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.

Phenotypic characterization of an international Pseudomonas aeruginosa reference panel: strains of cystic fibrosis (CF) origin show less in vivo virulence than non-CF strains

Pseudomonas aeruginosa causes chronic lung infections in people with cystic fibrosis (CF) and acute opportunistic infections in people without CF. Forty two P. aeruginosa strains from a range of clinical and environmental sources were collated into a single reference strain panel to harmonise research on this diverse opportunistic pathogen. To facilitate further harmonized and comparable research on P. aeruginosa, we characterised the panel strains for growth rates, motility, virulence in the Galleria mellonella infection model, pyocyanin and alginate production, mucoid phenotype, lipopolysaccharide (LPS) pattern, biofilm formation, urease activity, antimicrobial and phage susceptibilities. Phenotypic diversity across the P. aeruginosa panel was apparent for all phenotypes examined agreeing with the marked variability seen in this species. However, except for growth rate, the phenotypic diversity among strains from CF versus non-CF sources was comparable. CF strains were less virulent in the G. mellonella model than non-CF strains (p=0.037). Transmissible CF strains generally lacked O antigen, produced less pyocyanin, and had low virulence in G. mellonella. Further, in the three sets of sequential CF strains, virulence, O-antigen expression and pyocyanin production were higher in the earlier isolate compared to the isolate obtained later in infection. Overall, full phenotypic characterization of the defined panel of P. aeruginosa strains increases our understanding of the virulence and pathogenesis of P. aeruginosa and may provide a valuable resource for the testing of novel therapies against this problematic pathogen.

Fulfilling the 3Rs: Galleria mellonella as an In Vivo Model to Assess the Antimicrobial Efficacy of Self-assembling Peptide Hydrogels

The concept of ‘The Three Rs’ (The 3Rs: reduction, refinement and replacement) is an important consideration in the development of alternatives to animal testing in medical research. Invertebrate models such as Galleria mellonella are advantageous both economically and ethically.1 Galleria have proven to be effective alternatives to assess the antimicrobial activity of novel therapeutics.2
In this study Galleria mellonella are validated and used as an in vivo infection model to determine the antimicrobial activity of a novel self-assembling antimicrobial peptide NapFFKK.3 The peptide was considered as being non-toxic to the Galleria with 100% survival 120 hours post inoculation with NapFFKK. Following inoculation with Pseudomonas aeruginosa PAO1, Escherichia coli ATCC 11303, Staphylococcus epidermidis ATCC 35984 and Staphylococcus aureus ATCC 6538, the highest concentration allowing survival was selected and used as the test inoculum. Haemolymph was extracted from inoculated and peptide treated Galleria at either 24 or 72 hours post-treatment. Reduction in bacterial load was determined in comparison to a positive control. Bacterial load was decreased in all treated Galleria with decreasing antimicrobial activity demonstrated with a decreased concentration of peptide (2- log cycle reduction achieved in Escherichia coli inoculated Galleria treated with 2% NapFFKK). The results are promising regarding the use of Galleria mellonella as an infection model and NapFFKK as an effective novel antimicrobial.

Galleria mellonella as a novel in vivo model for assessment of the toxicity of 1-alkyl-3-methylimidazolium chloride ionic liquids

The larval form of the Greater Wax Moth (Galleria mellonella) was evaluated as a model system for the study of the acute in vivo toxicity of 1-alkyl-3-methylimidazolium chloride ionic liquids. 24-h median lethal dose (LD50) values for nine of these ionic liquids bearing alkyl chain substituents ranging from 2 to 18 carbon atoms were determined. The in vivo toxicity of the ionic liquids was found to correlate directly with the length of the alkyl chain substituent, and the pattern of toxicity observed was in accordance with previous studies of ionic liquid toxicity in other living systems, including a characteristic toxicity ‘cut-off’ effect. However, G. mellonella appeared to be more susceptible to the toxic effects of the ionic liquids tested, possibly as a result of their high body fat content. The results obtained in this study indicate that G. mellonella represents a sensitive, reliable and robust in vivo model organism for the evaluation of ionic liquid toxicity.

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.

Favorable effect on acute and chronic graft-versus-host disease with cyclophosphamide and in vivo anti-CD52 monoclonal antibodies for marrow transplantation from HLA-identical sibling donors for acquired aplastic anemia

Between August 1989 and November 2003, 33 patients at our center with acquired aplastic anemia underwent bone marrow transplantation (BMT) from HLA-identical sibling donors with cyclophosphamide and in vivo anti-CD52 monoclonal antibodies (MoAb) for conditioning. The median age at BMT was 17 years (range, 4-46 years). Before BMT, 58% were heavily transfused (>50 transfusions), and 42% had previously experienced treatment failure with antithymocyte globulin-based immunosuppressive therapy. Unmanipulated bone marrow was used as the source of stem cells in all patients except 1. Graft-versus-host disease (GVHD) prophylaxis was with cyclosporine alone in 19 (58%) patients; 14 received anti-CD52 MoAb in addition to cyclosporine. The conditioning regimen was well tolerated without significant acute toxicity. Graft failure was seen in 8 patients (primary, n = 4; secondary, n = 4). Of those whose grafts failed, 4 survived long-term (complete autologous recovery, n = 2; rescue with previously stored marrow, n = 1; second allograft, n = 1). The cumulative incidence of graft failure and grade II to IV acute and chronic GVHD was 24%, 14%, and 4%, respectively. None developed extensive chronic GVHD. With a median follow-up of 59 months, the 5-year survival was 81% (95% confidence interval, 68%-96%). No unexpected early or late infectious or noninfectious complications were observed. We conclude that the conditioning regimen containing cyclophosphamide and anti-CD52 MoAb is well tolerated and effective for acquired aplastic anemia with HLA-matched sibling donors. The favorable effect on the incidence and severity of GVHD is noteworthy in this study and warrants further investigation.

Campath-1G in vivo confers a low incidence of graft-versus-host disease associated with a high incidence of mixed chimaerism after bone marrow transplantation for severe aplastic anaemia using HLA-identical sibling donors

We have evaluated the effect of in vivo Campath-1G on engraftment and GVHD in 23 patients with severe aplastic anaemia transplanted from HLA-identical sibling donors. In 14 patients Campath 1g was given pre-transplant for up to 9 days in an attempt to overcome graft rejection (group 1). In nine patients Campath-1G was given pre-transplant, but also continued post-transplant until day +5 to reduce GVHD (group 2). There were three patients with late graft failure in group I following initial neutrophil engraftment, and four cases of grade II+ GVHD. In group II, two patients had early graft failure (no take), and there were no cases of acute GVHD out of seven evaluable patients. One patient in group I developed chronic GVHD of the liver, and two patients (one in each group) had transient localised chronic GVHD. PCR of short tandem repeats was used to evaluate chimaeric status in 13 patients. Of 11 patients with initial neutrophil engraftment, only one had 100% donor haemopoiesis at all times. The remaining patients had either transient mixed chimaerism or persistence of recipient (< 20%) cells. We conclude that in vivo Campath-1G is associated with a high incidence of mixed chimaerism which tips the balance away from GVHD but towards graft rejection.

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.

Assessing the in vivo efficacy of biologic antiangiogenic therapies

PURPOSE: To review key clinical issues underlying the assessment of in vivo efficacy when using antiangiogenic therapies for cancer treatment.

METHODS: Literature relevant to use of antiangiogenic therapies in cancer was reviewed, with particular emphasis on the assessment of in vivo efficacy of these agents, as well as additional angiogenic factors that could play a role in escape from angiogenesis inhibition.

RESULTS: In order to grow and metastasize, tumors need to continually acquire new blood supplies; therefore, therapeutic inhibition of angiogenesis has become a component of anticancer treatment for many tumor types. Bevacizumab, a humanized monoclonal antibody directed at vascular endothelial growth factor A (VEGF-A), has shown activity in combination with chemotherapy in metastatic colorectal cancer. Nevertheless, the use of antiangiogenic therapies remains suboptimal; specifically, optimal dose, duration of therapy, and combination of agents remain unknown. Also, at present, it is not possible to determine which patients are most likely to respond to a given form of antiangiogenic therapy. There has been increased recognition of alternative pathways possibly associated with disease progression in patients undergoing antiangiogenic therapy targeted at VEGF-A. Multiligand-targeted antiangiogenic therapies, such as ziv-aflibercept (formerly known as aflibercept, VEGF Trap), are currently undergoing clinical evaluation. Ziv-aflibercept forms monomeric complexes with VEGF-A, VEGF-B, and PlGF, which have a long half-life, allowing optimization of ziv-aflibercept doses and angiogenic blockage.

CONCLUSIONS: Although antiangiogenic therapies have increased treatment options for cancer patients, their use is limited by a lack of established and standardized methodology to evaluate their efficacy in vivo. Circulating endothelial cells, hypertension, and several molecular and imaging-based markers have potential for use as biomarkers in these patients and may better define appropriate patient populations.

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. 

Hydrogel-Forming Microneedle Arrays Allow Detection of Drugs and Glucose In Vivo: Potential for Use in Diagnosis and Therapeutic Drug Monitoring

We describe, for the first time the use of hydrogel-forming microneedle (MN) arrays for minimally-invasive extraction and quantification of drug substances and glucose from skin in vitro and in vivo. MN prepared from aqueous blends of hydrolysed poly(methyl-vinylether-co-maleic anhydride) (11.1% w/w) and poly(ethyleneglycol) 10,000 daltons (5.6% w/w) and crosslinked by esterification swelled upon skin insertion by uptake of fluid. Post-removal, theophylline and caffeine were extracted from MN and determined using HPLC, with glucose quantified using a proprietary kit. In vitro studies using excised neonatal porcine skin bathed on the underside by physiologically-relevant analyte concentrations showed rapid (5 min) analyte uptake. For example, mean concentrations of 0.16 μg/mL and 0.85 μg/mL, respectively, were detected for the lowest (5 μg/mL) and highest (35 μg/mL) Franz cell concentrations of theophylline after 5 min insertion. A mean concentration of 0.10 μg/mL was obtained by extraction of MN inserted for 5 min into skin bathed with 5 μg/mL caffeine, while the mean concentration obtained by extraction of MN inserted into skin bathed with 15 μg/mL caffeine was 0.33 μg/mL. The mean detected glucose concentration after 5 min insertion into skin bathed with 4 mmol/L was 19.46 nmol/L. The highest theophylline concentration detected following extraction from a hydrogel-forming MN inserted for 1 h into the skin of a rat dosed orally with 10 mg/kg was of 0.363 μg/mL, whilst a maximum concentration of 0.063 μg/mL was detected following extraction from a MN inserted for 1 h into the skin of a rat dosed with 5 mg/kg theophylline. In human volunteers, the highest mean concentration of caffeine detected using MN was 91.31 μg/mL over the period from 1 to 2 h post-consumption of 100 mg Proplus® tablets. The highest mean blood glucose level was 7.89 nmol/L detected 1 h following ingestion of 75 g of glucose, while the highest mean glucose concentration extracted from MN was 4.29 nmol/L, detected after 3 hours skin insertion in human volunteers. Whilst not directly correlated, concentrations extracted from MN were clearly indicative of trends in blood in both rats and human volunteers. This work strongly illustrates the potential of hydrogel-forming MN in minimally-invasive patient monitoring and diagnosis. Further studies are now ongoing to reduce clinical insertion times and develop mathematical algorithms enabling determination of blood levels directly from MN measurements.