Chemokine/cytokine profiling after rituximab: reciprocal expression of BCA-1/CXCL13 and BAFF in childhood OMS

The aim of the study was to test the hypothesis that B-cell repopulation following rituximab (anti-CD20) therapy is orchestrated by chemokines and non-chemokine cytokines. Twenty-five children with opsoclonus-myoclonus syndrome (OMS) received rituximab with or without conventional agents. A comprehensive panel of 40 chemokines and other cytokines were measured in serum by ELISA and multiplexed fluorescent bead-based immunoassay. Serum BAFF concentration changed dramatically (even after first infusion) and inversely with B-cell depletion/repopulation and CXCL13 concentration at 1, 3, and 6 months. Negative correlations were found for BAFF concentration vs blood B cell percentage and serum CXCL13 concentration; positive correlations with serum rituximab concentrations. Six months after initiation of therapy, no significant difference in the levels of APRIL, CXCL10, IL-6, or 17 other cytokines/chemokines were detected. These data reveal a major role for BAFF in peripheral B cell repopulation following rituximab-induced B-cell depletion, and novel changes in CXCL13. ClinicalTrials.gov NCT0024436.     

Recipient leukocyte infusion enhances the local and systemic graft-versus-neuroblastoma effect of allogeneic bone marrow transplantation in mice

Allogeneic hematopoietic stem cell transplantation and donor leukocyte infusion (DLI) may hold potential as a novel form of immunotherapy for high-risk neuroblastoma. DLI, however, carries the risk of graft-versus-host disease (GvHD). Recipient leukocyte infusion (RLI) induces graft-versus-leukemia responses without GvHD in mice and is currently being explored clinically. Here, we demonstrate that both DLI and RLI, when given to mixed C57BL/6 → A/J radiation chimeras carrying subcutaneous Neuro2A neuroblastoma implants, can slow the local growth of such tumors. DLI provoked full donor chimerism and GvHD; RLI produced graft rejection but left mice healthy. Flow cytometric studies showed that the chimerism of intratumoral leukocytes paralleled the systemic chimerism. This was associated with increased CD8/CD4 ratios, CD8⁺ T-cell IFN-γ expression and NK-cell Granzyme B expression within the tumor, following both DLI and RLI. The clinically safe anti-tumor effect of RLI was further enhanced by adoptively transferred naïve recipient-type NK cells. In models of intravenous Neuro2A tumor challenge, allogeneic chimeras showed superior overall survival over syngeneic chimeras. Bioluminescence imaging in allogeneic chimeras challenged with luciferase-transduced Neuro2A cells showed both DLI and RLI to prolong metastasis-free survival. This is the first experimental evidence that RLI can safely produce a local and systemic anti-tumor effect against a solid tumor. Our data indicate that RLI may provide combined T-cell and NK-cell reactivity effectively targeting Neuro2A neuroblastoma.     

Increasing the accuracy and precision of relative telomere length estimates by RT qPCR

As attrition of telomeres, DNA caps that protect chromosome integrity, is accelerated by various forms of stress, telomere length (TL) has been proposed as an indicator of lifetime accumulated stress. In ecological studies, it has been used to provide insights into ageing, life history trade‐offs, the costs of reproduction and disease. qPCR is a high‐throughput and cost‐effective tool to measure relative TL (rTL) that can be applied to newly collected and archived ecological samples. However, qPCR is susceptible to error both from the method itself and pre‐analytical steps. Here, repeatability was assessed overall and separately across multiple levels (intra‐assay, inter‐assay and inter‐extraction) to elucidate the causes of measurement error, as a step towards improving precision. We also tested how accuracy, defined as the correlation between the “gold standard” for TL estimation (telomere restriction fragment length analysis with in‐gel hybridization), could be improved. We find qPCR repeatability (intra‐ and inter‐assay levels) to be at similar levels across three common storage media (ethanol, Longmire's and Queen's). However, inter‐extraction repeatability was 50% lower for samples stored in Queen's lysis buffer, indicating storage medium can influence precision. Precision as well as accuracy could be increased by estimating rTL from multiple qPCR reactions and from multiple extractions. Repetition increased statistical power equivalent to a 25% (single extraction analysed twice) and 17% (two extractions) increase in sample size. Overall, this study identifies novel sources of variability in high‐throughput telomere quantification and provides guidance on sampling strategy design and how to increase rTL precision and accuracy.     

Antileishmanial activity, cytotoxicity and QSAR analysis of synthetic dihydrobenzofuran lignans and related benzofurans

A series of synthetic dihydrobenzofuran lignans and related benzofurans were evaluated for their cytotoxicity in a screening panel consisting of various human tumour cell lines, and for their antiprotozoal activity against L. donovani (axenic amastigotes), chloroquine resistant Plasmodium falciparum (strain K1), Trypanosoma brucei rhodesiense and T. cruzi, and for cytotoxicity on L6 cells. No promising cytotoxicities against human tumour cell lines were observed for newly synthesised compounds, but the dimerisation product of some lipophylic esters of caffeic acid, such as compound 2g, showed a high activity against chloroquine-resistant P. falciparum (strain K1) (IC50 0.43 microg/mL) and L. donovani (axenic amastigotes) (IC50 0.12 microg/mL), which was confirmed in an infected macrophage assay (IC50 0.19 microg/mL). QSAR models for the cytotoxic and antileishmanial activity were generated using Quasar receptor surface modelling.     

Substance Use and Depression Symptomatology: Measurement Invariance of the Beck Depression Inventory (BDI-II) among Non-Users and Frequent-Users of Alcohol,Nicotine and Cannabis

Depression is a highly heterogeneous condition, and identifying how symptoms present in various groups may greatly increase our understanding of its etiology. Importantly, Major Depressive Disorder is strongly linked with Substance Use Disorders, which may ameliorate or exacerbate specific depression symptoms. It is therefore quite plausible that depression may present with different symptom profiles depending on an individual’s substance use status. Given these observations, it is important to examine the underlying construct of depression in groups of substance users compared to non-users. In this study we use a non-clinical sample to examine the measurement structure of the Beck Depression Inventory (BDI-II) in non-users and frequent-users of various substances. Specifically, measurement invariance was examined across those who do vs. do not use alcohol, nicotine, and cannabis. Results indicate strict factorial invariance across non-users and frequent-users of alcohol and cannabis, and metric invariance across non-users and frequent-users of nicotine. This implies that the factor structure of the BDI-II is similar across all substance use groups     

A New 3D Tool for Assessing the Accuracy of Bimaxillary Surgery: The OrthoGnathicAnalyser

Aim

The purpose of this study was to present and validate an innovative semi-automatic approach to quantify the accuracy of the surgical outcome in relation to 3D virtual orthognathic planning among patients who underwent bimaxillary surgery.

Material and Method

For the validation of this new semi-automatic approach, CBCT scans of ten patients who underwent bimaxillary surgery were acquired pre-operatively. Individualized 3D virtual operation plans were made for all patients prior to surgery. During surgery, the maxillary and mandibular segments were positioned as planned by using 3D milled interocclusal wafers. Consequently, post-operative CBCT scan were acquired. The 3D rendered pre- and postoperative virtual head models were aligned by voxel-based registration upon the anterior cranial base. To calculate the discrepancies between the 3D planning and the actual surgical outcome, the 3D planned maxillary and mandibular segments were segmented and superimposed upon the postoperative maxillary and mandibular segments. The translation matrices obtained from this registration process were translated into translational and rotational discrepancies between the 3D planning and the surgical outcome, by using the newly developed tool, the OrthoGnathicAnalyser. To evaluate the reproducibility of this method, the process was performed by two independent observers multiple times.

Results

Low intra-observer and inter-observer variations in measurement error (mean error < 0.25 mm) and high intraclass correlation coefficients (> 0.97) were found, supportive of the observer independent character of the OrthoGnathicAnalyser. The pitch of the maxilla and mandible showed the highest discrepancy between the 3D planning and the postoperative results, 2.72° and 2.75° respectively.

Conclusion

This novel method provides a reproducible tool for the evaluation of bimaxillary surgery, making it possible to compare larger patient groups in an objective and time-efficient manner in order to optimize the current workflow in orthognathic surgery.     

Nucleotide variation of sFRP5 gene is not associated with obesity in children and adolescents

Because sFRP5 was shown to be an important extracellular modulator of the Wnt pathway, regulating adipogenesis, we wanted to investigate the role of sFRP5 variants in human, monogenic obesity by performing mutation analysis. We screened the complete sFRP5 coding region in 622 obese children and adolescents and 503 lean control individuals by high-resolution melting curve analysis and direct sequencing. We found a total of 15 sequence variants in sFRP5, 10 of which resulted in a non-synonymous amino acid change. Five of these variants were, to our knowledge, not previously reported. For one of the variants (c.-3G>A), we identified a trend towards association between the variant frequency and the obese phenotype. We argue that, when looking at conservation and location inside known protein domains, several of the identified variants (D103N, A113V, K212N and H317L), may affect sFRP5 protein function. In addition, we found c.-3G>A, residing in the Kozak sequence, with a lower frequency in cases compared to controls. However, functional studies investigating the effect of sFRP5 variants on protein function are necessary to determine the true role of sFRP5 genetic variation in human, monogenic obesity.     

Line FRAP with the confocal laser scanning microscope for diffusion measurements in small regions of 3-D samples

We present a truly quantitative fluorescence recovery after photobleaching (FRAP) model for use with the confocal laser scanning microscope based on the photobleaching of a long line segment. The line FRAP method is developed to complement the disk FRAP method reported before. Although being more subject to the influence of noise, the line FRAP model has the advantage of a smaller bleach region, thus allowing for faster and more localized measurements of the diffusion coefficient and mobile fraction. The line FRAP model is also very well suited to examine directly the influence of the bleaching power on the effective bleaching resolution. We present the outline of the mathematical derivation, leading to a final analytical expression to calculate the fluorescence recovery. We examine the influence of the confocal aperture and the bleaching power on the measured diffusion coefficient to find the optimal experimental conditions for the line FRAP method. This will be done for R-phycoerythrin and FITC-dextrans of various molecular weights. The ability of the line FRAP method to measure correctly absolute diffusion coefficients in three-dimensional samples will be evaluated as well. Finally we show the application of the method to the simultaneous measurement of free green fluorescent protein diffusion in the cytoplasm and nucleus of living A549 cells.