Regulation of T Cell Responses by Ionic Salt Signals.

T helper cell responses are tailored to their respective antigens and adapted to their specific tissue microenvironment. While a great proportion of T cells acquire a resident identity, a significant proportion of T cells continue circulating, thus encountering changing microenvironmental signals during immune surveillance. One signal, which has previously been largely overlooked, is sodium chloride. It has been proposed to have potent effects on T cell responses in the context of autoimmune, allergic and infectious tissue inflammation in mouse models and humans. Sodium chloride is stringently regulated in the blood by the kidneys but displays differential deposition patterns in peripheral tissues. Sodium chloride accumulation might furthermore be regulated by dietary intake and thus by intentional behavior. Together, these results make sodium chloride an interesting but still controversial signal for immune modulation. Its downstream cellular activities represent a potential therapeutic target given its effects on T cell cytokine production. In this review article, we provide an overview and critical evaluation of the impact of this ionic signal on T helper cell polarization and T helper cell effector functions. In addition, the impact of sodium chloride from the tissue microenvironment is assessed for human health and disease and for its therapeutic potential.

Antiviral innate immune response in non-myeloid cells is augmented by chloride ions via an increase in intracellular hypochlorous acid levels.

Phagocytes destroy ingested microbes by producing hypochlorous acid (HOCl) from chloride ions (Cl-) and hydrogen peroxide within phagolysosomes, using the enzyme myeloperoxidase. HOCl, the active ingredient in bleach, has antibacterial/antiviral properties. As myeloperoxidase is needed for HOCl production, non-myeloid cells are considered incapable of producing HOCl. Here, we show that epithelial, fibroblast and hepatic cells have enhanced antiviral activity in the presence of increasing concentrations of sodium chloride (NaCl). Replication of enveloped/non-enveloped, DNA (herpes simplex virus-1, murine gammaherpesvirus 68) and RNA (respiratory syncytial virus, influenza A virus, human coronavirus 229E, coxsackievirus B3) viruses are inhibited in a dose-dependent manner. Whilst treatment with sodium channel inhibitors did not prevent NaCl-mediated virus inhibition, a chloride channel inhibitor reversed inhibition by NaCl, suggesting intracellular chloride is required for antiviral activity. Inhibition is also reversed in the presence of 4-aminobenzoic hydrazide, a myeloperoxidase inhibitor, suggesting epithelial cells have a peroxidase to convert Cl- to HOCl. A significant increase in intracellular HOCl production is seen early in infection. These data suggest that non-myeloid cells possess an innate antiviral mechanism dependent on the availability of Cl- to produce HOCl. Antiviral activity against a broad range of viral infections can be augmented by increasing availability of NaCl.

The influence of sodium on pathophysiology of multiple sclerosis.

Multiple sclerosis (MS) is a chronic, inflammatory, autoimmune disease of the central nervous system, and is an important cause of disability in young adults. In genetically susceptible individuals, several environmental factors may play a partial role in the pathogenesis of MS. Some studies suggests that high-salt diet (>5 g/day) may contribute to the MS and other autoimmune disease development through the induction of pathogenic Th17 cells and pro-inflammatory cytokines in both humans and mice. However, the precise mechanisms of pro-inflammatory effect of sodium chloride intake are not yet explained. The purpose of this review was to discuss the present state of knowledge on the potential role of environmental and dietary factors, particularly sodium chloride on the development and course of MS.

Aldosterone Induces Renal Fibrosis and Inflammatory M1-Macrophage Subtype via Mineralocorticoid Receptor in Rats.

We aimed to evaluate macrophages heterogeneity and structural, functional and inflammatory alterations in rat kidney by aldosterone + salt administration. The effects of treatment with spironolactone on above parameters were also analyzed. Male Wistar rats received aldosterone (1 mgkg-1d-1) + 1% NaCl for 3 weeks. Half of the animals were treated with spironolactone (200 mg kg-1d-1). Systolic and diastolic blood pressures were elevated (p<0.05) in aldosterone + salt-treated rats. Relative kidney weight, collagen content, fibronectin, macrophage infiltrate, CTGF, Col I, MMP2, TNF-α, CD68, Arg2, and SGK-1 were increased (p<0.05) in aldosterone + salt-treated rats, being reduced by spironolactone (p<0.05). Increased iNOS and IFN-γ mRNA gene expression (M1 macrophage markers) was observed in aldosterone + salt rats, whereas no significant differences were observed in IL-10 and gene ArgI mRNA expression or ED2 protein content (M2 macrophage markers). All the observed changes were blocked with spironolactone treatment. Macrophage depletion with liposomal clodronate reduced macrophage influx and inflammatory M1 markers (INF-γ or iNOS), whereas interstitial fibrosis was only partially reduced after this intervention, in aldosterone plus salt-treated rats. In conclusion, aldosterone + salt administration mediates inflammatory M1 macrophage phenotype and increased fibrosis throughout mineralocorticoid receptors activation.

Salt, chloride, bleach, and innate host defense.

Salt provides 2 life-essential elements: sodium and chlorine. Chloride, the ionic form of chlorine, derived exclusively from dietary absorption and constituting the most abundant anion in the human body, plays critical roles in many vital physiologic functions, from fluid retention and secretion to osmotic maintenance and pH balance. However, an often overlooked role of chloride is its function in innate host defense against infection. Chloride serves as a substrate for the generation of the potent microbicide chlorine bleach by stimulated neutrophils and also contributes to regulation of ionic homeostasis for optimal antimicrobial activity within phagosomes. An inadequate supply of chloride to phagocytes and their phagosomes, such as in CF disease and other chloride channel disorders, severely compromises host defense against infection. We provide an overview of the roles that chloride plays in normal innate immunity, highlighting specific links between defective chloride channel function and failures in host defense.

Effect of an immune challenge on the functional performance of male weaponry.

Theories of parasite-mediated sexual selection predict a positive association between immune function and the expression of sexually selected ornaments. Few studies, however, have investigated how an immune challenge affects the performance of sexually selected weaponry. Male Wellington tree weta (Hemideina crassidens) (Orthoptera: Anostostomatidae) possess enlarged mandibles that are used as weapons in fights for access to females residing in tree galleries. Intense sexual competition appears to have favoured the evolution of alternative male mating strategies in this species as males have a trimorphic phenotype in which weapon size varies across morphotype: 8th instar males have the smallest jaws, 10th instar males have the largest and 9th instar males being intermediate to the other two. After injecting males and females with either lipopolysaccharide (LPS; immune challenge) or saline (control) I measured over a 24h period each weta's body mass to assess whether they responded immunologically to the LPS and their bite force to assess the functional performance of their jaws. Both sexes responded immunologically to the immune-challenge as LPS-injected individuals lost significantly more body mass than saline-injected controls with females losing more mass than males. Female bite force was significantly reduced 8h after LPS-injection whereas male bite force did not significantly decline. Both sexes regained pre-injection functional performance of their jaws 24h after the immune challenge. My results suggest that females trade-off bite force for immune function whereas males do not. This article is part of a Special Issue entitled: insert SI title.

Mizoribine ameliorates renal injury and hypertension along with the attenuation of renal caspase-1 expression in aldosterone-salt-treated rats.

Aldosterone-salt treatment induces not only hypertension but also extensive inflammation that contributes to fibrosis in the rat kidney. However, the mechanism underlying aldosterone-salt-induced renal inflammation remains unclear. Pyroptosis has recently been identified as a new type of cell death that is accompanied by the activation of inflammatory cytokines. We hypothesized that aldosterone-salt treatment could induce inflammation through pyroptosis and that mizoribine, an effective immunosuppressant, would ameliorate the renal inflammation that would otherwise cause renal fibrosis. Ten days after recovery from left uninephrectomy, rats were given drinking water with 1% sodium chloride. The animals were divided into three groups (n = 7 per group): (1) vehicle infusion group, (2) aldosterone infusion group, or (3) aldosterone infusion plus oral mizoribine group. Aldosterone-salt treatment increased the expression of the nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing 3 and caspase-1, and also increased the number of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells. However, the oral administration of mizoribine attenuated these alterations. Furthermore, mizoribine inhibited hypertension and renal fibrosis, and also attenuated the aldosterone-induced expression of serum/glucocorticoid-regulated kinase and α epithelial sodium channel. These results suggest that caspase-1 activation plays an important role in the development of inflammation induced by aldosterone-salt treatment and that it functions as an anti-inflammatory strategy that protects against renal injury and hypertension.

Mortalin/GRP75 binds to complement C9 and plays a role in resistance to complement-dependent cytotoxicity.

Mortalin/GRP75, the mitochondrial heat shock protein 70, plays a role in cell protection from complement-dependent cytotoxicity (CDC). As shown here, interference with mortalin synthesis enhances sensitivity of K562 erythroleukemia cells to CDC, whereas overexpression of mortalin leads to their resistance to CDC. Quantification of the binding of the C5b-9 membrane attack complex to cells during complement activation shows an inverse correlation between C5b-9 deposition and the level of mortalin in the cell. Following transfection, mortalin-enhanced GFP (EGFP) is located primarily in mitochondria, whereas mortalinΔ51-EGFP lacking the mitochondrial targeting sequence is distributed throughout the cytoplasm. Overexpressed cytosolic mortalinΔ51-EGFP has a reduced protective capacity against CDC relative to mitochondrial mortalin-EGFP. Mortalin was previously shown by us to bind to components of the C5b-9 complex. Two functional domains of mortalin, the N-terminal ATPase domain and the C-terminal substrate-binding domain, were purified after expression in bacteria. Similar to intact mortalin, the ATPase domain, but not the substrate-binding domain, was found to bind to complement proteins C8 and C9 and to inhibit zinc-induced polymerization of C9. Binding of mortalin to complement C9 and C8 occurs through an ionic interaction that is nucleotide-sensitive. We suggest that to express its full protective effect from CDC, mortalin must first reach the mitochondria. In addition, mortalin can potentially target the C8 and C9 complement components through its ATPase domain and inhibit C5b-9 assembly and stability.

Total hepatic ischemia and reperfusion after state controlled hemorrhagic shock, with used of different solutions: effects of neutrophils sequestration in kidney of rats

OBJETIVO: Avaliar e comparar o seqüestro de neutrófilos no rim de rato, como efeito da isquemia e reperfusão hepática total após estado de choque hemorrágico controlado, com uso de diferentes soluções eletrolíticas.MÉTODOS: Utilizou-se 18 ratos Wistar, machos, adultos, divididos em três grupos conforme a solução utilizada para reanimação: Grupo SF: solução fisiológica; Grupo SH: solução hipertônica de NaCl a 7,5% seguido pela solução de ringer com lactato; Grupo RL: solução de ringer com lactato. Todos os animais foram submetidos à sangria controlada até pressão arterial média (PAM) atingir 40 mmHg, permanecendo por 20 minutos. Realizou-se reanimação volêmica até PAM=80 mmHg com a solução conforme o grupo estudado. Em seguida realizou-se uma laparotomia e a manobra de Pringle por 15 minutos. Os animais foram acompanhados até duas horas. Para comparações estatísticas entre as contagens de neutrófilos, no interstício do córtex renal, foram efetuados os testes ANOVA e a análise de covariância, ajustando-se para o tempo de sobrevida. Os parâmetros hemodinâmicos avaliados foram: PAM, freqüência cardíaca, índice cardíaco, índice de resistência vascular sistêmica. As variáveis metabólicas analisadas foram: pH, bicarbonato, reserva de base e lactato, além de eletrólitos. RESULTADOS: Os valores médios de tempo de sobrevida, em minutos, por grupo foram: Grupo SF 79,0±12,0; Grupo RL 97,0±11,0; Grupo SH 67,0±10. Os valores médios da contagem de neutrófilos/campo no córtex renal foram: Grupo SF 0,55±0,68; Grupo RL 1,68±0,53; Grupo SH 1,33±0,43. E quando são ajustados para o tempo de sobrevida encontram-se: Grupo SF 0,55; Grupo RL 1,62; Grupo SH 1,39. Houve diferença estatisticamente significativa, na contagem de neutrófilo entre o Grupo SF com os demais, usando-se ou não o ajuste pelo tempo de sobrevida (p=0,016 e p=0,0128). CONCLUSAO: As duas situações críticas, choque hemorrágico controlado e manobra de Pringle, promoveram seqüestro de neutrófilos no interstício renal do rato, sendo a solução fisiológica com a menor média, diferenciando estatisticamente das demais soluções, neste modelo.

Inhibition of inflammatory responses by ambroxol, a mucolytic agent, in a murine model of acute lung injury induced by lipopolysaccharide.

OBJECTIVE: The aim of this study is to investigate whether ambroxol inhibits inflammatory responses in a murine model of lipopolysaccharide-induced acute lung injury (ALI). METHODS: Mice (n=295) were first intratracheally instilled with lipopolysaccharide (LPS) to induce ALI and then received an intraperitoneal (i.p.) injection of either normal saline (NS), ambroxol (30 or 90 mg/kg per day) or dexamethasone (2.5 or 5 mg/kg per day) for 7 days. Metabolism (n=10, each), lung morphology (n=5, each) and wet-to-dry lung weight ratio (n=10, each) were studied. The levels of tumor necrosis factor (TNF-alpha), interleukin-6 (IL-6) and transforming growth factor (TGF-beta1) and the protein concentration (n=5 or 7, each) in bronchoalveolar lavage (BAL) were measured. RESULTS: Mice with LPS-induced ALI that were treated with ambroxol at a dosage of 90 mg/kg per day significantly gained weight compared to the control and dexamethasone-treated groups. Ambroxol and dexamethasone significantly reduced the lung hemorrhage, edema, exudation, neutrophil infiltration and total lung injury histology score at 24 and 48 h. In addition, ambroxol and dexamethasone significantly attenuated the lung wet-to-dry weight ratio at 24 and 48 h (p<0.05). Compared to the control group, TNF-alpha, IL-6 and TGF-beta1 levels in the BAL in both ambroxol- and dexamethasone-treated groups were significantly reduced at 24 and 48 h. The protein in BAL, an index of vascular permeability, was also significantly decreased in the ambroxol- and dexamethasone-treated groups (p<0.05). CONCLUSION: Ambroxol inhibited proinflammatory cytokines, reduced lung inflammation and accelerated recovery from LPS-induced ALI.

Mast cells can amplify airway reactivity and features of chronic inflammation in an asthma model in mice.

The importance of mast cells in the development of the allergen-induced airway hyperreactivity and inflammation associated with asthma remains controversial. We found that genetically mast cell-deficient WBB6F(1)-W/W(v) mice that were sensitized to ovalbumin (OVA) without adjuvant, then challenged repetitively with antigen intranasally, exhibited much weaker responses in terms of bronchial hyperreactivity to aerosolized methacholine, lung tissue eosinophil infiltration, and numbers of proliferating cells within the airway epithelium than did identically treated WBB6F(1)-+/+ normal mice. However, W/W(v) mice that had undergone selective reconstitution of tissue mast cells with in vitro-derived mast cells of congenic +/+ mouse origin exhibited airway responses that were very similar to those of the +/+ mice. By contrast, W/W(v) mice that were sensitized with OVA emulsified in alum and challenged with aerosolized OVA exhibited levels of airway hyperreactivity and lung tissue eosinophil infiltration that were similar to those of the corresponding +/+ mice. Nevertheless, these W/W(v) mice exhibited significantly fewer proliferating cells within the airway epithelium than did identically treated +/+ mice. These results show that, depending on the "asthma model" investigated, mast cells can either have a critical role in, or not be essential for, multiple features of allergic airway responses in mice.

Inducible nitric oxide synthase inhibitors suppress airway inflammation in mice through down-regulation of chemokine expression.

Growing evidence demonstrates that inducible NO synthase (iNOS) is induced in the airways of asthmatic patients. However, the precise role of NO in the lung inflammation is unknown. This study investigated the effect of both selective and nonselective iNOS inhibitors in an allergen-driven murine lung inflammation model. OVA challenge resulted in an accumulation of eosinophils and neutrophils in the airways. Expression of iNOS immunostaining in lung sections together with an increase in calcium-independent NOS activity in lung homogenates was also observed after OVA challenge. Treatment with iNOS inhibitors from the day of challenge to the day of sacrifice resulted in an inhibition of the inflammatory cell influx together with a down-regulation of macrophage inflammatory protein-2 and monocyte chemoattractant protein-1 production. In contrast, eosinophilic and neutrophilic inhibition was not observed with treatment during the sensitization. Both treatments induced an increased production of Th2-type cytokines (IL-4 and IL-5) with a concomitant decrease in production of Th1-type cytokine (IFN-gamma). In vitro exposure of primary cultures of murine lung fibroblasts to a NO donor, hydroxylamine, induced a dose-dependent release of macrophage inflammatory protein-2 and monocyte chemoattractant protein-1. Our results suggest that lung inflammation after allergen challenge in mice is partially dependent on NO produced mainly by iNOS. NO appears to increase lung chemokine expression and, thereby, to facilitate influx of inflammatory cells into the airways.

Anti-phospholipid antibodies in HIV infection and SLE with or without anti-phospholipid syndrome: comparisons of phospholipid specificity, avidity and reactivity with beta2-GPI.

Increased prevalence of anti-phospholipid antibodies (aPL) and increased levels of lipid peroxidation have been described in patients with HIV infection. To assess the binding specificity and avidity of aPL antibodies in HIV infection, sera from 44 HIV-1 infected patients were evaluated for antibodies to cardiolipin (aCL), phosphatidyl serine (aPS), phosphatidyl inositol (aPI) and phosphatidyl choline (aPC) using enzyme linked immunosorbent assay (ELISA) methods. Sera from 30 patients with systemic lupus erythematosus (SLE), but without features of anti-phospholipid syndrome (APS) (SLE/non APS), six with SLE and secondary APS, (SLE/APS) and 11 with primary APS (PAPS) were also evaluated as controls. The resistance of the aPL antibody binding to dissociating agents was evaluated by treating the ELISA wells, after serum incubation with 2 M urea or 0.6 M NaCl for 10 min. An anti-beta2-glycoprotein-I (beta2-GPI) ELISA was used to assess serum reactivity against beta2-GPI, a plasma protein considered as the true antigen of aCL antibodies occurring in APS and SLE patients. The prevalence of aCL, aPS, aPI and aPC antibodies in HIV-1 infection was 36%, 56%, 34% and 43% respectively, which was comparable to that found in SLE/APS and PAPS patients and significantly higher than that observed in SLE/non-APS patients. Anti-beta2-GPI antibodies occurred in 5% of HIV-1 infected vs. 17% in SLE/non-APS (P=0.11), 50% in SLE/APS (P=0.009) and 70% in PAPS patients (P=0.0014). A significant decrease of aPL binding after urea and NaCl treatment was observed in the sera of HIV-1-infected, compared to that of APS patients, indicating that aPL antibodies from HIV-1 infected individuals have low resistance to dissociating agents. In conclusion, aPL antibodies (1) occur in HIV-1 infection; (2) tend to recognize various phospholipids but not beta2-GPI; and (3) are of low resistance to dissociating agents-a finding probably reflecting low antibody avidity. Finally, these, like the autoimmune-type aCL antibodies, tend to recognize the oxidized CL-a finding probably indicating autoantibody generation as a result of neoepitope formation by oxidized PLs.

A novel method for induction and detection of anaphylactic reaction using the mouse abdominal wall (AW method).

We found that an antigen-specific anaphylaxis was induced by antigen challenge to the abdominal wall, ear auricle, or subcutaneous tissue in mice sensitized 9 days previously with antigen and adjuvant. The anaphylactic reaction was detected by vascular permeability at the injected site 7 minutes after challenge, which was the best time for estimation. A novel method (AW method) for induction and detection of the anaphylactic reaction in mice was established using the abdominal wall as the challenge site. This method could detect the anaphylactic response in mice 1 to 3 weeks after sensitization. The increase in vascular permeability was completely inhibited by administration of diphenhydramine.

Different T helper cell types and antibody isotypes generated by saline and gene gun DNA immunization.

Several routes and methods of DNA immunization have been shown to generate Ab, Th cells, and CTL responses. However, few studies have directly compared the immune responses generated by different routes and methods of DNA immunization. Utilizing an influenza hemagglutinin (H1)-expressing plasmid, we compared the immune response produced by saline injection of DNA into skin or muscle, and gene gun immunization of skin or muscle. We found that saline-DNA immunization raised a predominantly Th1 response with mostly IgG2a anti-H1 Ab, while gene gun DNA immunization produced a predominantly Th2 response with mostly IgG1 anti-H1 Abs. These distinct types of immune responses were generated by the method, not the route, of DNA immunization. The initial immunization established the Th cell-type of the immune response. The Th cell-type did not change with further DNA immunizations by the same or the alternate method, or after a viral challenge. The ability to generate different Th types was not due to differences in the doses of DNA used in saline and gene gun DNA immunization. These findings have important implications for vaccine design and studies of the mechanism of Th cell differentiation.

Immunogenicity and safety of a liquid combination of DTP-PRP-T [corrected] vs lyophilized PRP-T reconstituted with DTP.

The immunogenicity and safety of a combined diphtheria, tetanus, pertussis and Haemophilus influenzae type b-tetanus conjugate vaccine (DTP-PRP-T) was compared to the same combination obtained by the reconstitution of H. influenzae type b-tetanus conjugate vaccine lyophilized (PRP-T) with liquid diphtheria-tetanus-pertussis vaccine (DTP). Two hundred and sixty-two healthy infants were randomized to receive a intramuscular injection of 0.5 ml of one of the above combination vaccines at 2, 4 and 6 months of age, and a subgroup of 134 infants received a booster dose at 12 months. Serum antibody levels to each vaccine component were measured at ages 2, 6, 7, 12 and 13 months. Systemic and local reactions were assessed during the first 3 days after each injection by diary cards distributed to the parents. After the third dose and booster administered at 12 months of age, significant equivalence between the groups was observed, and the geometric mean titer of anti H. influenzae type b capsular polysaccharide (Hib-CP) antibodies were 5.9 and 32.6 micrograms ml-1 for the liquid combination group and 5.8 and 19.4 for the lyophilized group, respectively. After the third dose, anti-Hib-PC antibody levels of > or = 1.0 microgram ml-1 and 0.15 microgram ml-1 were seen in 94% and 100%, respectively, of the liquid combination group and 90 and 99%, respectively of the lyophilized group. After the booster dose, levels of > or = 1.0 microgram ml-1 were observed in 100% and 93.5% of the liquid combination group and the lyophilized combination group, respectively. Systemic and local reactions to the vaccination were generally mild and did not differ significantly between the groups. We conclude that the liquid combination of DTP-PRP-T is safe and at least as immunogenic as the lyophilized preparation. This liquid preparation, like other combined vaccines may be helpful for planning vaccination programs with a reduced number of injections.

Anti-interleukin-4 inhibits immunoglobulin E production in a murine model of atopic asthma.

Immunoglobulin E (IgE) plays an important role in allergy, acting as an initiating factor and being involved in its persistence and exacerbations. As interleukin-4 (IL-4) is critical in IgE synthesis, we propose that treatment of mice with monoclonal anti-IL-4 (11B11) prior to active sensitization with ovalbumin will inhibit IgE synthesis, therefore arresting the allergic process at an early stage. Mice treated with 11B11 and sensitized with saline or ovalbumin had significantly less serum IgE than their respective control groups which were treated with saline (p < 0.05). This study suggests that anti-IL-4 may be a prophylactic agent in asthma and allergic disease.

Comparison of two disposable plastic skin test devices with the bifurcated needle for epicutaneous allergy testing.

BACKGROUND: The Greer DermaPIK and the Lincoln Diagnostics Duotip-Test are frequently used plastic, disposable, allergy skin testing devices. OBJECTIVES: To compare the prick method of using the bifurcated needle and DermaPIK with the Duotip-Test using both the scratch (rotation) and prick methods for sensitivity, precision, and level of discomfort. METHODS: Skin-testing was done with histamine and saline on the back in triplicate on 24 volunteers (mean age 32.8, seven males). Wheal and erythema were measured and a photograph was taken. Discomfort was rated on an analog scale. RESULTS: The bifurcated needle and the Duotip-Test prick technique had significantly smaller histamine wheal and erythema responses than either the DermaPIK prick or Duotip-Test scratch techniques (P < .05). The Duotip-Test scratch produced significantly larger wheals (mean 1.1 mm, P < .001) to saline than the other three methods. Erythema to saline by Duotip-Test scratch (mean 3.16 mm) was significantly larger than the bifurcated needle (mean 1.2 mm, P < .001) and Duotip-Test prick method (mean 1.6 mm, P < .01). There was no statistical difference in the histamine coefficient of variation among the four methods. The Duotip-Test scratch method was rated significantly higher in patient discomfort (mean 21.6, P < .05) than the bifurcated needle (mean 7.8). No differences in discomfort were noted between the other methods. CONCLUSIONS: The Duotip-Test scratch method had the largest mean wheal/erythema to histamine and the lowest CV. It had the most dermatographism and was more uncomfortable than the other methods. The other devices and methods were very similar in response to histamine and saline, and to precision and discomfort.

Wheal-and-flare responses to intradermally injected adenosine 5'-monophosphate, hypertonic saline, and histamine: comparison of atopic and nonatopic subjects.

Adenosine 5'-monophosphate (AMP) in increasing concentrations, and saline solutions of corresponding tonicity, were injected intradermally in seven atopic and seven normal subjects. Skin wheal-and-flare responses were elicited in a dose-dependent fashion in all subjects, and no difference was found between responses produced by AMP and responses produced by saline of corresponding tonicity. Also, no difference in response to AMP and saline was found between atopic and nonatopic subjects. We further investigated, in seven atopic subjects, whether the skin wheal-and-flare response to the single, highest dose of AMP, saline, and histamine could be inhibited by preadministration of 180 mg of terfenadine, a potent H1 antagonist. A significant inhibition of the wheal-and-flare response to histamine and no significant inhibition to AMP were found. There was a significant inhibition of the flare response caused by hypertonic saline but no inhibition of the wheal response. We interpret these findings as indicating that AMP does not specifically lead to mast cell degranulation in the skin and that there are functional differences between cutaneous and lung mast cells. The observation that terfenadine significantly inhibited the flare response to hypertonic saline suggests that this stimulus produced histamine release.