Bone marrow-derived mesenchymal stromal cells obstruct AML-targeting CD8+ clonal effector and CAR T-cell function while promoting a senescence-associated phenotype.

Bone marrow mesenchymal stromal cells (MSCs) have been described as potent regulators of T-cell function, though whether they could impede the effectiveness of immunotherapy against acute myeloid leukemia (AML) is still under investigation. We examine whether they could interfere with the activity of leukemia-specific clonal cytotoxic T-lymphocytes (CTLs) and chimeric antigen receptor (CAR) T cells, as well as whether the immunomodulatory properties of MSCs could be associated with the induction of T-cell senescence. Co-cultures of leukemia-associated Wilm's tumor protein 1 (WT1) and tyrosine-protein kinase transmembrane receptor 1 (ROR1)-reactive CTLs and of CD123-redirected switchable CAR T cells were prepared in the presence of MSCs and assessed for cytotoxic potential, cytokine secretion, and expansion. T-cell senescence within functional memory sub-compartments was investigated for the senescence-associated phenotype CD28-CD57+ using unmodified peripheral blood mononuclear cells. We describe inhibition of expansion of AML-redirected switchable CAR T cells by MSCs via indoleamine 2,3-dioxygenase 1 (IDO-1) activity, as well as reduction of interferon gamma (IFNγ) and interleukin-2 (IL-2) release. In addition, MSCs interfered with the secretory potential of leukemia-associated WT1- and ROR1-targeting CTL clones, inhibiting the release of IFNγ, tumor necrosis factor alpha, and IL-2. Abrogated T cells were shown to retain their cytolytic activity. Moreover, we demonstrate induction of a CD28loCD27loCD57+KLRG1+ senescent T-cell phenotype by MSCs. In summary, we show that MSCs are potent modulators of anti-leukemic T cells, and targeting their modes of action would likely be beneficial in a combinatorial approach with AML-directed immunotherapy.

Do preschoolers trust a competent robot pointer?

How young children learn from different informants has been widely studied. However, most studies investigate how children learn verbally conveyed information. Furthermore, most studies investigate how children learn from humans. This study sought to investigate how 3-year-old children learn from, and come to trust, a competent robot versus an incompetent human when competency is established using a pointing paradigm. During an induction phase, a robot informant pointed at a toy inside a transparent box, whereas a human pointed at an empty box. During the test phase, both agents pointed at opaque boxes. We found that young children asked the robot for help to locate a hidden toy more than the human (ask questions) and correctly identified the robot to be accurate (judgment questions). However, children equally endorsed the locations pointed at by both the robot and the human (endorse questions). This suggests that 3-year-olds are sensitive to the epistemic characteristics of the informant even when its displayed social properties are minimal.

Of children and social robots.

In the target article, Clark and Fischer argue that little is known about children's perceptions of social robots. By reviewing the existing literature we demonstrate that infants and young children interact with robots in the same ways they do with other social agents. Importantly, we conclude children's understanding that robots are artifacts (e.g., not alive) develops gradually during the preschool years.

Chemical Zymogens: Specificity and Steroidal Control of Reactivation.

Activating and masking enzymatic activity on demand is of the highest importance in nature. It is achieved by chemical interconversion of enzymes and the corresponding zymogens through, for example, proteolytic processing or reversible phosphorylation, and affords on-demand activation of enzymes, controlled in space and/or time. In stark contrast, examples of chemical zymogens are very few, and in most cases these are based on disulfide chemistry, which is largely indiscriminate as to the nature of the activating thiol. In this work, we address an outstanding challenge of specificity of reactivation of chemical zymogens. We achieve this through engineering affinity between the chemical zymogen and the activator. Additional, higher-level control over zymogen reactivation is installed in a nature-mimicking approach using steroidal hormones. Taken together, the results of this study take a step towards establishing the specificity of reactivation of synthetic, chemical zymogens. We anticipate that the results of this study will contribute significantly to the development of chemical zymogens as tools for diverse use in chemical biology and biotechnology.

Elevated fractional donor-derived cell-free DNA during subclinical graft injury after liver transplantation.

Personalized immunosuppression (IS) promises to improve the balance of necessary control of alloreactivity and dose-dependent adverse effects of long-term IS such as kidney insufficiency, infections, and malignancies. The majority of liver transplantation (LT) recipients exhibit graft injuries (graft inflammation and/or fibrosis) that are not eligible for an IS reduction according to current Banff criteria, even when liver enzymes are normal or only marginally elevated. This cross-sectional study evaluated the noninvasive prediction of such subclinical graft injuries in surveillance liver biopsies via donor-derived cell-free DNA (dd-cfDNA). Absolute and fractional dd-cfDNA increased stepwise from patients without histological signs of rejection (n = 26) over subclinical graft injury (n = 61), including subclinical T cell-mediated rejection to clinical overt T cell-mediated rejection (n = 21). Thus, fractional plasma dd-cfDNA was significantly elevated paired to surveillance biopsies with relevant subclinical graft injury according to 2016 Banff criteria compared with those with minimal or absent histological graft injury. In contrast, the presence of donor-specific anti-human leukocyte antigen antibodies was not associated with the amount of dd-cfDNA. The sensitivity and specificity of fractional dd-cfDNA to noninvasively predict relevant subclinical graft injury was rather limited with 73% and 52% at the cutoff value of 2.1% fractional dd-cfDNA. The positive predictive value of fractional dd-cfDNA above 2.1% was 76% to noninvasively predict subclinical graft injury, calculated on the prevalence of graft injury in our prospective surveillance biopsy program, whereas the negative predictive values was not predictive (47%). In conclusion, dd-cfDNA has a rather limited diagnostic fidelity in addition to other noninvasive markers for the assessment of subclinical graft injury in personalized IS approaches after LT in a cross-sectional setting.

Preschoolers' anthropomorphizing of robots: Do human-like properties matter?

Prior work has yielded contradicting evidence regarding the age at which children consistently and correctly categorize things as living or non-living. The present study tested children's animacy judgments about robots with a Naïve Biology task. In the Naïve Biology task, 3- and 5-year-olds were asked if robots, animals, or artifacts possessed mechanical or biological internal parts. To gauge how much children anthropomorphize robots in comparison to animals and artifacts, children also responded to a set of interview questions. To examine the role of morphology, two robots were used: a humanoid robot (Nao) and a non-humanoid robot (Dash). To investigate the role of dynamic characteristics, children saw one robot behave in a goal-directed manner (i.e., moving towards a ball) and one robot exhibit non-goal-directed behavior (i.e., moving away from a ball). Children of both age groups correctly attributed biological insides to the animal and mechanical insides to the artifact. However, 3-year-olds seemed confused about what belonged inside both robots and assigned biological and mechanical insides equally. In contrast, 5-year-olds correctly assigned mechanical insides to both robots, regardless of the robot's morphology or goal-directedness. Regarding the Animacy Interview, 3-year-olds performed at chance level when asked about the animacy of robots, animals, and artifacts. In contrast, 5-year-olds correctly attributed animacy to animals and accurately refrained from anthropomorphizing artifacts and the non-humanoid robot Dash. However, 5-year-olds performed at chance for Nao, suggesting they may be confused about the psychological properties of a human-looking robot. Taken together, these findings reveal a developmental transition during the preschool years in the attribution of biological and psychological properties to social robot.

[Disinfectants during the COVID-19 pandemic: a challenge]. / Desinfektionsmittel in der COVID-19-Pandemie: eine Herausforderung.

Disinfection measures have become more important as a result of the COVID-19 pandemic in Germany. The increased need for disinfectants at the beginning of the pandemic required temporary legal regulations in order to provide a sufficient quantity of products for the necessary disinfection in the medical sector on the one hand and for the additional demand in the population on the other. For this purpose, the Federal Institute for Drugs and Medical Devices (BfArM) and the Federal Institute for Occupational Safety and Health (BAuA) issued a general ruling, which is explained in more detail in this article. The focus was on measures for hygienic hand disinfection. However, other applications such as surface disinfection in relation to pandemic respiratory diseases are also addressed. The experience gained in ensuring the supply of disinfectants that are effective and safe to use should be used to prepare for further pandemics.

Role of Sialyl-O-Acetyltransferase CASD1 on GD2 Ganglioside O-Acetylation in Breast Cancer Cells.

The O-acetylated form of GD2, almost exclusively expressed in cancerous tissues, is considered to be a promising therapeutic target for neuroectoderm-derived tumors, especially for breast cancer. Our recent data have shown that 9-O-acetylated GD2 (9-OAcGD2) is the major O-acetylated ganglioside species in breast cancer cells. In 2015, Baumann et al. proposed that Cas 1 domain containing 1 (CASD1), which is the only known human sialyl-O-acetyltransferase, plays a role in GD3 O-acetylation. However, the mechanisms of ganglioside O-acetylation remain poorly understood. The aim of this study was to determine the involvement of CASD1 in GD2 O-acetylation in breast cancer. The role of CASD1 in OAcGD2 synthesis was first demonstrated using wild type CHO and CHOΔCasd1 cells as cellular models. Overexpression using plasmid transfection and siRNA strategies was used to modulate CASD1 expression in SUM159PT breast cancer cell line. Our results showed that OAcGD2 expression was reduced in SUM159PT that was transiently depleted for CASD1 expression. Additionally, OAcGD2 expression was increased in SUM159PT cells transiently overexpressing CASD1. The modulation of CASD1 expression using transient transfection strategies provided interesting insights into the role of CASD1 in OAcGD2 and OAcGD3 biosynthesis, and it highlights the importance of further studies on O-acetylation mechanisms.

Virological Outcome of Patients With HIV Drug Resistance Attending an Urban Outpatient Clinic in Uganda: A Need for Structured Adherence Counseling and Third-Line Treatment Options.

BACKGROUND: HIV drug resistance and suboptimal adherence are the main reasons for treatment failure among HIV-infected individuals. As genotypic resistance testing is not routinely available in resource-limited settings such as Uganda, data on transmitted and acquired resistance are sparse. METHODS: This observational follow-up study assessed the virological outcomes of patients diagnosed with virological failure or transmitted HIV drug resistance in 2015 at the adults' outpatient clinic of the Infectious Diseases Institute in Kampala, Uganda. Initially, 2430 patients on antiretroviral therapy (ART) underwent virological monitoring, of which 190 had virological failure and were subsequently eligible for this follow-up study. Nine patients diagnosed with transmitted drug resistance were eligible. In patients with a viral load > 1000 copies/mL, genotypic resistance testing was performed. RESULTS: Of 190 eligible patients, 30 (15.8%) had either died or were lost to follow-up. A total of 148 (77.9%) were included, of which 98 had had a change of ART regimen, and 50 had received adherence counseling only. The majority was now on second-line ART (N = 130, 87.8%). The median age was 39 years (interquartile range: 32-46), and 109 (73.6%) were women. Virological failure was diagnosed in 29 (19.6%) patients, of which 24 (82.8%) were on second-line ART. Relevant drug resistance was found in 25 (86.2%) cases, of which 12 (41.3%) carried dual and 7 (24.1%) triple drug resistance. CONCLUSION: Two years after initial virological failure, most patients followed up by this study had a successful virological outcome. However, a significant proportion either continued to fail or died or was lost to follow-up.

[Career prospects of graduate Bachelor nursing degree programs - Results of a nationwide study in Germany]. / Berufsperspektiven von Absolventinnen und Absolventen grundständig qualifizierender Pflegestudiengänge − Ergebnisse einer bundesweiten Verbleibstudie.

Career prospects of graduate Bachelor nursing degree programs - Results of a nationwide study in Germany Abstract. BACKGROUND: Graduates of Bachelor nursing degree programs are available on the labour market for a few years. Currently, there is no empirical evidence or reports on different fields of these academically prepared nurses in Germany. AIM: Aim of this study was to assess the whereabouts of academically prepared nurses in the German labour market, their satisfaction with their current working conditions, and future prospects of the graduates. METHODS: A cross-sectional design was conducted using an online survey to assess whereabouts of academically prepared nurses (n = 273) after graduation from all German Bachelor nursing degree programs. RESULTS: The majority of 77.6 % of participating nurses worked in direct acute settings or in elderly care. Those, working in direct patient care appeared to be significantly more dissatisfied with their working conditions in comparison to those in extended nursing practice (p = 0.000). High satisfaction scores were reported for qualification-adjusted activities, and working conditions in general. Positive perspectives for personal growth and development see 81.3 %. However, 31.1 % of this sample were enrolled in Master programs or plan to enrol in a further education study program into the next future. CONCLUSIONS: In order to become implicit, the process of academic nursing education needs to continue in all practice settings. However, innovative human resources development strategies are needed to use the benefit and full scope of practice in academically prepared nurses in Germany.

Ten years of antiretroviral therapy: Incidences, patterns and risk factors of opportunistic infections in an urban Ugandan cohort.

BACKGROUND: Despite increased antiretroviral therapy (ART) coverage and the raised CD4 threshold for starting ART, opportunistic infections (OIs) are still one of the leading causes of death in sub-Saharan Africa. There are few studies from resource-limited settings on long-term reporting of OIs other than tuberculosis. METHODS: Patients starting ART between April 2004 and April 2005 were enrolled and followed-up for 10 years in Kampala, Uganda. We report incidences, patterns and risk factors using Cox proportional hazards models of OIs among all patients and among patients with CD4 cell counts >200 cells/µL. RESULTS: Of the 559 patients starting ART, 164 patients developed a total of 241 OIs during 10 years of follow-up. The overall incidence was highest for oral candidiasis (25.4, 95% confidence interval (CI): 20.5-31.6 per 1000 person-years of follow-up), followed by tuberculosis (15.3, 95% CI: 11.7-20.1), herpes zoster (12.3, 95% CI: 9.1-16.6) and cryptococcal meningitis (3.0, 95% CI: 1.7-5.5). Incidence rates for all OIs were highest in the first year after ART initiation and decreased with the increase of the current CD4 cell count. Factors independently associated with development of OIs were baseline nevirapine-based regimens, time-varying higher viral load, time-varying lower CD4 cell count and time-varying lower hemoglobin. In patients developing OIs at a current CD4 cell count >200 cells/µL, factors independently associated with OI development were time-varying increase in viral load and time-varying decrease in hemoglobin, whereas a baseline CD4 cell count <50 cells/µL was protective. CONCLUSION: We report high early incidences of OIs, decreasing with increasing CD4 cell count and time spent on ART. Ongoing HIV replication and anemia were strong predictors for OI development independent of the CD4 cell count. Our findings support the recommendation for early initiation of ART and suggest close monitoring for OIs among patients recently started on ART, with low CD4 cell count, high viral load and anemia.

Multiple off-axis fiber Bragg gratings for 3D shape sensing.

Point-by-point femtosecond laser processed fiber Bragg gratings are arranged around the edge of a standard single-mode optical fiber core. The relative amplitudes of at least three such fiber Bragg gratings are utilized to detect the central position of the mode field within the fiber core and calculate the local curvature of the fiber. An analytical approximation is given, and an experimental validation is performed.

Is predictability salient? A study of attentional capture by auditory patterns.

In this series of behavioural and electroencephalography (EEG) experiments, we investigate the extent to which repeating patterns of sounds capture attention. Work in the visual domain has revealed attentional capture by statistically predictable stimuli, consistent with predictive coding accounts which suggest that attention is drawn to sensory regularities. Here, stimuli comprised rapid sequences of tone pips, arranged in regular (REG) or random (RAND) patterns. EEG data demonstrate that the brain rapidly recognizes predictable patterns manifested as a rapid increase in responses to REG relative to RAND sequences. This increase is reminiscent of the increase in gain on neural responses to attended stimuli often seen in the neuroimaging literature, and thus consistent with the hypothesis that predictable sequences draw attention. To study potential attentional capture by auditory regularities, we used REG and RAND sequences in two different behavioural tasks designed to reveal effects of attentional capture by regularity. Overall, the pattern of results suggests that regularity does not capture attention.This article is part of the themed issue 'Auditory and visual scene analysis'.

Preferential accumulation of T helper cells but not cytotoxic T cells characterizes benign subclinical rejection of human liver allografts.

Subclinical rejection (SCR) is a common event in protocol biopsies after liver transplantation (LT). So far the interpretation of the underlying histological changes and clinical significance is limited. Previous studies were restricted to SCR manifestations within the first weeks after transplantation with limited follow-up. We analyzed clinical data from our prospective protocol biopsy program and found late SCR (at least 3 months after transplantation) to be a common event (41/94 patients). SCR manifested much later than acute cellular rejection (ACR). In the second year after transplantation, the SCR incidence in protocol biopsies reached a plateau of approximately 25% and remained at this level until the latest observed manifestations more than 5 years after transplantation. During a median follow-up of 32 months after SCR, no acute or chronic rejection, relevant graft fibrosis, graft loss, or liver-related death occurred even without specific therapy for SCR. Immunophenotyping of liver biopsies during SCR showed that similar to ACR, the composition of intrahepatic T cells depended on the severity of histological rejection. However, SCR showed a different pattern of infiltrating T cells with a stronger accumulation of CD4(+) cells, an increasing CD4(+) /CD8(+) ratio, and an increasing CD4(+) forkhead box P3 (FOXP3)(+) regulatory T cell (Treg)/CD8(+) ratio, which was not seen in ACR. These intrahepatic T cell patterns were not reflected in the peripheral blood. In conclusion, late SCR after LT has a good clinical prognosis, and it seems safe to leave it untreated. This benign clinical course compared to ACR is associated with intrahepatic T cell infiltration patterns showing less cytotoxic T cells and more CD4(+) FOXP3(+) Tregs. Liver Transplantation 22 943-955 2016 AASLD.

TriFabs--Trivalent IgG-Shaped Bispecific Antibody Derivatives: Design, Generation, Characterization and Application for Targeted Payload Delivery.

TriFabs are IgG-shaped bispecific antibodies (bsAbs) composed of two regular Fab arms fused via flexible linker peptides to one asymmetric third Fab-sized binding module. This third module replaces the IgG Fc region and is composed of the variable region of the heavy chain (VH) fused to CH3 with "knob"-mutations, and the variable region of the light chain (VL) fused to CH3 with matching "holes". The hinge region does not contain disulfides to facilitate antigen access to the third binding site. To compensate for the loss of hinge-disulfides between heavy chains, CH3 knob-hole heterodimers are linked by S354C-Y349C disulphides, and VH and VL of the stem region may be linked via VH44C-VL100C disulphides. TriFabs which bind one antigen bivalent in the same manner as IgGs and the second antigen monovalent "in between" these Fabs can be applied to simultaneously engage two antigens, or for targeted delivery of small and large (fluorescent or cytotoxic) payloads.

9-O-Acetylation of sialic acids is catalysed by CASD1 via a covalent acetyl-enzyme intermediate.

Sialic acids, terminal sugars of glycoproteins and glycolipids, play important roles in development, cellular recognition processes and host-pathogen interactions. A common modification of sialic acids is 9-O-acetylation, which has been implicated in sialoglycan recognition, ganglioside biology, and the survival and drug resistance of acute lymphoblastic leukaemia cells. Despite many functional implications, the molecular basis of 9-O-acetylation has remained elusive thus far. Following cellular approaches, including selective gene knockout by CRISPR/Cas genome editing, we here show that CASD1--a previously identified human candidate gene--is essential for sialic acid 9-O-acetylation. In vitro assays with the purified N-terminal luminal domain of CASD1 demonstrate transfer of acetyl groups from acetyl-coenzyme A to CMP-activated sialic acid and formation of a covalent acetyl-enzyme intermediate. Our study provides direct evidence that CASD1 is a sialate O-acetyltransferase and serves as key enzyme in the biosynthesis of 9-O-acetylated sialoglycans.

Induction of heat shock protein HSPA6 (HSP70B') upon HSP90 inhibition in cancer cell lines.

Genome-wide transcript profiling to elucidate responses to HSP90 inhibition revealed strong induction of HSPA6 in MCF-7 cells treated with 17-AAG. Time- and dose dependent induction of HSPA6 (confirmed by qPCR and Western Blots) occurred also upon treatment with Radicicol, another HSP90 inhibitor. HSPA6 was not detectable in untreated cells or cells treated with toxins that do not inhibit HSP90, or upon applying oxidative stress. Thus, HSPA6 induction is not a general response to cytotoxic insults. Modulation of HSPA6 levels by siRNA-mediated inhibition or recombinant expression did not influence 17-AAG mediated cell death. HSPA6 induction as a consequence of HSP90 inhibition occurs in various (but not all) cell lines and may be a more specific marker for HSP90 inhibition than induction of other HSP70 proteins.

Intrahepatic regulatory T cells in autoimmune hepatitis are associated with treatment response and depleted with current therapies.

BACKGROUND & AIMS: Autoimmune hepatitis (AIH) is a chronic autoimmune liver disease usually requiring life-long immunosuppression. The mechanisms for disease initiation and chronicity are largely unknown. A contribution of deficient regulatory T cells (Tregs) in the blood was controversially discussed recently. So far investigations in the target organ have been limited to single parameter analysis in untreated AIH. METHODS: We retrospectively analysed the pattern of liver infiltrating T, B and regulatory T cells quantitatively with simultaneous multicolour immunofluorescence before (n=45) and under (n=31) therapy in adult AIH type 1 (AIH-1) patients. RESULTS: Intrahepatic CD4(+) cells dominate over CD8(+) at diagnosis, but with increasing disease activity the CD4(+)/CD8(+) ratio approached one. While there is no change of Tregs in the blood, they are enriched with effector T cells (Teffs) within the liver of patients with untreated AIH-1 with a constant Treg/Teff ratio. Even more importantly, immunosuppression mostly with steroids and azathioprine caused a disproportional loss of intrahepatic Tregs. Patients reaching biochemical remission had higher intrahepatic Treg/Teff and Treg/B cell ratios compared to patients failing to reach remission. In vitro proliferation of Tregs seemed to be more suppressed by prednisolone than expansion of Teffs. Furthermore, intraportal B cells correlated with serum IgG suggesting an autochthonous intrahepatic IgG production. CONCLUSIONS: Intrahepatic Tregs are rather enriched than numerically deficient in untreated AIH-1. The disproportional decrease of intrahepatic Tregs during therapy might explain high relapse rates after discontinuation of immunosuppression. Thus, future therapies increasing intrahepatic immunoregulation might be better suited for long-term control of AIH.

West syndrome caused by ST3Gal-III deficiency.

West syndrome consists of infantile spasms, hypsarrhythmia, and developmental arrest. Most patients remain mentally retarded and many develop Lennox-Gastaut syndrome. Using homozygosity mapping followed by exome sequencing we identified an ST3GAL3 mutation in three infants with West syndrome. ST3GAL3 encodes a sialyltransferase involved in the biosynthesis of sialyl-Lewis epitopes on cell surface-expressed glycoproteins. The mutation affected an essential sialyl-motif and abolished enzymatic activity. Abnormalities in proteins involved in forebrain γ-aminobutyric acid (GABA)ergic synaptic growth and function were recently proposed to account for infantile spasms. Dysfunctional ST3GAL3 may thus result in perturbation of the posttranslational sialylation of proteins in these pathways.

[Dysentery and amoebiasis in Poland in 2008]. / Czerwonka bakteryjna i pelzakowa w Polsce w 2008 roku.

The number as well as incidence rate of notified as shigellosis, bacillary dysentery cases in XXI century are every year lower. Only 33 cases were notified in the year 2008 (incidence rate 0.09/100 000 population), while the 64 cases were notified in 2007 (incidence rate 0.17/100 000), but 35 (incidence rate 0.09) were notified in 2006 and median in the 2002-2006 years was 75 cases, incidence rate 0.2/100 000 population. Since 2000 no one-death case was notified. Several imported cases were notified as they got infection being abroad, polish citizens on holiday abroad, and foreign children coming for vacations in Poland from countries with higher incidence rate for ex. from Ukraine. Out of 11 imported cases three were due to Shigella sp., eight to S. sonnei. Two persons were found infected by S. boydii 8-11 but no one of S. dysentery. In the period of low frequency of Shigella infections, the external quality assessment control of the quality of bacteriological media, laboratory and prae-laboratory procedures for detection of different Shigella groups and types should be executed. A lower number of amoebic dysentery cases were registered 5 cases in 2008 (incidence rate 0.013/100 000 population), but in 2007: the 19 cases, incidence rate 0.05/100 000, and even more in 2006 the 21 cases, incidence rate 0.055/100 000. The cases were registered in 3 voivodeships; infection was probably imported from underdeveloped countries.