptide (CGRP) in visceral and somatic nociception is incompletely understood. CGRPα is highly expressed in sensory neurons of dorsal root ganglia and particularly in neurons that also express the transient receptor potential cation channel subfamily V member 1 (Trpv1). Therefore, we investigated changes in visceral and somatic nociception following deletion of CGRPα from the Trpv1-Cre population using the Cre/lox system. In control mice, acetic acid injection (0.6%, ip) caused significant immobility (time stationary), an established indicator of visceral pain. In CGRPα-mCherrylx/lx;Trpv1-Cre mice, the duration of immobility was significantly less than controls, and the distance CGRPα-mCherrylx/lx;Trpv1-Cre mice traveled over 20 min following acetic acid was significantly greater than controls. However, following acetic acid injection, there was no difference between genotypes in the writhing reflex, number of abdominal licks, or forepaw wipes of the cheek. CGRPα-mCherrylx/lx;Trpv1-Cre mice developed more pronounced inflammation-induced heat hypersensitivity above baseline values compared with controls. However, analyses of noxious acute heat or cold transmission revealed no difference between genotypes. Also, odor avoidance test, odor preference test, and buried food test for olfaction revealed no differences between genotypes. Our findings suggest that CGRPα-mediated transmission within the Trpv1-Cre population plays a significant role in visceral nociceptive pathways underlying voluntary movement. Monitoring changes in movement over time is a sensitive parameter to identify differences in visceral nociception, compared with writhing reflexes, abdominal licks, or forepaw wipes of the cheek that were unaffected by deletion of CGRPα- from Trpv1-Cre population and likely utilize different mechanisms. NEW & NOTEWORTHY The neuropeptide calcitonin gene-related peptide (CGRP) is highly colocalized with transient receptor potential cation channel subfamily V member 1 (TRPV1)-expressing primary afferent neurons, but the functional role of CGRPα specifically in these neurons is unknown in pain processing from visceral and somatic afferents. We used cre-lox recombination to conditionally delete CGRPα from TRPV1-expressing neurons in mice. We show that CGRPα from within TRPV1-cre population plays an important role in visceral nociception but less so in somatic nociception.

Identification of a Neuronal Receptor Controlling Anaphylaxis.

Allergic reactions can in severe cases induce a state of circulatory shock referred to as anaphylaxis. Histamine, the primary mediator of this condition, is released from immune cells, and, therefore, anaphylaxis has so far been considered an immune system disorder. However, we here show that the glutamatergic receptor mGluR7, expressed on a subpopulation of both peripheral and spinal cord neurons, controls histamine-induced communication through calcium-dependent autoinhibition with implications for anaphylaxis. Genetic ablation of mGluR7, and thus altered regulation of histamine-sensing neurons, caused an anaphylaxis-like state in mGluR7(-/-) mice, which could be reversed by antagonizing signaling between neurons and mast cells but not by antagonizing a central itch pathway. Our findings demonstrate the vital role of nervous system control by mGluR7 in anaphylaxis and open up possibilities for preventive strategies for this life-threatening condition.

Temporal trends of contaminants in cod from Icelandic waters.

Contaminants have been analyzed in cod (Gadus morhua) since 1990 as part of the national monitoring program for the environmental conditions in the sea around Iceland. The aim of this study was to determine the temporal trends of persistent organic pollutants (POPs: polychlorinated biphenyls (PCBs), p,p'-dichlorodiphenyl dichloroethene (p,p'-DDE), hexachlorobenzene (HCB), hexachlorocyclohexanes (HCHs), chlordanes (CHLs) and toxaphenes (Tox)) and trace elements (As, Cd, Cu, Hg, Pb, Se and Zn) in cod over the last two decades at two different locations in the Arctic Ocean north of Iceland. The relationship between the contaminant concentrations and biological covariates was also determined. All of the POPs showed decreasing trends but the trace elements showed no clear signs of trend except arsenic which showed an increasing trend and zinc which showed a decreasing trend. The concentration of the POPs were lower or similar in the Icelandic cod compared to cod sampled in Norway, the Barents Sea and in the Baltic Sea, except for HCB which was higher in the Icelandic cod compared to the Norwegian cod. The concentration of the trace elements As, Cu, Hg and Zn were similar in the Icelandic cod compared to cod sampled in Norway and Greenland but the concentration of Cd was higher in the Icelandic cod. The inclusion of the biological covariates was found to be important for the statistical analysis. The POPs had a positive relationship with liver fat content but negative relationship with liver weight. The trace elements had a negative relationship with liver fat and liver weight except As which had positive relationship with liver weight. Only positive relationships were observed between the contaminant concentrations and length.

Spatial and temporal trends of contaminants in mussel sampled around the Icelandic coastline.

Contaminants have been determined in blue mussels (Mytilus edulis) at 11 locations around the Icelandic coastline from 1990 to 2010. The aim of the present study was to investigate if there has been a change in concentration of contaminants around the Icelandic coastline for the last two decades and if the concentrations and changes, if present, were consistent between locations. Concentrations of the persistent organic pollutants, p,p'-dichlorodiphenyldichloroethene (p,p'-DDE), hexachlorobenzene (HCB), α-hexachlorocyclohexane (α-HCH), polychlorinated biphenyl (PCB-153) and trans-nonachlor, have decreased at most of the sampling locations in Iceland in recent years. However, an increasing trend was found at a few locations that could be explained by anthropogenic activity. The concentration levels of the persistent organics were much lower than found at the Norwegian, USA and Chinese coasts, especially levels of p,p'-DDE. The concentration of copper and selenium had a consistent pattern of change and concentration between locations over the period which showed a decreasing trend in recent years. The trace elements arsenic, cadmium, mercury and zinc showed more variation in concentration between locations, the concentration of arsenic, mercury and zinc was fairly stable over the period, whereas there were fluctuations in cadmium concentrations. The concentrations of cadmium and zinc were observed to be somewhat higher than found in mussels from Norway, USA and China but values of mercury and lead were much lower in the mussel sampled in Iceland. The higher concentrations of cadmium and zinc can be explained by the volcanic activity in Iceland but no major anthropogenic sources of trace elements are known in Iceland.

Immunization prevents DDT buildup in mouse tissues.

DDT is used for pest control, causing health and environmental hazards in some parts of the world. The goal of this study was to assess whether immunization against a toxic compound could reduce the toxicant uptake of an organism, specifically to develop a DDT immunization that promotes the production of specific antibodies and assess whether it reduces DDT levels in the bodies of mice that are exposed to DDT by intake. BALB/c mice were immunized with DDT-keyhole limpet hemocyanine (DDT-KLH) conjugate (n=10) or unconjugated KLH (n=10), which was used as a control. After the immunization specific DDT antibodies in the mouse serum were determined by ELISA and then the mice were fed chow containing 40 mg/kg of DDT for 45 days. Finally, the concentration of DDT and its metabolites, DDE and DDD, in various tissues was measured by gas chromatography. Specific DDT antibody levels were significantly higher in the DDT immunized group than in the control group. DDT, DDE and DDD levels in adipose tissue, blood, brain and spleen were significantly reduced in the DDT immunized animals relative to control animals. However, DDT and DDD levels were higher in the liver compared to the control group. The findings indicate that the DDT immunization reduces the total uptake of DDT in animal tissues, which is reflected by the lower levels in adipose tissue, blood, brain and spleen. The elevated levels in liver suggest that DDT-antibody complexes in mouse serum are delivered to the liver.

Temporal trends of organochlorine contamination in Black Guillemots in Iceland from 1976 to 1996.

The levels of several different persistent organochlorines (OCs) in Black Guillemots Cepphus grylle, collected during the summers of 1976-1996 at Breioafjörour in W-Iceland, were investigated. The levels of about 40 different organochlorines (PCBs, DDTs, chlordanes, toxaphenes, HCH, HCB) were compared with respect to age, sex, fat content, and year of collection. The levels of PCBs correlated very closely with those of DDE, indicating long-range transport as the major source of these contaminants in Iceland, with the ratio PCBs/DDE mostly in the range of 2-5. Unlike the Gyrfalcon Falco rusticolus, the organochlorine levels did not seem to accumulate substantially with age, neither in males nor females. The variation in the levels of OCs at the age of 2 years was even greater than the variation in OC levels over an age range of 12 years. In immature birds the levels of PCBs, DDE, HCB and beta-HCH declined very slowly (T(1/2) from 12 to 20 years) over the years 1976-1996, whereas the levels of alpha-HCH and p,p'-DDT declined much faster. The levels of trans-nonachlor, alpha-chlordane, gamma-chlordane, oxychlordane, and toxaphene did not correlate with the year of collection. As the Black Guillemot is mostly a resident seabird, feeding mainly on small fish and invertebrates, this investigation should give a good indication of the temporal trends of organochlorine pollution at Breioafjörour, Iceland, during this 20 year period and is likely to reflect baseline trends in the marine environment of the North-Atlantic Ocean.

Persistent organochlorines, sedentary occupation, obesity and human male subfertility.

BACKGROUND: Studies have suggested that the quality of human semen has been declining over recent decades, presumably because of lifestyle or environmental factors. METHODS: Polychlorinated biphenyls and organochlorine pesticides were analysed in the plasma of 25 men with poor semen quality, 20 men with normal semen quality and idiopathic subfertility and 27 men with normal semen quality and female factor subfertility. Samples of seminal fluid were also analysed to assess the relationship between the levels in blood and semen. RESULTS: The results indicate no difference in the levels of organochlorines between the groups. The levels of organochlorines in seminal fluid were proportional to the levels in plasma, but approximately 40 times lower. Men with poor semen quality were three times more likely to be obese than men with normal semen quality. There was also a significant negative correlation between semen quality parameters and body mass index among men with normal semen quality. The prevalence of sedentary work was lowest among men with the best semen quality. CONCLUSIONS: Poor semen quality was found to be associated with sedentary work and obesity but not with plasma levels of persistent organochlorines. More research is needed to assess whether sedentary lifestyle and obesity are causal factors in the decline of semen quality.

[Phytoestrogens and human health.].

Recently there has been a growing interest in phytoestrogens, because they have been proven to play a protective role against many diseases that have been related to lifestyle in the westernized countries. The four main classes of phytoestrogens are: isoflavones, flavones, coumestrans and lignans. Known mechanisms of action include estrogen receptor agonism and possibly antagonism, influences on sex hormone binding globulin and/or key enzymes in sex hormone production and metabolism. Other effects not linked to sex hormones, such as antioxidant activity, antiproliferative activity and inhibition of angiogenesis, have also been suggested. A short review of the most studied health effects that have been linked to phytoestrogens is presented, such as prevention of certain types of cancer, osteoporosis and coronary heart disease.