Altered gene expression in immunogenic poorly differentiated thyroid carcinomas from RET/PTC3p53-/- mice.

Cancers develop and progress via activation of oncogenes and loss of tumor suppressor genes, a progression that can be recapitulated through cross breeding mouse strains harboring genetic mutations. To define the role of RET/PTC3, p53 and Fhit in thyroid carcinogenesis, we intercrossed RET/PTC3 transgenics with p53-/- mice. This new strain, RET/PTC3p53-/-, succumb to rapidly growing and strikingly large multilobed thyroid tumors containing mixtures of both well and poorly differentiated, highly proliferative follicular epithelial cells. Interestingly, transplanted tumors from RET/PTC3p53-/- mice grew in SCID but not syngeneic immunocompetent mice indicating that these advanced tumors were immunogenic. RET/PTC3 protein expression was reduced to undetectable levels in tumors of older mice suggesting that the continued elevated expression of RET/PTC3 may not be necessary for tumor progression. Similarly, expression of Fhit protein was reduced in early tumors and undetected in older tumors irrespective of tumor histopathology. In contrast to RET/PTC3p53-/- mice, RET/PTC3Fhit-/- mice did not develop advanced thyroid carcinomas. These studies support a model of human thyroid cancer whereby thyroid epithelium expresses RET/PTC3 protein at early stages of tumor development, followed by the reduction of RET/PTC3 and loss of p53 function with progressive reduction of Fhit protein expression coincident with malignant progression.

The TRK-T1 fusion protein induces neoplastic transformation of thyroid epithelium.

Genetic analysis of human papillary thyroid carcinomas (PTC) has revealed unique chromosomal translocations that form oncogenic fusion proteins and promote thyroid tumorigenesis in up to 60% of tumors examined. Although, the majority of thyroid specific translocations involve the growth factor receptor c-RET, variant rearrangements of the receptor for nerve growth factor, NTRK1 have also been described. One such translocation, TRK-T1, forms a fusion protein composed of the carboxyl terminal tyrosine kinase domain of NTRK1 and the amino terminal portion of TPR (Translocated Promoter Region). To determine if TRK-T1 expression can cause thyroid cancer in vivo, we developed transgenic mice that express the human TRK-T1 fusion protein in the thyroid. Immunohistochemical analysis of TRK-T1 transgenic mouse thyroids revealed TRK-T1 staining within the thyroid follicular epithelium. In contrast to nontransgenic littermates, 54% of transgenic mice developed thyroid abnormalities that included follicular hyperplasia and papillary carcinoma. Furthermore, all transgenic mice examined greater than 7 months of age developed thyroid hyperplasia and/or carcinoma. These data support the conclusion that TRK-T1 is oncogenic in vivo and contributes to the neoplastic transformation of the thyroid.

Scale-up of sonic energy for a major industrial application: ash conditioning for power utilities that use fluidized bed combustion.

The paper describes a new, powerful, patented piece of sonic equipment that operates in the low sonic frequency range, at 104 Hz, with nominal power of 75 kW. It has been tested for both physical and chemical sonic effects. A large-scale industrial application is described--the conditioning and pacification of ash from utility coal combustion by fluidized bed combustion. A variety of additional applications is suggested, most of which have been briefly tested.

Transcriptional regulation of beta-defensin gene expression in tracheal epithelial cells.

Innate immunity provides an ever-present or rapidly inducible initial defense against microbial infection. Among the effector molecules of this defense in many species are broad-spectrum antimicrobial peptides. Tracheal antimicrobial peptide (TAP) was the first discovered member of the beta-defensin family of mammalian antimicrobial peptides. TAP is expressed in the ciliated epithelium of the bovine trachea, and its mRNA levels are dramatically increased upon stimulation with bacteria or bacterial lipopolysaccharide (LPS). We report here that this induction by LPS is regulated at the level of transcription. Furthermore, the transfection of reporter gene constructs into tracheal epithelial cells indicates that DNA sequences in the 5' flanking region of the TAP gene, within 324 nucleotides of the transcription start site, are responsible in part for mediating gene induction. This region includes consensus binding sites for NF-kappaB and nuclear factor interleukin-6 (NF IL-6) transcription factors. Gel mobility shift assays indicate that LPS induces NF-kappaB binding activity in the nuclei of these cells, while NF IL-6 binding activity is constitutively present. The gene encoding human beta-defensin 2, a human homologue of TAP with similar inducible expression patterns in the airway, was cloned and found to have conserved NF-kappaB and NF IL-6 consensus binding sites in its 5' flanking region. Previous studies of antimicrobial peptides from insects indicated that their induction by infectious microbes and microbial products also occurs via activation of NF-kappaB-like and NF IL-6-like transcription factors. Together, these observations indicate that a strategy for the induction of peptide-based antimicrobial innate immunity is conserved among evolutionarily diverse organisms.

Persistence of an encephalitogenic T cell clone in the spinal cord during chronic, relapsing experimental autoimmune encephalomyelitis.

The CDR3 region of the TCR beta-chain of a CD4+, Th1, Vbeta2+ encephalitogenic T cell clone was used as an idiotypic marker to track the location of the clone in vivo. cDNA prepared from the spinal cord, thymus, lymph nodes, spleen, and liver of the recipients at various stages of EAE was amplified using Vbeta2 and Cbeta-region primers, and the products immobilized. The membrane was probed with a 32P-labeled oligonucleotide complementary to the CDR3 region of the T cell clone. The probe reacted strongly with products from the spinal cord, spleen and liver and less strongly with products from lymph nodes and thymus of mice with acute EAE. The signal was greatly diminished in the spinal cord and other tissues during recovery from acute disease and reappeared in the spinal cord at each relapse.

Enteric beta-defensin: molecular cloning and characterization of a gene with inducible intestinal epithelial cell expression associated with Cryptosporidium parvum infection.

A growing body of evidence suggests that endogenous antibiotics contribute to the innate defense of mammalian mucosal surfaces. In the cow, beta-defensins constitute a large family of antibiotic peptides whose members have been previously isolated from the respiratory and oral mucosa, as well as circulating phagocytic cells. A novel bovine genomic clone with beta-defensin-related sequence [corrected] related to those of these alpha-defensins was isolated and characterized. The corresponding cDNA was isolated from a small intestinal library; its open reading frame predicts a deduced sequence of a novel beta-defensin, which we designate enteric beta-defensin (EBD). Northern blot analysis of a variety of bovine tissues revealed that EBD mRNA is highly expressed in the distal small intestine and colon, anatomic locations distinct from those for previously characterized beta-defensins. EBD mRNA was further localized by in situ hybridization to epithelial cells of the colon and small intestinal crypts. Infection of two calves with the intestinal parasite Cryptosporidium parvum induced 5- and 10-fold increases above control levels of EBD mRNA in intestinal tissues. An anchored-PCR strategy was used to identify other beta-defensin mRNAs expressed in the intestine. In addition to that of EBD, several low-abundance cDNAs which corresponded to other beta-defensin mRNAs were cloned. Most of these clones encoded previously characterized beta-defensins or closely related isoforms, but two encoded a previously uncharacterized prepro-beta-defensin. Northern blot evidence supported that all of these other beta-defensin genes are expressed at levels lower than that of the EBD gene in enteric tissue. Furthermore, some of these beta-defensin mRNAs were abundant in bone marrow, suggesting that in enteric tissue their expression may be in cells of hematopoietic origin. Extracts of small intestinal mucosa obtained from healthy cows have numerous active chromatographic fractions as determined by an antibacterial assay, and one peptide was partially purified. The peptide corresponded to one of the low-abundance cDNAs. This study provides evidence of beta-defensin expression in enteric tissue and that the mRNA encoding a major beta-defensin of enteric tissue, EBD, is inducibly expressed in enteric epithelial cells. These findings support the proposal that beta-defensins may contribute to host defense of enteric mucosa.

Coordinate induction of two antibiotic genes in tracheal epithelial cells exposed to the inflammatory mediators lipopolysaccharide and tumor necrosis factor alpha.

Peptides with potent broad-spectrum antibiotic activity have been identified in many animal species. Recent investigations have demonstrated that epithelial cells are a site of antibiotic peptide expression, suggesting that these peptides contribute to host defense at mucosal surfaces. Expression of tracheal antimicrobial peptide (TAP), a member of the beta-defensin family of peptides, is inducible in cultured tracheal epithelial cells (TEC) upon challenge with bacterial lipopolysaccharide (LPS) (G. Diamond, J.P. Russell, and C.L. Bevins, Proc. Natl. Acad. Sci. USA, in press). In this study, an anchored reverse transcriptase PCR strategy was used to determine if TAP was the sole beta-defensin isoform expressed upon stimulation of the cells with LPS. In addition to TAP, a second class of cDNA clones which encoded lingual antimicrobial peptide (LAP), a beta-defensin peptide recently isolated from a different mucosal site, the bovine tongue, was identified (B.S. Schonwetter, E.D. Stolzenberg, and M. Zasloff, Science 267:1645-1648, 1995). Northern (RNA) blot analysis demonstrated in vivo expression of LAP mRNA in tracheal mucosa. Levels of LAP mRNA were higher in cultured TEC challenged with either LPS or tumor necrosis factor alpha than in control cells. Thus, a response of TEC exposed to inflammatory mediators is induction of antibiotic-encoding genes, including both TAP and LAP. This work complements the in vivo studies of Schonwetter et al. (cited above), which showed elevated levels of LAP mRNA in squamous epithelial cells of the tongue near sites of tissue injury and inflammation, by suggesting possible mediators of the in vivo observation. Together these lines of investigations support the hypothesis that inducible expression of endogenous antibiotic peptides by inflammatory mediators characterizes local defense of mammalian mucosal surfaces.

Inducible expression of an antibiotic peptide gene in lipopolysaccharide-challenged tracheal epithelial cells.

Mammals continually confront microbes at mucosal surfaces. A current model suggests that epithelial cells contribute to defense at these sites, in part through the production of broad-spectrum antibiotic peptides. Previous studies have shown that invertebrates can mount a host defense response characterized by the induction in epithelia] cells of a variety of antibiotic proteins and peptides when they are challenged with microorganisms, bacterial cell wall/membrane components, or traumatic injury [Boman, H.G. & Hultmark, D. (1987) Annu. Rev. Microbiol. 41, 103-126J. However, factors that govern the expression of similar defense molecules in mammalian epithelial cells are poorly understood. Here, a 13-fold induction of the endogenous gene encoding tracheal antimicrobial peptide was found to characterize a host response of tracheal epithelia] cells (TECs) exposed to bacterial lipopolysaccharide (LPS). Northern blot data indicated that TECs express CD14, a well-characterized LPS-binding protein known to mediate many LPS responses. A monoclonal antibody to CD14 blocked the observed tracheal antimicrobial peptide induction by LPS under serum-free conditions. Together the data support that CD14 of epithelial cell origin mediates the LPS induction of an antibiotic peptide gene in TECs, providing evidence for the active participation of epithelial cells in the host's local defense response to bacteria. Furthermore, the data allude to a conservation of this host response in evolution and suggest that a similar inducible pathway of host defense is prevalent at mucosal surfaces of mammals.

Human enteric defensins. Gene structure and developmental expression.

Paneth cells, secretory epithelial cells of the small intestinal crypts, are proposed to contribute to local host defense. Both mouse and human Paneth cells express a collection of antimicrobial proteins, including members of a family of antimicrobial peptides named defensins. In this study, data from an anchored polymerase chain reaction (PCR) strategy suggest that only two defensin mRNA isoforms are expressed in the human small intestine, far fewer than the number expressed in the mouse. The two isoforms detected by this PCR approach were human defensin family members, HD-5 and HD-6. The gene encoding HD-6 was cloned and characterized. HD-6 has a genomic organization similar to HD-5, and the two genes have a striking pattern of sequence similarity localized chiefly in their proximal 5'-flanking regions. Analysis of human fetal RNA by reverse transcriptase-PCR detected enteric defensin HD-5 mRNA at 13.5 weeks of gestation in the small intestine and the colon, but by 17 weeks HD-5 was restricted to the small intestine. HD-6 mRNA was detectable at 13.5-17 weeks of gestation in the small intestine but not in the colon. This pattern of expression coincides with the previously described appearance of Paneth cells as determined by ultrastructural approaches. Northern analysis of total RNA from small intestine revealed quantifiable enteric defensin mRNA in five samples from 19 24 weeks of gestation at levels approximately 40-250-fold less than those observed in the adult, with HD-5 mRNA levels greater than those of HD-6 in all samples. In situ hybridization analysis localized expression of enteric defensin mRNA to Paneth cells at 24 weeks of gestation, as is seen in the newborn term infant and the adult. Consistent with earlier morphological studies, the ratio of Paneth cell number per crypt was reduced in samples at 24 weeks of gestation compared with the adult, and this lower cell number partially accounts for the lower defensin mRNA levels as determined by Northern analysis. Low levels of enteric defensin expression in the fetus may be characteristic of an immaturity of local defense, which is thought to predispose infants born prematurely to infection from intestinal microorganisms.

Organization of the ribosomal ribonucleic acid genes in various wild-type strains and wild-collected strains of Neurospora.

The organization of the ribosomal DNA (rDNA) repeat unit in the standard wild-type strain of Neurospora crassa, 74-OR23-1A, and in 30 other wild-type strains and wild-collected strains of N. crassa, . tetrasperma, N. sitophila, N. intermedia, and N. discreta isolated from nature, was investigated by restriction enzyme digestion of genomic DNA, and probing of the Southern-blotted DNA fragments with specific cloned pieces of the rDNA unit from 74-OR23-1A. The size of the rDNA unit in 74-OR23-1A was shown to be 9.20 kilobase pairs (kb) from blotting data, and the average for all strains was 9.11 + 0.21 kb; standard error = 0.038; coefficient of variation (C.V.) = 2.34%. These data indicate that the rDNA repeat unit size has been highly conserved among the Neurospora strains investigated. However, while all strains have a conserved HindIII site near the 5' end of the 25 S rDNA coding sequence, a polymorphism in the number and/or position of HindIII sites in the nontranscribed spacer region was found between strains. The 74-OR23-1A strain has two HindIII sites in the spacer, while others have from 0 to at least 3. This restriction site polymorphism is strain-specific and not species-specific. It was confirmed for some strains by restriction analysis of clones containing most of the rDNA repeat unit. The current restriction map of the 74-OR23-1A rDNA repeat unit is presented.