Interlaboratory studies on in vitro test methods for estimating in vivo resorption of calcium phosphate ceramics

A potential standard method for measuring the relative dissolution rate to estimate the resorbability of calcium-phosphate-based ceramics is proposed. Tricalcium phosphate (TCP), magnesium-substituted TCP (MgTCP) and zinc-substituted TCP (ZnTCP) were dissolved in a buffer solution free of calcium and phosphate ions at pH 4.0, 5.5 or 7.3 at nine research centers. Relative values of the initial dissolution rate (relative dissolution rates) were in good agreement among the centers. The relative dissolution rate coincided with the relative volume of resorption pits of ZnTCP in vitro. The relative dissolution rate coincided with the relative resorbed volume in vivo in the case of comparison between microporous MgTCPs with different Mg contents and similar porosity. However, the relative dissolution rate was in poor agreement with the relative resorbed volume in vivo in the case of comparison between microporous TCP and MgTCP due to the superimposition of the Mg-mediated decrease in TCP solubility on the Mg-mediated increase in the amount of resorption. An unambiguous conclusion could not be made as to whether the relative dissolution rate is predictive of the relative resorbed volume in vivo in the case of comparison between TCPs with different porosity. The relative dissolution rate may be useful for predicting the relative amount of resorption for calcium-phosphate-based ceramics having different solubility under the condition that the differences in the materials compared have little impact on the resorption process such as the number and activity of resorbing cells.

Toxic interactions of metal ions (Cd2+, Pb2+, Zn2+ and Sb3- on in vitro biomass production of ectomycorrhizal fungi

A number of ectomycorrhizal (ECM) fungi, from sites uncontaminated by toxic metals, were investigated to determine their sensitivity to Cd^2-, Pb²⁺, Zn²⁺ and Sb^3-, measured as an inhibition of fungal biomass production. Isolates were grown in liquid media amended with the metals, individually (over a range of concentrations) and in combination (at single concentrations) to determine any significant interactions between the metals. Significant interspecific variation in sensitivity to Cd²⁺ and Zn²⁺ was recorded, while Pb²⁺ and Sb^3- individually had little effect. The presence of Pb²⁺ and Sb^3- in the media did however, ameliorate Cd²⁺ and Zn²⁺ toxicity in some circumstances. Interactions between Cd²⁺ and Zn²⁺ were investigated further over a range of concentrations. Zn²⁺ was found to significantly ameliorate the toxicity of Cd²⁺ to three of the four isolates tested. The influence of Zn²⁺ varied between ECM species and with the concentrations of metals tested.

Microtubule-targeting-compound PBOX-15 radiosensitizes cancer cells in vitro

BACKGROUND: We proposed to investigate the radiosensitizing properties of PBOX-15, a novel microtubule-disrupting agent, in a panel of cancer cell lines.

RESULTS: PBOX-15 treatment was associated with significant cell kill and increased radiosensitivity in all three cell lines tested. The number of surviving cells in response to the combined treatment was significantly less than PBOX -15 alone in 22Rv1 cells. In these cells, radiosensitisation correlated with induction of G2/M cell cycle arrest by PBOX-15. The compound sustained its activity and increased HIF-1Α expression under hypoxic conditions. PBOX-15 prevented onset of hypoxia-induced radioresistance in hypoxic prostate cells and reduced the surviving fraction of irradiated hypoxic cells to levels similar to those achieved under aerobic conditions.

METHODS: Clonogenic assays were used to determine sensitivity of a panel of cancer cell lines (22Rv1, A549, U87) to PBOX-15 alone or in combination with a single 2Gy dose fraction. Induction of cell cycle arrest and apoptosis was investigated in 22Rv1 prostate cancer cells. The cytotoxic properties of the compound under hypoxic conditions were correlated with Hypoxia Inducible Factor 1 alpha (HIF-1Α) gene and protein expression levels and its radiosensitisation potential was investigated in hypoxic 22Rv1 using clonogenic assays.

CONCLUSIONS: This preliminary data identifies the potential of PBOX-15 as a novel radiosensitising agent for the management of solid tumours and eradication of hypoxic cells.

Hypoxia response element-driven cytosine deaminase/5-fluorocytosine gene therapy system:a highly effective approach to overcome the dynamics of tumour hypoxia and enhance the radiosensitivity of prostate cancer cells in vitro

BACKGROUND: We proposed to exploit hypoxia-inducible factor (HIF)-1alpha overexpression in prostate tumours and use this transcriptional machinery to control the expression of the suicide gene cytosine deaminase (CD) through binding of HIF-1alpha to arrangements of hypoxia response elements. CD is a prodrug activation enzyme, which converts inactive 5-fluorocytosine to active 5-fluorouracil (5-FU), allowing selective killing of vector containing cells.

METHODS: We developed a pair of vectors, containing either five or eight copies of the hypoxia response element (HRE) isolated from the vascular endothelial growth factor (pH5VCD) or glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (pH8GCD) gene, respectively. The kinetics of the hypoxic induction of the vectors and sensitization effects were evaluated in 22Rv1 and DU145 cells in vitro.

RESULTS: The CD protein as selectively detected in lysates of transiently transfected 22Rv1 and DU145 cells following hypoxic exposure. This is the first evidence of GAPDH HREs being used to control a suicide gene therapy strategy. Detectable CD levels were sustained upon reoxygenation and prolonged hypoxic exposures. Hypoxia-induced chemoresistance to 5-FU was overcome in both cell lines treated with this suicide gene therapy approach. Hypoxic transfectants were sensitized to prodrug concentrations that were ten-fold lower than those that are clinically relevant. Moreover, the surviving fraction of reoxygenated transfectants could be further reduced with the concomitant delivery of clinically relevant single radiation doses.

CONCLUSIONS: This strategy thus has the potential to sensitize the hypoxic compartment of prostate tumours and improve the outcome of current therapies.

The pyrrolo-1,5-benzoxazepine, PBOX-6, inhibits the growth of breast cancer cells in vitro independent of estrogen receptor status and inhibits breast tumour growth in vivo

Members of a novel series of pyrrolo-1,5-benzoxazepine (PBOX) compounds have been shown to induce apoptosis in a number of human leukemia cell lines of different haematological lineage, suggesting their potential as anti-cancer agents. In this study, we sought to determine if PBOX-6, a well characterised member of the PBOX series of compounds, is also an effective inhibitor of breast cancer growth. Two estrogen receptor (ER)-positive (MCF-7 and T-47-D) and two ER-negative (MDA-MB-231 and SK-BR-3) cell lines were examined. The 3,4,5-dimethylthiazol-2-yl-2,5-diphenyl-tetrazolium bromide (MTT) assay was used to determine reduction in cell viability. PBOX-6 reduced the cell viability of all four cell lines tested, regardless of ER status, with IC(50) values ranging from 1.0 to 2.3 microM. PBOX-6 was most effective in the SK-BR-3 cells, which express high endogenous levels of the HER-2 oncogene. Overexpression of the HER-2 oncogene has been associated with aggressive disease and resistance to chemotherapy. The mechanism of PBOX-6-induced cell death was due to apoptosis, as indicated by the increased proportion of cells in the pre-G1 peak and poly(ADP-ribose) polymerase (PARP) cleavage. Moreover, intratumoural administration of PBOX-6 (7.5 mg/kg) significantly inhibited tumour growth in vivo in a mouse mammary carcinoma model (p=0.04, n=5, Student's t-test). Thus, PBOX-6 could be a promising anti-cancer agent for both hormone-dependent and -independent breast cancers.

Evaluation of mixed chimerism by in vitro amplification of dinucleotide repeat sequences using the polymerase chain reaction

The influence of mixed hematopoietic chimerism (MC) after allogeneic bone marrow transplantation remains unknown. Increasingly sensitive detection methods have shown that MC occurs frequently. We report a highly sensitive novel method to assess MC based on the polymerase chain reaction (PCR). Simple dinucleotide repeat sequences called microsatellites have been found to vary in their repeat number between individuals. We use this variation to type donor-recipient pairs following allogeneic BMT. A panel of seven microsatellites was used to distinguish between donor and recipient cells of 32 transplants. Informative microsatellites were subsequently used to assess MC after BMT in this group of patients. Seventeen of the 32 transplants involved a donor of opposite sex; hence, cytogenetics and Y chromosome-specific PCR were also used as an index of chimerism in these patients. MC was detected in bone marrow aspirates and peripheral blood in 18 of 32 patients (56%) by PCR. In several cases, only stored slide material was available for analysis but PCR of microsatellites or Y chromosomal material could be used successfully to assess the origin of cells in this archival material. Cytogenetic analysis was possible in 17 patients and MC was detected in three patients. Twelve patients received T-cell-depleted marrow and showed a high incidence of MC as revealed by PCR (greater than 80%). Twenty patients received unmanipulated marrow, and while the incidence of MC was lower (44%), this was a high percentage when compared with other studies. Once MC was detected, the percentages of recipient cells tended to increase. However, in patients exhibiting MC who subsequently relapsed, this increase was relatively sudden. The overall level of recipient cells in the group of MC patients who subsequently relapsed was higher than in those who exhibited stable MC. Thus, while the occurrence of MC was not indicative of a poor prognosis per se, sudden increases in the proportions of recipient cells may be a prelude to graft rejection or relapse.

In vitro and in vivo methodologies for studying the Sigma 54-dependent transcription.

Here we describe approaches and methods to assaying in vitro the major variant bacterial sigma factor, Sigma 54 (σ54), in a purified system. We include the complete transcription system, binding interactions between σ54 and its activators, as well as the self-assembly and the critical ATPase activity of the cognate activators which serve to remodel the closed promoter complexes. We also present in vivo methodologies that are used to study the impact of physiological processes, metabolic states, global signalling networks, and cellular architecture on the control of σ54-dependent gene expression.

Interleukin-8 is associated with proliferation, migration, angiogenesis and chemosensitivity in vitro and in vivo in colon cancer cell line models

Interleukin-8 (IL-8), a chemokine with a defining CXC amino acid motif, is known to possess tumorigenic and proangiogenic properties. Overexpression of IL-8 has been detected in many human tumors, including colorectal cancer (CRC), and is associated with poor prognosis. The goal of our study was to determine the role of IL-8 overexpression in CRC cells in vitro and in vivo. We stably transfected the IL-8 cDNA into two human colon cancer cell lines, HCT116 and Caco2, and selected IL-8-secreting transfectants. Real-time RT-PCR confirmed that IL-8 mRNA was overexpressed in IL-8 transfectants with 45- to 85-fold higher than parental cells. The IL-8-transfected clones secreted 19- to 28-fold more IL-8 protein than control and parental cells as detected by ELISA. The IL-8 transfectants demonstrated increased cellular proliferation, cell migration and invasion based on functional assays. Growth inhibition studies showed that IL-8 overexpression lead to a significant resistance to oxaliplatin (p < 0.0001). Inhibition of IL-8 overexpression with small interfering RNA reversed the observed increases in tumorigenic functions and oxaliplatin resistance, suggesting that IL-8 not only provides a proliferative advantage but also promotes the metastatic potential of colon cancer cells. Using a tumor xenograft model, IL-8-expressing cells formed significantly larger tumors than the control cells with increased microvessel density. Together, these findings indicate that overexpression of IL-8 promotes tumor growth, metastasis, chemoresistance and angiogenesis, implying IL-8 to be an important therapeutic target in CRC.

Assessment of DNA double-strand breaks induced by intravascular iodinated contrast media following in vitro irradiation and in vivo, during paediatric cardiac catheterization

Paediatric cardiac catheterizations may result in the administration of substantial amounts of iodinated contrast media and ionizing radiation. The aim of this work was to investigate the effect of iodinated contrast media in combination with in vitro and in vivo X-ray radiation on lymphocyte DNA. Six concentrations of iodine (15, 17.5, 30, 35, 45, and 52.5 mg of iodine per mL blood) represented volumes of iodinated contrast media used in the clinical setting. Blood obtained from healthy volunteers was mixed with iodinated contrast media and exposed to radiation doses commonly used in paediatric cardiac catheterizations (0 mGy, 70 mGy, 140 mGy, 250 mGy and 450 mGy). Control samples contained no iodine. For in vivo experimentation, pre and post blood samples were collected from children undergoing cardiac catheterization, receiving iodine concentrations of up to 51 mg of iodine per mL blood and radiation doses of up to 400 mGy. Fluorescence microscopy was performed to assess γH2AX-foci induction, which corresponded to the number of DNA double-strand breaks. The presence of iodine in vitro resulted in significant increases of DNA double-strand breaks beyond that induced by radiation for ≥17.5 mg/mL iodine to blood. The in vivo effects of contrast media on children undergoing cardiac catheterization resulted in a 19% increase in DNA double-strand breaks in children receiving an average concentration of 19 mg/mL iodine to blood. A larger investigation is required to provide further information of the potential benefit of lowering the amount of iodinated contrast media received during X-ray radiation investigations. 

A facile system to evaluate in vitro drug release from dissolving microneedle arrays

The use of biological tissues in the in vitro assessments of dissolving (?) microneedle (MN) array mechanical strength and subsequent drug release profiles presents some fundamental difficulties, in part due to inherent variability of the biological tissues employed. As a result, these biological materials are not appropriate for routine used in industrial formulation development or quality control (QC) tests. In the present work a facile system using Parafilm M® (PF) to test drug permeation performance using dissolving MN arrays is proposed. Dissolving MN arrays containing 196 needles (600 μm needle height) were inserted into a single layer of PF and a hermetic “pouch” was created including the array inside. The resulting system was placed in a dissolution bath and the release of model molecules was evaluated. Different MN formulations were tested using this novel setup, releasing between 40 and 180 µg of their cargos after 6 hours. The proposed system is a more realistic approach for MN testing than the typical performance test described in the literature for conventional transdermal patches. Additionally, the use of PF membrane was tested either in the hermetic “pouch” and using Franz Cell methodology yielding comparable release curves. Microscopy was used in order to ascertain the insertion of the different MN arrays in the PF layer. The proposed system appears to be a good alternative to the use of Franz cells in order to compare different MN formulations. Given the increasing industrial interest in MN technology, the proposed system has potential as a standardised drug/active agent release test for quality control purposes.

An in vitro model of nociceptors in human trigeminal nerves

Background: The oro-facial region is densely innervated by the trigeminal nerve, which when stimulated can induce noxious pain sensation and contribute to neurogenic inflammation in local tissues. Recent research on the expression of specialised ion channels on the trigeminal nerve has highlighted the need to undertake more extensive studies on ion channel expression/functionality with the aim of elucidating their role in pain sensations. A major family of such ion channels is the transient receptor potential (TRP) channels which are activated by a wide variety of thermal, mechanical or chemical stimuli and merit investigation as possible druggable targets for future analgesics.
Objective: Study of TRP channel expression and regulation in oro-facial tissues is hindered by the fact that the cell bodies of neurons innervating these tissues are located in the trigeminal ganglion. Using dental pulp stem cells differentiated towards peripheral neuronal equivalents (PNEs), we sought to determine TRP channel expression, functionality and potential modulation by cytokines in this novel model.
Method: Dental pulp stem cells (DPSCs) were grown on substrate-coated tissue culture plates and differentiated towards a neuronal phenotype using neuronal induction media. Quantitative polymerase chain reaction (qPCR) was performed on PNEs +/-cytokine treatment. Ion channel functionality was investigated using whole cell patch clamping.
Result: qPCR analysis showed that PNEs expressed the TRP channels TRPA1, TRPV1, TRPV4 and TRPM8. TRPA1 was the most abundantly expressed TRP channel studied whereas TRPM8 was lowly expressed. TRP channel expression was shown to be regulated by treatment with inflammatory cytokines. Patch clamp studies using specific agonists and antagonists for TRPA1 and TRPV1 showed these channels were functional.
Conclusion: PNEs differentiated from DPSCs provide a suitable model for TRP channel expression, regulation, and sensistisation in oro-facial tissues. This human neuronal model has potential for use in pre-clinical studies of novel analgesics.