Induced recruitment of NK cells to lymph nodes provides IFN-gamma for T(H)1 priming

Naive T cells are stimulated by antigen-presenting dendritic cells (DCs) in secondary lymphoid organs, but whether other types of cell participate in T cell priming is unclear. Here we show in mice that natural killer (NK) cells, which are normally excluded from lymph nodes, are rapidly recruited in a CCR7-independent, CXCR3-dependent manner to lymph nodes on stimulation by the injection of mature DCs. Recruitment of NK cells is also induced by some, but not all, adjuvants and correlates with the induction of T helper cell type 1 (T(H)1) responses. NK cell depletion and reconstitution experiments show that NK cells provide an early source of interferon-gamma (IFN-gamma) that is necessary for T(H)1 polarization. Taken together, our results identify an induced pathway of NK cell migration in antigen-stimulated lymph nodes and a mechanism by which some adjuvants may facilitate T(H)1 responses.     

Antigenic characterisation of influenza B virus with a new microneutralisation assay: comparison to haemagglutination and sequence analysis

Although the haemagglutination inhibition assay is considered the "gold standard" for antigenic characterisation of influenza viruses, some limitations of this technique are well known. A new microneutralisation assay, as a tool for antigenic characterisation of influenza B viruses, has been standardised and its performance evaluated in comparison with the haemagglutination inhibition test in the light of molecular characterisation of the haemagglutinin. Twelve B viruses belonging to the two lineages and the four sub-lineages discriminated by phylogenetic analysis of HA were tested. The microneutralisation assay clearly distinguishes viruses belonging to different lineages and, in addition, discriminates strains belonging to different sub-lineages that are poorly or not discriminated using the haemagglutination inhibition test. This new microneutralisation assay could provide a useful tool for antigenic characterisation of circulating influenza viruses and contribute, together with the haemagglutination inhibition test and sequence analysis of the haemagglutinin and neuraminidase, in the choice of the strain for use in vaccine composition.     

Spontaneous and MNNG‐induced reversion of an EGFP construct in HeLa cells: An assay for observing mutations in living cells by fluorescent microscopy

A HeLa cell line stably expressing the enhanced green fluorescence protein (EGFP) gene, interrupted by the HBB IVS2‐654 intron, was studied without treatment and after treatment with a single standard dose of 15 μM of N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG). This assay was done in order to prove that such a construct can revert by a variety of mechanisms and that it produces a visible phenotype, i.e., green fluorescence. The system permits visual detection of living mutant cells among a background of non‐mutant cells and does not require a selective medium. The results show that the construct reverts by large deletions (–62, –100, and –162 bp), small insertions (+4 bp), small rearrangements (19 bp duplication), base substitutions at purines (G₆₅₂, G₆₅₃, A₆₅₅, G₅₇₉), and a pyrimidine (T₆₅₄) between nucleotide positions 579 and 837. Splice‐site mutations were recovered, and some of the mechanisms underlying these mutations are discussed. Because of the ease of detection of revertant cells under fluorescent light and the wide variety of mutations that can be recovered, further development of this system could make it a useful new mammalian cell mutagenicity assay. Hum Mutat 18:526–534, 2001. © 2001 Wiley‐Liss, Inc.     

Congenital malformations in twins: an international study

Data provided by nine registries based in European and Latin America countries were analyzed to assess whether there is an excess of malformations in twins compared to singletons. Specific congenital malformations were coded according to the ninth revision of the International Classification of Diseases (ICD). Malformation rates and rate ratios (RR) for twins compared to singletons were calculated for each registry, and the homogeneity of the RRs was tested using the test of Breslow and Day. If departure from homogeneity in the different registries was not significant, registry-adjusted RRs with 95% confidence intervals were calculated. Overall, among 260,865 twins, 5,572 malformations were reported. A total of 101 different types of malformations or groups of defects was identified, and a homogeneous estimate of the RRs among registries was found for 91.1% of the malformations. Thirty-nine of the 92 malformations with homogeneous estimates of RRs were more common in twins than in singletons. For the remaining nine malformations, heterogeneous estimates of RRs were obtained. This study confirms the majority of already known associations and further identifies previously unreported malformations associated with twins. In conclusion, there is an excess of malformations in twins compared with singletons, and all anatomical sites are involved. The number of specific malformations associated with twins is higher than that previously reported in smaller studies.     

Modulation of the constitutive or cytokine-induced bronchial epithelial cell functions in vitro by fluticasone propionate

When exposed to proinflammatory mediators, human bronchial epithelial cells (HBECs) upregulate the 'constitutive' adhesion molecule expression and cytokine/chemokine release. We tested whether and to what extent the inhibitory effect of fluticasone propionate on HBECs could involve the 'constitutive' and 'cytokine-induced' proinflammatory functions. Stimulation of the HBECs with interleukin (IL)-4 plus tumour necrosis factor (TNF)-alpha was more effective in upregulating intercellular adhesion molecule (ICAM)-1 ( approximately 2.2-fold increase) than vascular adhesion molecule (VCAM)-1 ( approximately 1.6-fold increase) expression (P<0.05) and in increasing the release of 'regulated on activation normal T cell expressed' (RANTES, 5.7-fold increase) than of IL-8 (3.5-fold increase) and granulocyte macrophage-colony stimulating factor (GM-CSF, 2.8-fold increase), (P<0.01). Fluticasone propionate, at the two concentrations tested (10 and 100 nM), was more effective in inhibiting the 'IL-4 plus TNF-alpha-induced' ICAM-1 expression than VCAM-1 expression (P<0.05) and in downregulating RANTES than IL-8 or GM-CSF secretion (P<0.05). The degree of inhibition demonstrated by fluticasone propionate appeared to be related to the degree of cell activation. In addition, for both adhesion molecules, the effect of fluticasone propionate at both concentrations tested appeared to be related to a complete inhibition of 'IL-4 plus TNF-alpha-induced' expression with no involvement of the 'constitutive' expression. Slightly different results were observed for cytokine/chemokine release. Indeed, evaluating RANTES, a complete inhibition of the 'IL-4 plus TNF-alpha-induced' release with a partial inhibition also of the 'constitutive' release at both concentrations of the drug tested was found, whereas for GM-CSF and IL-8, only a partial inhibition of the 'IL-4 plus TNF-alpha-induced' release in the presence of fluticasone propionate 10 and 100 nM. Thus, HBECs can constitutively or upon activation express adhesion molecules and secrete proinflammatory proteins at various levels and the different ability of fluticasone propionate to modulate the HBEC functions appears to be mostly related to the different inhibition of the various 'IL-4 plus TNF-alpha-induced' responses.     

FISH characterization of a supernumerary r(1)(::cen→q22::q22→sq21::) chromosome associated with multiple anomalies and bilateral cataracts

We describe the case of a 15‐year‐old girl with multiple congenital anomalies, dysmorphic features, severe kyphoscoliosis, growth and mental retardation, and the absence of speech, in whom 35% of the cells carried a supernumerary ring chromosome 1. Fluorescence in situ hybridization (FISH) analysis using YAC/BAC clones spanning the region from 1p13 to 1q21 made it possible to determine the genomic content and structure of the ring(1), which was found to consist of the cytogenetic bands 1q21–22. A complex structure was delineated in the ring chromosome with a partial inverted duplication delimited by markers WI‐7732 and WI‐607, with WI‐7396 and WI‐8386 being the boundaries of the single copy segment. Comparison of the clinical signs of other patients with mosaic r(1) reported in the literature allowed the identification of a patient sharing a number of clinical signs including cataracts. Given that mutations of the GJA8 gene encoding connexin 50 (Cx50) and mapping to 1q21 have been associated with the presence of cataracts, it is possible that a gain in copy number or a rearrangement of GJA8 may contribute to cataractogenesis.