Physicochemical Tools : Toward a Detailed Understanding of the Architecture of Targeted Radiotherapy Nanoparticles

Funded by Chief Scientists Office (TCS/16/07), TENOVUS Scotland (G15-03), and the University of Aberdeen Development Trust. K.C.N. gratefully acknowledges the University of Aberdeen for the Elphinstone PhD scholarship (RG13451-10) and to Postnova Analytics UK for training and loan of the AFFF system. Microscopy was performed in the Microscopy and Histology Core Facility at the University of Aberdeen. GC acknowledges D. Zaton for useful discussions.Peer reviewedPostprin

Oxaliplatin combined with irinotecan and 5-fluorouracil/leucovorin (OCFL) in metastatic colorectal cancer: a phase I-II study

Background:: A phase I-II multicenter trial was conducted to define the maximal tolerated dose and describe the activity of an OCFL combination using oxaliplatin (OHP), irinotecan (CPT-11) and 5-fluorouracil (FU)/leucovorin (LV) in metastatic colorectal cancer (CRC). Patients and methods:: CRC patients not pretreated with palliative chemotherapy, with performance status ≤1 and adequate haematological, kidney and liver function, were eligible. Treatment consisted in weekly 24-h infusion 5-FU (2300 mg/m2)/LV (30 mg) and alternating OHP (70-85 mg/m2, days 1 and 15) and CPT-11 (80-140 mg/m2, days 8 and 22) repeated every 5 weeks. OHP and CPT-11 were escalated in cohorts of three to six patients. Results:: Thirty patients received a median of five cycles. Dose-limiting toxicity occurred at dose level 3, and the recommended dose was OHP 70 mg/m2, CPT-11 100 mg/m2, LV 30 mg and 5-FU 2300 mg/m2/24 h. Grade ≥3 toxicities were diarrhea 23%, neutropenia 20%, fatigue 7%, and neurologic 7%. Two febrile neutropenia episodes (one fatal) were recorded. Among 28 patients with measurable disease (90%), we observed two complete and 20 partial responses; overall RR was 78% (95% CI, 59% to 92%). Median time to progression and overall survival were 9.5 and 25.4 months, respectively. Seven patients underwent liver metastases resection. Conclusion:: OCFL is an overall well tolerated regimen with very high efficacy, which makes it most suitable for tumour control before surgery of metastatic diseas

A Peptide-binding Motif for I-Ag7, the Class II Major Histocompatibility Complex (MHC) Molecule of NOD and Biozzi AB/H Mice

The class II major histocompatibility complex molecule I-Ag7 is strongly linked to the development of spontaneous insulin-dependent diabetes mellitus (IDDM) in non obese diabetic mice and to the induction of experimental allergic encephalomyelitis in Biozzi AB/H mice. Structurally, it resembles the HLA-DQ molecules associated with human IDDM, in having a non-Asp residue at position 57 in its β chain. To identify the requirements for peptide binding to I-Ag7 and thereby potentially pathogenic T cell epitopes, we analyzed a known I-Ag7-restricted T cell epitope, hen egg white lysozyme (HEL) amino acids 9–27. NH2- and COOH-terminal truncations demonstrated that the minimal epitope for activation of the T cell hybridoma 2D12.1 was M12-R21 and the minimum sequence for direct binding to purified I-Ag7 M12-Y20/ K13-R21. Alanine (A) scanning revealed two primary anchors for binding at relative positions (p) 6 (L) and 9 (Y) in the HEL epitope. The critical role of both anchors was demonstrated by incorporating L and Y in poly(A) backbones at the same relative positions as in the HEL epitope. Well-tolerated, weakly tolerated, and nontolerated residues were identified by analyzing the binding of peptides containing multiple substitutions at individual positions. Optimally, p6 was a large, hydrophobic residue (L, I, V, M), whereas p9 was aromatic and hydrophobic (Y or F) or positively charged (K, R). Specific residues were not tolerated at these and some other positions. A motif for binding to I-Ag7 deduced from analysis of the model HEL epitope was present in 27/30 (90%) of peptides reported to be I-Ag7–restricted T cell epitopes or eluted from I-Ag7. Scanning a set of overlapping peptides encompassing human proinsulin revealed the motif in 6/6 good binders (sensitivity = 100%) and 4/13 weak or non-binders (specificity = 70%). This motif should facilitate identification of autoantigenic epitopes relevant to the pathogenesis and immunotherapy of IDDM

Oxaliplatin combined with irinotecan and 5-fluorouracil/leucovorin (OCFL) in metastatic colorectal cancer: a phase I-II study

BACKGROUND: A phase I-II multicenter trial was conducted to define the maximal tolerated dose and describe the activity of an OCFL combination using oxaliplatin (OHP), irinotecan (CPT-11) and 5-fluorouracil (FU)/leucovorin (LV) in metastatic colorectal cancer (CRC). PATIENTS AND METHODS: CRC patients not pretreated with palliative chemotherapy, with performance status < or =1 and adequate haematological, kidney and liver function, were eligible. Treatment consisted in weekly 24-h infusion 5-FU (2300 mg/m(2))/LV (30 mg) and alternating OHP (70-85 mg/m(2), days 1 and 15) and CPT-11 (80-140 mg/m(2), days 8 and 22) repeated every 5 weeks. OHP and CPT-11 were escalated in cohorts of three to six patients. RESULTS: Thirty patients received a median of five cycles. Dose-limiting toxicity occurred at dose level 3, and the recommended dose was OHP 70 mg/m(2), CPT-11 100 mg/m(2), LV 30 mg and 5-FU 2300 mg/m(2)/24 h. Grade > or =3 toxicities were diarrhea 23%, neutropenia 20%, fatigue 7%, and neurologic 7%. Two febrile neutropenia episodes (one fatal) were recorded. Among 28 patients with measurable disease (90%), we observed two complete and 20 partial responses; overall RR was 78% (95% CI, 59% to 92%). Median time to progression and overall survival were 9.5 and 25.4 months, respectively. Seven patients underwent liver metastases resection. CONCLUSION: OCFL is an overall well tolerated regimen with very high efficacy, which makes it most suitable for tumour control before surgery of metastatic disease

IL-1β Promotes TGF-β1 and IL-2 Dependent Foxp3 Expression in Regulatory T Cells

Earlier, we have shown that GM-CSF-exposed CD8α− DCs that express low levels of pro-inflammatory cytokines IL-12 and IL-1β can induce Foxp3+ Tregs leading to suppression of autoimmunity. Here, we examined the differential effects of IL-12 and IL-1β on Foxp3 expression in T cells when activated in the presence and absence of DCs. Exogenous IL-12 abolished, but IL-1β enhanced, the ability of GM-CSF-exposed tolerogenic DCs to promote Foxp3 expression. Pre-exposure of DCs to IL-1β and IL-12 had only a modest effect on Foxp3− expressing T cells; however, T cells activated in the absence of DCs but in the presence of IL-1β or IL-12 showed highly significant increase and decrease in Foxp3+ T cell frequencies respectively suggesting direct effects of these cytokines on T cells and a role for IL-1β in promoting Foxp3 expression. Importantly, purified CD4+CD25+ cells showed a significantly higher ability to maintain Foxp3 expression when activated in the presence of IL-1β. Further analyses showed that the ability of IL-1β to maintain Foxp3 expression in CD25+ T cells was dependent on TGF-β1 and IL-2 expression in Foxp3+Tregs and CD25− effectors T cells respectively. Exposure of CD4+CD25+ T cells to IL-1β enhanced their ability to suppress effector T cell response in vitro and ongoing experimental autoimmune thyroidits in vivo. These results show that IL-1β can help enhance/maintain Tregs, which may play an important role in maintaining peripheral tolerance during inflammation to prevent and/or suppress autoimmunity

25th annual computational neuroscience meeting: CNS-2016

The same neuron may play different functional roles in the neural circuits to which it belongs. For example, neurons in the Tritonia pedal ganglia may participate in variable phases of the swim motor rhythms [1]. While such neuronal functional variability is likely to play a major role the delivery of the functionality of neural systems, it is difficult to study it in most nervous systems. We work on the pyloric rhythm network of the crustacean stomatogastric ganglion (STG) [2]. Typically network models of the STG treat neurons of the same functional type as a single model neuron (e.g. PD neurons), assuming the same conductance parameters for these neurons and implying their synchronous firing [3, 4]. However, simultaneous recording of PD neurons shows differences between the timings of spikes of these neurons. This may indicate functional variability of these neurons. Here we modelled separately the two PD neurons of the STG in a multi-neuron model of the pyloric network. Our neuron models comply with known correlations between conductance parameters of ionic currents. Our results reproduce the experimental finding of increasing spike time distance between spikes originating from the two model PD neurons during their synchronised burst phase. The PD neuron with the larger calcium conductance generates its spikes before the other PD neuron. Larger potassium conductance values in the follower neuron imply longer delays between spikes, see Fig. 17.Neuromodulators change the conductance parameters of neurons and maintain the ratios of these parameters [5]. Our results show that such changes may shift the individual contribution of two PD neurons to the PD-phase of the pyloric rhythm altering their functionality within this rhythm. Our work paves the way towards an accessible experimental and computational framework for the analysis of the mechanisms and impact of functional variability of neurons within the neural circuits to which they belong

Ultrastructural non-radioactive in situ hybridization of GH mRNA in rat pituitary gland: pre-embedding vs ultra-thin frozen sections vs post-embedding.

International audienceIn situ hybridization at the ultrastructural level can be carried out using three different methods: on vibratome sections before embedding in epoxy resin, on ultra-thin frozen sections, or on ultra-thin sections of tissues embedded in hydrophilic resin such as Lowicryl. With the purpose of comparing the sensitivity, resolution, and ultrastructural preservation of these three methods, we examined the expression of the growth hormone (GH) gene in anterior pituitary cells by in situ hybridization at the ultrastructural level, using a synthetic oligonucleotide complementary to the codons of the mRNA from Gln 45 to Ser 54 labeled at the 3' end of biotin-21dUTP. All these methods gave similar results: mRNA was located on the lamellar endoplasmic reticulum of somatotrophs. The pre-embedding method gave the best ultrastructural preservation, with low resolution with the enzymatic detection system and an intermediate sensitivity. A probe concentration of 10 pmol/ml was sufficient to obtain a signal. With this method gold particles could not be used without pre-treatment. The frozen section method gave the best sensitivity (a signal was observed with 4 pmol/ml of probe) but the lowest ultrastructural preservation. On ultra-thin Lowicryl sections, resolution was as high as with the frozen-section method, ultrastructural conservation was intermediate, and sensitivity was low. These results indicate that the last method seems to be a good compromise between sensitivity and ultrastructural preservation.In situ hybridization at the ultrastructural level can be carried out using three different methods: on vibratome sections before embedding in epoxy resin, on ultra-thin frozen sections, or on ultra-thin sections of tissues embedded in hydrophilic resin such as Lowicryl. With the purpose of comparing the sensitivity, resolution, and ultrastructural preservation of these three methods, we examined the expression of the growth hormone (GH) gene in anterior pituitary cells by in situ hybridization at the ultrastructural level, using a synthetic oligonucleotide complementary to the codons of the mRNA from Gln 45 to Ser 54 labeled at the 3' end of biotin-21dUTP. All these methods gave similar results: mRNA was located on the lamellar endoplasmic reticulum of somatotrophs. The pre-embedding method gave the best ultrastructural preservation, with low resolution with the enzymatic detection system and an intermediate sensitivity. A probe concentration of 10 pmol/ml was sufficient to obtain a signal. With this method gold particles could not be used without pre-treatment. The frozen section method gave the best sensitivity (a signal was observed with 4 pmol/ml of probe) but the lowest ultrastructural preservation. On ultra-thin Lowicryl sections, resolution was as high as with the frozen-section method, ultrastructural conservation was intermediate, and sensitivity was low. These results indicate that the last method seems to be a good compromise between sensitivity and ultrastructural preservation