Effect of challenge method on sensitivity, reactivity, and maximal response to methacholine.

BACKGROUND: Recent data suggest that the tidal breathing method may produce methacholine provocation concentration that caused a decrease in forced expiratory volume in 1 second of 20% (PC20) values significantly lower than the dosimeter method; however, the effect of the challenge method on the shape of the concentration-response curve has not been investigated. OBJECTIVE: To determine the effect of the challenge method on sensitivity, reactivity, and maximal response to methacholine. METHODS: We measured airway responsiveness to methacholine using dosimeter and tidal breathing methods in 30 individuals with suspected asthma. Concentration-response curves were characterized by their PC20 (sensitivity), slope (reactivity), and, if possible, level of plateau. RESULTS: Dosimeter PC20 values were significantly higher than tidal breathing values (geometric mean, 8.9 and 5.2 mg/mL, respectively); the mean difference in PC20 values obtained using each method was 0.78 doubling concentrations (P = .01). The mean slopes were 22.7%/log mg/mL using the tidal breathing method and 24.9%/log mg/mL using the dosimeter method; the mean difference in the slopes obtained using each method was -2.17%/log mg/mL (P = .18). In 10 individuals who showed a plateau with the 2 methacholine challenge tests, the mean level of plateau was 19.8% using the tidal breathing method and 19.5% using the dosimeter method; the mean difference in the plateau values obtained with each method was 0.3% (P = .87). CONCLUSIONS: Although the tidal breathing method produces methacholine PC20 values significantly lower than the dosimeter method, both methods provide similar values for slope and level of plateau. These results suggest that the technical factors that affect methacholine sensitivity and the shape of the curve are different.     

Cohesin component dynamics during meiotic prophase I in mammalian oocytes

Cohesins are chromosomal proteins that form complexes involved in the maintenance of sister chromatid cohesion during division of somatic and germ cells. Three meiosis-specific cohesin subunits have been reported in mammals, REC8, STAG3 and SMC1 beta; their expression in mouse spermatocytes has also been described. Here we studied the localization of different meiotic and mitotic cohesin components during prophase I in human and murine female germ cells. In normal and atretic human fetal oocytes, from leptotene to diplotene stages, REC8 and STAG3 colocalize in fibers. In murine oocytes, SMC1beta, SMC3 and STAG3 are localized along fibers that correspond first to the chromosome axis and then to the synaptonemal complex in pachytene. Mitotic cohesin subunit RAD21 is also found in fibers that decorate the SC during prophase I in mouse oocytes, suggesting a role for this cohesin in mammalian sister chromatid cohesion in female meiosis. We observed that, unlike human oocytes, murine synaptonemal complex protein SYCP3 localizes to nucleoli throughout prophase I stages, and centromeres cluster in discrete locations from leptotene to dictyate. At difference from meiosis in male mice, the cohesin axis is progressively lost during the first week after birth in females with a parallel destruction of the axial elements at dictyate arrest, demonstrating sexual dimorphism in sister chromatid cohesion in meiosis.     

Migration of human blood dendritic cells across endothelial cell monolayers: adhesion molecules and chemokines involved in subset-specific transmigration

Distinct subsets of dendritic cells (DCs) are present in blood, probably "en route" to different tissues. We have investigated the chemokines and adhesion molecules involved in the migration of myeloid (CD11c(+)) and plasmacytoid (CD123(+)) human peripheral blood DCs across vascular endothelium. Among blood DCs, the CD11c(+) subset vigorously migrated across endothelium in the absence of any chemotactic stimuli, whereas spontaneous migration of CD123(+) DCs was limited. In bare cell migration assays, myeloid DCs responded with great potency to several inflammatory and homeostatic chemokines, whereas plasmacytoid DCs responded poorly to all chemokines tested. In contrast, the presence of endothelium greatly favored transmigration of plasmacytoid DCs in response to CXCL12 (stromal cell-derived factor-1) and CCL5 (regulated on activation, normal T expressed and secreted). Myeloid DCs exhibited a very potent transendothelial migration in response to CXCL12, CCL5, and CCL2 (monocyte chemoattractant protein-1). Furthermore, we explored whether blood DCs acutely switch their pattern of migration to the lymph node-derived chemokine CCL21 (secondary lymphoid-tissue chemokine) in response to microbial stimuli [viral double-stranded (ds)RNA or bacterial CpG-DNA]. A synthetic dsRNA rapidly enhanced the response of CD11c(+) DCs to CCL21, whereas a longer stimulation with CpG-DNA was needed to trigger CD123(+) DCs responsive to CCL21. Use of blocking monoclonal antibodies to adhesion molecules revealed that both DC subsets used platelet endothelial cell adhesion molecule-1 to move across activated endothelium. CD123(+) DCs required beta(2) and beta(1) integrins to transmigrate, whereas CD11c(+) DCs may use integrin-independent mechanisms to migrate across activated endothelium.     

Gene therapy of cancer based on interleukin 12

Tumor formation and growth depends mainly on the inability of the organism to elicit a potent immune response, and on the formation of new blood vessels that enable tumor nutrition. Interleukin-12 (IL-12) therapy can target both processes. And IL-12-based gene therapy may restrict IL-12 production to the relevant site in order to obtain enhanced antitumor activity and reduced toxicity. In the clinical setting, IL-12 gene transfer can be used either to improve the pharmacokinetic/pharmacodynamic profile of the cytokine, to transduce dendritic cells or to enhance the efficiency of antitumor vaccination. It can also synergize with other procedures involving the simultaneous transfer of other transgenes or non-gene based strategies. The strong anti-tumoral power shown in many different animal models has not been found in early clinical trials in which cancer patients were treated by peritumoral injections of autologous fibroblasts producing IL-12, intratumoral injections of an adenoviral vector encoding human IL-12 genes, or intratumoral injection of autologous dendritic cells transduced ex vivo with this same adenoviral vector. However, these trials have set the proof-of-concept that local production of IL-12 inside a tumor can stimulate tumor infiltration by effector immune cells and that in some cases it is followed by tumor regression. From the many questions that arise after these disappointing results the most relevant concerns the duration and intensity of transgene expression and the capability to monitor this topics in vivo. New vectors that might achieve regulated, long-term production of this cytokine might have better results and merit clinical testing.     

Exhaled nitric oxide levels and airway responsiveness to adenosine 5'-monophosphate in subjects with nasal polyposis

BACKGROUND:It is widely appreciated that asthma is an inflammatory disease of the airways associated with airway hyperresponsiveness, and that nasal polyposis and asthma are related diseases. The objective of this study was to determine differences in exhaled nitric oxide (ENO) levels and airway responsiveness to adenosine 5'-monophosphate (AMP) between nonasthmatic patients with nasal polyposis and healthy controls. METHODS: Twenty patients without asthma with nasal polyposis and 16 healthy control subjects were enrolled in the study. Participants were challenged with increasing concentrations of AMP and methacholine. ENO was measured with the single-exhalation method. RESULTS: Bronchoconstriction in response to AMP was detected in 7 (35%) subjects with nasal polyposis. The geometric mean (95% CI) of ENO for subjects with nasal polyposis was 33.1 parts per billion (ppb) (24.0-45.7 ppb) compared with 12.3 ppb (8.5-18.2 ppb) for the healthy controls (p = 0.0002). ENO values were significantly higher in atopic than in nonatopic subjects with nasal polyposis [51.3 ppb (32.3-83.2 ppb) vs. 24.5 ppb (16.2-37.1 ppb), p = 0.02]. Nonatopic subjects with nasal polyposis also had higher concentrations of ENO than healthy control subjects (p = 0.016). CONCLUSIONS: Inhaled AMP causes airway narrowing in a significantly higher proportion of nonasthmatic subjects with nasal polyposis than in healthy controls. Furthermore, increased concentrations of ENO are detected in atopic and nonatopic subjects with nasal polyposis. These results suggest that bronchial inflammation is present in nonasthmatic subjects with nasal polyposis.     

Synergistic effect of azithromycin on the phagocytic killing ofStaphylococcus aureus by human polymorphonuclear leukocytes

Many macrolides have been shown to affect the interaction between bacteria and various immune defense mechanisms, such as chemotaxis, accumulation, and bioactivity within phagocytic cells. The interaction of azithromycin with human polymorphonuclear leukocytes (PMNs) was studied in vitro and compared with the interactions between other macrolides and PMNs. The opsonophagocytic killing ofStaphylococcus aureus was synergistically enhanced by azithromycin at concentrations below and above the minimal inhibitory concentration, with a reduction of up to 2.82 log₁₀ cfu/ml with 2 mg/ml of azithromycin. Other macrolides were effective only at subinhibitory concentrations. The beneficial azithromycin-leukocyte interaction may explain azithromycin's efficacy against intracellular pathogens.     

Localization of non-specific X-linked mental retardation gene (MRX73) to Xp22.2

Clinical and molecular studies are reported on a family (MRX73) of five males with non-specific X-linked mental retardation (XLMR). A total of 33 microsatellite and RFLP markers was typed. The gene for this XLMR condition was been linked to DXS1195, with a lod score of 2.36 at theta = 0. The haplotype and multipoint linkage analyses suggest localization of the MRX73 locus to an interval of 2 cM defined by markers DXS8019 and DXS365, in Xp22.2. This interval contains the gene of Coffin-Lowry syndrome (RSK2), where a missense mutation has been associated with a form of non-specific mental retardation. Therefore, a search for RSK2 mutations was performed in the MRX73 family, but no causal mutation was found. We hypothesize that another unidentified XLMR gene is located near RSK2.     

Analysis of ABCA4 in mixed Spanish families segregating different retinal dystrophies

Genotype-phenotype correlations highlighted the function of ABCA4 in retinitis pigmentosa (RP),cone-rod dystrophy (CRD) and Stargardt/Fundus Flavimaculatus disease (STGD/FFM). Initial screening of ABCA4 variants showed a correlation between the type of mutation and the severity of the disease. In the present study we have undertaken mutational and haplotype analysis of ABCA4 in three mixed pedigrees segregating different retinal dystrophies. In family I, we have shown cosegregation of different ABCA4 alleles with CRD (homozygosity for L1940P) and three subtypes of STGD/FFM. The first, a mild form, consisting on fundus flavimaculatus-like distribution of flecks, but good visual acuity and absence of dark choroid, was found to cosegregate with alleles R1097C and F553L; the second, a conventional Stargardt phenotype was associated to alleles L1940P/R1097C and the third, displaying severely reduced visual acuity and dark choroid (named FFM), was associated to L1940P/F553L. In family II, segregating STGD and RP phenotypes, while the involvement of ABCA4 in STGD seems clear this is not the case for RP. Finally, in family III, also segregating STGD and RP, ABCA4 fails to explain either phenotype. Our data highlight the wide allelic heterogeneity involving this gene and support the genetic variability (beyond ABCA4) of mixed STGD/RP pedigrees.