Notes: After immunization of Wistar rats with different concentrations of ovalbumin (OA) emulsified in complete H37Ra adjuvant, the optimal antigen dose for specific IgM, IgG and IgE production and for delayed-type hypersensitivity appeared to be the same. It is therefore concluded that in the rat these function tests can be combined, which decreases the number of experimental animals needed. For the determination of IgM, IgG and IgE antibodies to OA, the enzyme-linked immunosorbent assay (ELISA) was used, which proved to be a specific method. In addition, using a multi-channel photometer, it is shown that specific antibody concentrations can be readily quantified by micro-ELISA instead of using end-point titrations.

Notes: Levels of exhaled nitric oxide (eNO) are flow-dependent, and the choice of an optimal flow rate for off-line and on-line eNO measurement has raised much debate. Recently, a flow rate of 50 ml/s was recommended, but children younger than 5–6 years are not capable of stabilizing their expiratory flow at low flow rates. The power of off-line eNO values to discriminate between normal and atopic children was therefore evaluated at different exhalation flow rates. At flow rates of both 8.3 ml/s and of 350 ml/s, children (8–12 years) sensitive to house dust mite have two-fold higher eNO values (p 〈 0.001) as compared with children lacking such a sensitivity. The power of eNO to discriminate between normal and atopic subjects was similar at the two flow rates (no difference in AUC of receiver operation curves, p = 0.89). All children from 4.5 to 5 years of age (n = 29) could perform a single off-line exhalation manoeuvre at high (〉350 ml/s) but not at low (8.3 ml/s) flow rates. At high exhalation flow rate, eNO was 7.1 ± 2.4 (mean ± SD) median, 6.5 p.p.b. with a mean variation coefficient of 5.5%. Depending on their developmental level, about half of the younger children (35–46 months of age) could perform an off-line exhalation manoeuvre at high flow rate with good reproducibility (mean variation coefficient of 6.6%). It is concluded that an exhalation flow rate of 350 ml/s is feasible to determine off-line eNO-values in children from 3.5 years of age, and that this high flow rate does not compromise the power of eNO to detect allergic disease.

Notes: Background Exhaled nitric oxide (NO) has been proposed as novel a non-invasive marker of airway inflammation.Objective The level of exhaled NO was determined in a random sample of school children (7–12 years old) with the aim of investigating the relationship between exhaled NO and sensitization to common allergens.Results In the 450 children tested by skin prick tests (SPT), the prevalence of sensitization was 29.5% (overall), 21.9% (sensitization to indoor allergens), and 15.0% (sensitization to outdoor allergens). Regression analysis showed that levels of exhaled nitric oxide were closely associated with various measures of sensitization to aeroallergens. Sensitization to indoor allergens was associated with higher levels of exhaled NO (eNO) than sensitization to outdoor allergens when assessed by IgE but not when assessed by SPT. Children with reported wheeze in the past 12 months had much stronger associations between sensitization and eNO than children without wheeze.Conclusion We conclude that allergic sensitization is strongly associated with increased levels of exhaled NO, especially in children with wheeze.

Notes: Background Timothy grass (Phleum pratense) pollen allergens are an important cause of allergic symptoms. However, pollen grains are too large to penetrate the deeper airways. Grass pollen is known to release allergen-bearing starch granules (SG) upon contact with water. These granules can create an inhalable allergenic aerosol capable of triggering an early asthmatic response and are implicated in thunderstorm-associated asthma.Objective We studied the humoral (IgE) and bronchial lymph node cells reactivities to SG from timothy grass pollen in pollen-sensitized rats.Methods Brown–Norway rats were sensitized (day 0) and challenged (day 21) intratracheally with intact pollen and kept immunized by pollen intranasal instillation by 4 weeks intervals during 3 months. Blood and bronchial lymph nodes were collected 7 days after the last intranasal challenge. SG were purified from fresh timothy grass pollen using 5 μm mesh filters. To determine the humoral response (IgE) to SG, we developed an original ELISA inhibition test, based on competition between pollen allergens and purified SG. The cell-mediated response to SG in the bronchial lymph node cells was determined by measuring the uptake of [3H]thymidine in a proliferation assay.Results An antibody response to SG was induced, and purified SG were able to inhibit the IgE ELISA absorbance by 45%. Pollen extract and intact pollen gave inhibitions of 55% and 52%, respectively. A cell-mediated response was also found, as pollen extract, intact pollen and SG triggered proliferation of bronchial lymph node cells.Conclusions It was confirmed that timothy grass pollen contains allergen-loaded SG, which are released upon contact with water. These granules were shown to be recognized by pollen-sensitized rats sera and to trigger lymph node cell proliferation in these rats. These data provide new arguments supporting the implication of grass pollen SG in allergic asthma.

Notes: Background:  Exhaled nitric oxide (eNO) has shown good validity for the assessment of airway inflammation in asthmatic children. In large-scale epidemiological studies, this method would be preferred above airway challenge tests, because it is a quick and easy applicable tool.Objective:  In this study, we aimed to assess the discriminatory capacity of eNO, and prechallenge FEV1 for airway hyper-responsiveness (AHR) in 8–13-year old schoolchildren.Materials and methods:  Parents completed the ISAAC questionnaire, and children were tested for atopy, AHR to hypertonic (4.5%) saline (HS), and eNO. Diagnostic value was assessed by the area under the receiver operating curves (ROC), and calculation of positive and negative predicted values at different cut-off points for eNO and prechallenge FEV1.Results:  Areas under the ROC-curves of AHR were 0.65 for eNO and 0.62 for FEV1. Values increased to 0.71 and respectively 0.75 for a combined occurrence of AHR and current wheeze. Highest sensitivity and specificity were obtained at a cut-off value of 43 ppb for eNO and 103% predicted for FEV1. At these cut-off values, the positive predictive values for the presence of AHR in symptomatic children were respectively 83% (eNO) and 33% (FEV1), and negative predictive values in asymptomatic children were, respectively, 90 (eNO) and 80% (FEV1).Conclusion:  Exhaled nitric oxide is a valid screening tool for AHR to HS in children that present with current wheeze, and it outperforms FEV1 as a predictor of AHR.

Notes: Summary Liposome encapsulation of doxorubicin (DXR) has been shown to increase the therapeutic index of the drug in several animal systems. The prevention of peak plasma concentrations of free drug might be a major factor contributing to the beneficial effects resulting from liposome encapsulation. If so, the administration of DXR as a continuous infusion should also lead to an improved therapeutic index. In the present paper, the administration of liposome-encapsulated DXR is compared with the infusion of DXR with regard to their potential to preserve antitumor activity, enhance survival and reduce cardiomyo- and nephropathy in IgM immunocytoma-bearing Lou/M Wsl rats. Plasma concentrations of DXR were determined to correlate the biological results with pharmacokinetic parameters. Liposomes containing phosphatidylcholine, phosphatidylserine and cholesterol (extrusion-multilamellar vesicles) were used. Bolus injections of free DXR (free DXR) and DXR liposomes (lip-DXR) in a multiple-dose regimen were compared with 24-h infusions of the same cumulative doses of DXR (inf-DXR). The antitumor activity of inf-DXR equalled that of free DXR as well as that of lip-DXR at doses of 〉0.25 mg/kg. The overall survival of tumor-bearing animals treated with 2.0 mg/kg lip-DXR was significantly prolonged (P〈0.01) in comparison with that of animals treated with 2.0 mg/kg free DXR; however, treatment with 2.0 mg/kg inf-DXR did not induce a significant prolongation of survival. At a cumulative dose of 12 mg/kg, inf-DXR appeared to be as effective as lip-DXR in reducing the severity of cardiomyopathy induced by free DXR. However, for the reduction of nephropathy, only therapy with lip-DXR was effective Inf-DXR induced high nephropathy scores comparable with those obtained with free DXR. For the first 24 h after an injection of 2.0 mg/kg or after the start of a continuous infusion of 2.0 mg/kg given over 24 h, similar areas under the plasma concentration-time curves (AUC) were calculated for free DXR and inf-DXR. However, for lip-DXR a much higher value was calculated. The higher plasma levels of lip-DXR did not result in higher cardiac levels. After five daily doses of 2.0 mg/kg, a much lower DXR concentration was found in cardiac tissue after the administration of lip-DXR than after the administration of free DXR or inf-DXR. This suggests that an important parameter to be determined and correlated with biological results is the free (i.e. not bound to liposomes) circulating fraction of DXR in lip-DXR-injected animals. In conclusion, in the IgM immunocytoma system the administration of DXR as a continuous infusion was as effective as DXR encapsulated in liposomes in reducing cardiotoxicity while preserving the antitumor effect; this indicates that the avoidance of peak plasma levels is an important component of the mode of action of DXR-containing liposomes. However, if both antitumor efficacy and nephrotoxicity are taken into account, lip-DXR could be considered to be superior to inf-DXR.

Notes: Abstract  An IgM immunocytoma cell line sensitive to cis-diamminedichloroplatinum(II) (CDDP) and a subline with acquired resistance were grown in LOU/M rats. In a previous study with such rats that had been treated with a high dose of CDDP (10 mg/kg) the tumors did not show differences in cellular platinum content or DNA-adduct levels, either immediately after treatment or 24 h later. Recently, this high dose was found to overcome resistance. Therefore, the study was repeated with a 10-fold lower dose (1 mg/kg, i.v.). At 1 and 24 h after treatment, tumor and kidney tissue were assayed for cellular platinum (atomic absorption spectroscopy, AAS) and DNA platination (immunochemical detection of the four CDDP-DNA adducts). The results were compared with previous data. All tissues showed a linear response to dose with regard to platinum uptake as well as adduct formation, with no quantitative difference being seen between the tumors. Also the relative occurrence of the four adducts was very similar. Between 1 and 24 h, in tumors a substantial decrease occurred in both platinum content and adduct level; the kidneys showed little reduction, if any. At the lower CDDP dose a somewhat larger loss of platinum and removal of DNA adducts was observed for the resistant tumor, but these differences could be explained by “dilution”, as this tumor continues to grow after low-dose treatment (about 20% within 24 h). Since the strong difference observed between the tumors in sensitivity to CDDP cannot be attributed to differences in CDDP uptake, efficiency of adduct formation, or repair capability, other mechanisms are held responsible.

Notes: Summary Intralesional injection of BCG into an established line-10 hepatocellular carcinoma in the strain-2 guinea pig causes regression of the tumor and induction of line-10 immunity. We found that the animals were already protected for a second challenge with line-10 tumor cells 7 days after BCG treatment. We studied whether this early induction of immunity was correlated with the expression of MHC class II antigens on line-10 tumor cells and was correlated with an increased expression of MHC class II antigens on leukocytes in the primary tumor and in the regional lymph node (Ln. axillaris accessorius). The MHC class II antigens and the leukocyte subpopulations were measured with monoclonal antibodies and flow cytofluorometry. In the draining lymph node the number of nucleated cells increased about 10-fold during the first 5 days after intralesional injection of BCG. At this time the MHC class II antigen expression of these cells was increased from 21%–32% in the naive controls to 39%–53% in animals with BCG-treated tumors. This implies that the number of MHC-class-II-positive cells increased about 20-fold in the draining lymph node. Surprisingly, the increase in percentage of MHC-class-II-antigen-positive cells was mainly due to an increase of IgM-positive B cells from 8%–11% to 22%–41% and an increase of IgG-positive B cells from 7%–27% to 25%–44%. In the tumor, BCG treatment induced a small increase of MHC-class-II-antigen-positive cells from 11%–12% to 15%–20%. Probably this increase came not from tumor cells but mainly from a BCG-induced infiltration of mononuclear cells, as an increase of T cells from 14% to 20%, an increase of macrophages from 8% to 18%, and an increase of B cells from 0 to 6% was observed. We conclude that the potentiation of anti-(line-10 tumor cell) immunity correlated with a 20-fold increase of MHC-class-II-antigen-positive cells in the lymph nodes and a small increase in the number of MHC-class-II-antigen-positive tumor-infiltrating cells.

Notes: Summary Five cows bearing bovine ocular squamous cell carcinoma (BOSCC) were treated with low doses of recombinant human interleukin-2 (rhIL-2). A dose of 2500 U rhIL-2 was injected intralesionally and another 2500 U were injected into the subparotid regional lymph node once a day during a period of 5 consecutive days. This cycle of 5 days was repeated after an interval of 2 days. Total regression of the tumor was observed in three out of five animals. One cow showed tumor regression (〉 80%) accompanied by metastases to the regional lymph node that were observed from the fifth week after the beginning of the treatment. Growth of the tumor of the fifth animal was retarded after treatment. In vitro proliferation of peripheral blood lymphocytes was investigated in two animals and tumor-infiltrating lymphocytes in one animal during incubation in various rhIL-2 concentrations. Cytotoxic activity of both cell populations against P815, Yac-1 and BOSCC-derived cell lines increased during incubation with rhIL-2. Cultured BOSCC-infiltrating lymphocytes showed predominant killing of the BOSCC-derived autologous cell line after 4 weeks of culture. Preliminary phenotype analysis did not give conclusive results with respect to the types of cells responsible for killing.