Targeted optical fluorescence imaging: a meta-narrative review and future perspectives.

PURPOSE: The aim of this review is to give an overview of the current status of targeted optical fluorescence imaging in the field of oncology, cardiovascular, infectious and inflammatory diseases to further promote clinical translation. METHODS: A meta-narrative approach was taken to systematically describe the relevant literature. Consecutively, each field was assigned a developmental stage regarding the clinical implementation of optical fluorescence imaging. RESULTS: Optical fluorescence imaging is leaning towards clinical implementation in gastrointestinal and head and neck cancers, closely followed by pulmonary, neuro, breast and gynaecological oncology. In cardiovascular and infectious disease, optical imaging is in a less advanced/proof of concept stage. CONCLUSION: Targeted optical fluorescence imaging is rapidly evolving and expanding into the clinic, especially in the field of oncology. However, the imaging modality still has to overcome some major challenges before it can be part of the standard of care in the clinic, such as the provision of pivotal trial data. Intensive multidisciplinary (pre-)clinical joined forces are essential to overcome the delivery of such compelling phase III registration trial data and subsequent regulatory approval and reimbursement hurdles to advance clinical implementation of targeted optical fluorescence imaging as part of standard practice.

Magnetic transition and spin fluctuations in the unconventional antiferromagnetic compound Yb3Pt4.

Muon spin rotation and relaxation measurements have been carried out on the unconventional antiferromagnet Yb3Pt4. Oscillations are observed below T(N) = 2.22(1) K, consistent with the antiferromagnetic (AFM) Néel temperature observed in bulk experiments. In agreement with neutron diffraction experiments the oscillation frequency ω(µ)(T)/2π follows an S = 1/2 mean-field temperature dependence, yielding a quasistatic local field of 1.71(2) kOe at T = 0. A crude estimate gives an ordered moment of ∼ 0.66 µ(B) at T = 0, comparable to 0.81 µ(B) from neutron diffraction. As T-->T(N) from above the dynamic relaxation rate λ(d) exhibits no critical slowing down, consistent with a mean-field transition. In the AFM phase a T-linear fit to λ(d)(T), appropriate to a Fermi liquid, yields highly enhanced values of λ(d)/T and the Korringa constant K(µ)(2)T/λ(d), with K(µ) the estimated muon Knight shift. A strong suppression of λ(d) by applied field is observed in the AFM phase. These properties are consistent with the observed large Sommerfeld-Wilson and Kadowaki-Woods ratios in Yb3Pt4 (although the data do not discriminate between Fermi-liquid and non-Fermi-liquid states), and suggest strong enhancement of q≈0 spin correlations between large-Fermi-volume band quasiparticles in the AFM phase of Yb3Pt4.

Six months of recombinant human GH therapy in patients with ischemic cardiac failure does not influence left ventricular function and mass.

Beneficial effects of recombinant human GH on cardiac function have been reported in humans with GH deficiency and in patients with idiopathic dilated cardiomyopathy. No randomized controlled trial has been performed on the effects of recombinant human GH on cardiac function in patients with ischemic cardiac failure. We therefore randomly assigned 22 patients with ischemic cardiac failure (left ventricular ejection fraction, <40%; 19 men and 3 women; mean age, 64 yr) to receive 6 months of unblinded therapy with recombinant human GH (2.0 IU/d) or no treatment. Primary end points were left ventricular ejection fraction and left ventricular mass. Left ventricular end-diastolic volume, left ventricular end-systolic volume, and myocardial perfusion, both at rest and during exercise, were assessed as well. Cardiac imaging techniques were electrocardiographically gated single photon emission computer tomography and magnetic resonance imaging. In addition, biochemical and biometric measurements were performed. Nineteen patients completed the study (10 controls and 9 GH-treated subjects). IGF-I and IGF-binding protein-3 increased significantly after recombinant human GH treatment (+24% and +58%, respectively) compared with control values (-14% and +5%; P < 0.05). Left ventricular ejection fraction, left ventricular end-diastolic volume, left ventricular end-systolic volume, left ventricular mass, and myocardial perfusion were not influenced by recombinant human GH therapy. We conclude that 6 months of recombinant human GH treatment in patients with ischemic cardiac failure had no beneficial effect on left ventricular function and mass.

Additional beneficial effects of alendronate in growth hormone (GH)-deficient adults with osteoporosis receiving long-term recombinant human GH replacement therapy: a randomized controlled trial.

We conducted a randomized controlled trial in osteoporotic adult GH-deficient (GHD) patients to assess whether additional treatment with a bisphosphonate would further favorably influence parameters of bone turnover and bone mineral density measurements (BMD). All patients were receiving stable recombinant human (rhGH) replacement therapy for 4 yr at the start of the study. Eighteen GHD patients with osteoporosis were randomized to continue their rhGH maintenance dose or to receive combination therapy with rhGH and alendronate for 12 months. All patients were calcium and vitamin replete, and there were no changes in calcium, vitamin D, or hormone replacement therapy for the duration of the study. At baseline there were no significant differences between the alendronate and the control group in parameters of bone turnover, BMD, or prevalence of vertebral fractures. Childhood-onset and adult-onset GHD were equally distributed between the groups, with no statistical differences in age and gender or other parameters between groups. Mean serum osteocalcin, serum bone-specific alkaline phosphatase, and urinary N-telopeptide/creatinine ratio were within the normal range at the start of the study. In the alendronate group all measured parameters of bone turnover, i.e. bone-specific alkaline phosphatase, osteocalcin, and urinary N-telopeptide/creatinine ratio, significantly decreased after 6 months, with no further decrease thereafter. No changes were observed in the control group. In the alendronate-treated patients serum bone-specific alkaline phosphatase decreased from 10.9 +/- 0.9 to 6.8 +/- 0.7 microg/L at 6 months (P < 0.001), serum osteocalcin decreased from 3.9 +/- 0.4 to 1.7 +/- 0.3 microg/L (P < 0.001), and the urinary N-telopeptide/creatinine ratio decreased from 27.3 +/- 7.0 to 6.4 +/- 0.8 nmol/mmol (P = 0.01). In this group, lumbar spine BMD significantly increased from baseline by 3.4% at 6 months (P = 0.001) and by 4.4% at 12 months (P < 0.001) of treatment, with no further significant increase between 6 and 12 months (P = 0.217). No changes in lumbar spine BMD were observed in the control group. There were no significant changes in femoral neck BMD in either group for the duration of the study. No incident vertebral or peripheral fractures were documented in either group at the end of the study. In summary, this is the first report indicating that treatment with alendronate was able to significantly increase BMD at the lumbar spine in GHD patients with osteoporosis receiving stable rhGH replacement for 4 yr. This increase was significantly greater in alendronate-treated patients than in patients maintained on rhGH. The increase in lumbar spine BMD in the alendronate-treated patients was associated with a decrease in the measured markers of bone turnover, whereas these markers did not change further in the patients maintained on rhGH. This controlled study suggests that additional treatment with alendronate in GHD patients with osteoporosis receiving stable rhGH replacement therapy is effective in increasing BMD at the lumbar spine. Further investigation is required to assess whether rhGH replacement alone or combined treatment with rhGH and alendronate is able to reduce the increased fracture risk associated with GHD.

Mitochondrial-derived oxidants and quartz activation of chemokine gene expression.

Macrophage inflammatory protein 2 (MIP-2) is a chemotactic cytokine which mediates neutrophil recruitment in the lung and other tissues. Pneumotoxic particles such as quartz increase MIP-2 expression in rat lung and rat alveolar type II epithelial cells. Deletion mutant analysis of the rat MIP-2 promoter demonstrated quartz-induction depended on a single NFkappaB consensus binding site. Quartz activation of NFkappaB and MIP-2 gene expression in RLE-6TN cells was inhibited by anti-oxidants suggesting the responses were dependent on oxidative stress. Consistent with anti-oxidant effects, quartz was demonstrated to increase RLE-6TN cell production of hydrogen peroxide. Rotenone treatment of RLE-6TN cells attenuated hydrogen peroxide production, NFkappaB activation and MIP-2 gene expression induced by quartz indicating that mitochondria-derived oxidants were contributing to these responses. Collectively, these findings indicate that quartz and crocidolite induction of MIP-2 gene expression in rat alveolar type II cells results from stimulation of an intracellular signaling pathway involving increased generation of hydrogen peroxide by mitochondria and subsequent activation of NFkappaB.

Beta-catenin, an inducer of uncontrolled cell proliferation and migration in malignancies, is localized in the cytoplasm of vascular endothelium during neovascularization after myocardial infarction.

Beta-catenin is a protein involved in cell-cell adhesion and proliferation. In neoplastic diseases, defects in the regulation of the cellular beta-catenin content and cytoplasmic accumulation of the protein contribute to the uncontrolled cell proliferation and migration. Whether beta-catenin plays a role in the controlled proliferative and migratory responses to injury, eg, of vascular endothelial cells during neovascularization after myocardial infarction (MI), is not known. In the present study, we examined the localization of beta-catenin in the infarcted rat heart at different time points after MI. Cytoplasmic beta-catenin was observed in the endothelial cells of the newly formed and pre-existing blood vessels in the infarct area in the first week after MI, but not in the uninjured parts of the heart and not at later time points. Adenomatous polyposis coli (APC) protein was also detected; interaction of APC with beta-catenin has been reported to be critical in epithelial tube formation in vitro. Moreover, the expression of dishevelled-1, an upstream regulatory molecule of the cellular beta-catenin content, was observed in vascular endothelial cells in the infarct area. These findings suggest a role for the beta-catenin-APC complex in the proliferation and migration of vascular endothelial cells during neovascularization of the infarct area.

Production of the acute-phase protein lipopolysaccharide-binding protein by respiratory type II epithelial cells: implications for local defense to bacterial endotoxins.

This study demonstrates for the first time that respiratory epithelial cells are able to produce the acute phase protein lipopolysaccharide (LPS)-binding protein (LBP), which is known to play a central role in the defense to bacterial endotoxins (or LPS). Indications for local presence of LBP in human lung was obtained via reverse transcriptase/polymerase chain reaction that showed LBP messenger RNA (mRNA) expression. Therefore, LBP production by the human lung epithelial cell line A549, a human adenocarcinoma with features of type II pneumocytes, was studied. These cells produced LBP in response to interleukin (IL)-1beta, IL-6, and tumor necrosis factor- alpha, a response that was strongly enhanced by dexamethasone. In addition, LBP mRNA was detected in A549 cells, in increasing amounts as a result of stimulation. The pattern of cytokine-induced LBP production in A549 cells was similar to the pattern in the human liver epithelial cell line HuH-7. Moreover, the molecular weight of A549-derived LBP was approximately 60 kD, which is similar to HuH-7-derived LBP. Biologic activity of LBP produced by A549 cells was evaluated on the basis of its ability to interact with LPS. Further indications that type II alveolar epithelial cells are able to produce LBP were obtained from the observations that the murine lung type II epithelial cell line C10 produced murine LBP, and that isolated human primary type II pneumocytes expressed LBP mRNA, which was enhanced after stimulation of cells. The local production of this endotoxin binding protein by lung epithelial cells might contribute to a highly specific response at the site of exposure to bacteria and bacterial endotoxins.

A switch from oral (2 mg/day) to transdermal (50 microg/day) 17beta-estradiol therapy increases serum insulin-like growth factor-I levels in recombinant human growth hormone (GH)-substituted women with GH deficiency.

The response to GH therapy in adults with GH deficiency (GHD) is considerably variable. Generally, the response with regard to serum insulin-like growth factor (IGF)-I concentrations is significantly lower in females compared with males with GHD, which could at least partly be explained by the use of oral estrogen replacement therapy. In the present study, we investigated whether a switch from oral to transdermal estrogen therapy alters serum IGF-I concentrations in women with GHD on stable GH therapy. Six females with GHD and LH deficiency were investigated. During cycles 1 and 2, an oral dose of estradiol was given (2 mg/day), whereas during cycles 3, 4, and 5 estradiol was administered via the transdermal route at a dose of 50 microg/day. Serum estrone levels significantly decreased (2470+/-475 to 110+/-26 pmol/L, P = 0.005), serum sex hormone-binding globulin levels significantly decreased (102+/-13 to 63+/-7 nmol/L, P = 0.004), and serum estradiol levels also decreased albeit nonsignificantly with transdermal therapy (273+/-81 to 114+/-18, P = 0.083). Serum IGF-I levels significantly increased after the switch from oral to transdermal estrogen therapy (18.7+/-1.6 and 23.4+/-2.5 nmol/L, respectively, P = 0.008). Two of the six patients experienced fluid retention-related side effects, which disappeared after a reduction in dose at the end of the study. The results of the present study suggest that the potency of GH is altered in patients on transdermal compared to oral estradiol therapy. Further investigation should be undertaken to answer the question whether the increase in serum IGF-I levels is due to lower serum levels of estradiol or to differences in the mode of administration of estradiol.

Nitrotyrosine formation in the airways and lung parenchyma of patients with asthma.

BACKGROUND: Recent evidence has shown that nitric oxide (NO) levels are increased in asthmatic airways. Although the role of NO in asthma is unknown, reactive metabolites of NO may lead to nitrotyrosine formation and promote airway dysfunction. OBJECTIVE: The aim of this study was to determine whether nitrotyrosine, as a marker of nitrating species, could be found in the airways and lung parenchyma of subjects with asthma who died of status asthmaticus or other nonrespiratory causes. METHODS: Lung tissue specimens were obtained from 5 patients who died of status asthmaticus, 2 asthmatic patients who died of nonrespiratory causes, and 6 nonasthmatic control subjects who died of nonrespiratory causes. Lung sections were stained for immunofluorescence with use of an antinitrotyrosine antibody, followed by a indiocarbocyanine (Cy5, Jackson Immunochemicals, Westgrove, Pa)-conjugated secondary antibody. RESULTS: Nonasthmatic lungs showed little or no nitrotyrosine staining, whereas asthmatic lungs demonstrated significantly more staining of nitrotyrosine residues distributed in both the airways and lung parenchyma. CONCLUSION: This study demonstrates the presence of nitrotyrosine, and hence evidence of formation of nitrating species, in the airways and lung parenchyma of patients with asthma who died of status asthmaticus or other nonrespiratory causes. This finding supports the concept that widespread airway and parenchymal inflammation occurs in asthma, and, more specifically, that NO and its reactive metabolites may play a pathophysiologic role in asthma.

The absorption profile and availability of a physiological subcutaneously administered dose of recombinant human growth hormone (GH) in adults with GH deficiency.

AIMS: To investigate the absorption profile and estimate the bioavailability of three doses of recombinant human growth hormone (rhGH) smaller than 2 IU in females with GH deficiency (GHD). A second aim of the study was to compare the mean 24 h GH concentrations after s.c. injection of rhGH with the physiological mean 24 h GH concentration of healthy females of comparable age, height, and BMI. METHODS: Fourteen female patients with substituted GHD, and 14 healthy females of comparable age, height, and BMI were studied. All GHD patients underwent 24 h GH sampling after s.c. injection of rhGH in doses of 0.6, 1.2, or 1.8 IU. In addition, these patients underwent a 4 h GH sampling after i.v. injection of rhGH (1 IU). In healthy subjects, blood was withdrawn every 10 min for 24 h to determine the physiological GH profile. RESULTS: A s.c. dose of 0.6 IU resulted in a mean and maximum GH concentration of 0.95+/-0.04 mU l(-1) and 2.62+/-0.09 mU l(-1). A doubling (or tripling) of the rhGH dose resulted in a doubling (or tripling) of the mean and maximum GH concentration. The time of maximum GH concentration was reached on average after 261+/-27 min. Mean GH concentration in healthy females was comparable with the mean GH concentration after a s.c. dose of 1.2 IU. Mean availability of the s.c. injected dose was 63%+/-4%. CONCLUSIONS: A dose of 1.2 IU resulted in a mean GH concentration comparable with the mean physiological GH concentration in healthy females of comparable age, height, and BMI.

Nuclear factor kappa B activity in response to oxidants and antioxidants.

The goal of this chapter was to review the current protocols that are available to measure the activation of NF-kappa B. The methods discussed all have their pitfalls when used in isolation. To obtain meaningful information, nuclear translocation and transcriptional activation should be studied in conjunction. Study of NF-kappa B regulated protein expression is the most physiologically relevant approach to monitoring the transcription regulatory effect of NF-kappa B. Because of the limitations of transcriptional analysis in primary cell cultures or tissues, incorporation of multiple approaches is recommended when the involvement of NF-kappa B in a disease process is evaluated.

Changes in muscle volume, strength, and bioenergetics during recombinant human growth hormone (GH) therapy in adults with GH deficiency.

Adults with GH deficiency (GHD) suffer from muscle weakness, which can be caused by the frequently reported decrease in muscle mass. However, measurements of both muscle strength and mass of muscle tested are scarce in adults with GHD. The aim of the present study was, therefore, to investigate intrinsic muscle strength (strength expressed per muscle volume unit) in adults with GHD at baseline and after 52 weeks of recombinant human GH (rhGH) therapy given in low, more physiological doses. A second objective was to investigate the influence of GH on muscle bioenergetics in the resting muscle. Isometric and isokinetic quadriceps strengths were measured in 28 males with GHD and in healthy controls matched for age and height. Quadriceps mass, determined by magnetic resonance imaging, and muscle bioenergetics, determined by phosphorus nuclear magnetic resonance spectroscopy, were measured in 20 of 28 patients with GHD and in controls matched for age and height. All patients were treated with doses of rhGH ranging from 0.6-1.8 IU/day, given for 52 weeks. Measurements of muscle mass, strength, and bioenergetics were repeated after 52 weeks of treatment with rhGH. The mean GH dose at 52 weeks of rhGH treatment was 1.3 +/- 0.8 IU/day. The mean serum insulin-like growth factor I level at baseline was 9.4 +/- 0.7 nmol/L and significantly increased to 26.4 +/- 1.2 nmol/L after 52 weeks of rhGH treatment. Adults with GHD had significantly reduced quadriceps muscle mass (P = 0.034) and reduced isometric muscle strength (P = 0.002) and tended to have low isokinetic muscle strength (P = 0.06), which all improved after rhGH therapy. Intrinsic muscle strength was not significantly different in adults with GHD compared with that in healthy controls and did not change during rhGH therapy. No bioenergetic abnormalities at baseline or after rhGH therapy were found in males with GHD. In conclusion, quadriceps muscle mass is decreased in adults with GHD and increased with rhGH therapy. These changes in muscle mass account for the changes in muscle strength found in these patients, as no changes in intrinsic muscle strength were found.

Depletion of nitric oxide causes cell cycle alterations, apoptosis, and oxidative stress in pulmonary cells.

Nitric oxide (NO.) is important in the regulation of mitochondrial function, cell signaling, and gene expression. To elucidate how endogenous NO. regulates the function of airway epithelial cells, we used carboxy-PTIO, a hydrophilic, negatively charged NO. trap, to scavenge NO. from rat lung epithelial (RLE) and rat pleural mesothelial (RPM) cells and to determine the elicitation of cell cycle alterations, apoptosis, and oxidative stress. The reaction of NO. with PTIO causes the formation of PTI, which is measured by electron spin resonance (ESR) and is a quantitative measure of NO. formation. ESR spectroscopy revealed the production of NO. in RLE or RPM cells over a period from 1 to 24 h of exposure, indicating scavenging of NO. by PTIO. Cycle analyses in confluent RLE or RPM cells revealed two- to threefold increases in S and G2/M phases after exposure to 100-200 microM PTIO as well as increases in the fraction of cells undergoing apoptosis. Direct addition of PTI to cells failed to elicit cell cycle perturbations or apoptosis. The guanylyl cyclase inhibitor ODQ mimicked the effects of PTIO. 8-Bromo-cGMP but not 8-bromo-cAMP ameliorated the PTIO- or ODQ-mediated cell cycle perturbations and apoptosis, suggesting that cGMP-dependent pathways are involved in these cell cycle perturbations. Treatment of log-phase cells with PTIO resulted in more dramatic cell cycle perturbations compared with cells treated at confluence. Assessment of 5-bromo-2'-deoxyuridine incorporation to measure DNA synthesis demonstrated decreases in PTIO-treated compared with sham cells in addition to a cell cycle arrest in late S or G2/M phase. Last, incubation with dichlorofluorescin diacetate revealed oxidative stress in PTIO- but not in PTI-exposed RLE or RPM cells. We conclude that the depletion of endogenous NO. induces oxidative stress, cell cycle perturbations, and apoptosis. Our findings illustrate the importance of endogenous NO. in the control of cell cycle progression and survival of pulmonary and pleural cells and that a critical balance between NO. and superoxide may be necessary for these physiological events.

Crocidolite activates NF-kappa B and MIP-2 gene expression in rat alveolar epithelial cells. Role of mitochondrial-derived oxidants.

Nuclear factor kappa B (NF-kappa B) is a transcription factor that regulates expression of several genes coding for inflammatory and immunoregulatory proteins including the neutrophil chemotactic cytokine MIP-2. In previous studies we found that crocidolite asbestos activates the nuclear translocation of NF-kappa B as well as MIP-2 gene expression in rat alveolar type II cells. Here we report that both crocidolite-induced NF-kappa B activation of MIP-2 gene expression can be attenuated by the antioxidant tetramethylthiourea, suggesting the dependence of these responses on oxidative stress. Crocidolite exposure of RLE-TN cells also increased production of H2O2, a response that was inhibited by the mitochondrial respiratory chain inhibitor thenoyltrifluoroacetone (TTFA). TTFA treatment of RLE-6TN cells also inhibited crocidolite-induced nuclear translocation of NF-kappa B and MIP-2 gene expression. These results indicate crocidolite exposure of rat alveolar type II cells results in increased production of mitochondrial-derived hydrogen peroxide and that mitochondrial-derived oxidants contribute to crocidolite activation of NF-kappa B and increases in MIP-2 gene expression.

Modulation of mitochondrial gene expression in pulmonary epithelial cells exposed to oxidants.

Oxidants are important in the regulation of signal transduction and gene expression. Multiple classes of genes are transcriptionally activated by oxidants and are implicated in different phenotypic responses. In the present study, we performed differential mRNA display to elucidate genes that are induced or repressed after exposure of rat lung epithelial (RLE) cells to H2O2 or crocidolite asbestos, a pathogenic mineral that generates oxidants. After 8 or 24 hr of exposure, RNA was extracted, reverse transcribed, and amplified by polymerase chain reaction with degenerate primers to visualize alterations in gene expression. The seven clones obtained were sequenced and encoded the mitochondrial genes, NADH dehydrogenase subunits ND5 and ND6, and 16S ribosomal RNA. Evaluation of their expression by Northern blot analysis revealed increased expression of 16S rRNA after 1 or 2 hr of exposure to H2O2. At later time periods (4 and 24 hr), mRNA levels of 16S rRNA and NADH dehydrogenase were decreased in H2O2-treated RLE cells when compared to sham controls. Crocidolite asbestos caused increases in 16S rRNA levels after 8 hr of exposure, whereas after 24 hr of exposure to asbestos, 16S rRNA levels were decreased in comparison to sham controls. In addition to these oxidants, the nitric oxide generator spermine NONOate caused similar decreases in NADH dehydrogenase mRNA levels after 4 hr of exposure. The present data and previous studies demonstrated that all oxidants examined resulted in apoptosis in RLE cells during the time frame where alterations of mitochondrial gene expression were observed. As the mitochondrion is a major organelle that controls apoptosis, alterations in expression of mitochondrial genes may be involved in the regulation of apoptosis.

Patterns of c-fos and c-jun proto-oncogene expression, apoptosis, and proliferation in rat pleural mesothelial cells exposed to erionite or asbestos fibers.

Erionite, a naturally occurring fibrous zeolite, is associated with the development of nonmalignant and malignant lung diseases and is more carcinogenic than asbestos fibers in man and rodent inhalation models of disease. To investigate the possible molecular mechanisms of erionite-induced toxicity and carcinogenesis and whether cationic content of erionite fibers was important, we examined c-fos and c-jun mRNA levels, activator protein-1 (AP-1) binding to DNA, and changes in cell proliferation and apoptosis in rat pleural mesothelial (RPM) cells exposed to different cation-substituted erionite fibers or crocidolite asbestos at various concentrations (1, 5, or 10 microg/cm2 dish) at time periods from 8 to 48 h after addition of minerals. c-fos mRNA levels in cells exposed to equal weight concentrations of various erionites and crocidolite fibers were increased comparably. When compared to other fibers, Na-erionite caused significantly increased levels of c-jun mRNA at lower mass concentrations (1 and 5 microg/cm2) than crocidolite asbestos, but comparable AP-1 binding to DNA. In comparison to untreated controls, numbers of RPM cells incorporating 5'-bromodeoxyuridine (BrdU) were increased dramatically after exposure to asbestos or Na-erionite at 5 and 10 microg/cm2. Significant dose-dependent increases in apoptosis were observed with asbestos at all time points, whereas erionites failed to induce apoptosis at 8 or 24 h, with minimal induction at higher concentrations than asbestos at 48 h. These data suggest that erionite increases the balance between cell proliferation (and/or abnormal DNA repair) and apoptosis, a normal mechanism of elimination of transformed or proliferating cells.

Skeletal effects of two years of treatment with low physiological doses of recombinant human growth hormone (GH) in patients with adult-onset GH deficiency.

A low bone mass in adults with childhood-onset GH deficiency (GHD) is likely to be caused by deficient bone accretion during childhood and early adulthood, whereas a decreased bone mass in patients with adult-onset GHD is likely to be caused by an imbalance in bone remodeling. Data on bone mineral density (BMD) and biochemical parameters of bone metabolism and data on response of these parameters to treatment with GH are scarce in patients with adult-onset GHD. It has been suggested that in patients with GHD, GH at the relatively high dose originally used may have beneficial effects on the skeleton. To address the question as whether lower, more physiological doses would have similar effects on the skeleton, we studied 47 patients with adult-onset GHD (27 women and 20 men, range 26-70 yr) randomized to receive one of three recombinant human GH (rhGH) dose regimens: 0.6 IU/day, 1.2 IU/day, or 1.8 IU/day as part of a study examining optimal GH dose replacement therapy. After 24 weeks of treatment, the dose of rhGH was individually adjusted to maintain the concentration of serum insulin growth factor-I within the normal laboratory reference range. Biochemical parameters of bone metabolism were measured at baseline and after 24 and 52 weeks and 2 yr of treatment. BMD of the lumbar spine was measured at baseline and after 52 weeks and 2 yr of treatment. Parameters of bone metabolism generally fell within the low-normal range and increased in a dose-dependent manner at 24 weeks of treatment. Between 24 and 52 weeks of rhGH treatment, mean serum osteocalcin levels and alkaline phosphatase activity further increased, whereas mean 24-h urine hydroxyproline/creatinine and N-telopeptide/creatinine excretion remained unchanged. After 52 weeks of treatment, serum alkaline phosphatase activity and 24-h urine hydroxyproline/ creatinine excretion decreased, although not to pretreatment levels. Mean BMD at the lumbar spine (Z-score) was normal at baseline (-0.20 +/- 0.16) and increased during treatment (at 2 yr of treatment: 0 +/- 0.20; P < 0.005). Our data suggest that a low physiological dose of rhGH, individually adjusted to maintain serum insulin-like growth factor I levels within the normal laboratory reference range, increased bone turnover in favor of bone formation, as suggested by the significant, albeit small increase in BMD observed after 2 yr of treatment. Further studies are required to establish whether in patients with adult-onset GHD the preservation and/or increase in bone mass observed with the use of physiological doses of rhGH could be maintained with longer-term treatment.

GRP78, HSP72/73, and cJun stress protein levels in lung epithelial cells exposed to asbestos, cadmium, or H2O2.

Occupational exposure to crocidolite asbestos is associated with the development of nonmalignant and malignant pulmonary disease. Considerable evidence indicates that the mechanisms of asbestos-induced toxicity involve the production of active oxygen species (AOS). Production of AOS in excess of cellular defenses creates an environment of oxidative stress and stimulates the expression of a number of different genes whose products may be involved in mediating responses from oxidant injury. To further investigate the mechanisms of asbestos-induced pathogenicity, we have examined by Western blot analyses the induction of the stress response proteins GRP78 and HSP72/73 in rat lung epithelial cells (RLE) exposed to crocidolite asbestos. In comparative studies, we also examined GRP78, HSP72/73, and cJun expression in RLE cells exposed to equitoxic concentrations of cadmium chloride (CdCl2) and hydrogen peroxide (H2O2). Our results demonstrate that asbestos and H2O2 do not alter GRP78 or HSP72/73 protein levels in RLE cells, but do increase levels of cJun protein. Increases by asbestos and H2O2 were not accompanied by alterations in cellular glutathione levels in this cell type, but asbestos caused elevations in protein levels of manganese-containing superoxide dismutase (MnSOD), an indirect indicator of oxidant stress. In contrast, exposure of cells to CdCl2 led to no changes in MnSOD protein levels, but increases in GRP78, HSP72/73, and cJun proteins as well as significant increases in oxidized and reduced thiol pools. Results suggest that environmental agents causing oxidative injury to lung epithelium elicit different patterns of stress responses.

Increased epidermal growth factor-receptor protein in a human mesothelial cell line in response to long asbestos fibers.

Epidermal growth factor (EGF) is a potent mitogen for human mesothelial cells, and autophosphorylation of the EGF receptor (EGF-R) occurs in these cell types after exposure to asbestos, a carcinogen associated with the development of mesothelioma. Here, the intensity and distribution of EGF-R protein was documented by immunocytochemistry in a human mesothelial cell line (MET5A) exposed to various concentrations of crocidolite asbestos and man-made vitreous fibers (MMVF-10). Whereas cells in contact with or phagocytizing shorter asbestos fibers (<60 microm length) or MMVF-10 at a range of concentrations showed no increase in EGF-R protein as determined by immunofluorescence, elongated cells phagocytizing and surrounding longer fibers (> or =60 microm) showed intense staining for EGF-R. In contrast, human A549 lung carcinoma cells showed neither elongation nor increased accumulation of EGF-R protein in response to long fibers. Patterns of aggregation and increases in EGF-R protein in mesothelial cells phagocytizing long asbestos fibers were distinct from diffuse staining of phosphotyrosine residues observed in asbestos-exposed cultures. These studies indicate that aggregation of EGF-R by long fibers may initiate cell signaling cascades important in asbestos-induced mitogenesis and carcinogenesis.

Asbestos increases mammalian AP-endonuclease gene expression, protein levels, and enzyme activity in mesothelial cells.

Only two DNA repair enzymes, DNA polymerase beta and O6-methylguanine-DNA methyltransferase, have been shown to be inducible in mammalian cells by genotoxic agents. We show here that crocidolite asbestos induces the DNA repair enzyme, apurinic/apyrimidinic (AP)-endonuclease, in isolated mesothelial cells, the progenitor cells of malignant mesothelioma. Asbestos at nontoxic concentrations of 1.25 and 2.5 microg/cm2 significantly increased AP-endonuclease mRNA and protein levels as well as enzyme activity (P < 0.05) in a dose-dependent manner in rat pleural mesothelial cells. These increases were persistent from 24 to 72 h after initial exposure to fibers. Changes were not observed with glass beads, a noncarcinogenic particle. Confocal scanning laser microscopy showed that AP-endonuclease was primarily localized in the nucleus but also in mitochondria. Our data are the first to demonstrate the inducibility of AP-endonuclease by a human class I carcinogen associated with oxidant stress in normal cells of the lung.