Microchimerism in the rheumatoid nodules of patients with rheumatoid arthritis.

OBJECTIVE: The rheumatoid nodule is a lesion commonly found on extraarticular areas prone to mechanic trauma. When present with inflammatory symmetric polyarthritis, it is pathognomonic of rheumatoid arthritis (RA), an autoimmune disease in which naturally acquired microchimerism has previously been described and can sometimes contribute to RA risk. Since RA patients harbor microchimerism in the blood, we hypothesized that microchimerism is also present in rheumatoid nodules and could play a role in rheumatoid nodule formation. This study was undertaken to investigate rheumatoid nodules for microchimerism. METHODS: Rheumatoid nodules were tested for microchimerism by real-time quantitative polymerase chain reaction (qPCR). The rheumatoid nodules of 29 female patients were tested for a Y chromosome-specific sequence. After HLA genotyping of patients and family members, rheumatoid nodules from 1 man and 14 women were tested by HLA-specific qPCR, targeting a nonshared HLA allele of the potential microchimerism source. Results were expressed as genome equivalents of microchimeric cells per 10(5) patient genome equivalents (GE/10(5)). RESULTS: Rheumatoid nodules from 21% of the female patients contained male DNA (range <0.5, 10.3 GE/10(5)). By HLA-specific qPCR, 60% of patients were microchimeric (range 0, 18.5 GE/10(5)). Combined microchimerism prevalence was 47%. A fetal or maternal source was identified in all patients who tested positive by HLA-specific qPCR. Unexpectedly, a few rheumatoid nodules also contained microchimerism without evidence of a fetal or maternal source, suggesting alternative sources. CONCLUSION: Our findings indicate that microchimerism is frequently present in the rheumatoid nodules of RA patients. Since microchimerism is genetically disparate, whether microchimerism in rheumatoid nodules serves as an allogeneic stimulus or allogeneic target warrants further investigation.

Characterization of cationic liposome formulations designed to exhibit extended plasma residence times and tumor vasculature targeting properties.

Cationic liposomes exhibit a propensity to selectively target tumor-associated blood vessels demonstrating potential value as anti-cancer drug delivery vehicles. Their utility however, is hampered by their biological instability and rapid elimination following i.v. administration. Efforts to circumvent rapid plasma elimination have, to date, focused on decreasing cationic lipid content and incorporating polyethylene glycol (PEG)-modified lipids. In this study we wanted to determine whether highly charged cationic liposomes with surface-associated PEG could be designed to exhibit extended circulation lifetimes, while retaining tumor vascular targeting properties in an HT29 colorectal cancer xenograft model. Cationic liposomes prepared of DSPC, cationic lipids (DODAC, DOTAP, or DC-CHOL), and DSPE-PEG(2000) were studied. Our results demonstrate that formulations prepared with 50 mol% DODAC or DC-CHOL, and 20 mol% DSPE-PEG(2000) exhibited circulation half-lives ranging from 6.5 to 12.5 h. Biodistribution studies demonstrated that DC-CHOL formulations prepared with DSPE-PEG(2000) accumulated threefold higher in s.c. HT29 tumors than its PEG-free counterpart. Fluorescence microscopy studies suggested that the presence of DSPE-PEG(2000) did not adversely affect liposomal tumor vasculature targeting. We show for the first time that it is achievable to design highly charged, highly pegylated (20 mol% DSPE-PEG(2000)) cationic liposomes which exhibit both extended circulation lifetimes and tumor vascular targeting properties.

Suppression of VEGF secretion and changes in glioblastoma multiforme microenvironment by inhibition of integrin-linked kinase (ILK).

Integrin-linked kinase (ILK) was assesed as a therapeutic target in glioblastoma xenograft models through multiple endpoints including treatment related changes in the tumor microenvironment. Glioblastoma cell lines were tested in vitro for sensitivity toward the small-molecule inhibitors QLT0254 and QLT0267. Cell viability, cell cycle, and apoptosis were evaluated using MTT assay, flow cytometry, caspase activation, and DAPI staining. Western blotting and ELISA were used for protein analysis (ILK, PKB/Akt, VEGF, and HIF-1alpha). In vivo assessment of growth rate, cell proliferation, BrdUrd, blood vessel mass (CD31 labeling), vessel perfusion (Hoechst 33342), and hypoxia (EF-5) was done using U87MG glioblastoma xenografts in RAG2-M mice treated orally with QLT0267 (200 mg/kg q.d.). ILK inhibition in vitro with QLT0254 and QLT0267 resulted in decreased levels of phospho-PKB/Akt (Ser473), secreted VEGF, G2-M block, and apoptosis induction. Mice treated with QLT0267 exhibited significant delays in tumor growth (treated 213 mm3 versus control 549 mm3). In situ analysis of U87MG tumor cell proliferation from QLT0267-treated mice was significantly lower relative to untreated mice. Importantly, VEGF and HIF-1alpha expression decreased in QLT0267-treated tumors as did the percentage of blood vessel mass and numbers of Hoechst 33342 perfused tumor vessels compared with control tumors (35% versus 83%). ILK inhibition with novel small-molecule inhibitors leads to treatment-associated delays in tumor growth, decreased tumor angiogenesis, and functionality of tumor vasculature. The therapeutic effects of a selected ILK inhibitor (QLT0267) should be determined in the clinic in cancers that exhibit dysregulated ILK, such as PTEN-null glioblastomas.

Use of a passive equilibration methodology to encapsulate cisplatin into preformed thermosensitive liposomes.

A conventional, cholesterol-containing liposome formulation of cisplatin has demonstrated insignificant activity in clinical trials, due in part, to insufficient release of encapsulated content following localization within solid tumors. For this reason, the development of a triggered release liposome formulation is desirable. In this report, cisplatin was encapsulated into lysolipid-containing thermosensitive liposomes (LTSL) using a novel technique, which relies on the equilibration of cisplatin across the liposomal membrane at temperatures above the gel-to-liquid crystalline phase transition temperature (TC) of the bulk phospholipid. Mild heating and drug loading into LTSL did not induce morphological changes of the liposomes. In vitro data demonstrated that >95% of encapsulated cisplatin was released from LTSL within 5 min following mild heating at 42 degrees C, while <5% was released at 37 degrees C. Under similar conditions, lysolipid-free thermosensitive liposomes exhibited 70% release of cisplatin at 42 degrees C, and cholesterol-containing liposomes exhibited negligible drug release at 42 degrees C. The pharmacokinetic profiles of LTSL- and TSL-cisplatin indicated that these formulations were rapidly eliminated from circulation (terminal t(1/2) of 1.09 and 2.83 h, respectively). The therapeutic utility of LTSL-cisplatin formulation will be based on strategies where hyperthermia is applied prior to the administration of the liposomal drug-a strategy similar to that used in the clinical assessment of LTSL-doxorubicin formulation.

Non-invasive evaluation of tumour hypoxia in the Shionogi tumour model for prostate cancer with 18F-EF5 and positron emission tomography.

OBJECTIVE: To evaluate hypoxia non-invasively in androgen-dependent (AD), regressing (6-days after castration, RG) and androgen-independent (AI) Shionogi tumours, using the radiolabelled tracer for hypoxia, 18F-EF5, and positron emission tomography (PET). MATERIALS AND METHODS: Groups of mice bearing AD, RG and AI Shionogi tumours were co-injected with 18F-EF5 and unlabelled EF5. The mice were imaged non-invasively with PET to examine the accumulation of 18F-EF5 in hypoxic regions of the tumour. The tumours were subsequently placed in a gamma-counter, or disaggregated for flow cytometry, to determine the levels of 18F-EF5 and the percentage of hypoxic cells present in the tumour, respectively. RESULTS: The mean (sd) levels of hypoxia in AD Shionogi tumours decreased significantly 6 days after androgen ablation as measured by flow cytometry, from 17.1 (4.77) to 1.74 (0.46)% (P=0.003). There were no significant differences in the levels of 18F-EF5 in the tissue between AD and RG tumours using region-of-interest analysis of PET images or gamma-counting, although the differences were significant when measured by flow cytometry. However, mean (sd) levels of hypoxia in AI Shionogi tumours were significantly higher than in AD tumours regardless of the analysis method; PET, 10.5 (4.93)x10(-5)) Bq/cm2 (P=0.017), flow cytometry, 42.98 (3.35)% (P<0.001), well count, 6.81 (1.17)x10(4) and 13.1 (1.99)x10(4) cpm/g, for AD and AI tumours, respectively (P<0.001). CONCLUSIONS: Differences in hypoxia between AD and AI, but not RG, Shionogi tumours can be detected non-invasively with 18F-EF5 and PET. As prostate tumours are hypoxic and the oxygen levels can change with androgen ablation, noninvasive imaging of hypoxia with PET and 18F-EF5 might ultimately have a prognostic and/or diagnostic role in the clinical management of the disease.

Magnetic resonance imaging of temperature-sensitive liposome release: drug dose painting and antitumor effects.

BACKGROUND: In preclinical studies, lysolipid-based temperature-sensitive liposomes (LTSLs) containing chemotherapy drugs administered in combination with local hyperthermia have been found to increase tumor drug concentrations and improve antitumor efficacy of the drugs. We used a novel magnetic resonance imaging (MRI) method to measure the temporal and spatial patterns of drug delivery in a rat fibrosarcoma model during treatment with LTSLs containing doxorubicin and an MRI contrast agent (manganese) (Dox/Mn-LTSLs) administered at different times with respect to hyperthermia. METHODS: Rats bearing 10- to 12-mm fibrosarcomas (n = 6-7 per group) were treated with Dox/Mn-LTSLs (at a dose of 5 mg doxorubicin/kg body weight) before and/or during 60 minutes of local tumor hyperthermia administered via a catheter inserted at the center of the tumor. Drug distribution was monitored continuously via MRI. Magnetic resonance changes were used to calculate intratumoral doxorubicin concentrations throughout treatment. Tumors were monitored until they reached five times their volume on the day of treatment or 60 days. Doxorubicin concentrations and times for tumors to reach five times their volume on the day of treatment were analyzed using the Kruskal-Wallis test and the Kaplan-Meier product-limit method, respectively. All statistical tests were two-sided. RESULTS: Administration of Dox/Mn-LTSLs before, during, and both before and during hyperthermia yielded central, peripheral, and uniform drug distributions, respectively. Doxorubicin accumulated more quickly and reached higher concentrations in the tumor when Dox/Mn-LTSLs were administered during hyperthermia than when administered before hyperthermia (rate: 9.8 versus 1.8 microg/min, difference = 8.0 microg/min, 95% confidence interval [CI] = 6.8 to 12.8 microg/min, P = .003; concentration: 15.1 versus 8.0 ng/mg, difference = 7.1 ng/mg, 95% CI = 3.6 to 10.6 ng/mg, P = .028). LTSL administered during hyperthermia also yielded the greatest antitumor effect, with a median time for tumors to reach five times their volume on the day of treatment of 34 days (95% CI = 30 days to infinity) compared with 18.5 days (95% CI = 16 to 23 days) for LTSL before hyperthermia and 22.5 days (95% CI = 15 to 25 days) for LTSL before and during hyperthermia. CONCLUSIONS: In this rat fibrosarcoma model, LTSLs were most effective when delivered during hyperthermia, which resulted in a peripheral drug distribution.

Characterisation and expression analysis of an interleukin 6 homologue in the Japanese pufferfish, Fugu rubripes.

The first IL-6 sequence in fish has been determined in Fugu rubripes by exploiting the synteny that is found between some regions of the human and Fugu genomes. The predicted 227 aa IL-6 homologue contains the IL-6/G-CSF/MGF motif, has a predicted secondary structure of four alpha-helixes but only contains two of the four cysteines important in disulphide bond formation. It shows low amino acid identities (20-29%) with known IL-6 sequences, although phylogenetic analysis groups the Fugu molecule with the other IL-6 molecules. The gene organisation of Fugu IL-6 and the level of synteny between the human and Fugu genomes has been well conserved during evolution with the order and orientation of the genes matching exactly to human chromosome 7. PHA stimulation of Fugu kidney cells resulted in a large increase in the Fugu IL-6 transcript, whereas LPS and Poly I:C resulted in a significant increase within spleen cells. The discovery of IL-6 in fish will now allow more detailed investigations of local inflammatory responses.

Hypoxia in the androgen-dependent Shionogi model for prostate cancer at three stages.

The objective of this study was to investigate a possible relationship between androgen status and hypoxia in the Shionogi murine prostate tumor model, which is widely used to study the effects of androgen withdrawal on hormone resistance and radiation response. Binding of the nitroimidazole hypoxia marker EF5 was assessed using the Cy3-tagged monoclonal antibody ELK3-51. Three hours after injection of EF5 (30 mg/kg), tumors from the following three stages were excised: androgen-dependent, regressed tumors 7 days after castration, and androgen-independent. Half of each tumor was disaggregated for analysis by flow cytometry and the remainder was flash frozen. Statistically significant differences (P < 0.01) were found between androgen-dependent, regressed and androgen-dependent tumors: approximately 30, approximately 2 and approximately 50% hypoxic cells, respectively. Frozen sections from androgen-dependent tumors exhibited highly variable EF5 binding; regressed tumors showed very little or no binding; each section from androgen-dependent tumors showed high levels and uniformly distributed binding of EF5. There was no correlation between the degree of hypoxia and tumor weight (P > 0.1). The results from this preliminary study indicate that hypoxia may play an important role with respect to the timing of irradiation in prostate cancer treatments and possibly may be a useful prognostic tool. In addition, hypoxia may also be relevant to progression in this disease after androgen ablation.

HER-2/neu overexpression increases the viable hypoxic cell population within solid tumors without causing changes in tumor vascularization.

The effects of HER-2/neu overexpression on the tumor microenvironment in an aggressive breast cancer xenograft model were investigated. These studies focused on tumors derived following the subcutaneous injection of MDA-MB-435/LCC6 cells transfected with human c-erbB2 (LCC6(HER-2)) into SCID-Rag2M mice. LCC6(HER-2) tumors were more viable (H&E-stained tumor sections) than isogenic vector control tumors (LCC6(Vector)). Correspondingly, a 2.7-fold increase in trypan blue-excluding cells (P = 0.00056) and a 4.8-fold increase in clonogenic cells (P = 0.00146) were noted in cell suspensions derived from disaggregated LCC6(HER-2) versus LCC6(Vector) tumors. Tumor sections stained with the antibody detecting 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)-acetamide (EF5), a marker of hypoxia, showed a greater fraction of hypoxic tissue in LCC6(HER-2) tumors compared with control tumors. Flow cytometric analyses based on viable tumor cells (DNA content >/= 2N) in cell suspensions from disaggregated tumors confirmed that there were significantly more EF5-positive cells (i.e., hypoxic) in LCC6(HER-2) than in LCC6(Vector) tumors (16.41 +/- 8.1% and 5.96 +/- 4.1%, respectively; P = 0.0015). Protein levels of phosphorylated (Ser(536)) nuclear factor-kappaB p65 were significantly elevated in LCC6(HER-2) tumors (P = 0.00048), and a trend in increased hypoxia-inducible factor-1alpha protein levels was observed in LCC6(HER-2) compared with LCC6(Vector) tumors. Despite the substantial viable hypoxic cell fraction and a 1.7-fold increase of vascular endothelial growth factor protein (P = 0.05) in LCC6(HER-2) tumors, no significant differences were found (P > 0.05) between LCC6(HER-2) and LCC6(Vector) vasculature (CD31 staining and Hoechst 33342 perfusion). These results suggest that HER-2/neu overexpression may be linked with overall increased tumor viability and a significant increase in the population of viable hypoxic cells, which is not due to differences in tumor vascularization.