A multi-body model for comparative study of cervical traction simulation – development,improvement and validation

A computer simulation model was developed to study the dynamic behavior of the cervical spine during cervical traction therapy in inclined and sitting traction positions. The model improved upon an old model with additional components to represent the behavior of the intervertebral discs and the posterior ligaments. The simulation result of the new model was compared against the cervical traction data from a radiographic experiment in both positions. The simulation results of the old model and new model were compared to illustrate the improvement. Using the new model, we compared the timing response of cervical traction in the inclined and sitting positions.     

Manufacturing of highly functional and specific T cells for adoptive immunotherapy against virus from granulocyte colony-stimulating factor–mobilized donors

Background aimsCytomegalovirus (CMV) reactivation remains an important risk after hematopoietic stem cell transplantation, which can be effectively controlled through adoptive transfer of donor-derived CMV-specific T cells (CMV-T). CMV-T are usually obtained from donor peripheral blood mononuclear cells (PBMCs) collected before G-CSF mobilization. Despite previous studies that showed impaired T-cell function after granulocyte colony-stimulating factor (G-CSF) mobilization, recent publications suggest that G-CSF-primed PBMCs retain anti-viral function and are a suitable starting material for CMV-T manufacturing. The objective of this study was to assess the feasibility of generating CMV-T from G-CSF–mobilized donors by use of the activation marker CD137 in comparison with conventional non-primed PBMCs.MethodsCMV-T were isolated from G-CSF–mobilized and non-mobilized donor PBMCs on the basis of CMVpp65 activation-induced CD137 expression and expanded during 3 weeks. Functional assays were performed to assess antigen-specific activation, cytokine release, cytotoxic activity and proliferation after anti-genic re-stimulation.ResultsWe successfully manufactured highly specific, functional and cytotoxic CMV-T from G-CSF–mobilized donor PBMCs. Their anti-viral function was equivalent to non-mobilized CMV-T, and memory phenotype would suggest their long-term maintenance after adoptive transfer.ConclusionsWe confirm that the use of an aliquot from G-CSF–mobilized donor samples is suitable for the manufacturing of CMV cellular therapies and thereby abrogates the need for successive donations and ensures the availability for patients with unrelated donors.     

Protection or damage: a dual role for the virus-specific cytotoxic T lymphocyte response in hepatitis B and C infection?

During infection with hepatitis B or C viruses, cytotoxic T lymphocytes (CTLs) have been implicated as both the mediators of protection and the principal effectors of liver pathology. Recent studies have allowed an investigation of the relationship between virus-specific CTL responses, liver damage and viral replication. In the presence of an efficient virus-specific CTL response, a scenario is emerging where inhibition of viral replication can be independent of liver pathology. We discuss the possibility that an inadequate CTL response--unable to control viral replication--may contribute to liver pathology not only directly but also via the recruitment of non-virus-specific T cells.     

Seed banks of a river–reservoir wetland system and their implications for vegetation development

The Danjiangkou Reservoir, constructed in 1970s, is the water source area of the middle route of China's interbasin South-to-North Water Transfer Project. To serve such purpose, the Danjiangkou Reservoir Dam will be increased from its present 162.0 m to 176.6 m, and its regular water level from 157 m to 170 m above mean sea level. Vegetation development in the new reservoir margins is therefore one of great environmental concerns. To explore the potential origin of species in the present reservoir margin vegetation, we investigated and quantified the composition in the soil seed banks and established vegetations of the reservoir margins and its upstream- and downstream-wetlands. In both existent vegetation and seed banks, most species and seedlings were found in upstream wetlands, followed by reservoir margins and downstream wetlands. Seedling density of downstream wetlands was reduced by 75–80% compared to upstream wetlands and reservoir margins. This suggests that presence of the dam reduced the diversity and abundance of downstream propagules. Sørensen's coefficient and the comparisons of rare species indicated that the seed bank composition of reservoir margins was evidently associated with upstream wetlands. It implies that hydrochorous transport of seeds from the upstream catchment is critical for plant colonization of the reservoir margins.     

Manufacturing development and clinical production of NKG2D chimeric antigen receptor–expressing T cells for autologous adoptive cell therapy

Background aims

Adoptive cell therapy employing natural killer group 2D (NKG2D) chimeric antigen receptor (CAR)-modified T cells has demonstrated preclinical efficacy in several model systems, including hematological and solid tumors. We present comprehensive data on manufacturing development and clinical production of autologous NKG2D CAR T cells for treatment of acute myeloid leukemia and multiple myeloma (ClinicalTrials.gov Identifier: NCT02203825). An NKG2D CAR was generated by fusing native full-length human NKG2D to the human CD3ζ cytoplasmic signaling domain. NKG2D naturally associates with native costimulatory molecule DAP10, effectively generating a second-generation CAR against multiple ligands upregulated during malignant transformation including MIC-A, MIC-B and the UL-16 binding proteins.

Lack of validation of genetic variants associated with anti–tumor necrosis factor therapy response in rheumatoid arthritis: a genome-wide association study replication and meta-analysis

Introduction

In this study, our aim was to elucidate the role of four polymorphisms identified in a prior large genome-wide association study (GWAS) in which the investigators analyzed the responses of patients with rheumatoid arthritis (RA) to treatment with tumor necrosis factor inhibitors (TNFi). The authors of that study reported that the four genetic variants were significantly associated. However, none of the associations reached GWAS significance, and two subsequent studies failed to replicate these associations.

Methods

The four polymorphisms (rs12081765, rs1532269, rs17301249 and rs7305646) were genotyped in a total of 634 TNFi-treated RA patients of Spanish Caucasian origin. Four outcomes were evaluated: changes in the Disease Activity Score in 28 joints (DAS28) after 6 and 12 months of treatment and classification according to the European League Against Rheumatism (EULAR) response criteria at the same time points. Association with DAS28 changes was assessed by linear regression using an additive genetic model. Contingency tables of genotype and allele frequencies between EULAR responder and nonresponder patients were compared. In addition, we combined our data with those of previously reported studies in a meta-analysis including 2,998 RA patients.

Results

None of the four genetic variants showed an association with response to TNFi in any of the four outcomes analyzed in our Spanish patients. In addition, only rs1532269 yielded a suggestive association (P = 0.0033) with the response to TNFi when available data from previous studies were combined in the meta-analysis.

Conclusion

Our data suggest that the rs12081765, rs1532269, rs17301249 and rs7305646 genetic variants do not have a role as genetic predictors of TNFi treatment outcomes.     

Metaheuristics and Pontryagin’s minimum principle for optimal therapeutic protocols in cancer immunotherapy: a case study and methods comparison

Journal of Mathematical Biology - In this paper, the performance appropriateness of population-based metaheuristics for immunotherapy protocols is investigated on a comparative basis while the goal...     

Weevils and camellias in a Darwin’s race: model system for the study of eco-evolutionary interactions between species

Organisms are surrounded by their predators, parasites, hosts, and mutualists, being involved in reciprocal adaptation processes with such “biotic environment”. The concept of “coevolution”, therefore, provides a basis for the comprehensive understanding of evolutionary and ecological dynamics in biological communities and ecosystems. Recent studies have shown that coevolutionary processes are spatially heterogeneous and that traits mediating interspecific interactions can evolve rapidly in natural communities. Here, I discuss factors promoting the geographic differentiation of coevolutionary interactions, the spatial scales of the geographic structuring, and the pace of coevolutionary changes, reviewing findings in the arms race coevolution involving a long-mouthed weevil and its host camellia plant. Evolutionary, ecological, and population genetic studies on the system illuminated that viewpoints from the aspect of “coevolving biosphere” were important for predicting how ongoing anthropogenic change in global environment alter the spatiotemporal dynamics of biological communities.     

Development and validation of a sensitive and rapid non-aqueous LC–ESI-MS/MS method for measurement of diosgenin in the plasma of normal and hyperlipidemic rats: A comparative study

A sensitive and specific electrospray ionization liquid chromatography–tandem mass spectrometry method was developed to detect diosgenin in the plasma of normal and hyperlipidemic rats. Diosgenin was extracted with n-hexane–ethyl acetate (9:1, v/v) using sarsasapogenin as an internal standard. With multiple reaction monitoring modes, linear calibration curves were obtained in the range 10–1500 ng/mL (r ≥ 0.9979) and the limit of quantification was 10 ng/mL. Intra- and inter-assay variabilities were within 7.74%, and accuracies were between ?5.33% and 1.50%. The assay was successfully applied to study pharmacokinetics in rats after oral administration of diosgenin. Significantly different pharmacokinetics between normal and hyperlipidemic rats were observed, which would be beneficial for the clinical use of diosgenin.     

Development and validation of a LC–MS/MS method for the determination of clebopride and its application to a pharmacokinetics study in healthy Chinese volunteers

A sensitive and specific liquid chromatography–electrospray ionization-mass spectrometry (LC–ESI-MS/MS) method has been developed and validated for the identification and quantification of clebopride in human plasma using itopride as an internal standard. The method involves a simple liquid–liquid extraction. The analytes were separated by isocratic gradient elution on a CAPCELL MG-III C₁₈ (5 μm, 150 mm × 2.1 mm i.d.) column and analyzed in multiple reaction monitoring (MRM) mode with positive electrospray ionization (ESI) interface using the respective [M+H]⁺ ions, m/z 373.9 → m/z184.0 for clebopride, m/z 359.9 → m/z71.5 for itopride. The method was validated over the concentration range of 69.530–4450.0 pg/ml for clebopride. Within- and between-batch precision (RSD%) was all within 6.83% and accuracy ranged from −8.16 to 1.88%. The LLOQ was 69.530 pg/ml. The extraction recovery was on an average 77% for clebopride. The validated method was used to study the pharmacokinetics profile of clebopride in human plasma after oral administration of clebopride.     

Subsite mapping of enzymes: Collecting and processing experimental data—a case study of an amylase-malto-oligosaccharide system

Two research groups have independently developed the theory and experimental methodology for quantitatively assessing substrate monomer-subsite binding-energies for depolymerases. When the two approaches are applied to the same enzyme-substrate system they yield surprisingly divergent results. This paper outlines the application of the two approaches to an amylase-maltooligosaccharide system and points out the more important areas of disagreement. We show that by proper data-management, the conflicts between the two laboratories are basically resolved. The complexities of the subsite model demand extensive data-gathering and exacting data-processing and verification that the computed model-parameters can faithfully reproduce the experimental data.     

Development and validation of a liquid chromatography–mass spectrometry method for the determination of verticinone in rat plasma and its application to pharmacokinetic study

We have developed and validated a sensitive liquid chromatography–electrospray ionization-mass spectrometric (LC–ESI-MS) method for the quantification of verticinone, a major active constituent from Fritillaria hupehensis Hsiao et KC Hsia., in rat plasma. Verticinone and the internal standard (IS), hupehenine, were extracted from plasma samples by a simple liquid–liquid extraction with ethyl acetate after being alkalified by 1 M ammonia hydroxide. Chromatographic separation was achieved on a C₁₈ column using a gradient elution program with methanol and water as the mobile phase. The detection was performed by selected ion monitoring (SIM) mode via positive electrospray ionization (ESI) interface. The lower limit of quantification (LLOQ) was 0.1 ng/mL. The calibration curves were linear (r² > 0.998) over the concentration range of 0.1–200 ng/mL. Within- and between-run precision was less than 6.5% and accuracy was within ±10.7%. The validated method was applied to the pharmacokinetic study of verticinone in rats after a single oral administration of 1 mg/kg.     

Iodine concentration of milk in a dose–response study with dairy cows and implications for consumer iodine intake

Most feed is poor in iodine and iodine supplementation of cow's diets must guarantee milk iodine concentrations for humans that contribute to prevention of the deficiency and minimize the risk of exceeding an upper limit of iodine intake. Five Holstein cows were fed four iodine doses (via Ca(ΙO₃)₂·6H₂O). In four sequential 14-d periods, doses of 0.2 (basal diet), 1.3, 5.1, and 10.1 mg iodine kg^?1 diet dry matter (DM) were administered. Samples of milk were collected during each period; blood was also sampled from each cow for each iodine dosage. In an 18-d depletion period, a non-supplemented diet was provided. Iodine was determined by inductively coupled plasma-mass spectrometry. The iodine content of milk and serum reflected the iodine dosages in feed significantly. The levels for the four doses tested in milk were 101±32, 343±109, 1215±222, and 2762±852 μg iodine kg^?1. The total amount of iodine in milk per day was 30–40% of ingested supplemental iodine. Omitting additional iodine resulted in a short-term reduction of serum and milk iodine following an exponential decay function. The iodine supplementation of 0.5–1.5 mg kg^?1 diet DM represents the requirement of the cow, resulting in 100–300 μg iodine L^?1 milk, which optimally contributes to human supply. The maximum dietary levels of former and present EU legislations (10 and 5 mg iodine kg^?1 cow feed) increase the risk of iodine excess in humans.     

Clinical effect and safety of dendritic cell–cytokine-induced killer cell immunotherapy for pancreatic cancer: a systematic review and meta-analysis

BackgroundAlthough promising results have recently been reported using dendritic cells (DCs) and cytokine-induced killer cells (CIKs) to treat pancreatic cancer (PC), its clinical effect and safety are associated with some controversy, and lack sufficient evidence. Here, we conducted a meta-analysis of 21 clinical trials to better evaluate the efficacy of DC-CIK immunotherapy in clinical practice to treat PC.MethodsPubMed, Cochrane Library, China National Knowledge Infrastructure (CNKI) and Wanfang Data Knowledge Service Platform (WANFANG Data) were searched to identify clinical trials that used DC-CIK immunotherapy for PC. Meta-analysis was performed using RevMan 5.3 and Stata 12.0.ResultsA total of 21 clinical trials involving 1549 patients were included. Compared with traditional treatment, DC-CIK immunotherapy improved and increased the clinical indices such as complete remission, partial remission, overall response rate, disease control rate, overall survival (0.5-y OS, 1-y OS, 1.5-y OS, 2-y OS and 3-y OS), interferon γ and CD3+, CD4+, CD4+/CD8+ and CD3+CD56+ lymphocyte. Additionally, DC-CIK immunotherapy reduced stable disease, progression disease, mortality, CD8+, CD4+CD25+CD127 low lymphocyte and interleukin-4. Furthermore, it showed a low incidence of adverse reactions (22%).ConclusionIn contrast to traditional therapy, DC-CIK immunotherapy not only shows improved short-term effect, long-term effect and immunologic function, but also reduces mortality and negative immunoregulatory index, and shows mild adverse reactions. This is the first study to evaluate the clinical effect and safety of DC-CIK immunotherapy for PC, and it indicated that DC-CIK immunotherapy may be suitable for patients with advanced PC or intolerance to radiotherapy and chemotherapy.     

The Tübingen approach: identification,selection, and validation of tumor-associated HLA peptides for cancer therapy

There is substantial need for molecularly defined tumor antigens to prime cytotoxic T cells in vivo for cancer immunotherapy, especially in the case of tumor entities for which only a few tumor antigens have been defined so far. In this review, we present the Tübingen approach to identify, select, and validate large numbers of MHC/HLA class I–associated peptides derived from tumor-associated antigens. Step 1 is the identification of naturally presented HLA-associated peptides directly from primary tumor cells. Step 2 is selection of tumor-associated peptides from step 1 by differential gene expression analysis and data mining. Step 3 is validation of selected candidates by monitoring in vivo T-cell responses in the context of patient-individualized immunizations. Our approach combines methods from genomics, proteomics, bioinformatics, and T-cell immunology. The aim is to develop effective immunotherapeutics consisting of multiple tumor-associated epitopes in order to induce a broad and specific immune response against cancer cells.This work was presented at the first Cancer Immunology and Immunotherapy Summer School, 8–13 September 2003, Ionian Village, Bartholomeio, Peloponnese, Greece.     

Development and validation of a high throughput and robust LC–MS/MS with electrospray ionization method for simultaneous quantitation of diltiazem and its two metabolites in human plasma: Application to a bioequivalence study

A high throughput and specific method using ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) was developed for the simultaneous determination of diltiazem and its two metabolite (N-desmethyldiltiazem and O-desacetyldiltiazem) in human plasma. A one-step liquid–liquid extraction (LLE) with methyl-t-butyl ether (MTBE) involved for the extraction of diltiazem (DLTZ), metabolites (DMeD and DAcD) and internal standard. Analytes were chromatographed on a ACQUITY UPLC? BEH C₁₈ column (100 mm × 2.1 mm, i.d., 1.7 μm) with isocratic elution at a flow rate of 0.2 mL/min using 10 mM ammonium acetate buffer–acetonitrile (25:75, v/v). The Quattro Premier XE LC–MS/MS was operated under the multiple reaction-monitoring mode (MRM) using the electrospray ionization technique. Using 300 μL plasma, the method was validated over the concentration range 0.48–639.9 ng/mL for DLTZ and 0.24–320.1 for DMeD and 0.24–320.7 ng/mL for DAcD, with a lower limit of quantification of 0.48 ng/mL for DLTZ and 0.24 ng/mL for metabolites. The intra- and inter-day precision and accuracy were within 10.0%. The recovery was 77.4%, 76.0%, 74.5% and 74.1% for DLTZ, DMeD, DAcD and Ziprasidone, respectively. Total run time was 2.0 min only.     

Fit for purpose? A case study: validation of immunological endpoint assays for the detection of cellular and humoral responses to anti-tumour DNA fusion vaccines

Clinical trials are governed by an increasingly stringent regulatory framework, which applies to all levels of trial conduct. Study critical immunological endpoints, which define success or failure in early phase clinical immunological trials, require formal pre-trial validation. In this case study, we describe the assay validation process, during which the sensitivity, and precision of immunological endpoint assays were defined. The purpose was the evaluation of two multicentre phase I/II clinical trials from our unit in Southampton, UK, which assess the effects of DNA fusion vaccines on immune responses in HLA-A2+ patients with carcinoembryonic antigen (CEA)-expressing malignancies and prostate cancer. Validated immunomonitoring is being performed using ELISA and IFNγ ELISPOTs to assess humoral and cellular responses to the vaccines over time. The validated primary endpoint assay, a peptide-specific CD8+ IFNγ ELISPOT, was tested in a pre-trial study and found to be suitable for the detection of low frequency naturally occurring CEA- and prostate-derived tumour-antigen-specific T cells in patients with CEA-expressing malignancies and prostate cancer. This paper is a Focussed Research Review based on a presentation given at the Sixth Annual Meeting of the Association for Immunotherapy of Cancer (CIMT), held in Mainz, Germany, 15–16 May 2008.     

Ionic mechanisms and Ca2+ dynamics underlying the glucose response of pancreatic β cells: a simulation study

To clarify the mechanisms underlying the pancreatic β-cell response to varying glucose concentrations ([G]), electrophysiological findings were integrated into a mathematical cell model. The Ca(2+) dynamics of the endoplasmic reticulum (ER) were also improved. The model was validated by demonstrating quiescent potential, burst-interburst electrical events accompanied by Ca(2+) transients, and continuous firing of action potentials over [G] ranges of 0-6, 7-18, and >19 mM, respectively. These responses to glucose were completely reversible. The action potential, input impedance, and Ca(2+) transients were in good agreement with experimental measurements. The ionic mechanisms underlying the burst-interburst rhythm were investigated by lead potential analysis, which quantified the contributions of individual current components. This analysis demonstrated that slow potential changes during the interburst period were attributable to modifications of ion channels or transporters by intracellular ions and/or metabolites to different degrees depending on [G]. The predominant role of adenosine triphosphate-sensitive K(+) current in switching on and off the repetitive firing of action potentials at 8 mM [G] was taken over at a higher [G] by Ca(2+)- or Na(+)-dependent currents, which were generated by the plasma membrane Ca(2+) pump, Na(+)/K(+) pump, Na(+)/Ca(2+) exchanger, and TRPM channel. Accumulation and release of Ca(2+) by the ER also had a strong influence on the slow electrical rhythm. We conclude that the present mathematical model is useful for quantifying the role of individual functional components in the whole cell responses based on experimental findings.