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Notes: Abstract. Objective and Design: A comprehensive study to standardise interleukin (IL)-1〈alpha〉, –1〈beta〉, -6, -10, -12 and tumour necrosis factor alpha (TNF〈alpha〉) mRNA detection in murine peritoneal macrophages, using the reverse transcription polymerase chain reaction (RT-PCR) was carried out. ¶Subjects: Thioglycollate-elicited peritoneal exudate cells were harvested from female BALB/c mice and the adherent macrophage fraction isolated for use. ¶Treatment: Peritoneal macrophages (1 × 106) were incubated in the presence or absence of lipopolysaccharide (LPS; at a final concentration of 1 〈mu〉g/ml) for 0, 1, 2, 3, 4, 5 and 24 h. ¶Methods: Culture supernatants and cells were harvested at each time point, the secreted cytokine protein levels quantified by sandwich immunoassays and the cytokine mRNA levels assessed by RT-PCR. ¶Results: The IL-6 mRNA was not expressed in detectable amounts in the macrophages, unless challenged with LPS. TNF〈alpha〉, IL-1〈alpha〉, IL-1〈beta〉, IL-10 and IL-12 mRNAs were expressed in both stimulated and unstimulated macrophages. The levels of the PCR products and thus mRNAs of all the cytokines increased with LPS stimulation, maximal levels being achieved 3 to 5 h post stimulation. ¶Conclusions: RT-PCR produced consistent results, indicating that this technique could be used to investigate the effect of biological mediators and novel pharmacological agents on cytokine mRNA levels in macrophages.

Notes: Abstract Many aquatic species, including the estuarine fish Fundulus heteroclitus (mummichogs), adapt to local environmental conditions. We conducted studies to evaluate whether highly exposed populations of mummichogs adapt to toxic environmental contaminants. These fish populations are indigenous to an urban estuary contaminated with persistent and bioaccumulative contaminants (dioxin-like compounds, or DLCs) that are particularly toxic to the early development of fish. We conducted laboratory challenge experiments to compare mummichog embryos and larvae from reference sites and this highly contaminated site [New Bedford Harbor (NBH), Massachusetts, USA] for their sensitivity to DLCs. While there was variation in DLC-responsiveness within each group, fish from NBH were profoundly less sensitive to DLCs than reference fish. Specifically, concentrations of DLCs similar to those measured in NBH-collected mummichog eggs were lethal to reference embryos. Further, DLC-responsiveness was inherited and independent of maternal contaminant contributions. These findings are consistent with the conclusion that DLC contamination in NBH has contributed to the selection of fish that are resistant to the short-term toxic effects of these environmental-contaminant exposures. This adaptation may be a critical mechanism by which fish populations persist in this highly contaminated site. Further evaluation of this ecosystem may provide important information concerning the direct and indirect consequences of this “unnatural” selection.

Notes: Summary Patch-clamp recording from the apical surface of the epithelium of frog lens reveals a cation-selective channel after pressure (about ±30 mm Hg) is applied to the pipette. The open state of this channel has a conductance of some 50 pS near the resting potential (−56.1±2.3 mV) when 107mm NaCl and 10 HEPES (pH 7.3) is outside the channel. The probability of the channel being open depends strongly on pressure but the current-voltage relation of the open state does not. With minimal Ca2+ (55±2 μm) outside the channel, the current-voltage relation is nonlinear even in symmetrical salt solutions, allowing more current to flow into the cell than out. The channel, in minimal Ca2+ solution, is selective among the monovalent cations in the following sequence K+〉Rb+〉Cs+〉Na+〉Li+. The conductance depends monotonically on the mole fraction of K+ when the other ion present is Li+ or Na+. The single-channel current is a saturating function of [K+] when K+ is the permeant ion, for [K+]≤214mm. When [Ca2+]=2mm, the currentvoltage relation is linearized and the channel cannot distinguish Na+ and K+.

Notes: Summary Since cyclosporin A (CsA) is an immuno-suppressive agent, its beneficial effect in psoriasis suggests that immune cells may play a role in the pathogenesis and resolution of psoriasis. To determine early effects of CsA in psoriasis, we quantitated immune cells using double immunofluorescence microscopy on biopsy specimens obtained prior to therapy and after 3,7, and 14 days of CsA therapy. CsA therapy resulted in significant reductions in the absolute number of immune cells (including T cells, monocytes/macrophages, and antigen presenting cells) contained within psoriatic skin. The effect was rapid, with over one-half of the reduction in the density of HLe1+ (human leukocyte antigen-1 positive or bone marrow derived) cells, including T cells, activated T cells, monocytes, and Langerhans cells (LCs), occurring within 3 days. Despite the overall reduction in the numbers of immunocytes in the skin, the proportion of T cells, Langerhans cells, and monocytes in relation to the total number of immune cells was unchanged with therapy, reflecting equally proportional losses of each subtype. Dermal CD1+DR+ cells (putative Langerhans cells), which are not found in normal skin but are present in lesional psoriasis skin, were virtually cleared from the papillary dermis after CsA therapy. Although absolute numbers of epidermal Langerhans cells, defined as cells expressing both CD1 (T6) and DR molecules (CD1+DR+), were also reduced after CsA, epidermal non-Langerhans CD1-DR+ cells (macrophages, activated T cells, DR- keratinocytes) demonstrated a proportionally greater decrease, with the ratio of CD1+DR+ Langerhans cells/non-Langerhans CD1-DR+ epidermal cells changing from a mean of 0.82 at baseline to 1.92 at day 14. Thus, early in the course of therapy, CsA appears to be effective at clearing CD1-DR+ cells while leaving LC relatively intact in the epidermis.

Notes: Abstract The acute safety of the alternative chlorofluorocarbon-free (CFC-free) propellant HFA-134a from a pressurized metered-dose inhaler (MDI) was assessed in 12 healthy male subjects according to a double-blind, randomized, crossover design. On each of three consecutive days, cumulative doses of 1, 2, 4, 8 and 16 inhalations were administered 30 min apart from one of three MDIs. The three MDIs contained either the HFA-134a CFC-free system without drug (HFA-Placebo), the CFC-free system with salbutamol sulphate (HFA-Salbutamol), or a conventional CFC propellant mixture without drug (CFC-Placebo). Pulmonary function (FEV1, FEF25–75%), cardiovascular performance (heart rate and blood pressure), objective tremor measurements and serum potassium were measured after each incremental dose. Similar responses for pulmonary function, cardiovascular performance, tremor and serum potassium were observed between the HFA-Placebo and CFC-Placebo groups. No statistically significant difference was seen in change from baseline of any parameter between the two propellant systems. The administration of HFA-Salbutamol produced statistically significant dose-related increases in heart rate, systolic blood pressure and tremor and a significant dose-related decrease in serum potassium; these responses were expected based on cumulative doses of active drug. Blood samples for HFA-134a analysis were collected to measure systemic absorption of this propellant. Levels of HFA-134a between 200 and 700 ng · ml−1 were detected in all subjects given the CFC-free system. This study shows that acute inhalation of HFA-134a in a CFC-free system is as safe as a CFC propellant system. Salbutamol sulphate in the CFC-free system can be delivered in a dose-linear fashion, without any noticeable change in the safety profile of active drug.

Notes: Synopsis The murine local lymph node assay (LLNA) can be used to determine the relative skin sensitizing potency of chemicals via interpolation of the quantitative dose response data generated. Using this approach we have demonstrated previously that the vehicle matrix in which a chemical allergen is encountered on the skin can have a significant influence on sensitizing potency. Estimates of relative potency are calculated from LLNA dose responses as a function of the mathematically derived EC3 value, this being the concentration estimated to induce a stimulation index (SI) of 3. To investigate further the influence of application vehicle on sensitizing potency, the LLNA has been used to examine the activity of four recognized human contact allergens: isoeugenol and cinnamic aldehyde, two fragrance chemicals; 3-dimethylaminopropylamine (a sensitizing impurity of cocamidopropyl betaine, a surfactant used in shower gel) and dibromodicyanobutane (the sensitizing component of Euxyl K 400, a preservative used in cosmetics). The four chemicals were applied in each of seven different vehicles (acetone: olive oil [4 : 1]; dimethylsulphoxide; methylethylketone; dimethyl formamide; propylene glycol; and both 50 : 50 and 90 : 10 mixtures of ethanol and water). It was found that the vehicle in which a chemical is presented to the epidermis can have a marked effect on sensitizing activity. EC3 values ranged from 0.9 to 4.9% for isoeugenol, from 0.5 to 1.7% for cinnamic aldehyde, from 1.7 to 〉 10% for dimethylaminopropylamine and from 0.4 to 6.4% for dibromodicyanobutane. These data confirm that the vehicle in which a chemical is encountered on the skin has an important influence on the relative skin sensitizing potency of chemicals and may have a significant impact on the acquisition of allergic contact dermatitis. The data also demonstrate the utility of the LLNA as a method for the prediction of these effects and thus for the development of more accurate risk assessments. Résumé Le test local des ganglions lymphatiques murins (LLNA) peut être utilisé pour déterminer le potentiel relatif de sensibilisation de la peau de produits chimiques, par interpolation des données quantitatives de dose/réponse obtenues. En utilisant cette approche, nous avions démontré précédemment que la matrice vecteur par laquelle un allergène chimique est mis en contact avec la peau peut avoir une influence significative sur le potentiel de sensibilisation. Des estimations d’activité relative sont calculées à partir des doses/réponses de LLNA en fonction de la valeur EC3 dérivée mathématiquement, celle-ci étant la concentration estimée comme induisant un indice de stimulation (IS) de 3. Pour examiner plus avant l’influence du vecteur d’application sur l’activité de sensibilisation, on a utilisé le LLNA pour déterminer l’activité de quatre allergènes de contact humains reconnus: isoeugénol et aldéhyde cinnamique, deux substances chimiques de parfumerie; la 3-diméthylaminopropylamine (une impureté sensibilisante de la cocamidopropyl bétaïne, un tensioactif utilisé dans les gels douches) et le dibromodicyanobutane (le composant sensibilisant de Euxyl K 400, un conservateur utilisé dans les cosmétiques). Les quatre produits chimiques ont été appliqués dans chacun de sept vecteurs différents (acétone: huile d’olive [4: 1]; diméthylsulfoxyde; méthyléthylcétone; diméthylformamide; propy- lène glycol; et deux mélanges 50: 50 et 90: 10 d’éthanol et d’eau). On observe que le vecteur dans lequel le produit chimique est présentéà l’épiderme peut avoir un effet marqué sur l’activité sensibilisatrice. Les valeurs EC3 vont de 0,9 à 4,9 % pour l’isoeugénol, de 0,5 à 1,7 % pour l’aldéhyde cinnamique, de 1,7 à 〉 10 % pour la diméthylaminopropylamine et de 0,4 à 6,4 % pour le dibromodicyanobutane. Ces données confirment que le vecteur dans lequel un produit chimique est mis en contact avec la peau a une influence importante sur le potentiel relatif de sensibilisation de la peau des produits chimiques, et peut avoir un impact significatif sur l’apparition de dermatite allergique par contact. Les données démontrent aussi l’utilité du LLNA comme méthode de prévision de ces effets et donc pour le développement d’évaluations plus précises des risques.