Notes: The nontoxic B subunit of cholera toxin (CTB) has been used as an adjuvant in experimental systems of mucosal vaccination. However, the mechanisms behind its adjuvant effects remain unclear. Here, we have used an ex vivo system to elucidate these mechanisms in antigen-specific T cells. Using splenocytes from keyhole limpet haemocyanin (KLH)-immunized mice, initial experiments showed that recombinant CTB (rCTB) did not affect the KLH-specific proliferation of splenocytes isolated from mice immunized 2 weeks earlier. However, rCTB strongly enhanced the KLH-specific proliferation of splenocytes from mice immunized with KLH 4 weeks prior. This adjuvant effect was dose-dependent, with maximum at 30–300 ng/ml rCTB. At higher doses of CTB this effect declined because of the induction of apoptosis. Using antibody depletion and coculture systems, we show that rCTB directly costimulates KLH-specific CD4+ and CD8+ T-cell proliferation but not B cells. Enzyme-linked immunospot (ELISPOT) assays revealed that rCTB also enhanced the KLH-specific CD4+ T-cell-mediated production of interleukin-2 (IL-2), IL-4 and interferon-γ(IFN-γ) by four to fivefold. Characterizing the adjuvant effect of rCTB in vivo confirmed the results above, i.e. rCTB mediated a twofold increase in the ex vivo T-cell response when used as a classical adjuvant in a secondary, but not in a primary KLH-immunization regimen. Together these data show that rCTB can act as a strong adjuvant, by directly costimulating antigen-primed CD4+ and CD8+ T cell in a dose-dependent manner. This new insight might be valuable in the future rational design of bacterial toxin-based vaccines.

Notes: When conjugated to various proteins, the nontoxic B-chain of cholera toxin (CTB) significantly increases the ability of these proteins to induce immunological tolerance after oral administration. Here, we investigated if a nonconjugated form of CTB enhances the induction of immune tolerance after oral insulin administration. Induction of immunological tolerance was studied after oral administration of insulin preparations in three mouse models; an insulin/ovalbumin coimmunization model, a model of virus-induced diabetes in transgenic RIP-LCMV-NP mice and in nonobese diabetic (NOD) mice serving as a model of spontaneous diabetes. In the immunization model, we demonstrate that mixing with CTB increases the tolerogenic potential of insulin, approximately 10 fold. Titration of the CTB concentration in this system revealed that an insulin : CTB ratio of 100 : 1 was optimal for the induction of bystander suppression. Further studies revealed that this insulin : CTB ratio also was optimal for the prevention of diabetes in a virus-induced, transgenic diabetes model. In addition, the administration of this optimal insulin–CTB preparation significantly prevented the onset of diabetes in old NOD mice with established islet infiltration. The data presented here demonstrate that CTB, even in its unconjugated form, functions as a mucosal adjuvant, increasing the specific tolerogenic effect of oral insulin.

Notes: The changes in weed:crop competition as affected by the interaction of slurry distribution pattern with crop sowing pattern was examined, using the parameters dry matter (DM) production, nitrogen (N) uptake and recovery of applied N in spring wheat and weeds. Pig slurry enriched with isotopic nitrogen (15N) was applied either fully incorporated or in concentrated bands. The two slurry distribution patterns were combined with either crop density or distance between the slurry band and seed row. Spring wheat and weeds were sampled separately seven times during the season. A high and fast crop recovery of applied N coincident with a low recovery in weeds may be obtained by at least three management elements: sowing the crop with a high seed density, sowing the crop row close to the band-applied slurry N, and early sowing. The effects on crop DM, N-uptake and 15N recovery of a high crop density and a narrow distance between the slurry band and crop row appear clearly at the beginning of the spring wheat elongation phase. For weeds the effects were most pronounced for 15N recovery, and using DM as the only response variable may cause insignificant results. However, methods for N application may be part of a strategy controlling weeds, but to obtain satisfactory weed control the N management needs to be supplemented with chemical or mechanical weed management.

Notes: Under field conditions the efficacy of glufosinate-ammonium on Galium aparine L. is quite often variable. Experiments under controlled conditions with G. aparine and the more susceptible species Brassica rapa L. showed that especially low relative humidity (r.h.) and low light intensity reduced the accumulation of ammonia in the plants and glufosinate-ammonium performance. As the light intensity under field conditions always exceeds the intensities applied in the experiments this parameter may not be of practical importance. In contrast, low r.h. often occurs under field conditions and could explain the variable G. aparine control. A retention study showed that retention on G. aparine was significantly higher than on B. rapa but ammonia accumulation in the plants indicated that glufosinate-ammonium uptake by G. aparine was lower than that by B. rapa. Glufosinate-ammonium performance was improved on both species by adding ammonium sulphate to the spray solution; however, the increase in efficacy by ammonium sulphate was two times higher for G. aparine than for B. rapa. Ammonia accumulation in untreated plant parts after glufosinate-ammonium application indicated some translocation of glufosinate-ammonium in B. rapa but none in G. aparine.

Notes: Summary The experiments were carried out in the two spring barley fields of the organic six-course cattle:crop rotation at Foulumgaard, Denmark. The weed density was 300 and 1800 plants m−2 respectively. Pig slurry was applied by hand in microplots by four methods: broadcasting followed by incorporation, or injected in bands to depths of 5, 10 or 15 cm. Spring barley and weeds were sampled separately six times during the tillering and elongation phase of the spring barley. The effect of application method on dry-matter (d.m.) production, nitrogen uptake and recovery of applied nitrogen in the spring barley and the weeds is reported. Slurry banding halved the weed d.m. and weed N uptake compared with broadcasting, irrespective of weed density. Weeds recovered up to 12% of the applied nitrogen, which made them a significant competitor when the slurry was broadcast and incorporated. Banding by direct injection reduced the slurry:soil contact and the weed:crop competition balance for applied nitrogen moved in favour of the crop. Thus, the crop recovery of applied nitrogen at the end of the sampling period was increased from approximately 45% for broadcast and incorporated to approximately 50% for injected slurry, and coincidental weed recovery was reduced to a maximum of 5%. As the nitrogen supply normally affects plant d.m. production, banding of slurry might well improve crop competitiveness and its tolerance to mechanical weed control.

Notes: Background Several studies in patient populations have reported a positive association between alcohol consumption and serum total IgE. Furthermore, we have previously reported a positive association between alcohol consumption and the prevalence of skin prick test (SPT positivity) to inhalant allergens in a population-based cross-sectional study.Objective To investigate the relationship of alcohol consumption to levels of serum total IgE and the development of IgE sensitization to inhalant allergens.Methods In 1990, self-reported consumption of alcohol, serum total IgE, SPT positivity and specific IgE positivity to inhalant allergens were assessed in 1112 subjects, aged 15–69 years, participating in a population-based cross-sectional study in Copenhagen, Denmark. In 1998, they were invited to a follow-up and 734 were re-examined (participation rate 69.0%). Adjustment for potential confounders was performed by using multivariable regression analyses.Results In non-atopic (specific IgE negative) subjects there was a positive association between alcohol consumption and the concentration of total IgE (P = 0.001). During the follow-up period, 45 and 33 subjects developed SPT positivity and specific IgE positivity, respectively. There was no significant association between alcohol consumption and the development of SPT positivity or specific IgE positivity. However, the risk of developing SPT positivity tended to increase with increasing consumption of alcohol (P = 0.055).Conclusions This epidemiological study confirms that alcohol consumption has an influence on levels of serum total IgE. A significant association between alcohol consumption and the development of IgE sensitization was not established. However, there seems to be a lack epidemiological data on this issue.

Notes: Background There is evidence that antenatal factors play a role in the development of atopic dermatitis (AD). However, little is known about the effects of maternal lifestyle factors during pregnancy on the risk of AD in the offspring.Objective To investigate the effect of alcohol consumption during pregnancy on the incidence of AD in the offspring.Methods A total of 24 341 mother–child pairs enrolled in the Danish National Birth Cohort were followed prospectively. Information about alcohol consumption was obtained by interview at 12 and 30 weeks of gestation. Information about symptoms, time of onset, and doctor's diagnosis of AD in the offspring was obtained by interview at 18 months of age. The effect of alcohol consumption during pregnancy on the incidence of AD was analysed by Cox regression allowing for different effects of alcohol before (early infancy) and after 2 months (60 days) of age.Results Alcohol during pregnancy was associated with a significant and dose-dependent increased risk of AD in early infancy. This effect was mainly seen in high-risk infants (two parents with allergic disease). Thus, the highest risk of AD in early infancy was seen in high-risk infants of mothers who consumed four or more drinks per week at 30 weeks of gestation (adjusted relative risk 4.2, 95% confidence interval 1.7–10.1). There was no effect of alcohol during pregnancy beyond early infancy.Conclusions The results suggest that alcohol during pregnancy increases the risk of early-onset AD in predisposed infants.

Notes: The objective of this study was to detail the topographical and zonal distribution of the cell adhesion molecule CD44 in normal and osteoarthritic cartilage.〈section xml:id="abs1-2"〉〈title type="main"〉Methods and results:Immunohistochemistry utilizing well characterized anti-CD44 antibodies (clones A3D8, Bric 235, 2C5) was performed on cryostat and paraffin sections of human articular cartilage from macroscopically normal (n = 18) and osteoarthritic (n = 11) femoral heads. Samples for cryostat sections were obtained from 12 topographically different sites. Sections were divided into zones (superficial, middle, deep) and the CD44 staining scored. Chondrocytes in normal articular cartilage and cartilage from osteoarthritic femoral heads stained positive for CD44 in both cryostat and paraffin sections. Normal cartilage showed a significant decrease in CD44 staining in the deep zone as compared to the superficial zone (P 〈 0.05). However, cryostat sections of residual cartilage from osteoarthritic femoral heads showed increased CD44 staining in the deep zone as compared to normal articular cartilage. The CD44 staining showed no topographical variation in either the normal cartilage or the osteoarthritic residual cartilage.〈section xml:id="abs1-3"〉〈title type="main"〉Conclusions:CD44 expression displays a distinct zonal variation in normal articular cartilage which is lost in osteoarthritic cartilage due to an up-regulated expression in the deep zone. CD44 expression does not exhibit topographical variation.

Notes: Background SDZ ASM 981 is a selective inhibitor of the production of pro-inflammatory cytokines from T cells and mast cells in vitro. It is the first ascomycin macrolactam derivative under development for the treatment of inflammatory skin diseases. Objectives This study was designed to determine the safety and efficacy of SDZ ASM 981 cream at concentrations of 0·05%, 0·2%, 0·6% and 1·0% in the treatment of patients with atopic dermatitis and to select the concentration to be used in phase III studies. Methods This was a double-blind, randomized, parallel-group, multicentre dose-finding study. A total of 260 patients were randomly assigned to treatment with SDZ ASM 981 cream at concentrations of 0·05%, 0·2%, 0·6%, or 1·0%, matching vehicle cream, or the internal control 0·1% betamethasone-17-valerate cream (BMV). Treatment was given twice daily for up to 3 weeks. Results A clear dose–response relationship for SDZ ASM 981 was evident, with 0·2%, 0·6% and 1·0% SDZ ASM 981 creams all being significantly more effective than vehicle (P = 0·041, 0·001 and 0·008, respectively) in terms of baseline to end-point changes in the Eczema Area Severity Index (EASI) and pruritus score. The 1·0% cream was the most effective SDZ ASM 981 concentration. BMV was more effective than the SDZ ASM 981 creams tested in this study. It appears that the efficacy plateau was not reached with the SDZ ASM 981 creams within 3 weeks treatment. SDZ ASM 981 was well tolerated. Burning or a feeling of warmth were the only adverse events reported more frequently in the 0·6% and 1·0% SDZ ASM 981 treatment groups than in the vehicle treatment group (42·9%, 48·9% and 34·9%, respectively). Few systemic adverse events were reported during the study (headache was the most frequent systemic event reported by 15 of 252 patients) and none was considered to be related to treatment. The local tolerability profile of the 1·0% cream was similar to that of the lower concentrations. Conclusions 1·0% SDZ ASM 981 cream, which was shown to be safe, well tolerated and the most effective concentration in this study, was selected as the concentration to be further developed in phase III studies.

Notes: Background  Head and neck dermatitis (HND) is a variant of atopic dermatitis often seen in young adults. A hypersensitivity to Malassezi antigens is considered to be of pathogenic importance. Previous mostly uncontrolled studies have shown that oral antimycotics might be of use in this condition.Objective  To evaluate the efficacy of itraconazole in the treatment of HND in a randomized, double-blind, placebo-controlled trial.Patients  Adult patients with HND were included. Systemic steroids and oral/topical antimycotics were avoided 1 and 2 months prior to the trial. Topical steroids were not allowed in the head and neck area within 2 weeks. Patients in generally good health were included and female patients had to have had a negative urine pregnancy test. The patients signed an informed consent.Study design  The study included a 7-day treatment period and a follow-up period of 105 days. Control visits were carried out on days 3, 7 and 14 and after 15 weeks.Methods  The SCORAD index (one for the head and neck area and one for the remaining surface area) and global evaluations by patient and investigator were used for clinical evaluation at each visit. Prick tests with Malassezia antigens and Candida albicans antigen were carried out at the start of the trial and included positive and negative controls. The patients were randomized into three groups, which were treated with 400 mg itraconazole daily, 200 mg itraconazole daily or placebo, respectively, for 7 days.Results  The number of patients included was 53: 18 had 200 mg itraconazole daily, 17 had 400 mg itraconazole daily and 18 placebo. At days 7 and 14, significant improvement was seen in the SCORAD of the head and neck area for the groups given 400 mg itraconazole daily (P = 0.0385 and P = 0.0134), and 200 mg daily (P = 0.0104 and P = 0.0006). Patients in the placebo group improved slightly (P = 0.0785). At day 14, comparison of improvement of SCORAD of the head and neck area between all three groups showed a significant difference in favour of the 200 mg itraconazole group compared to the placebo group (P = 0.0318). The prick test was positive for Pityrosporum ovale in 37% and negative for C. albicans in all patients.Conclusions  One week of treatment with 200 or 400 mg itraconazole as a single treatment has a significant effect on the head and neck area. Compared to placebo there was a significant improvement in SCORAD of the head and neck area in favour of the 200 mg itraconazole group after 14 days. The important observation seems to be that antifungal systemic treatment has a significant SCORAD reduction of atopic dermatitis, irrespective of the presence of allergy.