IL-15-deficient mice develop enhanced allergic responses to airway allergen exposure.

BACKGROUND: Interleukin-15 is a pleiotropic cytokine that is critical for the development and survival of multiple haematopoietic lineages. Mice lacking IL-15 have selective defects in populations of several pro-allergic immune cells including natural killer (NK) cells, NKT cells, and memory CD8+ T cells. We therefore hypothesized that IL-15-/- mice will have reduced inflammatory responses during the development of allergic airway disease (AAD). OBJECTIVE: To determine whether IL-15-/- mice have attenuated allergic responses in a mouse model of AAD. METHODS: C57BL/6 wild-type (WT) and IL-15-/- mice were sensitized and challenged with ovalbumin (OVA), and the development of AAD was ascertained by examining changes in airway inflammatory responses, Th2 responses, and lung histopathology. RESULTS: Here, we report that IL-15-/- mice developed enhanced allergic responses in an OVA-induced model of AAD. In the absence of IL-15, OVA-challenged mice exhibited enhanced bronchial eosinophilic inflammation, elevated IL-13 production, and severe lung histopathology in comparison with WT mice. In addition, increased numbers of CD4+ T and B cells in the spleens and bronchoalveolar lavage (BAL) were also observed. Examination of OVA-challenged IL-15Rα-/- animals revealed a similar phenotype resulting in enhanced airway eosinophilia compared to WT mice. Adoptive transfer of splenic CD8+ T cells from OVA-sensitized WT mice suppressed the enhancement of eosinophilia in IL-15-/- animals to levels observed in WT mice, but had no further effects. CONCLUSION AND CLINICAL RELEVANCE: These data demonstrate that mice with an endogenous IL-15 deficiency are susceptible to the development of severe, enhanced Th2-mediated AAD, which can be regulated by CD8+ T cells. Furthermore, the development of disease as well as allergen-specific Th2 responses occurs despite deficiencies in several IL-15-dependent cell types including NK, NKT, and γδ T cells, suggesting that these cells or their subsets are dispensable for the induction of AAD in IL-15-deficient mice.

The influence of hybridization between African and European honeybees, Apis mellifera, on asymmetries in wing size and shape.

We examined the possible role of hybridization in the invasion process of the African honeybee by testing two hypotheses regarding fluctuating asymmetry (FA), a measure of developmental stability, in wing characteristics: (1) FA should be higher in hybrid versus parental genotypes of African and European races; (2) FA should be lower in African bees compared to hybrid and European workers. Parental and reciprocal hybrid worker genotypes were cross fostered in common-hive rearing environments. We did not find greater FA for wing size and shape in the hybrids compared to both parental types. However, we did find significantly lower FA of shape in the African workers compared to the European and hybrid workers, suggesting that European bees and their hybrids may have compromised fitness relative to African bees. We also found that the two hybrid genotypes significantly differed in overall wing size and shape. If these differences affect wing aerodynamics, then the paternity of hybrids may influence worker performance and could potentially contribute to the loss of European matrilines. Hybridization had few consistent effects on directional asymmetry for wing size and shape. Genotypic factors played a far greater role in determining the effect of hybridization on wing morphology than did differences in rearing environment. Thus, African bees may have lower FA for wing shape (and by inference greater developmental stability) relative to European and hybrid workers, which may contribute to the ability of African bees to displace European honeybee races in invaded regions.

An 18q- syndrome breakpoint resides between the duplicated serpins SCCA1 and SCCA2 and arises via a cryptic rearrangement with satellite III DNA.

The 18q-syndrome is representative of a group of terminal deficiency or macrodeletion syndromes characterized by mental retardation and congenital malformations. To gain insight into the mechanism of chromosomal loss and stabilization in these disorders, we cloned a putative terminal deletion breakpoint from an 18q-syndrome patient. The 18q21.3 breakpoint occurred between two nearly identical serine protease inhibitor (serpin) genes, SCCA1 and SCCA2. Although cytogenetic studies suggested that this chromosomal aberration was formed by a simple terminal deletion, DNA sequence analysis, pulsed-field gel electrophoresis and fluorescence in situ hybridization showed that the breakpoint was contiguous with a 35 bp filler sequence followed by a satellite III DNA-containing telomeric fragment of 475-1000 kb. This type of satellite III DNA sequence was not detected on the normal chromosome 18, but was highly homologous with types of satellite III DNA sequences normally located on the short arms (p11) of the acrocentric chromosomes and other heterochromatic regions. This DNA sequence analysis suggested that the terminal deficiency in this 18q-syndrome patient arose via illegitimate (non-homologous) recombination. Moreover, these data raise the possibility that a subset of chromosomal aberrations appearing cytogenetically and molecularly as simple terminal truncations or deletions are caused by small (<1000 kb) cryptic rearrangements.

Construction and validation of yeast artificial chromosome contig maps by RecA-assisted restriction endonuclease cleavage.

RecA-assisted restriction endonuclease (RARE) cleavage is an "Achilles' heel" approach to restriction mapping whereby a RecA-protein-oligodeoxynucleotide complex protects an individual restriction site from methylation, thus limiting subsequent digestion to a single, predetermined site. We have used RARE cleavage to cut yeast artificial chromosomes (YACs) at specific EcoRI sites located within or adjacent to sequence-tagged sites (STSs). Each cleavage reaction produces two YAC fragments whose sizes are a direct measure of the position of the STS in the YAC. In this fashion, we have positioned 45 STSs within a contig of 19 independent YACs and constructed a detailed RARE-cleavage map that represents 8.4 Mbp of human chromosome 6p21.3-22. By comparing maps of overlapping YACs, we were able to detect seven internal deletions that ranged from approximately 75 kbp to approximately 1 Mbp in size. Thirteen pairs of EcoRI sites were targeted for double RARE cleavage in uncloned total human DNA. The excised fragments, up to 2 Mbp in size, were resolved by pulsed-field gel electrophoresis and were detected by hybridization. In general, the genomic RARE-cleavage results support the YAC-based map. In one case, the distance in uncloned DNA between the two terminal EcoRI sites of a YAC insert was approximately 1 Mbp larger than the YAC itself, indicating a major deletion. The general concept of RARE-cleavage mapping as well as its applications and limitations are discussed.

Training and generalized production of wh- and NP-movement structures in agrammatic aphasia.

The present research examines production of "complex" sentences, which involve movement of noun phrases (NPs), in 2 agrammatic aphasic subjects. According to linguistic theory (Chomsky, 1991, 1993), such sentences are derived using one of two movement operations, either wh- or NP-movement, subsumed under the general rule "move alpha." In this experiment recovery of both wh- and NP-movement derived sentences was investigated using a treatment research paradigm. Subjects were sequentially trained to produce either wh-movement (i.e., who questions, object clefts) or NP-movement (i.e., passives, subject-raising structures) derived sentences. Throughout training, generalization to untrained sentences relying on both types of movement was tested. The influence of training on aspects of narrative discourse also was examined. Results showed generalization patterns constrained to type of movement. Training wh-movement structures resulted in generalized production of untrained wh-movement structures without influencing production of NP-movement structures. Similarly, training of NP-movement structures resulted in generalization only to other sentence types also relying on NP-movement. Aspects of sentence production in narrative contexts also was improved with treatment. These data indicate that movement to an argument (A) position as in NP-movement is distinct from movement to a non-argument (A-bar) position, required in wh-movement. The site where movement terminates in the s-structure of noncanonical sentences appears to influence sentence production. These findings show that linguistic properties of sentences influence sentence production breakdown and recovery in aphasia.

A serine proteinase inhibitor locus at 18q21.3 contains a tandem duplication of the human squamous cell carcinoma antigen gene.

The squamous cell carcinoma antigen (SCCA) is a member of the ovalbumin family of serine proteinase inhibitors (serpins). A neutral form of the protein is found in normal and some malignant squamous cells, whereas an acidic form is detected exclusively in tumor cells and in the circulation of patients with squamous cell tumors. In this report, we describe the cloning of the SCCA gene from normal genomic DNA. Surprisingly, two genes were found. They were tandemly arrayed and flanked by two other closely related serpins, plasminogen activator inhibitor type 2 (PAI2) and maspin at 18q21.3. The genomic structure of the two genes, SCCA1 and SCCA2, was highly conserved. The predicted amino acid sequences were 92% identical and suggested that the neutral form of the protein was encoded by SCCA1 and the acidic form was encoded by SCCA2. Further characterization of the region should determine whether the differential expression of the SCCA genes plays a causal role in development of more aggressive squamous cell carcinomas.

The 18q- syndrome: analysis of chromosomes by bivariate flow karyotyping and the PCR reveals a successive set of deletion breakpoints within 18q21.2-q22.2.

The 18q- syndrome is one of several terminal deletion disorders that occur in humans. Previous G-banding studies suggest that the loss of a critical band, 18q21.3, results in mental retardation, craniofacial anomalies, and metabolic defects. However, it is difficult to reconcile the consistent loss of a single region with the large variability in clinical phenotype. The purpose of this study was to reassess the extent of chromosomal loss in a cohort of 17 18q- syndrome patients by using fluorescent-activated chromosome sorting, PCR, and FISH. Bivariate flow karyotypes revealed heterogeneity among the deletions; they ranged in size from 9 to 26 Mb. To confirm this heterogeneity at a molecular level, deleted and normal chromosomes 18 of six patients were collected by flow sorting, preamplified by random priming, and assayed for marker content by the PCR. This analysis defined five unique breakpoints among the six patients. We conclude that the terminal deletions in the 18q- syndrome occur over a broad region spanning the interval from 18q21.2 to 18q22.2. Our results suggest that the variability in clinical phenotype may be more representative of a contiguous-gene syndrome with a baseline deficit of 18q22.2-qter than of the loss of a single critical region within 18q21.3.

Preferential amplification of the paternal allele in neuroblastomas with N-myc amplification.

There is increasing evidence that imprinting plays an important role in influencing the parental origin of genes involved in cancer-specific rearrangements. In advanced stage neuroblastomas, both allelic loss of the short arm of chromosome 1 (1p) and amplification of the proto-oncogene N-myc are often seen. Therefore, we analyzed 22 human neuroblastomas with N-myc amplification to determine the parental origin of the N-myc allele that is amplified and the 1p allele that is deleted. We used at least three polymorphisms for both the 1p and the N-myc locus to analyze blood and tumor samples from neuroblastoma patients, as well as blood samples from their parents. We determined that the paternal allele of N-myc was amplified in 12 of 15 informative cases (P = 0.02), and the paternal allele on 1p was lost in 6 of 11 informative cases (P > 0.2). These results suggest that parental imprinting does not appear to affect the 1p allele that is deleted, at least in the cases that we have examined. However, imprinting has an important influence on determining the N-myc allele that is amplified in these tumors, suggesting that the paternal allele is either more highly expressed or more likely to undergo amplification.

High-resolution mapping of the N-myc amplicon core domain in neuroblastomas.

The N-myc proto-oncogene is amplified in 25% of neuroblastomas. Amplification is strongly correlated with advanced disease stage and rapid tumor progression. We have constructed a detailed restriction map of the amplified core region in neuroblastomas which will allow the identification of structural features such as joint fragments, rearrangements and CpG islands. Using probes that had been obtained previously, twenty YACs were isolated from a library constructed from a double-minute-containing neuroblastoma cell line with 150-fold amplification of N-myc. Twenty-one YACs also were isolated from two normal human libraries. Normal and neuroblastoma YAC contiguous arrays (contigs), each spanning over 1 Mb of DNA, have been assembled (Molec Cell Biol 12:5563, 1992). A high-resolution restriction map of over 200 kb of contiguous DNA containing N-myc has been generated by subcloning YACs into cosmids. Using cosmids from this region plus additional amplified probes, we have determined that the amplicons from 33 neuroblastomas range in size from 350 kb to over 1 Mb. Rearrangements and deletions were identified in both tumors and cell lines. However joint fragments were not always amplified to the same level as the major amplicon, and may therefore represent a subset of amplicons. We have defined a 130 kb region which was amplified in 32 of the 33 tumors, and one additional tumor had deleted 65 kb of this region from its amplicon. The only CpG island found in this region was within the N-myc gene. Cosmids were screened with radiolabeled total cDNA probes and no highly expressed genes other than N-myc were found in the amplicon.

Preferential amplification of the paternal allele of the N-myc gene in human neuroblastomas.

Genomic imprinting plays a role in influencing the parental origin of genes involved in cancer-specific rearrangements. We have analysed 22 neuroblastomas with N-myc amplification to determine the parental origin of the amplified N-myc allele and the allele that is deleted from chromosome 1p. We analysed DNA from neuroblastoma patients and their parents, using four polymorphisms for 1p and three for the N-myc amplicon. We determined that the paternal allele of N-myc was preferentially amplified (12 out of 13 cases; P = 0.002). However, the paternal allele was lost from 1p in six out of ten cases, consistent with a random distribution (P > 0.2). These results suggest that parental imprinting influences which N-myc allele is amplified in neuroblastomas, but it does not appear to affect the 1p allele that is deleted in the cases that we have examined.

Isolation and structural analysis of a 1.2-megabase N-myc amplicon from a human neuroblastoma.

Oncogene amplification is observed frequently in human cancers, but little is known about the mechanism of gene amplification or the structure of amplified DNA in tumor cells. We have studied the N-myc amplified domain from a representative neuroblastoma cell line, SMS-KAN, and compared the map of the amplicon in this cell line with that seen in normal DNA. The SMS-KAN cell line DNA was cloned into yeast artificial chromosomes (YACs), and clones were identified by screening the YAC library with amplified DNA probes that were obtained previously (B. Zehnbauer, D. Small, G. M. Brodeur, R. Seeger, and B. Vogelstein, Mol. Cell. Biol. 8:522-530, 1988). In addition, YAC clones corresponding to the normal N-myc locus on chromosome 2 were obtained by screening two normal human YAC libraries with these probes, and the restriction maps of the two sets of overlapping YACs were compared. Our results suggest that the amplified domain in this cell line is a approximately 1.2-Mb circular molecule with a head-to-tail configuration, and the physical map of the normal N-myc locus generally is conserved in the amplicon. These results provide a physical map of the amplified domain of a neuroblastoma cell line that has de novo amplification of an oncogene. The head-to-tail organization, the general conservation of the normal physical map in the amplicon, and the extrachromosomal location of the amplified DNA are most consistent with the episome formation-plus-segregation mechanism of gene amplification in these tumors.

Yeast artificial chromosome (YAC) vector cloning of the MYCN amplified domain in neuroblastomas.

About 25% of human neuroblastomas have amplification of the MYCN proto-oncogene, and this feature is associated with advanced stages of disease and rapid tumor progression. Estimates of the size of the amplicon range from 300 to 3,000 kb, with MYCN at or near the center. It has been determined previously that there is considerable conservation of the amplified sequences among different neuroblastomas, and very few rearrangements have been found. We decided to clone the entire amplified domain in YACs to assess the size, structure and organization of the amplified sequences in neuroblastomas and to identify novel or junctional sequences at the ends of an amplicon in double minutes (dmins) or in the germline. A YAC library was constructed from the SMS-KAN cell line, which contains dmins and has about 150 copies of MYCN. About 5,000 clones from this library were screened with MYCN as well as other probes from the amplified domain. To date, 16 YACs have been identified, with a median size of 240 kb (range 50-460 kb). These YAC clones can be arranged in a contiguous, linear map of greater than or equal to 1 megabase. Our data suggest that most neuroblastomas with MYCN amplification also amplify a region that is a megabase or more in size, making this the largest amplified domain cloned to date. The very large size suggests that there are other genes near MYCN whose expression is important in mediating the aggressive phenotype associated with MYCN amplification, or, alternatively, that the nearest origin of replication may be a considerable distance from MYCN.

Identification by immunobinding assay of the polypeptide coded by the DNA polymerase gene of bacteriophage T5 and its amber mutants and the direction of transcription of the gene.

We identified by immunobinding assay the polypeptides synthesized as the result of amber mutations in the DNA polymerase gene of bacteriophage T5. Comparison of the size of such polypeptides revealed the order of mutagenic loci of these mutations and the direction of transcription of the gene. Extracts of cells infected with wild-type T5 and with five amber mutants of the polymerase gene (D7, D8, D9, am1, and am6) were prepared, and the proteins were resolved by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. After transfer of the proteins to a nitrocellulose sheet, a radioimmunolabeling technique was used to identify the T5 DNA polymerase and its amber mutant polypeptides. Based on the relative sizes of the polypeptides, the transcription of the T5 DNA polymerase gene was determined to proceed in the order D7, D8, am1, D9, and am6. The molecular weights of the DNA polymerase polypeptides coded by D8, am1, D9, am6, and T5+ were 23,000, 45,000, 75,000, 83,000, and 96,000, respectively. The D7-coded polypeptide was not detectable. These data suggest that the carboxyl-terminal region of the enzyme is essential for the polymerase function.

Surgical management of ankylosed impacted maxillary canines.

Impacted and ankylosed maxillary canines were repositioned to replace maxillary lateral incisors lost after root resorption. Direct bonding of orthodontic brackets and arch wire fixation were performed at the time of surgery.