Simultaneous Inhibition of mTOR-Containing Complex 1 (mTORC1) and MNK Induces Apoptosis of Cutaneous T-Cell Lymphoma (CTCL) Cells

BACKGROUND: mTOR kinase forms the mTORC1 complex by associating with raptor and other proteins and affects a number of key cell functions. mTORC1 activates p70S6kinase 1 (p70S6K1) and inhibits 4E-binding protein 1 (4E-BP1). In turn, p70S6K1 phosphorylates a S6 protein of the 40S ribosomal subunit (S6rp) and 4E-BP1, with the latter negatively regulating eukaryotic initiation factor 4E (eIF-4E). MNK1 and MNK2 kinases phosphorylate and augment activity of eIF4E. Rapamycin and its analogs are highly specific, potent, and relatively non-toxic inhibitors of mTORC1. Although mTORC1 activation is present in many types of malignancies, rapamycin-type inhibitors shows relatively limited clinical efficacy as single agents. Initially usually indolent, CTCL displays a tendency to progress to the aggressive forms with limited response to therapy and poor prognosis. Our previous study (M. Marzec et al. 2008) has demonstrated that CTCL cells display mTORC1 activation and short-term treatment of CTCL-derived cells with rapamycin suppressed their proliferation and had little effect on the cell survival. METHODS: Cells derived from CTCL were treated with mTORC1 inhibitor rapamycin and MNK inhibitor and evaluated for inhibition of the mTORC1 signaling pathway and cell growth and survival. RESULTS: Whereas the treatment with rapamycin persistently inhibited mTORC1 signaling, it suppressed only partially the cell growth. MNK kinase mediated the eIF4E phosphorylation and inhibition or depletion of MNK markedly suppressed proliferation of the CTCL cells when combined with the rapamycin-mediated inhibition of mTORC1. While MNK inhibition alone mildly suppressed the CTCL cell growth, the combined MNK and mTORC1 inhibition totally abrogated the growth. Similarly, MNK inhibitor alone displayed a minimal pro-apoptotic effect; in combination with rapamycin it triggered profound cell apoptosis. CONCLUSIONS: These findings indicate that the combined inhibition of mTORC1 and MNK may prove beneficial in the treatment of CTCL and other malignancies

Increasing integrated testing in community settings through interventions for change, including the Spring European Testing Week

Background: Maximising access to testing by targeting more than one infection is effective in identifying new infections in settings or populations. Within the EU funded Joint Action INTEGRATE, this paper examined the feasibility and impact of expanding integrated testing for HIV, hepatitis C (HCV), chlamydia, gonorrhoea and/or syphilis in four community-based pilots through targeted interventions in Croatia, Italy and Poland and the Spring European Testing Week since community settings are key in detecting new infections and reaching key populations. Methods: Pilots led by local INTEGRATE partners prioritised testing for other infections or key populations. The Croatian pilot expanded testing for men who have sex with men to syphilis, chlamydia and gonorrhoea. Italian partners implemented a HIV and HCV testing/information event at a migrant centre. A second Italian pilot tested migrants for HIV and HCV through outreach and a low-threshold service for people who use drugs. Polish partners tested for HIV, HCV and syphilis among people who inject drugs in unstable housing via a mobile van. Pilots monitored the number of individuals tested for each infection and reactive results. The pilot Spring European Testing Week from 18 to 25 May 2018 was an INTEGRATE-driven initiative to create more testing awareness and opportunities throughout Europe. Results: The Croatian pilot found a high prevalence for each syphilis, chlamydia and gonorrhoea respectively, 2.1%, 12.4% and 6.7%. The Italian migrant centre pilot found low proportions who were previously tested for HIV (24%) or HCV (11%) and the second Italian pilot found an HCV prevalence of 6.2%, with low proportions previously tested for HIV (33%) or HCV (31%). The Polish pilot found rates of being previously tested for HIV, HCV and syphilis at 39%, 37%, and 38%, respectively. Results from the Spring European Testing Week pilot showed it was acceptable with increased integrated testing, from 50% in 2018 to 71% in 2019 in participants. Conclusions: Results show that integrated testing is feasible and effective in community settings, in reaching key populations and minimising missed testing opportunities, and the pilots made feasible because of the European collaboration and funding. For sustainability and expansion of integrated community testing across Europe, local government investment in legislation, financial and structural support are crucial.The INTEGRATE Joint Action was co-funded by the 3rd Health Programme of the European Union under grant agreement no 761319. The EuroTEST/European Testing Week initiative has received funding and grants from Gilead Sciences, ViiV Healthcare, Janssen, Merck/MSD and the European Commission under the 3rd and 2nd Health Programmes. HUHIV: CheckPoint Zagreb is funded by cooperation programs by the City of Zagreb and Ministry of Health incl. HIV, HCV and syphilis rapid tests, CT/NG tests are donated by Cepheid with the contribution of the Department of Immunological and Molecular Diagnostics of the Clinic for Infectious Diseases Dr Fran Mihaljević during the pilot project. CRI/FVM: For the pilot activity in the migrant centre, HIV and HCV rapid tests were donated by FVM/CRI. Moreover, FVM contributed with the staff and equipment (mobile unit). FVM: The medical centre and outreach street unit are funded by the Health Department of Lazio Regional Administration of Italy. NAC/FES: Funding for FES pilot in 2019 was provided by NAC. FES secured their staff, mobile unit and tests. Daniel Simões is the recipient of PhD Grant PD/BD/128008/2016 from Fundação para a Ciência e Tecnologia (FCT). All funders had no role in the study design, analysis, decision to publish, or preparation of the manuscript

Raman-based spectrophenotyping of the most important cells of the immune system

INTRODUCTION: Human peripheral blood mononuclear cells (PBMCs) are a heterogeneous population of cells that includes T and B lymphocytes. The total number of lymphocytes and their percentage in the blood can be a marker for the diagnosis of several human diseases. Currently, cytometric methods are widely used to distinguish subtypes of leukocytes and quantify their number. These techniques use cell immunophenotyping, which is limited by the number of fluorochrome-labeled antibodies that can be applied simultaneously. OBJECTIVE: B and T lymphocytes were isolated from peripheral blood obtained from healthy human donors. METHODS: The immunomagnetic negative selection was used for the enrichment of B and T cells fractions, and their purity was assessed by flow cytometry. Isolated cells were fixed with 0.5% glutaraldehyde and measured using confocal Raman imaging. K-means cluster analysis, principal component analysis and partial least squares discriminant methods were applied for the identification of spectroscopic markers to distinguish B and T cells. HPLC was the reference method for identifying carotene in T cells. RESULTS: Reliable discrimination between T and B lymphocytes based on their spectral profile has been demonstrated using label-free Raman imaging and chemometric analysis. The presence of carotene in T lymphocytes (in addition to the previously reported in plasma) was confirmed and for the first time unequivocally identified as β-carotene. In addition, the molecular features of the lymphocytes nuclei were found to support the discriminant analysis. It has been shown that although the presence of carotenoids in T cells depends on individual donor variability, the reliable differentiation between lymphocytes is possible based on Raman spectra collected from individual cells. CONCLUSIONS: This proves the potential of Raman spectroscopy in clinical diagnostics to automatically differentiate between cells that are an important component of our immune system

Alternative technique of intrauterine myelomeningocele repair to decrease the incidence of unfavorable maternal and fetal outcomes

Objectives: The aim of the study was to determine the effectiveness of an alternative method of open fetal surgery to prevent severe unfavorable prenatal events, both for the mother and the fetus. Material and methods: In this study, the previously published results for a cohort of 46 patients, who had undergone intrauterine myelomeningocele repair (IUMR) at our Center by 2014, constituted the retrospective control group (CG). The MOMS protocol had been applied for hysterotomy, with an automatic uterine stapling device. The study group (SG) n = 57 was assembled during a prospective observation. IUMR was performed using an alternative method of hysterotomy, with the typical opening and closure of the uterus, without automatic stapling device, as described by Moron et al. Additionally, our single-center results were compared with the post-MOMS findings of other centers: CHOP (Children’s Hospital of Philadelphia) and VUMC (Vanderbilt University Medical Center). Results: No cases of delivery before 30 weeks of gestation (0%, 0/55) were observed in the study group, which is a statistically significant difference (p < 0.05) as compared to controls (15/44). Statistically significantly lower incidence of chorioamniotic separation (5.4% (3/55) vs CHOP 22.9% (22/96), p < 0.001) and contractile activity resulting in preterm labor (16.3% (9/55) vs CHOP 37.5% (36/96), p < 0.05) was found in the study group. Premature rupture of the membranes was statistically significantly less common in the study group as compared to controls, CHOP and VUMC (SG 12.7% (7/55) vs CG 52.2% (24/46), p < 0.001; vs CHOP 32.3% (31/96), p < 0.001; vs VUMC 22% (9/43), p < 0.01, respectively). Conclusions: The presented IUMR method is associated with improved perinatal outcomes, i.e., lower rates of preterm delivery at < 30 weeks of gestation, preterm premature rupture of membranes, and uterine contractility resulting in preterm delivery. That, in turn, results in lower prematurity rates and, consequently, more favorable neonatal outcomes

Inhibition of caspase-1 prolongs survival of mice infected with rabies virus

Rabies virus infects almost all mammals resulting in lethal disease. To date there is no treatment available for symptomatic rabies and there is an urgent need to develop treatment strategies that would prolong survival, thereby providing a window of opportunity for the host to mount a protective immune response. We hypothesized that both virus and excessive immune response contribute to disease and that interfering with both is necessary to prevent lethal disease. Here, we have inhibited the pro-inflammatory response associated with pyroptosis and showed that inhibition of CASP-1 had a beneficial effect on survival time. Our results confirm that some inflammatory responses may be involved in the pathogenesis of severe disease and the results suggest that effective intervention includes inhibition of virus and host response

Scintillator counters with WLS fiber/MPPC readout for the side muon range detector (SMRD)of the T2K experiment

The T2K neutrino experiment at J-PARC uses a set of near detectors to measure the properties of an unoscillated neutrino beam and neutrino interaction cross-sections. One of the sub-detectors of the near-detector complex, the side muon range detector (SMRD), is described in the paper. The detector is designed to help measure the neutrino energy spectrum, to identify background and to calibrate the other detectors. The active elements of the SMRD consist of 0.7 cm thick extruded scintillator slabs inserted into air gaps of the UA1 magnet yokes. The readout of each scintillator slab is provided through a single WLS fiber embedded into a serpentine shaped groove. Two Hamamatsu multi-pixel avalanche photodiodes (MPPC's) are coupled to both ends of the WLS fiber. This design allows us to achieve a high MIP detection efficiency of greater than 99%. A light yield of 25-50 p.e./MIP, a time resolution of about 1 ns and a spatial resolution along the slab better than 10 cm were obtained for the SMRD counters.Comment: 7 pages, 4 figures; talk at TIPP09, March 12-17, Tsukuba, Japan; to be published in the conference proceeding

Oxidative Stress in Cells with Extra Centrosomes Drives Non-Cell-Autonomous Invasion

This work was supported by a Cancer Research UK Centre Grant to Barts Cancer Institute (C16420/A18066). P.R.C. was funded by BBSRC (BB/M006174/1) and Barts and The London Charity (297/2249). S.A.G. is a fellow of the Lister Institute and is supported by the Medical Research Council (MR/M010414/1)