Reduced incidence of insect-bite hypersensitivity in Icelandic horses is associated with a down-regulation of interleukin-4 by interleukin-10 and transforming growth factor-beta1

Insect bite hypersensitivity (IBH) is an allergic dermatitis of horses caused by IgE-mediated reactions to bites of insects of the genus Culicoides. IBH does not occur in Iceland due to the absence of Culicoides. However, Icelandic horses exported to mainland Europe as adults (1st generation) have a >/=50% incidence of developing IBH. In contrast, their progeny (2nd generation) has a <10% incidence of IBH. Here we show that peripheral blood mononuclear cells (PBMC) from Icelandic horses born in mainland Europe and belonging either to the IBH or healthy subgroup produce less interleukin (IL)-4 after polyclonal or allergen-specific stimulation when compared with counterparts from horses born in Iceland. We examined a role of IL-10 and transforming growth factor (TGF)-beta1 in down-regulation of IL-4 in healthy 2nd generation Icelandic horses. Supernatants of PBMC from 2nd generation healthy horses down-regulated the proportion of IL-4-producing cells and IL-4 production in stimulated cultures of PBMC from 1st generation IBH. This inhibition was mimicked by a combination of IL-10 and TGF-beta1 but not by the single cytokines. Cultures of stimulated PBMC of healthy 2nd generation horses produced a low level of IL-4, but IL-4 production was increased by anti-equine IL-10 and anti-human TGF-beta1. This shows for the first time that in horses, IL-10 and TGF-beta1 combined regulate IL-4 production in vitro. It is suggested that in this naturally occurring IgE-mediated allergy, IL-10 and TGF-beta1 have a role in the down-regulation of IL-4-induced allergen-specific Th2 cells, thereby reducing the incidence of IBH.

CD69 upregulation on T cells as an in vitro marker for delayed-type drug hypersensitivity

BACKGROUND: T cells play a key role in delayed-type drug hypersensitivity reactions. Their reactivity can be assessed by their proliferation in response to the drug in the lymphocyte transformation test (LTT). However, the LTT imposes limitations in terms of practicability, and an alternative method that is easier to implement than the LTT would be desirable. METHODS: Four months to 12 years after acute drug hypersensitivity reactions, CD69 upregulation on T cells of 15 patients and five healthy controls was analyzed by flow cytometry. RESULTS: All 15 LTT-positive patients showed a significant increase of CD69 expression on T cells after 48 h of drug-stimulation exclusively with the drugs incriminated in drug-hypersensitivities. A stimulation index of 2 as cut-off value allowed discrimination between nonreactive and reactive T cells in LTT and CD69 upregulation. T cells (0.5-3%) showed CD69 up-regulation. The reactive cell population consisted of a minority of truly drug reactive T cells secreting cytokines and a higher number of bystander T cells activated by IL-2 and possibly other cytokines. CONCLUSIONS: CD69 upregulation was observed after 2 days in all patients with a positive LTT after 6 days, thus appearing to be a promising tool to identify drug-reactive T cells in the peripheral blood of patients with drug-hypersensitivity reactions.

Drug-specific in vitro release of IL-2, IL-5, IL-13 and IFN-gamma in patients with delayed-type drug hypersensitivity

BACKGROUND: The most prevalent drug hypersensitivity reactions are T-cell mediated. The only established in vitro test for detecting T-cell sensitization to drugs is the lymphocyte transformation test, which is of limited practicability. To find an alternative in vitro method to detect drug-sensitized T cells, we screened the in vitro secretion of 17 cytokines/chemokines by peripheral blood mononuclear cells (PBMC) of patients with well-documented drug allergies, in order to identify the most promising cytokines/chemokines for detection of T-cell sensitization to drugs. METHODS: Peripheral blood mononuclear cell of 10 patients, five allergic to beta-lactams and five to sulfanilamides, and of five healthy controls were incubated for 3 days with the drug antigen. Cytokine concentrations were measured in the supernatants using commercially available 17-plex bead-based immunoassay kits. RESULTS: Among the 17 cytokines/chemokines analysed, interleukin-2 (IL-2), IL-5, IL-13 and interferon-gamma (IFN-gamma) secretion in response to the drugs were significantly increased in patients when compared with healthy controls. No difference in cytokine secretion patterns between sulfonamide- and beta-lactam-reactive PBMC could be observed. The secretion of other cytokines/chemokines showed a high variability among patients. CONCLUSION: The measurement of IL-2, IL-5, IL-13 or IFN-gamma or a combination thereof might be a useful in vitro tool for detection of T-cell sensitization to drugs. Secretion of these cytokines seems independent of the type of drug antigen and the phenotype of the drug reaction. A study including a higher number of patients and controls will be needed to determine the exact sensitivity and specificity of this test.

In vitro induction of functional allergen-specific CD4+ CD25high Treg cells in horses affected with insect bite hypersensitivity

BACKGROUND Insect bite hypersensitivity (IBH) is a recurrent allergic dermatitis of horses with similarities to human atopic eczema, caused by bites of insects of the genus Culicoides. Previous studies suggested a dysregulated T cell tolerance to Culicoides allergen in IBH-affected horses. OBJECTIVE We have investigated whether the suppressive function of CD4(+) CD25(high) cells is impaired in IBH-affected horses and possible ways to restore it. METHODS CD4(+) CD25(-) cells sorted from peripheral blood mononuclear cells (PBMC) were stimulated with irradiated autologous PBMC pulsed with Culicoides or tetanus toxoid as control antigen, in the presence of CD4(+) CD25(high) cells. Furthermore, Culicoides-specific CD4(+) CD25(high) regulatory cells were expanded or induced from CD4(+) CD25(-) cells in vitro in the presence of a combination of rIL-2 and rTGF-β1 (rIL-2/rTGF-β1) or of retinoic acid and rapamycin (RetA/Rapa). Proliferation was determined by [(3) H] thymidine incorporation and cytokine production measured by flow cytometry. RESULTS The ability of Culicoides- but not tetanus-stimulated CD4(+) CD25(high) cells to suppress proliferation of CD4(+) CD25(-) cells was significantly lower in IBH-affected horses (28%) than in healthy controls (86%). The decreased suppression in IBH-affected horses was associated with a significantly higher proportion of IL-4(+) cells and a lower percentage of FoxP3(+) IL-10(+) compared to controls. Addition of rIL-2/rTGF-β1 or of RetA/Rapa to Culicoides-stimulated CD4(+) CD25(high) cells from IBH-affected horses significantly increased the proportion of FoxP3(+) IL-10(+) cells. We also found that RetA/Rapa induced a more significant decrease in the frequency of IL-4(+) cells than rIL-2/rTGF-β1. Moreover, the suppressive activity of Culicoides-stimulated CD4(+) CD25(high) cells was significantly restored by both rIL-2/rTGF-β1and RetA/Rapa, albeit in an antigen-unspecific manner. In contrast, in vitro induced Culicoides-specific CD4(+) CD25(high) cells suppressed proliferation of CD4(+) CD25(-) cells in an antigen-specific manner. CONCLUSION AND CLINICAL RELEVANCE The in vitro induction of functional allergen-specific Treg cells in IBH-affected horses suggests a potential therapeutic use of these cells in allergy.

DAMAGE-INDUCED INFLAMMATION AND NOCICEPTIVE HYPERSENSITIVITY IN DROSOPHILA LARVAE

Mounting an effective response to tissue damage requires a concerted effort from a number of systems, including both the immune and nervous systems. Immune-responsive blood cells fight infection and clear debris from damaged tissues, and specialized pain receptors become hypersensitive to promote behavior that protects the damaged area while it heals. To uncover the cellular and molecular mechanisms underlying these processes, we have developed a genetically tractable invertebrate model of damage-induced inflammation and pain hypersensitivity using Drosophila larvae. To study wound-induced inflammation, we generated transgenic larvae with fluorescent epidermal cells and blood cells (hemocytes). Using live imaging, we monitored the circulatory dynamics of hemocytes and the methods by which they accumulate at epidermal wounds. We found that circulating hemocytes attach to wound sites directly from circulation, a mechanism once thought to work exclusively in species with a closed circulatory system. To study damage-induced pain hypersensitivity, we developed a “sunburn assay” and found that larvae have a lowered pain threshold (allodynia) and an exaggerated response to noxious stimuli (hyperalgesia) following UV damage. We screened for genes required for hypersensitivity in pain receptors (nociceptors), and discovered a number of novel mediators that have well conserved mammalian homologs. Together, these results help us to understand how various cell types in the immune and nervous systems both detect and respond to tissue damage.

Chromatin structure and DNase I hypersensitivity in the transcriptionally active and inactive porcine tumor necrosis factor gene locus

We have analyzed the chromatin structure of the porcine tumor necrosis factor gene locus (TNF-alpha and TNF-beta). Nuclei from porcine peripheral blood mononuclear cells were digested with different nucleases. As assessed with micrococcal nuclease, the two TNF genes displayed slightly faster digestion kinetics than bulk DNA. Studies with DNaseI revealed distinct DNaseI hypersensitive sites (DH-sites) within the porcine TNF locus. Four DH-sites could be observed in the promoter and mRNA leader regions of the TNF-beta gene. Two DH-sites could be observed for the TNF-alpha gene, one located in the promoter region close to the TATA-box and the other site in intron 3. This pattern of DH-sites was present independently of the activation state of the cells. Interestingly in a porcine macrophage-like cell line, we found that the TNF-alpha promoter DH-site disappeared and another DH-site appeared in the region of intron 1. Additionally, the DH-site of intron 3 could be enhanced by PMA-stimulation in these cells. TNF-beta sites were not detected in this cell line. However, DH-sites were totally absent in fibroblasts (freshly isolated from testicles) and in porcine kidney cells (PK15 cell line) both of which do not transcribe the TNF genes. Therefore, the pattern of DH-sites corresponds to the transcriptional activity of analyzed cells.

Flare-up reactions in severe drug hypersensitivity: infection or ongoing T-cell hyperresponsiveness

"Flare-up" reactions are late manifestations of severe T-cell-mediated drug hypersensitivity reactions. Management is anti-inflammatory treatment and avoiding unnecessary medicines. Symptoms like fever, lymph node swelling, and blood count abnormalities may lead to confusion with bacterial infections. For prompt recognition it is important to keep the differential diagnosis in mind.

Sustained hypersensitivity to angiotensin II and its mechanism in mice lacking the subtype-2 (AT2) angiotensin receptor

The vast majority of the known biological effects of the renin–angiotensin system are mediated by the type-1 (AT1) receptor, and the functions of the type-2 (AT2) receptor are largely unknown. We investigated the role of the AT2 receptor in the vascular and renal responses to physiological increases in angiotensin II (ANG II) in mice with targeted deletion of the AT2 receptor gene. Mice lacking the AT2 receptor (AT2-null mice) had slightly elevated systolic blood pressure (SBP) compared with that of wild-type (WT) control mice (P < 0.0001). In AT2-null mice, infusion of ANG II (4 pmol/kg/min) for 7 days produced a marked and sustained increase in SBP [from 116 ± 0.5 to 208 ± 1 mmHg (P < 0.0001) (1 mmHg = 133 Pa)] and reduction in urinary sodium excretion (UNaV) [from 0.6 ± 0.01 to 0.05 ± 0.002 mM/day (P < 0.0001)] whereas neither SBP nor UNaV changed in WT mice. AT2-null mice had low basal levels of renal interstitial fluid bradykinin (BK), and cyclic guanosine 3′,5′-monophosphate, an index of nitric oxide production, compared with WT mice. In WT mice, dietary sodium restriction or ANG II infusion increased renal interstitial fluid BK, and cyclic guanosine 3′,5′-monophosphate by ≈4-fold (P < 0.0001) whereas no changes were observed in AT2-null mice. These results demonstrate that the AT2 receptor is necessary for normal physiological responses of BK and nitric oxide to ANG II. Absence of the AT2 receptor leads to vascular and renal hypersensitivity to ANG II, including sustained antinatriuresis and hypertension. These results strongly suggest that the AT2 receptor plays a counterregulatory protective role mediated via BK and nitric oxide against the antinatriuretic and pressor actions of ANG II.

Early transplantation of mesenchymal stem cells after spinal cord injury relieves pain hypersensitivity through suppression of pain-related signaling cascades and reduced inflammatory cell recruitment

Bone marrow-derived mesenchymal stem cells (BMSC) modulate inflammatory/immune responses and promote motor functional recovery after spinal cord injury (SCI). However, the effects of BMSC transplantation on central neuropathic pain and neuronal hyperexcitability after SCI remain elusive. This is of importance because BMSC-based therapies have been proposed for clinical treatment. We investigated the effects of BMSC transplantation on pain hypersensitivity in green fluorescent protein (GFP)-positive bone marrow-chimeric mice subjected to a contusion SCI, and the mechanisms of such effects. BMSC transplantation at day 3 post-SCI improved motor function and relieved SCI-induced hypersensitivities to mechanical and thermal stimulation. The pain improvements were mediated by suppression of protein kinase C-γ and phosphocyclic AMP response element binding protein expression in dorsal horn neurons. BMSC transplants significantly reduced levels of p-p38 mitogen-activated protein kinase and extracellular signal-regulated kinase (p-ERK1/2) in both hematogenous macrophages and resident microglia and significantly reduced the infiltration of CD11b and GFP double-positive hematogenous macrophages without decreasing the CD11b-positive and GFP-negative activated spinal-microglia population. BMSC transplants prevented hematogenous macrophages recruitment by restoration of the blood-spinal cord barrier (BSCB), which was associated with decreased levels of (a) inflammatory cytokines (tumor necrosis factor-α, interleukin-6); (b) mediators of early secondary vascular pathogenesis (matrix metallopeptidase 9); (c) macrophage recruiting factors (CCL2, CCL5, and CXCL10), but increased levels of a microglial stimulating factor (granulocyte-macrophage colony-stimulating factor). These findings support the use of BMSC transplants for SCI treatment. Furthermore, they suggest that BMSC reduce neuropathic pain through a variety of related mechanisms that include neuronal sparing and restoration of the disturbed BSCB, mediated through modulation of the activity of spinal-resident microglia and the activity and recruitment of hematogenous macrophages.

Evidences Of Endocytosis Via Caveolae Following Blood-brain Barrier Breakdown By Phoneutria Nigriventer Spider Venom.

Spider venoms contain neurotoxic peptides aimed at paralyzing prey or for defense against predators; that is why they represent valuable tools for studies in neuroscience field. The present study aimed at identifying the process of internalization that occurs during the increased trafficking of vesicles caused by Phoneutria nigriventer spider venom (PNV)-induced blood-brain barrier (BBB) breakdown. Herein, we found that caveolin-1α is up-regulated in the cerebellar capillaries and Purkinje neurons of PNV-administered P14 (neonate) and 8- to 10-week-old (adult) rats. The white matter and granular layers were regions where caveolin-1α showed major upregulation. The variable age played a role in this effect. Caveolin-1 is the central protein that controls caveolae formation. Caveolar-specialized cholesterol- and sphingolipid-rich membrane sub-domains are involved in endocytosis, transcytosis, mechano-sensing, synapse formation and stabilization, signal transduction, intercellular communication, apoptosis, and various signaling events, including those related to calcium handling. PNV is extremely rich in neurotoxic peptides that affect glutamate handling and interferes with ion channels physiology. We suggest that the PNV-induced BBB opening is associated with a high expression of caveolae frame-forming caveolin-1α, and therefore in the process of internalization and enhanced transcytosis. Caveolin-1α up-regulation in Purkinje neurons could be related to a way of neurons to preserve, restore, and enhance function following PNV-induced excitotoxicity. The findings disclose interesting perspectives for further molecular studies of the interaction between PNV and caveolar specialized membrane domains. It proves PNV to be excellent tool for studies of transcytosis, the most common form of BBB-enhanced permeability.

Nebivolol Reduces Central Blood Pressure In Stage I Hypertensive Patients: Experimental Single Cohort Study.

Assessment of central blood pressure (BP) has grown substantially over recent years because evidence has shown that central BP is more relevant to cardiovascular outcomes than peripheral BP. Thus, different classes of antihypertensive drugs have different effects on central BP despite similar reductions in brachial BP. The aim of this study was to investigate the effect of nebivolol, a β-blocker with vasodilator properties, on the biochemical and hemodynamic parameters of hypertensive patients. Experimental single cohort study conducted in the outpatient clinic of a university hospital. Twenty-six patients were recruited. All of them underwent biochemical and hemodynamic evaluation (BP, heart rate (HR), central BP and augmentation index) before and after 3 months of using nebivolol. 88.5% of the patients were male; their mean age was 49.7 ± 9.3 years and most of them were overweight (29.6 ± 3.1 kg/m2) with large abdominal waist (102.1 ± 7.2 cm). There were significant decreases in peripheral systolic BP (P = 0.0020), diastolic BP (P = 0.0049), HR (P < 0.0001) and central BP (129.9 ± 12.3 versus 122.3 ± 10.3 mmHg; P = 0.0083) after treatment, in comparison with the baseline values. There was no statistical difference in the augmentation index or in the biochemical parameters, from before to after the treatment. Nebivolol use seems to be associated with significant reduction of central BP in stage I hypertensive patients, in addition to reductions in brachial systolic and diastolic BP.

Antibody Recognition Of Plasmodium Falciparum Infected Red Blood Cells By Symptomatic And Asymptomatic Individuals In The Brazilian Amazon.

In the Amazon Region, there is a virtual absence of severe malaria and few fatal cases of naturally occurring Plasmodium falciparum infections; this presents an intriguing and underexplored area of research. In addition to the rapid access of infected persons to effective treatment, one cause of this phenomenon might be the recognition of cytoadherent variant proteins on the infected red blood cell (IRBC) surface, including the var gene encoded P. falciparum erythrocyte membrane protein 1. In order to establish a link between cytoadherence, IRBC surface antibody recognition and the presence or absence of malaria symptoms, we phenotype-selected four Amazonian P. falciparum isolates and the laboratory strain 3D7 for their cytoadherence to CD36 and ICAM1 expressed on CHO cells. We then mapped the dominantly expressed var transcripts and tested whether antibodies from symptomatic or asymptomatic infections showed a differential recognition of the IRBC surface. As controls, the 3D7 lineages expressing severe disease-associated phenotypes were used. We showed that there was no profound difference between the frequency and intensity of antibody recognition of the IRBC-exposed P. falciparum proteins in symptomatic vs. asymptomatic infections. The 3D7 lineages, which expressed severe malaria-associated phenotypes, were strongly recognised by most, but not all plasmas, meaning that the recognition of these phenotypes is frequent in asymptomatic carriers, but is not necessarily a prerequisite to staying free of symptoms.

Taurine Supplementation Reduces Blood Pressure And Prevents Endothelial Dysfunction And Oxidative Stress In Post-weaning Protein-restricted Rats.

Taurine is a sulfur-containing amino acid that exerts protective effects on vascular function and structure in several models of cardiovascular diseases through its antioxidant and anti-inflammatory properties. Early protein malnutrition reprograms the cardiovascular system and is linked to hypertension in adulthood. This study assessed the effects of taurine supplementation in vascular alterations induced by protein restriction in post-weaning rats. Weaned male Wistar rats were fed normal- (12%, NP) or low-protein (6%, LP) diets for 90 days. Half of the NP and LP rats concomitantly received 2.5% taurine supplementation in the drinking water (NPT and LPT, respectively). LP rats showed elevated systolic, diastolic and mean arterial blood pressure versus NP rats; taurine supplementation partially prevented this increase. There was a reduced relaxation response to acetylcholine in isolated thoracic aortic rings from the LP group that was reversed by superoxide dismutase (SOD) or apocynin incubation. Protein expression of p47phox NADPH oxidase subunit was enhanced, whereas extracellular (EC)-SOD and endothelial nitric oxide synthase phosphorylation at Ser 1177 (p-eNOS) were reduced in aortas from LP rats. Furthermore, ROS production was enhanced while acetylcholine-induced NO release was reduced in aortas from the LP group. Taurine supplementation improved the relaxation response to acetylcholine and eNOS-derived NO production, increased EC-SOD and p-eNOS protein expression, as well as reduced ROS generation and p47phox expression in the aortas from LPT rats. LP rats showed an increased aortic wall/lumen ratio and taurine prevented this remodeling through a reduction in wall media thickness. Our data indicate a protective role of taurine supplementation on the high blood pressure, endothelial dysfunction and vascular remodeling induced by post-weaning protein restriction. The beneficial vascular effect of taurine was associated with restoration of vascular redox homeostasis and improvement of NO bioavailability.

Rh, Kell, Duffy, Kidd And Diego Blood Group System Polymorphism In Brazilian Japanese Descendants.

Polymorphisms of Rh, Kell, Duffy, Kidd and Diego blood group systems were studied in 209 unrelated Brazilian Japanese descendants from South of Brazil. The methods used were multiplex-PCR, AS-PCR and RFLP-PCR. The differences in frequencies among the populations were evaluated using chi-square test. The frequencies for Rh, Kell, Kidd and Diego system were similar to those of the Japanese. RHCE(*)CC, RHCE(*)EE genotypes and FY(*)01 allele were lower and FY(*)01N.01 was higher than Japanese. These differences in the frequencies between Brazilian Japanese descendants and Japanese could indicate a gene flow in Brazilian population and reinforce the importance of this knowledge to achieve safe red blood cells.

Understanding Red Blood Cell Parameters In The Context Of The Frailty Phenotype: Interpretations Of The Fibra (frailty In Brazilian Seniors) Study.

Frailty and anemia in the elderly appear to share a common pathophysiology associated with chronic inflammatory processes. This study uses an analytical, cross-sectional, population-based methodology to investigate the probable relationships between frailty, red blood cell parameters and inflammatory markers in 255 community-dwelling elders aged 65 years or older. The frailty phenotype was assessed by non-intentional weight loss, fatigue, low grip strength, low energy expenditure and reduced gait speed. Blood sample analyses were performed to determine hemoglobin level, hematocrit and reticulocyte count, as well as the inflammatory variables IL-6, IL-1ra and hsCRP. In the first multivariate analysis (model I), considering only the erythroid parameters, Hb concentration was a significant variable for both general frailty status and weight loss: a 1.0g/dL drop in serum Hb concentration represented a 2.02-fold increase (CI 1.12-3.63) in an individual's chance of being frail. In the second analysis (model II), which also included inflammatory cytokine levels, hsCRP was independently selected as a significant variable. Each additional year of age represented a 1.21-fold increase in the chance of being frail, and each 1-unit increase in serum hsCRP represented a 3.64-fold increase in the chance of having the frailty phenotype. In model II reticulocyte counts were associated with weight loss and reduced metabolic expenditure criteria. Our findings suggest that reduced Hb concentration, reduced RetAbs count and elevated serum hsCRP levels should be considered components of frailty, which in turn is correlated with sarcopenia, as evidenced by weight loss.