Monitoring Therapy Response of Experimental Arthritis with Radiolabeled Tracers Targeting Fibroblasts, Macrophages, or Integrin αvß3.

UNLABELLED: Rheumatoid arthritis is an autoimmune disease resulting in chronic synovial inflammation. Molecular imaging could be used to monitor therapy response, thus enabling tailored therapy regimens and enhancing therapeutic outcome. Here, we hypothesized that response to etanercept could be monitored by radionuclide imaging in arthritic mice. We tested 3 different targets, namely fibroblast activation protein (FAP), macrophages, and integrin αvß3. METHODS: Male DBA/1J mice with collagen-induced arthritis were treated with etanercept. SPECT/CT scans were acquired at 1, 24, and 48 h after injection of (111)In-RGD2 (integrin αvß3), (111)In-anti-F4/80-A3-1 (antimurine macrophage antibody), or (111)In-28H1 (anti-FAP antibody), respectively, with nonspecific controls included. Mice were dissected after the last scan, and scans were analyzed quantitatively and were correlated with macroscopic scoring. RESULTS: Experimental arthritis was imaged with (111)In-28H1 (anti-FAP), (111)In-anti-F4/80-A3-1, and (111)In-RGD2. Tracer uptake in joints correlated with arthritis score. Treatment decreased joint uptake of tracers from 23 ± 15, 8 ± 4, and 2 ± 1 percentage injected dose per gram (%ID/g) to 11 ± 11 (P < 0.001), 4 ± 4 (P < 0.001), and 1 ± 0.2 %ID/g (P < 0.01) for (111)In-28H1, (111)In-anti-F4/80-A3-1, and (111)In-RGD2, respectively. Arthritis-to-blood ratios (in mice with arthritis score 2 per joint) were higher for (111)In-28H1 (5.5 ± 1; excluding values > 25), (111)In-anti-F4/80-A3-1 (10.4 ± 4), and (111)In-RGD2 (7.2 ± 1) than for control (111)In-DP47GS (0.7 ± 0.5; P = 0.002), (111)In-rat IgG2b (0.5 ± 0.2; P = 0.002), or coinjection of excess RGD2 (3.5), indicating specific uptake of all tracers in arthritic joints. CONCLUSION: (111)In-28H1, (111)In-anti-F4/80-A3-1, and (111)In-RGD2 can be used to specifically monitor the response to therapy in experimental arthritis at the molecular level. Further studies, however, still need to be performed.

Evaluation of Glucose Uptake in Normal and Cancer Cell Lines by Positron Emission Tomography.

To date, there is no definitive demonstration of the utility of positron emission tomography (PET) in studying glucose metabolism in cultured cell lines. Thus, this study was designed to compare PET to more standardized methods for the quantitative assessment of glucose uptake in nontransformed and transformed living cells and to validate PET for metabolic studies in vitro. Human colon and breast carcinoma cell lines and mouse embryo fibroblasts were evaluated for [(18)F]fluorodeoxyglucose ([(18)F]FDG) uptake by PET and autoradiography and 2-deoxyglucose (2-DG) incorporation by colorimetric assay and analyzed for the radiotoxic effects of [(18)F]FDG and the expression levels of glucose transporters. Indeed, [(18)F]FDG incorporation on PET was comparable to [(18)F]FDG uptake by autoradiography and 2-DG incorporation by colorimetric assay, although radiotracer-based methods exhibited more pronounced differences between individual cell lines. As expected, these data correlated with glucose transporters 1 to 4 and hexokinase II expression in tumor cell lines and mouse fibroblasts. Notably, [(18)F]FDG incorporation resulted in low apoptotic rates, with fibroblasts being slightly more sensitive to radiotracer-induced cell death. The quantitative analysis of [(18)F]FDG uptake in living cells by PET represents a valuable and reproducible method to study tumor cell metabolism in vitro, being representative of the differences in the molecular profile of normal and tumor cell lines.

PCH-2 regulates Caenorhabditis elegans lifespan.

Components or downstream targets of many signaling pathways such as Insulin/IGF-1 and TOR, as well as genes involved in cellular metabolism and bioenergetics can extend worm lifespan 20% or more. The C. elegans gene pch-2 and its homologs, including TRIP13 in humans, have been studied for their functions in cell mitosis and meiosis, but have never been implicated in lifespan regulation. Here we show that over-expression of TRIP13 in human fibroblasts confers resistance to environmental stressors such as UV radiation and oxidative stress. Furthermore, pch-2 overexpression in C. elegans extends worm lifespan, and enhances worm survival in response to various stressors. Conversely, reducing pch-2 expression with RNAi shortens worm lifespan. Additional genetic epistasis analysis indicates that the molecular mechanism of pch-2 in worm longevity is tied to functions of the sirtuin family, implying that pch-2 is another chromatin regulator for worm longevity. These findings suggest a novel function of the pch-2 gene involved in lifespan determination.

In-vivo comparison of the acute retention of stem cell derivatives and fibroblasts after intramyocardial transplantation in the mouse model.

PURPOSE: Various strategies have been applied to increase the engraftment of an intramyocardial cell transplant (Tx) to treat ischemic myocardium. Thereby, co-transplanted fibroblasts (FB) improve the long-term survival of stem cell derivatives (SCD) in a murine model of myocardial infarction. For therapeutic use, the time frame in which FB exert putative supportive effects needs to be identified. Therefore, we tracked the biodistribution and retention of SCD and FB in vivo using highly sensitive positron emission tomography (PET) imaging. METHODS: Murine [(18)F]-fluorodeoxyglucose (FDG) labeled SCD and FB were transplanted after left anterior descending artery (LAD) ligation into the border zone of the ischemic area in female C57BL/6 mice. Cardiac retention and biodistribution during the initial 2 h after injection were measured via PET imaging. RESULTS: Massive initial cell loss occurred independently of the cell type. Thereby, FB were retained slightly, yet significantly better than SCD until 60 min post-injection (7.5 ± 1.7 vs. 5.2 ± 0.7% ID at 25 min and 7.0 ± 1.5 vs. 4.8 ± 0.8% ID at 60 min). Thereafter, a fraction of ∼ 5% that withstood the massive initial washout remained at the site of injection independently of the applied cell type (120 min, SCD vs. FB P = 0.64). Most of the lost cells were detected in the lungs (∼ 30 % ID). CONCLUSIONS: We were able to quantitatively define the retention and biodistribution of different cell types via PET imaging in a mouse model after intramyocardial Tx. The utmost accuracy was achieved through this cell- and organ-specific approach by correcting PET data for cellular FDG efflux. Thereby, we observed a massive initial cell loss of ∼ 95%, causing low rates of long-term engraftment for both SCD and FB. We conclude that FB are not privileged compared to SCD regarding their acute retention kinetics, and therefore exert their beneficial effects at a later time point.

Bystander effect induced by UVC radiation in Chinese hamster V79 cells.

In past decades, researches on radiation-induced bystander effect mainly focused on ionizing radiation such as α-particle, ß-particle, X-ray and γ-ray. But few researches have been conducted on the ability of ultraviolet (UV) radiation-induced bystander effect, and knowledge of UVC-induced bystander effect is far limited. Here, we adopted medium transfer experiment to detect whether UVC could cause bystander effect in Chinese hamster V79 cells. We determined the cell viability, apoptosis rate, chromosome aberration and ultrastructure changes, respectively. Our results showed that: (1) the viability of UVC-irradiated V79 cells declined significantly with the dosage of UVC; (2) similar to the irradiated cells, the main death type of bystander cells cultured in irradiation conditioned medium (ICMs) was also apoptosis; (3) soluble factors secreted by UVC-irradiated cells could induce bystander effect in V79 cells; (4) cells treated with 4 h ICM collected from 90 mJ cm(-2) UVC-irradiated cells displayed the strongest response. Our data revealed that UVC could cause bystander effect through the medium soluble factors excreted from irradiated cells and this bystander effect was a novel quantitative and kinetic response. These findings might provide a foundation to further explore the exact soluble bystander factors and detailed mechanism underlying UVC-induced bystander effect.

Fibroblastic rheumatism: an addition to fibromatosis.

Fibroblastic rheumatism (FR) is a rare rheumatologic entity of unknown etiology. It is characterized by symmetrical polyarthritis associated with multiple cutaneous nodules. Bone erosion can occur as the disease progresses and destructive arthropathy is not rare. We report on an 18-year-old man with FR who presented a 6-year history of cutaneous nodules localized at para-articular sites with only minimal oligoarthralgia on exertion. There was no visceral involvement, and all the routine and immunological tests were normal. The diagnosis of FR was confirmed by histological examination of a nodule, which composed of myofibroblastic proliferation and thickened collagen fibers. Most skin lesions resolved after treated with IFN-α, however there was sequelae of permanent disability due to the progressive bone erosion despite weekly methotrexate treatment.

A study of the biological effects of modulated 6 MV radiation fields.

The delivery of spatially modulated radiation fields has been shown to impact on in vitro cell survival responses. To study the effect of modulated fields on cell survival, dose response curves were determined for human DU-145 prostate, T98G glioma tumour cells and normal primary AGO-1552 fibroblast cells exposed to modulated and non-modulated field configurations delivered using a 6 MV Linac with multi-leaf collimator. When exposed to uniform fields delivered as a non-modulated or modulated configuration, no significant differences in survival were observed with the exception of DU-145 cells at a dose of 8 Gy (p = 0.024). Survival responses were determined for exposure to non-uniform-modulated beams in DU-145 and T98G and showed no deviation from the survival response observed following uniform non-modulated exposures. The results of these experiments indicate no major deviation in response to modulated fields compared to uniform exposures.

Hyaluronic acid modulates gene expression of connective tissue growth factor (CTGF), transforming growth factor-beta1 (TGF-beta1), and vascular endothelial growth factor (VEGF) in human fibroblast-like synovial cells from advanced-stage osteoarthritis in vitro.

Intraarticular injection of hyaluronan (hyaluronic acid; HA) is the common way to treat osteoarthritis (OA) of knees. This treatment cannot only maintain the viscoelastic properties of knee but also release the OA pain. However, the exact molecular mechanism is unknown. In this study, after human synovial cells were stimulated with HA and Hylan (Synvisc) for 24 h, real-time polymerase chain reaction (real-time PCR) was used to detect the alteration of connective tissue growth factor (CTGF), transforming growth factor-beta1 (TGF-beta1), and vascular endothelial growth factor (VEGF) gene expression, which were specific genes related to pathogenesis of OA knees. Our results illustrated that both HA and Hylan might not cause cytotoxicity or apoptosis of synovial cells in serum deprivation environment. The gene expressions of TGF-beta1 and VEGF were significantly increased at the concentration of 0.1 mg/mL HA and 0.1 mg/mL Hylan, respectively (alpha < 0.05). The synovial cells with treatment of 0.1 mg/mL Hylan decreased the CTGF gene expression (0.66-fold) and VEGF (0.78-fold) compared to 0.1 mg/mL HA (alpha < 0.05). We suggested that the profile of CTGF, TGF-beta1, and VEGF gene expressions in our study might provide the rational mechanism for the therapeutic effect of hyaluronan on OA knees.

Functional bone engineering using ex vivo gene therapy and topology-optimized, biodegradable polymer composite scaffolds.

Bone tissue engineering could provide an alternative to conventional treatments for fracture nonunion, spinal fusion, joint replacement, and pathological loss of bone. However, this approach will require a biocompatible matrix to allow progenitor cell delivery and support tissue invasion. The construct must also support physiological loads as it degrades to allow the regenerated tissue to bear an increasing load. To meet these complex requirements, we have employed topology-optimized design and solid free-form fabrication to manufacture biodegradable poly(propylene fumarate)/beta-tricalcium phosphate composites. These scaffolds were seeded with primary human fibroblasts transduced with an adenovirus expressing bone morphogenetic protein-7 and implanted subcutaneously in mice. Specimens were evaluated by microcomputed tomography, compressive testing, and histological staining. New bone was localized on the scaffold surface and closely followed its designed contours. Furthermore, the total stiffness of the constructs was retained for up to 12 weeks after implantation, as scaffold degradation and tissue invasion took place.

Primary cardiac fibrosarcoma in a dog.

A primary cardiac fibrosarcoma in the right atrium of a 6-year-old Chihuahua dog is described. At necropsy, there was a firm, whitish and spherical mass in the right atrium. Histopathologically, the mass had moderate cellularity composed of spindle-shaped cells with scattered multinucleated giant cells. The tumor cells were arranged in interwoven bundles and sheets in the collagenous stroma. No metastases were observed. Ultrastructurally, the tumor cells mainly consisted of fibroblasts. Multinucleated giant cells did not have any certain organelles that would indicate a higher order of differentiation. Primary cardiac sarcomas in dogs are extremely rare.

[18F]-2-fluoro-2-deoxyglucose transport kinetics as a function of extracellular glucose concentration in malignant glioma, fibroblast and macrophage cells in vitro.

FDG-PET is used to measure the metabolic rate of glucose. Transport and phosphorylation determine the amount of hexose analog that is phosphorylated and trapped. Competition occurs for both events, such that extracellular glucose concentration affects the FDG image. This study investigated the effect of glucose concentration on the rate of FDG accumulation in three cell lines. The results show that extracellular glucose concentration has a greater impact on the rate of FDG accumulation than the relative abundance of GLUT transporter subtypes.

3-[123I]Iodo-L-alpha-methyl tyrosine transport into human fibroblasts and comparison with Ewing's sarcoma cells.

The cellular transport systems and the transport kinetics of [123I]IMT uptake into non-malignant extracranial cells were characterized for the first time. Human fibroblasts were chosen as non-malignant extracranial cells as they are found ubiquitous in the body. [123I]IMT is exclusively transported into fibroblasts via the sodium independent system L. An apparent Michaelis constant K(m) = 116.2 +/- 18.9 microM and a maximum transport velocity V(max) = 191.6 +/- 13.9 pmol x (10(6) cells)(-1) x min(-1) were calculated for the sodium-independent transport. These results were compared with those determined in two malignantly transformed extracranial cell lines, the human Ewing's sarcoma cell lines VH-64 and CADO-ES-1.

Genomic alterations associated with loss of heterozygosity for TP53 in Li-Fraumeni syndrome fibroblasts.

Studies of Li-Fraumeni syndrome fibroblasts heterozygous for germline TP53 mutations have shown that loss of heterozygosity (LOH) occurs during passaging and is associated with genomic instability, such as chromosomal aberrations and aneuploidy to investigate the genomic changes associated with LOH in Li-Fraumeni (LF) fibroblasts, we have analysed cell strains at increasing population doublings (PD) using Comparative Genomic Hybridization (CGH). We have looked at three groups of cell strains: LF mutation-carrying strains which showed LOH for TP53, LF mutation-carrying strains which did not show LOH, and strains from normal individuals. Using CGH, we have detected loss of distinct chromosomal regions associated with LOH in 4 out of 5 mutation-carrying strains. In particular we have found loss of chromosomal regions containing genes involved in cell cycle control or senescence, including loss of 9p and 17p in these strains. Other recurrent changes included loss of chromosomes 4q and 6q, regions shown to contain one or more tumour suppressor genes. No genomic alterations were detected at cumulative PD in the normal strains or in the LF mutation-carrying strains which did not show LOH for TP53. We have also analysed the three groups of strains for microsatellite instability and somatic TP53 mutations, and have found genetic alterations in only one strain.

Ultraviolet A radiation induces immediate release of iron in human primary skin fibroblasts: the role of ferritin.

In mammalian cells, the level of the iron-storage protein ferritin (Ft) is tightly controlled by the iron-regulatory protein-1 (IRP-1) at the posttranscriptional level. This regulation prevents iron acting as a catalyst in reactions between reactive oxygen species and biomolecules. The ultraviolet A (UVA) radiation component of sunlight (320-400 nm) has been shown to be a source of oxidative stress to skin via generation of reactive oxygen species. We report here that the exposure of human primary skin fibroblasts, FEK4, to UVA radiation causes an immediate release of "free" iron in the cells via proteolysis of Ft. Within minutes of exposure to a range of doses of UVA at natural exposure levels, the binding activity of IRP-1, as well as Ft levels, decreases in a dose-dependent manner. This decrease coincides with a significant leakage of the lysosomal components into the cytosol. Stabilization of Ft molecules occurs only when cells are pretreated with lysosomal protease inhibitors after UVA treatment. We propose that the oxidative damage to lysosomes that leads to Ft degradation and the consequent rapid release of potentially harmful "free" iron to the cytosol might be a major factor in UVA-induced damage to the skin.

Cytotoxic and anti-proliferative effects of high-energy pulsed ultrasound (HEPUS) on human squamous cell carcinoma cells as compared to connective tissue fibroblasts.

The cytotoxic and anti-proliferative effects of high-energy pulsed ultrasound (HEPUS) on human squamous cell carcinoma cells cloned from the hypopharynx (FaDu) and benign connective tissue cells (fibroblasts) were investigated in vitro. Sonication was carried out using an experimental piezoelectric, self-focusing burst-signal transducer. To increase the induction of cavitation, the transducer used was specifically designed to produce multiple oscillations with a high negative pressure amplitude. In both cell lines tested, the application of 100, 800 and 2000 pulses resulted in a high reduction of vital cells. After 2000 pulses, 4.0 +/- 1.1% of the fibroblasts but only 2.0 +/- 0.4% of the FaDu cells survived HEPUS exposure. A postexposure inhibiting effect of HEPUS for 10 days on the proliferation of surviving cells was noted for the FaDu cells exposed to 2000 pulses, but not as much for the fibroblasts. These findings support the hypothesis that human squamous cell carcinoma cells of the hypopharynx might be more sensitive to HEPUS than fibroblasts and that total tumor cell ablation might be possible in vitro given a sufficient number of HEPUS pulses.

Biophysical effects of high-energy pulsed ultrasound on human cells.

Human benign and malignant cells of different human origin (pancreas, liver, kidney, pharynx, tongue, lip) were exposed to high-energy pulsed ultrasound (HEPUS) in vitro to evaluate the effects of various physical parameters and sonication conditions on cell viability. This included the number of pulses, focal pressure, pulse repetition rate, pulse shape, cell suspension volume, water level of the basin and cell density. Cell viability was found to depend significantly on the number of pulses (exponential), the focal pressure (linear) and the pulse repetition rate (minimum at 1 Hz). Other parameters showed no marked influence. Furthermore, electron microscopy revealed intracellular damage, and proliferation rates of cells surviving sonication were normal after HEPUS exposure. The experimental piezoelectric ultrasound transducer used in the experiments generated oscillating bipolar pulses with high negative pressure amplitudes. Measurements were made of the pulse shape and ultrasonic field of the experimental device and of a conventional lithotripter for comparison.

Inhaled radon-induced genotoxicity in Wistar rat, Syrian hamster, and Chinese hamster deep-lung fibroblasts in vivo.

This study was performed (1) to provide a comparison of the genotoxic effects of inhaled radon and radon progeny, referred to as radon in this paper, among three species of rodents: Wistar rats, Syrian hamsters, and Chinese hamsters; (2) to determine if initial chromosome damage was related to the risk of induction of lung cancer; and (3) to evaluate the tissue repair and long-term presence of cytogenetic damage in respiratory tract cells. These species were selected because Syrian hamsters are very resistant to radon induction of lung cancer and Wistar rats are sensitive; no literature is available on the in vivo effects of radon in the Chinese hamster. Exposure-response relationships were established for the rats and Syrian hamsters while the Chinese hamsters received a single exposure of radon. At 4 h (0.2 days), 15 days, and 30 days after the highest WLM exposure to radon, Wistar rats, Chinese hamsters, and Syrian hamsters were killed, and lung fibroblasts were isolated and grown in culture to determine the frequency of induced micronuclei. Animals at each level of exposure showed an increase in the frequency of micronuclei relative to that in controls (P < 0.05). The exposure-response relationship data for rats and Syrian hamsters killed 0.2 days after the end of exposure were fit to linear equations (micronuclei/1000 binucleated cells = 15.5 +/- 14.4 + 0.53 +/- 0.06 WLM and 38.3 +/- 15.1 + 0.80 +/- 0.08 WLM, respectively). For the single exposure level used (496 WLM) in Chinese hamsters killed at 0.2 days after exposure, the frequency of micronuclei/1000 binucleated cells/WLM was 1.83 +/- 0.02. A comparison of the sensitivity for induction of micronuclei/WLM illustrated that Chinese hamsters were three times more sensitive than rats. The Syrian hamsters also showed a significantly elevated response (P < 0.05) relative to rats. These data suggest that initial chromosome damage is not the major factor responsible for the high rate of radon-induced cancer in rats relative to Syrian hamsters. The frequency of micronuclei in radon-exposed rats, Syrian hamsters, and Chinese hamsters significantly decreased (P < 0.05) as a function of time after the exposure. The rate of loss of damaged cells from the lung was greatest in the Chinese hamsters, followed by Wistar rats and Syrian hamsters, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

The response of normal and malignant cells to ultrasound in vitro.

The effect of ultrasonic irradiation on the viability of normal and tumor cell cultures derived from human and mouse origins was investigated. The cells were irradiated with a frequency of 2 MHz and intensity of 0.33 W/cm2, up to 4 min and immediately tested for cell viability using four different parameters: vital staining for the determination of the rate of cell growth; [3H]-thymidine and [3H]-leucine incorporation as an indication of the rate of DNA and protein synthesis respectively; and cloning efficiency as a measurement of the cell ability to multiply. Two human normal cell lines used in our studies, FS11 foreskin fibroblasts and Wish cells, were relatively resistant to ultrasonic irradiation effect although the growth rate of the latter was somewhat affected, particularly after 2 or 4 min of irradiation. However, cells derived from either malignant melanoma or breast carcinoma were highly sensitive to irradiation as demonstrated by a reduction of 96% and 65%, respectively, in cloning efficiency even after irradiation for 1 min. A third tumor cell line derived from lung carcinoma was more resistant. Two normal clones derived from NIH/3T3 mouse fibroblasts were used. These clones revealed some degree of sensitivity, particularly after 4 min of irradiation. However, their murine-sarcoma-virus transformed counterparts were found to be even more sensitive at identical times of ultrasonic irradiation, although the differences are not as striking as demonstrated with cells from human origin.(ABSTRACT TRUNCATED AT 250 WORDS)