Analyses of regulatory CD4+ CD25+ FOXP3+ T cells and observations from peripheral T cell subpopulation markers during the development of type 1 diabetes in children.

Our aim was to study whether the aberrant amount or function of regulatory T cells is related to the development of type 1 diabetes (T1D) in children. We also set out to investigate the balance of different T cell subtype markers during the T1D autoimmune process. Treg cells were quantified with flow cytometric assay, and the suppression capacity was analysed with a carboxyfluorescein succinimidyl ester (CFSE)-based T cell suppression assay in children in various phases of T1D disease process and in healthy autoantibody-negative control children. The mRNA expression of different T cell subpopulation markers was analysed with real-time qPCR method. The proportion and suppression capacity of regulatory T cells were similar in seroconverted children at an early stage of beta cell autoimmunity and also in children with T1D when compared to healthy and autoantibody-negative children. Significant differences were observed in the mRNA expression of different T cell subpopulation markers in prediabetic children with multiple (≥ 2) autoantibodies and in children with newly diagnosed T1D when compared to the control children. In conclusion, there were no quantitative or functional differences in regulatory T cells between the case and control groups in any phase of the autoimmune process. Decreased mRNA expression levels of T cell subtype markers were observed in children with multiple islet autoantibodies and in those with newly diagnosed T1D, probably reflecting an exhaustion of the immune system after the strong immune activation during the autoimmune process or a generally aberrant immune response related to the progression of the disease.

Collagens I and III in a porcine bronchial model of obliterative bronchiolitis.

The main extracellular matrix components of the lung, type I and III collagens, were studied in chronic allograft rejection developing in a porcine heterotopic bronchial transplantation model. Specific porcine complementary DNA probes were constructed for detection of the expression of type I and III procollagen messenger RNAs in the bronchial wall structures and in the obliterative plug by in situ hybridization. In autografts, and in allografts immunosuppressed with 40-O-(2-hydroxyethyl)-rapamycin, cyclosporine A, and methylprednisolone, no histological changes of obliterative bronchiolitis (OB) developed, and the number of fibroblast-like cells expressing type I and III procollagen mRNA remained low. In nontreated allografts obliterating within 21 d, a preponderance of fibroblast-like cells showing positivity for type III procollagen mRNA existed in the obliterative plug and bronchial wall. This study shows for the first time the temporal and spatial activation of type I and III procollagen genes during the course of obliterative bronchiolitis. The number of cells expressing procollagen III mRNA increased parallel to developing obliteration and fibrosis in nontreated allografts, whereas autografts and immunosuppressed allografts exhibited no such trend. This finding suggests a positive association between type III collagen mRNA expression in fibroblast-like cells and development of obliterative bronchiolitis.

Surfactant proteins A and D in Eustachian tube epithelium.

Surfactant protein (SP) A and SP-D are collectins that have roles in host defense. The Eustachian tube (ET) maintains the patency between the upper airways and the middle ear. Dysfunction of local mucosal immunity in ET may predispose infants to recurrent otitis media. We recently described preliminary evidence of the expression of SP-A and SP-D in the ET. Our present aim was to establish the sites of SP-A and SP-D expression within the epithelium of the ET in vivo. With in situ hybridization, electron microscopy, and immunoelectron microscopy, the cells responsible for SP-A and SP-D expression and storage were identified. SP-A expression was localized within the ET epithelium, and the protein was found in the electron-dense granules of microvillar epithelial cells. Being concentrated in the epithelial lining, only a few cells revealed intracellular SP-D, and it was not associated with granules. The SP-A and SP-D immunoreactivities in ET lavage fluid, as shown by Western blot analyses, were similar to those in bronchoalveolar lavage fluid. We propose that there are specialized cells in the ET epithelium expressing and secreting SP-A and SP-D. SP-A and SP-D may be important for antibody-independent protection of the middle ear against infections.

Surfactant in respiratory distress syndrome and lung injury.

A deficiency in alveolar surfactant due to immaturity of alveolar type II epithelial cells causes respiratory distress syndrome (RDS). In contrast to animals, the fetal maturation of surfactant in human lungs takes place before term, exceptionally large quantities of surfactant accumulating in the amniotic fluid. The antenatal development of surfactant secretion is very variable but corresponds closely to the risk of RDS. The variation in SP-A and SP-B genes, race, sex and perinatal complications influence susceptibility to RDS. Surfactant therapy has improved the prognosis of RDS remarkably. Abnormalities in alveolar or airway surfactant characterize many lung and airway diseases. In the acute respiratory distress syndrome, deficiencies in surfactant components (phospholipids, SP-B, SP-A) are evident, and may be caused by pro-inflammatory cytokines (IL-1, TNF) that decrease surfactant components. The resultant atelectasis localizes the disease, possibly allowing healing (regeneration, increase in surfactant). In the immature fetus, cytokines accelerate the differentiation of surfactant, preventing RDS. After birth, however, persistent inflammation is associated with low SP-A and chronic lung disease. A future challenge is to understand how to inhibit or redirect the inflammatory response from tissue destruction and poor growth towards normal lung development and regeneration.

Expression and localization of lung surfactant protein B in Eustachian tube epithelium.

Surfactant protein (SP) B is an essential component of the pulmonary surfactant complex, which participates in reducing the surface tension across the alveolar air-liquid interface. The Eustachian tube (ET) connects the upper respiratory tract to the middle ear, serving as an intermittent airway between the pharynx and the middle ear. Recently, we described the expression of SP-A and SP-D in the ET, suggesting their role in middle ear host defense. Our present aim was to detect whether the expression of SP-B is evident in the porcine ET. With Northern blot analysis, RT-PCR, and in situ hybridizations, SP-B mRNA was identified and localized in the ET epithelium. The cellular localization of SP-B was revealed with immunohistochemistry, electron microscopy, and immunoelectron microscopy. The protein was found in the secretory granules of epithelial cells and also attached to the microvilli at the luminal side of these cells. The SP-B immunoreactivity of aggregates isolated from ET lavage fluid was similar to that isolated from bronchoalveolar lavage fluid. We conclude that there are specialized cells in the ET epithelium expressing and secreting SP-B and propose that SP-B may facilitate normal opening of the tube and mucociliary transport.

Stage-and tissue-specific expression of a Col2a1-Cre fusion gene in transgenic mice.

To achieve chondrocyte-specific deletion of floxed genes we generated a transgenic mouse line expressing the Cre recombinase under the control of the mouse type II collagen gene (Col2a1) regulatory regions. Northern and in situ hybridization analyses demonstrated the expression of the transgene (Col2a1-Cre) in cartilaginous tissues. To test the excision efficiency of Cre, the Col2a1-Cre strain was crossed with the ROSA26 reporter strain. LacZ staining of double transgenic mice revealed Cre activity in both chondrogenic and non-chondrogenic tissues. During early embryonic development (E9.5-11.5), LacZ expression was detected in tissues where the endogenous Col2a1 transcript is expressed such as the otic capsule, notochord, developing brain, sclerotome and mesenchymal condensations of future cartilage. At later stages, Cre activity was observed in all cartilaginous tissues with virtually 100% of chondrocytes being LacZ positive. These data suggest that the Col2a1-Cre mouse strain described here can be useful to achieve Cre-mediated recombination in Col2a1 expressing cells, especially in chondrocytes.

Ontogeny of Toll-like receptors Tlr2 and Tlr4 in mice.

Toll-like receptors (TLR:) have recently been linked to the immunostimulatory function of microbial toxins in human and mice. TLR: signals activation of nuclear factor kappaB that leads to the production of a number of proinflammatory mediators. TLR:4 mediates the endotoxin-induced inflammatory response, whereas TLR:2 may be involved in the response to yeast and Gram-positive bacterial products. To better understand age-related changes in acute inflammatory response, we studied the ontogeny of TLR:2 and TLR:4 mRNA in murine fetal lung, liver, and placenta by quantitative reverse transcriptase-PCR. Different expression patterns were seen between the tissues and between the TLR: This is in accordance with the evidence that there are differences in the receptors for different microbial toxins and that the response is organ specific. We additionally show that the expression of TLR: was dependent on the stage of differentiation. In the liver, the levels of Tlr2 and Tlr4 were high regardless of the age. In the lung, Tlr2 and Tlr4 expression levels were barely detectable in immature fetus (d 14-15). Tlr2 and Tlr4 were increased several-fold during prenatal development and further increased after birth. The present results support the finding of a deficient inflammatory response of the immature lung to microbial toxins.

Temporospatial expression of tissue inhibitors of matrix metalloproteinases-1, -2 and -3 during development, growth and aging of the mouse skeleton.

Proteolytic degradation of collagen-rich extracellular matrices is a key feature in the development, growth and aging of skeleton. Matrix metalloproteinases (MMPs) are a family of enzymes capable of performing this function, whereas tissue inhibitors of MMPs (TIMPs) are believed to play an important role in regulating their activity. To better understand the roles of TIMP-1, -2 and -3, we have studied their mRNA levels in several different mouse tissues with special emphasis on the skeleton and the developing eye. A systematic analysis of TIMP-1, -2 and -3 mRNA levels in mouse knee joints during growth and aging demonstrated markedly different expression patterns for each TIMP. Immunohistochemical analysis revealed several time-dependent changes in the distribution of TIMP-1 and -2 in articular and growth cartilages, synovial tissue and bone. The data suggest that upon aging synovial tissue becomes the major source of synovial fluid TIMPs. In articular cartilage these inhibitors were mainly found in the deep layer and in subchondral bone. Compared with epiphyseal growth plate, the amounts of TIMP-1 and -2 in articular cartilage were quite low. These findings suggest that the capacity of articular cartilage chondrocytes to inhibit MMP activities by local production of TIMPs is limited, which may be of consequence during osteoarthritic cartilage degeneration.

Degree of lung maturity determines the direction of the interleukin-1- induced effect on the expression of surfactant proteins.

Intra-amniotic interleukin (IL)-1 increases surfactant components in immature fetal lung, whereas high IL-1 after birth is associated with surfactant dysfunction. Our aim was to investigate whether the fetal age influences the responsiveness of surfactant proteins (SPs) to IL-1. Rabbit lung explants from fetuses at 19, 22, 27, and 30 d of gestation and 1-d-old newborns were cultured in serum-free medium in the presence of recombinant human (rh) IL-1alpha or vehicle. The influence of IL-1alpha on SP-A, -B, and -C messenger RNA (mRNA) content was dependent on the conceptional age. In very immature lung on Day 19, rhIL-1alpha (570 ng/ml for 20 h) increased SP-A, -B, and -C mRNA by 860+/-15%, 314+/-108%, and 64+/-17%, respectively. The increase in SP-A mRNA was evident within 4 to 6 h. IL-1alpha increased the SP-A concentration in alveolar epithelial cells and in the culture medium within 20 h. In contrast, at 27 to 30 d of gestation and in newborns, IL-1alpha decreased SP-C, -B, and -A mRNA by means of 64 to 67%, 48 to 59%, and 12 to 15%, respectively. SP-B protein decreased by 45 to 60%. The decrease in mRNA became evident within 8 to 12 h and was dependent on IL-1 concentration. On Day 27, IL-1alpha accelerated the degradation of SP-B mRNA in the presence of actinomycin D. IL-1 did not increase the degradation rate of SP-A mRNA unless both actinomycin D and cycloheximide were added to the explants. The present findings may explain some of the contrasting associations between inflammatory cytokines and lung diseases during the perinatal period. The determinants of the direction of the IL-1 effect on the expression of SPs remain to be identified.

Surfactant protein A and D expression in the porcine Eustachian tube.

Surfactant proteins A and D are collectins which are considered to play an important role in the innate immunity of lungs. Our aim was to investigate whether surfactant protein A or D is expressed in the porcine Eustachian tube originating from the upper airways. Both surfactant proteins A and D were present in the epithelial cells of the Eustachian tube, as shown by strong immunostaining. Using RT-PCR and Northern hybridization, these collectins were detected in the Eustachian tube. The present study is the first report demonstrating surfactant protein gene expression in the Eustachian tube. Surfactant proteins A and D may be important in the antibody-independent protection of the middle ear.

Cathepsin expression during skeletal development.

Cysteine proteinases, cathepsins B, H, K, L and S, have been implicated in several proteolytic processes during development, growth, remodeling and aging, as well as in a variety of pathological processes. For systematic analysis of cathepsin gene expression we have produced cDNA clones for mouse and human cysteine cathepsins. Northern analysis of a panel of total RNAs isolated from 16-19 different human and mouse tissues revealed the presence of mRNAs for cathepsin B, H, K, L and S in most tissues, but each with a distinct profile. Of the different cathepsin mRNAs, those for cathepsin K were clearly the highest in bone and cartilage. However, relatively high mRNA levels for the other cathepsins were also present in these tissues. To better understand the roles of different cathepsins during endochondral ossification in mouse long bones, cathepsin mRNAs were localized by in situ hybridization. Cathepsin K mRNAs were predominantly seen in multinucleated chondroclastic and osteoclastic cells at the osteochondral junction and on the surface of bone spicules. The other cathepsin mRNAs were also seen in osteoclasts, and in hypertrophic and proliferating chondrocytes. These observations were confirmed by immunohistochemistry and suggest that all cysteine cathepsins are involved in matrix degradation during endochondral ossification.

Effects of signal peptide mutations on processing of Bacillus stearothermophilus alpha-amylase in Escherichia coli.

Bacillus stearothermophilus alpha-amylase has a signal peptide typical for proteins exported by Gram-positive bacteria. There is only one signal peptidase processing site when the protein is exported from the original host, but when it is exported by Escherichia coli, two alternative sites are utilized. Site-directed mutagenesis was used to study the processing in E. coli. Processing sites for 13 B. stearothermophilus alpha-amylases carrying mutations in their signal peptide were determined. Processing of the signal peptide was remarkably tolerant to mutations, because switching between the alternative sites was possible. The length and the sequence of the region between the hydrophobic core and the cleavage site was crucial for determining the choice of the processing site. Some mutations more distal to the cleavage site also affected the site preference.

Analysis of aggrecan and tenascin gene expression in mouse skeletal tissues by northern and in situ hybridization using species specific cDNA probes.

Cartilage matrix is an interacting multicomponent system of collagen fibrils, fibril-associated small proteoglycans, and large proteoglycans and glycoproteins entrapped within the fibrillar network. In order to better understand the relationships between these different components we have constructed short cDNA clones for detection of mRNAs for two major noncollagenous macromolecules of cartilage matrix, aggrecan and tenascin. We subsequently determined their corresponding mRNA levels by Northern analysis in a panel of total RNAs isolated from several newborn mouse tissues. The expression of aggrecan was strictly restricted to cartilages while tenascin mRNA was present at variable levels in most of the tissues studied. The cDNA clones were also used to identify the cells responsible for aggrecan and tenascin production in newborn mouse tissues by in situ hybridization. With this technique aggrecan mRNA was detected in chondrocytes throughout the developing skeleton in a pattern very similar but not identical to those of type II and IX collagen mRNAs. In the newborn mouse skeleton tenascin and aggrecan mRNAs were expressed essentially in a mutually exclusive manner, tenascin transcripts being present in osteoblasts, periosteal and perichondrial cells, and in cells at articular surfaces. None of these cells expressed the cartilage specific collagen or aggrecan genes. The results further suggest different patterns of gene expression in chondrocytes based on their location in the different cartilages.

Molecular analysis of the Trichosporon cutaneum DSM 70698 argA gene and its use for DNA-mediated transformations.

Genomic clones capable of complementing a previously isolated arginine auxotrophic mutant strain of the filamentous yeast Trichosporon cutaneum DSM 70698 have been identified by DNA-mediated transformation, and a complementing 4,082-bp subfragment was sequenced. This analysis revealed an intact gene (arg4) showing a high degree of homology with the Saccharomyces cerevisiae CPA2 gene encoding the large subunit of carbamoyl-phosphate synthetase (CPS-A). The inferred amino acid sequence of the T. cutaneum argA-encoded protein contains 1,168 residues showing 62% identity with the sequence of the S. cerevisiae CPA2 protein, and the comparison of the two sequences uncovered a putative intron sequence of 81 nucleotides close to the 5' end of the coding region of the T. cutaneum argA gene. The presence of this intron was confirmed by nuclease protection studies and by direct DNA sequence analysis of a cDNA fragment which had been obtained by PCR amplification. The T. cutaneum intron shares the general characteristics of introns found in yeasts and filamentous fungi. A major transcript of around 4 kb was found in Northern (RNA) blots. The T. cutaneum argA coding region was expressed in Escherichia coli under the control of the regulatable tac promoter. A roughly 130-kDa protein which was found to cross-react with an anti-rat CPS antibody in Western blots (immunoblots) was observed. Two putative ATP-binding domains were identified, one in the amino-terminal half of the argA-encoded protein and the other in the carboxy-terminal half. These domains are highly conserved among the known CPS-A sequences from S. cerevisiae, E. coli, and the rat. From these results we conclude that the T. cutaneum argA gene encodes the large subunit of CPS. This is the first gene to be identified and analyzed in the T. cutaneum DSM 70698 strain.

Cloning of cDNA for rat pro alpha 1(III) collagen mRNA. Different expression patterns of type I and type III collagen and fibronectin genes in experimental granulation tissue.

A cDNA clone for rat pro alpha 1(III) collagen mRNA was isolated from a cDNA library constructed for poly(A)+ RNA from 15-day experimental granulation tissue. Two clones, pRGR1 and pRGR5, were characterized by restriction mapping and sequencing. Comparison with human type III collagen sequences revealed 92% identity at the level of translated amino acids, and 88% identity at nucleotide level in the coding region. In the 3'-untranslated sequence the identity was even higher (90%). The clones were used together with cDNA clones for type I collagen chains, fibronectin and gamma-actin to study the expression of the corresponding mRNAs during the development of experimental sponge-induced granulation tissue in rats. These studies revealed a marked activation of type I and type III collagen genes during the second week of granuloma development followed by a transient reduction in their levels during the third week. The mRNA levels for both collagen types remained relatively unchanged from day 25. The molar ratio of pro alpha 1(III) and pro alpha 1(I) collagen mRNAs was at a maximum on day 6, and then decreased to reach a plateau by the end of the third week. Fibronectin mRNA levels were found to increase slower; the maximum value was reached during the fifth week of granuloma development. The mRNA levels of gamma-actin increased continuously up to the end of the fourth week, thus following the cellular maturation of the tissue.

Nuclear factor binding to an AP-1 site is associated with the activation of pro-alpha 1(I)-collagen gene in dedifferentiating chondrocytes.

Isolated chondrocytes grown on plastic gradually lose their differentiated phenotype upon subculturing. This dedifferentiation is manifested by an altered production of extracellular-matrix molecules (ECM): e.g., the cartilage specific type II collagen is replaced by types I and III. We have studied the regulation of ECM gene expression in dedifferentiating human and murine fetal chondrocytes. Nuclear extracts from dedifferentiated cells, human fetal fibroblasts and 3T3 cells contained a protein that bound in an electrophoretic mobility shift assay to an AP-1 site in the first intron of the human alpha 1(I) collagen gene. This binding activity was not present in freshly isolated human or murine chondrocytes, which produced type II, but not type I, collagen mRNA in culture. Thus the binding activity was induced simultaneously with alpha 1(I)-collagen-gene expression during dedifferentiation. The specific interaction was sensitive to dephosphorylation of the nuclear extract and to chemical modification of reduced cysteine residues. The AP-1 site we studied had previously been shown to be a positive transcriptional contributor in the first intron to the expression of the alpha 1(I) collagen gene. In transient transfections into dedifferentiating chondrocytes, an alpha 1(I) collagen expression plasmid carrying a mutated AP-1 site in the first intron resulted in three-times-lower reporter gene RNA levels than a plasmid carrying the respective functional AP-1 site. These data suggest that the AP-1 sequence and its respective trans-acting factors may play a role in the transcriptional regulation of the alpha 1(I) collagen gene during dedifferentiation of chondrocytes.

Genetic transformation of auxotrophic mutants of the filamentous yeast Trichosporon cutaneum using homologous and heterologous marker genes.

A transformation system for the filamentous yeast Trichosporon cutaneum based on auxotrophic markers is presented and techniques for the induction, isolation and characterization of mutants are described. A number of auxotrophic mutants were isolated and characterized by using biosynthetic precursors and/or inhibitors. A mutant unable to grow in the presence of ornithine could be complemented successfully by spheroplast transformation experiments using the cloned Aspergillus nidulans ornithine transcarbamoylase gene (argB gene) as selection marker with an efficiency of 5-100 transformants per microgram of DNA. In these transformants the heterologous argB gene was present in multiple tandem copies and the transforming DNA was found to remain stable after more than 50 generations in non-selective media. The same mutant could be complemented by a T. cutaneum cosmid gene library and a complementary cosmid was subsequently isolated from this library by a sib-selection strategy. This cosmid transformed T. cutaneum spheroblasts with an efficiency of 50-200 colonies per microgram of DNA. Southern blot analyses were consistent with the view that the transforming sequences became stably integrated into the host genome at the homologous site.

Transfer and expression of heterologous genes in yeasts other than Saccharomyces cerevisiae.

In the past few years, yeasts other than those belonging to the genus Saccharomyces have become increasingly important for industrial applications. Species such as Pichia pastoris, Hansenula polymorpha, Schizosaccharomyces pombe, Yarrowia lipolytica and Kluyveromyces lactis have been modified genetically and used for the production of heterologous proteins. For a number of additional yeasts such as Schwanniomyces occidentalis, Zygosaccharomyces rouxii, Trichosporon cutaneum, Pachysolen tannophilus, Pichia guilliermondii and members of the genus Candida genetic transformation systems have been worked out. Transformation was achieved using either dominant selection markers based on antibiotic resistance genes or auxotrophic markers in conjunction with cloned biosynthetic genes involved in amino acid or nucleotide metabolism.

Genetic transformation of the filamentous yeast, Trichosporon cutaneum, using dominant selection markers.

An efficient transformation system for the filamentous yeast, Trichosporon cutaneum, has been developed. Transformation was obtained with plasmids carrying either the Escherichia coli hygromycin B phosphotransferase-encoding gene (hph) or the Streptoalloteichus hindustanus phleomycin-resistance gene (ble), as dominant selection markers. Expression of both resistance-conferring genes was controlled by the gpd promoter and the trpC terminator, from Aspergillus nidulans. The transformation frequency was up to 500 colonies/micrograms of transforming DNA, using the ble gene, and up to 100 colonies/micrograms of transforming DNA, using the hph gene. Co-transformation frequencies using unselected DNA varied between 50 and 65%. The transforming DNA was found to consist of multiple tandem plasmid copies of high Mr. This polymeric structure, in nonselective media, was mitotically unstable, possibly indicating that it existed in an episomal state.