Immune checkpoint therapy responders display early clonal expansion of tumor infiltrating lymphocytes.

Immune checkpoint therapy (ICT) causes durable tumour responses in a subgroup of patients, but it is not well known how T cell receptor beta (TCRß) repertoire dynamics contribute to the therapeutic response. Using murine models that exclude variation in host genetics, environmental factors and tumour mutation burden, limiting variation between animals to naturally diverse TCRß repertoires, we applied TCRseq, single cell RNAseq and flow cytometry to study TCRß repertoire dynamics in ICT responders and non-responders. Increased oligoclonal expansion of TCRß clonotypes was observed in responding tumours. Machine learning identified TCRß CDR3 signatures unique to each tumour model, and signatures associated with ICT response at various timepoints before or during ICT. Clonally expanded CD8+ T cells in responding tumours post ICT displayed effector T cell gene signatures and phenotype. An early burst of clonal expansion during ICT is associated with response, and we report unique dynamics in TCRß signatures associated with ICT response.

GAVeCeLT-WoCoVA Consensus on subcutaneously anchored securement devices for the securement of venous catheters: Current evidence and recommendations for future research.

BACKGROUND: Subcutaneously anchored securement devices (or subcutaneous engineered securement devices) have been introduced recently into the clinical practice, but the number of published studies is still scarce. The Italian Group of Long-Term Central Venous Access Devices (GAVeCeLT)-in collaboration with WoCoVA (World Congress on Vascular Access)-has developed a Consensus about the effectiveness, safety, and cost-effectiveness of such devices. METHODS: After the definition of a panel of experts, a systematic collection and review of the literature on subcutaneously anchored securement devices was performed. The panel has been divided in two working groups, one focusing on adult patients and the other on children and neonates. RESULTS: Although the quality of evidence is generally poor, since it is based mainly on non-controlled prospective studies, the panel has concluded that subcutaneously anchored securement devices are overall effective in reducing the risk of dislodgment and they appear to be safe in all categories of patients, being associated only with rare and negligible local adverse effects; cost-effectiveness is demonstrated-or highly likely-in specific populations of patients with long-term venous access and/or at high risk of dislodgment. CONCLUSION: Subcutaneously anchored securement is a very promising strategy for avoiding dislodgment. Further studies are warranted, in particular for the purpose of defining (a) the best management of the anchoring device so to avoid local problems, (b) the patient populations in which it may be considered highly cost-effective and even mandatory, (c) the possible benefit in terms of reduction of other catheter-related complications such as venous thrombosis and/or infection, and-last but not least-(d) their impact on the workload and stress level of nurses taking care of the devices.

Development of a novel noncapillary plasma microsampling device for ultra-low volume of blood collection.

The desire for serial microsampling in mice has led to extensive research in this field within the pharmaceutical industry. The ability to profile a compound's in vivo properties with less material and fewer mice has obvious advantages. A new device and workflow was developed at the Takeda Oncology site to allow scientists to isolate plasma from very low volumes of mouse blood (as low as 20 µl) collected using standard microsampling techniques. A side-by-side in vitro comparison of plasma concentrations was performed using this new device and conventional sampling methods with commercial and in-house molecules. The plasma concentrations of the molecules tested were very consistent between the conventional sampling techniques and this new device/workflow. In addition, several in-life studies have also been conducted to validate this new technique as a primary PK screening tool at the Takeda Boston. The new device is simple to use and very cost effective with the added benefit that no additional training is needed for the animal technicians and the same centrifuge equipment can be employed. This device can be used for blood volumes ranging from 20 to 100 µl enabling studies not just in rat and dog but more importantly in mice.

Retrograde optogenetic characterization of the pontospinal module of the locus coeruleus with a canine adenoviral vector.

Noradrenergic neurons of the brainstem extend projections throughout the neuraxis to modulate a wide range of processes including attention, arousal, autonomic control and sensory processing. A spinal projection from the locus coeruleus (LC) is thought to regulate nociceptive processing. To characterize and selectively manipulate the pontospinal noradrenergic neurons in rats, we implemented a retrograde targeting strategy using a canine adenoviral vector to express channelrhodopsin2 (CAV2-PRS-ChR2-mCherry). LC microinjection of CAV2-PRS-ChR2-mCherry produced selective, stable, transduction of noradrenergic neurons allowing reliable opto-activation in vitro. The ChR2-transduced LC neurons were opto-identifiable in vivo and functional control was demonstrated for >6 months by evoked sleep-wake transitions. Spinal injection of CAV2-PRS-ChR2-mCherry retrogradely transduced pontine noradrenergic neurons, predominantly in the LC but also in A5 and A7. A pontospinal LC (ps:LC) module was identifiable, with somata located more ventrally within the nucleus and with a discrete subset of projection targets. These ps:LC neurons had distinct electrophysiological properties with shorter action potentials and smaller afterhyperpolarizations compared to neurons located in the core of the LC. In vivo recordings of ps:LC neurons showed a lower spontaneous firing frequency than those in the core and they were all excited by noxious stimuli. Using this CAV2-based approach we have demonstrated the ability to retrogradely target, characterise and optogenetically manipulate a central noradrenergic circuit and show that the ps:LC module forms a discrete unit. This article is part of a Special Issue entitled SI: Noradrenergic System.

Combined activity of oridonin and wogonin in advanced-stage ovarian cancer cells: sensitivity of ovarian cancer cells to phyto-active chemicals.

The initial response rates of advanced-stage epithelial ovarian cancer to the chemotherapeutic agents carboplatin and paclitaxel are high. However, once drug resistance develops, further chemotherapy is less effective. The objective of this study is to investigate the anti-proliferative activity of the phyto-active chemicals (PACs) oridonin and wogonin in chemo-resistant epithelial ovarian cancer cells. Primary cell cultures from the ascitic fluid of three patients at diagnosis, two patients chemo-resistant to carboplatin and paclitaxel, and one patient treated with letrozole for breast cancer were studied and compared to the ovarian cancer cell lines A2780 and PTX10, by cell viability assay (MTS). Effects on cell cycle modulation and apoptosis were examined by flow cytometry and Western blot analysis (WB). WB was further conducted to investigate protein expressions altered by PACs. The results show that IC(50) of the primary cultures ranged from 0.6 to 5.4 µg/ml for oridonin and 0.3-12.7 µg/ml for wogonin. The paclitaxel-resistant cell line PTX10 was more sensitive to each of the PACs than the chemo-sensitive cell line A2780. Of particular interest is that in combination, the two PACs were synergistic in their cytotoxicity to five of six of the primary cultures and to both the cell lines (combination indices of 0.39-0.95). The inhibition is attributable to apoptosis and cell cycle modulation induced by the PACs as demonstrated in A2780 and PTX10. Up-regulation of the functional p53 protein in A2780 and down-regulation of Akt protein in PTX10 have in part contributed to the apoptosis. These findings suggest that oridonin and wogonin may have activity in ovarian cancer following its development of resistance to carboplatin and paclitaxel.

Stimulation of tumor growth and angiogenesis by low concentrations of RGD-mimetic integrin inhibitors.

Inhibitors of alpha(v)beta(3) and alpha(v)beta(5) integrin have entered clinical trials as antiangiogenic agents for cancer treatment but generally have been unsuccessful. Here we present in vivo evidence that low (nanomolar) concentrations of RGD-mimetic alpha(v)beta(3) and alpha(v)beta(5) inhibitors can paradoxically stimulate tumor growth and tumor angiogenesis. We show that low concentrations of these inhibitors promote VEGF-mediated angiogenesis by altering alpha(v)beta(3) integrin and vascular endothelial growth factor receptor-2 trafficking, thereby promoting endothelial cell migration to VEGF. The proangiogenic effects of low concentrations of RGD-mimetic integrin inhibitors could compromise their efficacy as anticancer agents and have major implications for the use of RGD-mimetic compounds in humans.

The proximate control of pupal color in swallowtail butterflies: implications for the evolution of environmentally cued pupal color in butterflies (Lepidoptera: Papilionidae).

The environmentally cued production of cryptic green/yellow or brown/melanized pupae is widespread in butterflies, occurring in the Nymphalidae, Pieridae, and the Papilionidae subfamily Papilioninae. The dimorphism is controlled by the hormone pupal melanization reducing factor (PMRF). In the nymphalid Inachis io dibutryl cAMP mimics PMRF, and inhibits pupal melanization. However, in the papilionid Papilio polyxenes PMRF stimulates browning, suggesting that the control of pupal color by PMRF has evolved independently in the swallowtail and nymphalid-pierid lineages. We examined this hypothesis by using ligatures to prevent hormone release in five species representing three Papilioninae tribes. One species, Papilio glaucus, produces only brown pupae. Ligatures resulted in green cuticle posterior to the ligature in all five swallowtail species, including P. glaucus, suggesting that the mode of action of PMRF is the same in the three tribes. We also found that in P. polyxenes injections of dibutryl cAMP into prepupal larvae mimic the effect of PMRF, by causing dose-dependent pupal browning. Our results support the hypothesis that the control of pupal color by PMRF has evolved independently in the two lineages. The observation that green pupal color can be induced in P. glaucus by ligature indicates that environmentally cued pupal color could evolve by facultative inhibition of PMRF release.

VEGF regulates the mobilization of VEGFR2/KDR from an intracellular endothelial storage compartment.

Endothelial cells respond to vascular endothelial growth factor (VEGF) to produce new blood vessels. This process of angiogenesis makes a critical contribution during embryogenesis and also in the response to ischemia in adult tissues. We have studied the intracellular trafficking of the major VEGF receptor KDR (VEGFR2). Unlike other related growth factor receptors, we find that a significant proportion of KDR is held in an endosomal storage pool within endothelial cells. We find that KDR can be delivered to the plasma membrane from this intracellular pool and that VEGF stimulates this recycling to the cell surface. KDR recycling appears to be distinct from the previously characterized Rab4- and Rab11-dependent pathways, but, instead, KDR(+) recycling vesicles contain Src tyrosine kinase and VEGF-stimulated recycling requires Src activation. Taken together, these data show that intracellular trafficking of KDR is markedly different from other receptor tyrosine kinases and suggest that the regulation of KDR trafficking by VEGF provides a novel mechanism for controlling the sensitivity of endothelial cells to proangiogenic signals.

The role of extracellular regulated kinases I/II in late-phase long-term potentiation.

Extracellular regulated kinases (ERKI/II), members of the mitogen-activated protein kinase family, play a role in long-term memory and long-term potentiation (LTP). ERKI/II is required for the induction of the early phase of LTP, and we show that it is also required for the late phase of LTP in area CA1 in vitro, induced by a protocol of brief, repeated 100 Hz trains. We also show that ERKI/II is necessary for the upregulation of the proteins encoded by the immediate early genes Zif268 and Homer after the induction of LTP in the dentate gyrus by tetanic stimulation of the perforant path in vivo or by BDNF stimulation of primary cortical cultures. To test whether the induction of persistent synaptic plasticity by stimuli such as BDNF is associated with nuclear translocation of ERKI/II, we expressed enhanced green fluorescent protein (EGFP)-ERKII in PC12 cell lines and primary cortical cultures. In both preparations, we observed translocation of EGFP-ERKII from the cytoplasm to the nucleus in cells exposed to neurotrophic factors. Our results suggest that the induction of late LTP involves translocation of ERKI/II to the nucleus in which it activates the transcription of immediate early genes. The ability to visualize the cellular redistribution of ERKII after induction of long-term synaptic plasticity may provide a method for visualizing neuronal circuits underlying information storage in the brain in vivo.