Do they stay or do they go? Acoustic monitoring of whale sharks at Ningaloo Marine Park, Western Australia.

Whale sharks Rhincodon typus were monitored via acoustic transmitters at the northern end of Western Australia's Ningaloo Marine Park to establish the extent to which the species inhabits the region beyond the whale-shark ecotourism industry season, which usually extends from March to August in each year. Despite the vast majority (c. 98%) of photographic submissions of R. typus from Ningaloo Reef being between March and August, acoustic detections from the tagged R. typus at Ningaloo were recorded in all months of the year, but do not preclude the occurrence of extended absences. It is concluded that as a species, R. typus occurs year round at Ningaloo, where it generally remains in close proximity to the reef edge, but that some individuals move outside of the detection range of the array for extended periods.

CRISPR-Cas9-mediated gene knockout in primary human airway epithelial cells reveals a proinflammatory role for MUC18.

Targeted knockout of genes in primary human cells using CRISPR-Cas9-mediated genome-editing represents a powerful approach to study gene function and to discern molecular mechanisms underlying complex human diseases. We used lentiviral delivery of CRISPR-Cas9 machinery and conditional reprogramming culture methods to knockout the MUC18 gene in human primary nasal airway epithelial cells (AECs). Massively parallel sequencing technology was used to confirm that the genome of essentially all cells in the edited AEC populations contained coding region insertions and deletions (indels). Correspondingly, we found mRNA expression of MUC18 was greatly reduced and protein expression was absent. Characterization of MUC18 knockout cell populations stimulated with TLR2, 3 and 4 agonists revealed that IL-8 (a proinflammatory chemokine) responses of AECs were greatly reduced in the absence of functional MUC18 protein. Our results show the feasibility of CRISPR-Cas9-mediated gene knockouts in AEC culture (both submerged and polarized), and suggest a proinflammatory role for MUC18 in airway epithelial response to bacterial and viral stimuli.

Elevation of susceptibility to ozone-induced acute tracheobronchial injury in transgenic mice deficient in Clara cell secretory protein.

Increases in Clara cell abundance or cellular expression of Clara cell secretory protein (CCSP) may cause increased tolerance of the lung to acute oxidant injury by repeated exposure to ozone (O3). This study defines how disruption of the gene for CCSP synthesis affects the susceptibility of tracheobronchial epithelium to acute oxidant injury. Mice homozygous for a null allele of the CCSP gene (CCSP-/-) and wild type (CCSP+/+) littermates were exposed to ozone (0.2 ppm, 8 h; 1 ppm, 8 h) or filtered air. Injury was evaluated by light and scanning electron microscopy, and the abundance of necrotic, ciliated, and nonciliated cells was estimated by morphometry. Proximal and midlevel intrapulmonary airways and terminal bronchioles were evaluated. There was no difference in airway epithelial composition between CCSP+/+ and CCSP-/- mice exposed to filtered air, and exposure to 0.2 ppm ozone caused little injury to the epithelium of both CCSP+/+ and CCSP-/- mice. After exposure to 1.0 ppm ozone, CCSP-/- mice suffered from a greater degree of epithelial injury throughout the airways compared to CCSP+/+ mice. CCSP-/- mice had both ciliated and nonciliated cell injury. Furthermore, lack of CCSP was associated with a shift in airway injury to include proximal airway generations. Therefore, we conclude that CCSP modulates the susceptibility of the epithelium to oxidant-induced injury. Whether this is due to the presence of CCSP on the acellular lining layer surface and/or its intracellular distribution in the secretory cell population needs to be defined.

Freeze drying--principles and practice for successful scale-up to manufacturing.

Freeze Drying involves transfer of heat and mass to and from the product under preparation, respectively, thus it is necessary to scale these transport phenomena appropriately from pilot plant to manufacturing-scale units to maintain product quality attributes. In this manuscript we describe the principal approach and tools utilized to successfully transfer the lyophilization process of a labile pharmaceutical product from pilot plant to manufacturing. Based on pilot plant data, the lyophilization cycle was tested during limited scale-up trials in manufacturing to identify parameter set-point values and test process parameter ranges. The limited data from manufacturing were then used in a single-vial mathematical model to determine manufacturing lyophilizer heat transfer coefficients, and subsequently evaluate the cycle robustness at scale-up operating conditions. The lyophilization cycle was then successfully demonstrated at target parameter set-point values.

Altered lung gene expression in CCSP-null mice suggests immunoregulatory roles for Clara cells.

Clara cell secretory protein (CCSP) is one of the most abundant proteins present in airway lining fluid of mammals. In an effort to elucidate the function of CCSP, we established CCSP-null [CCSP(-/-)] mice and demonstrated altered sensitivity to various environmental agents including oxidant pollutants and microorganisms. Although CCSP deficiency itself may be central to the observed changes in environmental susceptibility, altered lung gene expression associated with CCSP deficiency may contribute to the observed phenotype. To determine whether CCSP deficiency results in altered lung gene expression, high-density cDNA microarrays were used to profile gene expression in the total lung RNA of wild-type and CCSP(-/-) mice. Genes that were differentially expressed between wild-type and CCSP(-/-) mice included a previously non-annotated expressed sequence tag (EST W82219) and immunoglobulin A (IgA), both of which were elevated with CCSP deficiency. mRNA expression of EST W82219 and IgA was localized in the lungs of wild-type and CCSP(-/-) mice to airway Clara cells and peribronchial lymphoid tissues, respectively. We conclude that CCSP deficiency is associated with 1) altered gene expression in Clara cells of the conducting airway epithelium and 2) alterations to peribronchial B lymphocytes. These findings identify new roles for Clara cells and their secretions in airway homeostasis.

Clara cell secretory protein-expressing cells of the airway neuroepithelial body microenvironment include a label-retaining subset and are critical for epithelial renewal after progenitor cell depletion.

Stem cells with potential to contribute to the re-establishment of the normal bronchiolar epithelium have not been definitively demonstrated. We previously established that neuroepithelial bodies (NEBs) sequester regenerative cells that contribute to bronchiolar regeneration after selective chemical depletion of Clara cells, a major progenitor cell population. Two candidate stem cells were identified on the basis of proliferative potential after chemical ablation: a pollutant-resistant subpopulation of Clara cells that retain their expression of Clara cell secretory protein (CCSP) (variant CCSP-expressing [CE] cells or vCE cells) and calcitonin gene-related peptide (CGRP)-expressing pulmonary neuroendocrine cells (PNECs). In the present study, two populations of label-retaining cells were identified within the NEB: CGRP-expressing cells and a subpopulation of CE cells. To investigate contributions made by CE and CGRP-expressing cells to epithelial renewal, CE cells were ablated through acute administration of ganciclovir to transgenic mice expressing herpes simplex virus thymidine kinase under the regulatory control of the mouse CCSP promoter. CGRP-immunoreactive PNECs proliferated after depletion of CE cells, yet were unable to repopulate CE cell-depleted airways. These results support the notion that vCE cells represent either an airway stem cell or are critical for stem cell maintenance, and suggest that PNECs are not sufficient for epithelial renewal.

Phenotypic consequences of lung-specific inducible expression of FGF-3.

Members of the fibroblast growth factor (FGF) family play a critical role in embryonic lung development and adult lung physiology. The in vivo investigation of the role FGFs play in the adult lung has been hampered because the constitutive pulmonary expression of these factors often has deleterious effects and frequently results in neonatal lethality. To circumvent these shortcomings, we expressed FGF-3 in the lungs under the control of the progesterone antagonist-responsive binary transgenic system. Four binary transgenic lines were obtained that showed ligand-dependent induction of FGF-3 with induced levels of FGF-3 expression dependent on the levels of expression of the GLp65 regulator as well as the dose of the progesterone antagonist, RU486, administered. FGF-3 expression in the adult mouse lung resulted in two phenotypes depending on the levels of induction of FGF-3. Low levels of FGF-3 expression resulted in massive free alveolar macrophage infiltration. High levels of FGF-3 expression resulted in diffuse alveolar type II cell hyperplasia. Both phenotypes were reversible after the withdrawal of RU486. This system will be a valuable means of investigating the diverse roles of FGFs in the adult lung.

Conditional clara cell ablation reveals a self-renewing progenitor function of pulmonary neuroendocrine cells.

The neuroepithelial body (NEB) is a highly dynamic structure that responds to chronic airway injury through hyperplasia of associated pulmonary neuroendocrine (PNE) cells. Although NEB dysplasia is correlated with preneoplastic conditions and PNE cells are thought to serve as a precursor for development of small cell lung carcinoma, mechanisms regulating expansion of the PNE cell population are not well understood. Based on studies performed in animal models, it has been suggested that NEB-associated progenitor cells that are phenotypically distinct from PNE cells contribute to PNE cell hyperplasia. We have previously used a Clara cell-specific toxicant, naphthalene, to induce airway injury in mice and have demonstrated that naphthalene-resistant Clara cells, characterized by their expression of Clara cell secretory protein (CCSP), and PNE cells contribute to airway repair and associated hyperplasia of NEBs. This study was conducted to define the contribution of NEB-associated CCSP-expressing progenitor cells to PNE cell hyperplasia after Clara cell ablation. Transgenic (CCtk) mice were generated in which herpes simplex virus thymidine kinase was expressed within all CCSP-expressing cells of the conducting airway epithelium through the use of transcriptional regulatory elements from the mouse CCSP promoter. Chronic administration of ganciclovir (GCV) to CCtk transgenic mice resulted in selective ablation of CCSP-expressing cells within conducting airways. Proliferation and hyperplasia of PNE cells occurred in the absence of detectable proliferation among any other residual airway epithelial cell populations. These results demonstrate that PNE cells function as a self-renewing progenitor population and that NEB-associated Clara cells are not necessary for PNE cell hyperplasia.

Cloning of the chick BMP1/Tolloid cDNA and expression in skeletal tissues.

The astacin-related metalloproteases Bone Morphogenetic Protein-1 (BMP1) and Tolloid possess multiple functions in the maturation of extracellular matrices containing fibrillar collagens. We are interested in developing an in-vitro model system to study the role of BMP1 and Tolloid in chondrocytes and osteoblasts. Cloning of the cDNAs for chick BMP1 and Tolloid reveals that the two gene products are more than 80% identical to their human and mouse homologs and are similarly derived from the same genetic locus. Anti-BMP1/Tolloid antibodies have been developed, and detect two proteins of 80 and 116kDa. Chick BMP1 and Tolloid message and proteins are found in a variety of embryonic and juvenile tissues, including chondrocytes and osteoblasts. Tolloid message and protein are generally less abundant than BMP1 message; this discrepancy is greatest in growth plate chondrocytes. Tolloid protein is more tightly bound than BMP1 to the extracellular matrix produced by cultured osteoblasts. The Chordin gene is also expressed in chondrocytes and osteoblasts, suggesting that BMP1 and Tolloid influence BMP signaling as well as matrix maturation during skeletogenesis.

Neuroepithelial bodies of pulmonary airways serve as a reservoir of progenitor cells capable of epithelial regeneration.

Remodeling of the conducting airway epithelium is a common finding in the chronically injured lung and has been associated with increased risk for developing lung cancer. Pulmonary neuroendocrine cells and clusters of these cells termed neuroepithelial bodies (NEBs) play a central role in each of these processes. We previously developed an adult mouse model of airway injury and repair in which epithelial regeneration after naphthalene-induced Clara cell ablation occurred preferentially at airway branch points and gave rise to nascent Clara cells. Continued repair was accompanied by NEB hyperplasia. We now provide the following evidence that the NEB microenvironment serves as a source of airway progenitor cells that contribute to focal regeneration of the airway epithelium: 1) nascent Clara cells and NEBs localize to the same spatial domain; 2) within NEB, both Clara cell secretory protein- and calcitonin gene-related peptide-immunopositive cells are proliferative; 3) the NEB microenvironment of both the steady-state and repairing lung includes cells that are dually immunopositive for Clara cell secretory protein and calcitonin gene-related peptide, which were previously identified only within the embryonic lung; and 4) NEBs harbor variant Clara cells deficient in cytochrome P450 2F2-immunoreactive protein. These data suggest that the NEB microenvironment is a reservoir of pollutant-resistant progenitor cells responsive to depletion of an abundant airway progenitor such as the Clara cell.

Alteration of pulmonary neuroendocrine cells during epithelial repair of naphthalene-induced airway injury.

Whole-mount airway preparations isolated from the lungs of mice treated by intraperitoneal injection of naphthalene and allowed to recover for 5 days were examined for the distribution and abundance of solitary pulmonary neuroendocrine cells (PNECs) and neuroepithelial bodies (NEBs) along the main axial pathway of the right middle lobe. Sham mice treated with corn oil vehicle were examined in a similar manner. An antibody to calcitonin gene-related peptide, a neuroendocrine cell marker, was used to identify the location, size, and number of PNECs and NEBs in the airways. After naphthalene treatment and epithelial repair, NEBs were significantly increased along the walls of the airways as well as on branch point ridges. The surface area covered by NEBs composed of 20 or fewer PNECs was significantly enlarged after naphthalene treatment compared with control NEBs of an equivalent cell number. The PNEC number per square millimeter was also increased more than threefold above control values after naphthalene treatment. These findings provide further support for a key role of neuroendocrine cells in the reparative process of airway epithelial cell renewal after injury.

Pulmonary phenotype of CCSP/UG deficient mice: a consequence of CCSP deficiency or altered Clara cell function?

Clara cell secretory protein (CCSP) is the most abundant secreted protein within airways of the lung. Moreover, CCSP levels are modulated in human lung disease, supporting a potentially important role for CCSP and/or Clara cells in lung homeostasis. However, in vivo roles for CCSP remain elusive. A popular hypothesis is that CCSP is a regulator of the inflammatory response. The purpose of this review is to provide an overview of the phenotype of CCSP null mice and relate this phenotype to proposed functions for the protein. Phenotypic analysis of mice homozygous for the CCSP-1 null allele of the CCSP gene (CCSP-/-1) revealed susceptibility to inhaled oxidant gases. Sensitivity of CCSP-/-1 mice to inhaled ozone is unrelated to alterations in antioxidant defenses, but is associated with increased cellular injury. Additional studies investigating inflammatory control in CCSP deficient mice found no differences between wild-type and CCSP-/-1 mice in their inflammatory response to low-dose inhaled endotoxin exposure, arguing against a role for CCSP in regulation of pulmonary inflammation. The findings among CCSP-/-1 mice of ultrastructural alterations to Clara cell secretory apparatus, with associated changes in airway lining fluid protein composition, demonstrate that the CCSP-/-1 genotype results in more complex changes to airways than CCSP deficiency per se. It can be concluded that CCSP does not regulate endotoxin-induced pulmonary inflammation. Moreover, CCSP-/-1 mice represent a valuable tool for probing functional roles for Clara cells in regulation of airway lining fluid composition and lung pollutant susceptibility.

Normal function and lack of fibronectin accumulation in kidneys of Clara cell secretory protein/uteroglobin deficient mice.

Clara cell secretory protein (CCSP), also known as uteroglobin (Ug), is a 16-kDa homodimeric protein of unknown function. Within rodent species, CCSP is expressed predominantly by nonciliated Clara cells that line conducting airways of the lung. To investigate in vivo functions for CCSP, we established mice homozygous for a null allele of the CCSP gene (CCSP-/-). We previously showed no overt phenotypic consequences associated with CCSP deficiency when CCSP-/- mice are maintained in the absence of environmental stress. However, CCSP-/- mice show an oxidant-sensitive phenotype that cannot be attributed to alterations in the inflammatory response when challenged by inhaled oxidant gases. The current study was undertaken to determine whether CCSP deficiency results in pathological changes to the kidney. This study was prompted by the recent description of severe systemic disease and kidney fibrosis/dysfunction in an independent line of CCSP-deficient mice, termed Ug-/- (Zhang et al, Science 276:1408-1412, 1997). CCSP-/- mice show normal growth and reproductive performance when maintained in two independent genetic backgrounds, inbred 129 and congenic C57BL/6. Strain 129 CCSP-/- mice have normal kidney function, as assessed by urinary glucose, lactate dehydrogenase, and glomerular filtration rate; they show no kidney fibrosis or abnormalities in fibronectin accumulation and no histological abnormalities in proximal convoluted tubules or glomeruli at either light or electron microscopic levels. CCSP deficiency is associated with mild proteinurea involving a modest increase in mouse major urinary protein-1. We conclude that CCSP (Ug) deficiency, per se, is not the cause of severe renal pathology and systemic disease reported for Ug-/- mice.

Clara cell secretory protein-deficient mice differ from wild-type mice in inflammatory chemokine expression to oxygen and ozone, but not to endotoxin.

The in vivo function of Clara cell secretory protein (CCSP) is unknown. Biologic and biochemical properties associated with CCSP have led to speculation that it participates in pulmonary inflammatory control. Our earlier studies have demonstrated that CCSP-deficient mice are more sensitive to either hyperoxia or ozone toxicity and show altered oxidant-induced pulmonary proinflammatory responses. In this study we test the hypothesis that altered chemokine responses seen in CCSP-/- mice following oxidant stress are a direct consequence of altered immunoregulation associated with CCSP deficiency. To test this hypothesis we utilized three distinct models of inducing pulmonary toxicity: hyperoxia and ozone (O3), which cause epithelial cell injury, and endotoxin, which causes pulmonary inflammation independent of direct epithelial cell injury. Wild-type (WT) or CCSP-/- strain 129 mice were exposed to O3 at 1.0 ppm for 24 hours, oxygen (O2) > 99% for 68 hours or inhalation of 0.0575 microgram endotoxin per mouse for 10 minutes and examined 6 hours postexposure. Mice displayed increased sensitivity to O3, as demonstrated by increased abundance of mRNAs encoding Eotaxin, macrophage inflammatory protein (MIP)-1 alpha, and MIP-2, after 4 hours of exposure, whereas WT mice were unaltered from controls. Increased sensitivity to hyperoxia was also observed, as demonstrated by increased abundance of mRNAs encoding Eotaxin, MIP-1 alpha, MIP-1 beta, MIP-2, and interferon-gamma inducible (IP)-10 after 68 hours of exposure, whereas WT mice were unaltered from controls. In contrast, WT and CCSP-/- mice responded identically 6 hours postinhalation of 0.0575 microgram lipopolysaccharide (LPS) per mouse. PMN response was 63% and 64% in WT and CCSP-/- mice, respectively. Messenger RNAs encoding Eotaxin, MIP-1 alpha, MIP-1 beta, MIP-2, IP-10, and MCP-1 were increased identically. We conclude that CCSP does not participate in regulation of the endotoxin-elicited pulmonary inflammatory response. Identical inflammatory and chemokine responses of CCSP-/- and WT mice in response to a nonepithelial toxic agent (endotoxin) suggest that altered inflammatory control observed between WT and CCSP-/- mice following O2 and O3 exposure is not the result of altered immunoregulation.

Inflammatory and antioxidant gene expression in C57BL/6J mice after lethal and sublethal ozone exposures.

Ozone (O3) is a highly reactive and toxic oxidant pollutant. The objective of this study is to compare cytokine, chemokine, and metallothionein (Mt) changes elicited by lethal and sublethal exposure to ozone in a genetically sensitive strain of mice. Eight-week-old C57BL/6J mice were exposed to 0.3 ppm ozone for 0, 24, or 96 hours; 1.0 ppm ozone for 0, 1, 2, or 4 hours; or 2.5 ppm ozone for 0, 2, 4, or 24 hours. After 24 hours of exposure to 0.3 ppm ozone, increases in mRNA abundance were detected for messages encoding eotaxin, macrophage inflammatory protein (MIP)-1 alpha, and MIP-2. These increases persisted through 96 hours of exposure. At this time point messages encoding lymphotactin (Ltn) and metallothionein were also increased. After 4 hours of 1.0 ppm ozone exposure, increases in mRNA abundance were detected for messages encoding eotaxin, MIP-1 alpha, MIP-2, and interleukin (IL)-6. Mt mRNA abundance was increased after 1 hour of exposure and persisted through 4 hours, although the magnitude of the alterations increased. After 2 hours of 2.5 ppm ozone exposure, increases were detected for messages encoding eotaxin, MIP-1 alpha, MIP-2, IL-6, and Mt. These increases persisted through 4 hours of exposure. Lung weights of mice exposed to 2.5 ppm ozone for 24 hours were approximately 2 times greater than air-exposed mice. At this dose lethality occurred by 36 hours. Increased mRNAs for eotaxin, MIP-1 alpha, MIP-2, and Mt were to a higher magnitude than were detected after 2 and 4 hours of exposure. Messages encoding IL-12, IL-10, interferon (IFN)-gamma, IL-1 alpha, IL-1 beta, and IL-1Ra were unaltered at all time points and doses examined. Our results demonstrate dose- and time-dependent changes in chemokine, cytokine, and Mt mRNA abundance and that early acute changes may be predictive of subacute and chronic responses to ozone.

Clara cell secretory protein deficiency increases oxidant stress response in conducting airways.

Little is known about the molecular basis for differential pulmonary oxidant sensitivity observed between genetically disparate members of the same species. We have generated mice that are deficient in Clara cell secretory protein (CCSP -/-) and that exhibit an oxidant-sensitive phenotype. We characterized the kinetics and distribution of altered stress-response [interleukin-6 (IL-6) and metallothionein (MT)] and epithelial cell-specific [cytochrome P-450 2F2 (CYP2F2)] gene expression to further understand the cellular and molecular basis for altered oxidant sensitivity in 129 strain CCSP -/- mice. Increases in IL-6 and MT mRNA abundance were detected by 2 h of exposure to 1 part/million ozone and preceded reductions in Clara cell CYP2F2 mRNA expression. Despite being qualitatively similar, increases in IL-6 and MT mRNA expression were enhanced in CCSP -/- mice with respect to coexposed 129 strain wild-type mice. Increased MT mRNA expression, indicative of the stress response, localized to the airway epithelium, surrounding mesenchyme, and endothelium of blood vessels. These results demonstrate a protective role for Clara cells and their secretions and indicate potential genetic mechanisms that may influence susceptibility to oxidant stress.

Bone morphogenetic protein-7 in growth-plate chondrocytes: regulation by retinoic acid is dependent on the stage of chondrocyte maturation.

Although the bone morphogenetic proteins stimulate chondrogenesis, little is known regarding their expression and regulation in growth-plate chondrocytes. The expression of bone morphogenetic protein-7 was examined in chick growth-plate chondrocyte cultures. Low basal levels of bone morphogenetic protein-7 mRNA and protein expression were stimulated by increasing doses of all-trans retinoic acid, a metabolite of vitamin A. The addition of 10 microM retinoic acid resulted in approximately a 6-fold increase in bone morphogenetic protein-7 mRNA levels. In contrast, other growth regulators, including basic fibroblast growth factor, transforming growth factor-beta, vitamin D, bone morphogenetic protein-6, bone morphogenetic protein-7, and parathyroid hormone-related peptide, did not alter bone morphogenetic protein-7 transcript levels. The increase in bone morphogenetic protein-7 transcripts, although present at 6 hours, was maximal following a 12-hour exposure to retinoic acid. Retinoic acid induction of bone morphogenetic protein-7 transcript levels was dependent on protein synthesis because the induction could be blocked by cyclohexamide. In maturationally distinct subpopulations of chondrocytes separated by countercurrent centrifugal elutriation, retinoic acid markedly induced bone morphogenetic protein-7 mRNA levels in the least differentiated chondrocytes but had no effect in the most terminally differentiated hypertrophic chondrocytes. Immunohistochemical localization of bone morphogenetic protein-7 demonstrates its expression throughout the developing and adolescent growth plate consistent with the constitutive pattern of expression seen in isolated chondrocytes. The addition of exogenous bone morphogenetic protein-7 to chondrocyte cultures stimulated maturation in undifferentiated chondrocyte populations. The data support a role for bone morphogenetic protein-7 as an autocrine regulator of chondrocyte maturation in the growth plate. Regulation of bone morphogenetic protein-7 by retinoic acid may be important in normal growth and development as well as in pathologic conditions of an excess or deficiency of vitamin A.

Increased levels of tumor necrosis factor-alpha and interleukin-6 protein and messenger RNA in human peripheral blood monocytes due to titanium particles.

Cytokines produced by macrophages in the periprosthetic membranes surrounding joint replacements have been implicated as causal agents in osteolysis and prosthetic loosening. The present study characterizes the response of human peripheral blood monocytes to titanium particles. Monocytes were obtained from volunteers and blood that had been donated to the American Red Cross and were cultured in the presence of titanium particles (one to three micrometers in diameter). There were consistent dose-dependent increases in the production of TNF-alpha (tumor necrosis factor-alpha) and IL-6 (interleukin-6) protein, with the greatest stimulation generally observed with a concentration of 6 x 10(5) to 6 x 10(6) particles of titanium per milliliter. The level of TNF-alpha was the greatest (fifty to 1000 times greater than the control level) after eight hours of exposure to titanium particles; the level of IL-6 was two to five times greater than the control level after sixteen hours of exposure. These increases were similar to those observed after stimulation with lipopolysaccharide and depended on de novo synthesis rather than on release from intracellular stores. The production of TNF-alpha was inhibited in a dose-dependent manner by the translational inhibitor cycloheximide and the transcriptional inhibitor actinomycin D, indicating the requirement for both mRNA (messenger RNA) and protein synthesis for the induction of cytokine synthesis by titanium particles. Although the increase in the levels of cytokine mRNA in response to titanium was rapid (thirty to ninety minutes), the increase in the level of TNF-alpha mRNA preceded that of IL-6 mRNA. The level of TNF-alpha mRNA was the greatest at ninety minutes and the level of IL-6 mRNA was the greatest at three hours. After stimulation with titanium particles, the level of TNF-alpha mRNA was increased as much as fivefold and the level of IL-6 mRNA, as much as twelvefold.

Identification and characterization of a unique chondrocyte gene involved in transition to hypertrophy.

The character of differentiating chondrocytes in growing long bones has been defined by altered expression of a limited number of genes. To expand this set we have applied differential display to identify genes expressed in either mineralizing or nonmineralizing chondrocytes. One such gene, Band 17, has the following characteristics: (1) Band 17 expression is predominantly found in cartilage destined for mineralization. Band 17 mRNA is undetectable in articular cartilage and undetectable or weak in all other tissues tested. (2) Band 17 expression is spatially restricted to the lower proliferative/upper hypertrophic zone of chondrocytes in the growth plate of long bones and embryonic vertebrae. (3) Induction of a hypertrophic phenotype in progenitor sternal chondrocytes by treatment with ascorbate increases expression of Band 17. (4) Induction of hypertrophy in growth plate chondrocytes in short-term monolayer cultures correlates with a rapid but transient rise in Band 17 message. Our interpretation of these findings is that Band 17 expression is associated with the transition to hypertrophy, not maintenance of the hypertrophic phenotype. Molecular analysis of the 3' end of Band 17 cDNAs and genomic structure has shown that Band 17 is a single copy gene transcribed into four messages. Alternative splicing of these messages is predicted to result in two proteins that differ at the C-terminal by 131 amino acids. The longer protein contains a C-terminal consensus sequence that potentially targets this protein to the lumen of the endoplasmic reticulum. There is a Band 17 homologue in humans, suggesting conservation of Band 17 function in mammals. In summary, the pattern of expression and the predicted primary structure identify Band 17 as unique among all previously known chondrocyte genes.