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bioRxiv2018

Importance of vitamin D in critically ill children with subgroup analyses of sepsis and respiratory tract infections: a systematic review and meta-analysis

BackgroundCritical care and sepsis remain high priority concerns in children. Observational studies report high prevalence of vitamin D deficiency and present mixed results regarding the correlation between vitamin D status and adverse outcomes. Associations between deficiency and mortality, particularly in children with sepsis, remain unclear. We performed a systematic review and meta-analysis to address this uncertainty.\n\nMethodsPubMed, OVID and Google Scholar were searched for observational studies in critically ill children. We obtained pooled prevalence estimates for vitamin D deficiency and odds ratios for the association of mortality in critically ill children treated in intensive care units, with subgroup analysis for children with sepsis. Meta-regression and sensitivity analyses were used to investigate heterogeneity.\n\nFindingsForty-eight studies were included. The total sample size was 7,199, with 1,679 (23%) children acting as controls in case-control studies. Of 5,520 critically ill children, 2,664 (48%) were vitamin D deficient (< 50 nmol/L). Results of the random effects model demonstrated a pooled prevalence of vitamin D deficiency of 54.9% (95% CI 48.0-61.6, I2=95.0%, 95% CI 94.0-95.8, p < 0.0001). In subgroup analysis of children with sepsis (16 studies, 788 total individuals) we observed higher prevalence of deficiency (63.8%, 95% CI 49.9-75.7, I2=90.5%, 95% CI 86.2-93.5%, p < 0.0001). In patients admitted to intensive care for respiratory tract infections (24 studies, 1,683 total individuals), prevalence was 49.9% (95% CI 37.6-62.2; I2 = 93.9%, 95% CI 92.1-95.3, p < 0.0001). Only one identified study assessed vitamin D levels in sepsis and mortality. The meta-regression model with all available variables (year of publication, total study sample size, quality score, study design, country group and clinical setting) explained 37.52% of I2 (F = 5.1119, p = 0.0005) with clinical setting and country groups being significant predictors for prevalence.\n\nOur meta-analysis (18 studies, 2,463 total individuals) showed an increased risk of death in vitamin D deficient critically ill children both with the random (OR 1.81, 95% CI 1.24-2.64, p-value = 0.002) and fixed effects (OR 1.72, 95% CI 1.27-2.33, p= 0.0005) models with low heterogeneity (I2 = 25.7%, 95% CI 0.0-58.0, p = 0.153) and low evidence of publication bias (p = 0.084, Eggers test).\n\nInterpretationCirculating vitamin D deficiency is common amongst critically ill children, particularly in those with sepsis. Our results suggest that vitamin D deficiency in critically ill children is associated with increased mortality. Clinical trials, studies with larger sample sizes and standardized approaches are needed to further assess associations between circulating levels of vitamin D and mortality or other outcomes in the pediatric population.\n\nFundingNone\n\nRegistrationPROSPERO (CRD42016050638)\n\nCopyrightOpen access article under terms of CC BY

epidemiology↗

bioRxiv2018

High-dose rifamycins enable shorter oral treatment in a murine model of Mycobacterium ulcerans disease

Buruli ulcer (BU), caused by Mycobacterium ulcerans, is a neglected tropical skin and soft tissue infection that is associated with disability and social stigma. The mainstay of BU treatment is an eight-week course of 10 mg/kg rifampin (RIF) and 150 mg/kg streptomycin (STR). Recently, the injectable STR has been shown to be replaceable with oral clarithromycin (CLR) for smaller lesions for the last four weeks of treatment. A shorter, all-oral, highly efficient regimen for BU is needed, as the long treatment duration and indirect costs currently burden patients and health systems. Increasing the dose of RIF or replacing it with the more potent rifamycin drug rifapentine (RPT) could provide such a regimen. Here, we performed a dose-ranging experiment of RIF and RPT in combination with CLR over four weeks of treatment in a mouse model of M. ulcerans disease. A clear dose-dependent effect of RIF on both clinical and microbiological outcomes was found, with no ceiling effect observed with tested doses up to 40 mg/kg. RPT-containing regimens were more effective on M. ulcerans. All RPT-containing regimens achieved culture negativity after only four weeks while only the regimen with the highest RIF dose (40 mg/kg) did so. We conclude that there is dose-dependent efficacy of both RIF and RPT and that a ceiling effect is not reached with the current standard regimen used in the clinic. A regimen based on higher rifamycin doses that are currently being evaluated against tuberculosis in clinical trials could shorten and improve therapy of Buruli ulcer.

microbiology↗

bioRxiv2018

Engineered transfer RNAs for suppression of premature termination codons

Premature termination codons (PTCs) are responsible for 10-15% of all inherited disease. PTC suppression during translation offers a promising approach to treat a variety of genetic disorders, yet small molecules that promote PTC read-through have yielded mixed performance in clinical trials. We present a high-throughput, cell-based assay to identify anticodon engineered transfer RNAs (ACE-tRNA) which can effectively suppress in-frame PTCs and faithfully encode their cognate amino acid. In total, we identified ACE-tRNA with a high degree of suppression activity targeting the most common human disease-causing nonsense codons. Genome-wide transcriptome ribosome profiling of cells expressing ACE-tRNA at levels which repair PTC indicate that there are limited interactions with translation termination codons. These ACE-tRNAs display high suppression potency in mammalian cells, Xenopus oocytes and mice in vivo, producing PTC repair in multiple genes, including disease causing mutations within the cystic fibrosis transmembrane conductance regulator (CFTR).

molecular biology↗

bioRxiv2018

An HIV-1 broadly neutralizing antibody from a clade C infected pediatric elite neutralizer potently neutralizes the contemporaneous and autologous evolving viruses

Broadly neutralizing antibodies (bNAbs) have demonstrated protective effects against HIV-1 in primate studies and recent human clinical trials. Elite-neutralizers are potential candidates for isolation of HIV-1 bNAbs and coexistence of bNAbs such as BG18 with neutralization susceptible autologous viruses in an HIV-1 infected adult elite controller has been suggested to control viremia. Disease progression is faster in HIV-1 infected children than adults. Plasma bNAbs with multiple epitope specificities are developed in HIV-1 chronically infected children with more potency and breadth than in adults. Therefore, we evaluated the specificity of plasma neutralizing antibodies of an antiretroviral naive HIV-1 clade C chronically infected pediatric elite neutralizer AIIMS_330. The plasma antibodies showed broad and potent HIV-1 neutralizing activity with >87% (29/33) breadth, median inhibitory dilution (ID50) value of 1246 and presence of N160 and N332-supersite dependent HIV-1 bNAbs. The sorting of BG505.SOSIP.664.C2 T332N gp140 HIV-1 antigen-specific single B cells of AIIMS_330 resulted in the isolation of an HIV-1 N332-supersite dependent bNAb AIIMS-P01. The AIIMS-P01 neutralized 67% of HIV-1 cross-clade viruses; exhibited substantial indels despite limited somatic hypermutations; interacted with native-like HIV-1 trimer as observed in negative stain electron microscopy and demonstrated high binding affinity. In addition, AIIMS-P01 potently neutralized the coexisting and evolving autologous viruses suggesting the coexistence of vulnerable autologous viruses and HIV-1 bNAbs in AIIMS_330 pediatric elite neutralizer. Further studies on such pediatric elite-neutralizers and isolation of novel HIV-1 pediatric bNAbs may provide newer insights to guide vaccine design.\n\nImportanceMore than 50% of the HIV-1 infections globally are caused by clade C viruses. Till date, there is no effective vaccine to prevent HIV-1 infection. Based on the structural information of the currently available HIV-1 bNAbs, attempts are underway to design immunogens that can elicit correlates of protection upon vaccination. Here we report the isolation and characterization of an HIV-1 N332-supersite dependent bNAb AIIMS-P01 from a clade C chronically infected pediatric elite neutralizer. The N332-supersite is an important epitope and is one of the current HIV-1 vaccine targets. AIIMS-P01 potently neutralized the contemporaneous and autologous evolving viruses and exhibits substantial indels despite low somatic hypermutations. Taken together with the information on infant bNAbs, further isolation of bNAbs contributing to the plasma breadth in HIV-1 infected children may help to better understand their development and characteristics, which in turn may guide vaccine design.

immunology↗

bioRxiv2018

TP53 mutations and drug sensitivity in acute myeloid leukaemia cells with acquired MDM2 inhibitor resistance

BackgroundMDM2 inhibitors are under investigation for the treatment of acute myeloid leukaemia (AML) patients in phase III clinical trials. To study resistance formation to MDM2 inhibitors in AML cells, we here established 45 sub-lines of the AML TP53 wild-type cell lines MV4-11 (15 sub-lines), OCI-AML-2 (10 sub-lines), OCI-AML-3 (12 sub-lines), and SIG-M5 (8 sub-lines) with resistance to the MDM2 inhibitor nutlin-3.\n\nMethods: Nutlin-3-resistant sub-lines were established by continuous exposure to stepwise increasing drug concentrations. The TP53 status was determined by next generation sequencing, cell viability was measured by MTT assay, and p53 was depleted using lentiviral vectors encoding shRNA.\n\nResultsAll MV4-11 sub-lines harboured the same R248W mutation and all OCI-AML-2 sub-lines the same Y220C mutation, indicating the selection of pre-existing TP53-mutant subpopulations. In concordance, rare alleles harbouring the respective mutations could be detected in the parental MV4-11 and OCI-AML-2 cell lines. The OCI-AML-3 and SIG-M5 sub-lines were characterised by varying TP53 mutations or wild type TP53, indicating the induction of de novo TP53 mutations. Doxorubicin, etoposide, gemcitabine, cytarabine, and fludarabine resistance profiles revealed a noticeable heterogeneity among the sub-lines even of the same parental cell lines. Loss-of-p53 function was not generally associated with decreased sensitivity to cytotoxic drugs.\n\nConclusionWe introduce a substantial set of models of acquired MDM2 inhibitor resistance in AML. MDM2 inhibitors select, in dependence on the nature of a given AML cell population, pre-existing TP53-mutant subpopulations or induce de novo TP53 mutations. Although loss-of-p53 function has been associated with chemoresistance in AML, nutlin-3-adapted sub-lines displayed in the majority of experiments similar or increased drug sensitivity compared to the respective parental cells. Hence, chemotherapy may remain an option for AML patients after MDM2 inhibitor therapy failure. Even sub-lines of the same parental cancer cell line displayed considerable heterogeneity in their response to other anti-cancer drugs, indicating the need for the detailed understanding and monitoring of the evolutionary processes in cancer cell populations in response to therapy as part of future individualised treatment protocols.

pharmacology and toxicology↗

bioRxiv2018

Applicability of Drug Response Metrics for Cancer Studies using Biomaterials

Bioengineers have built increasingly sophisticated models of the tumor microenvironment in which to study cell-cell interactions, mechanisms of cancer growth and metastasis, and to test new potential therapies. These models allow researchers to culture cells in conditions that include features of the in vivo tumor microenvironment (TME) implicated in regulating cancer progression, such as ECM stiffness, integrin binding to the ECM, immune and stromal cells, growth factor and cytokine depots, and a 3D geometry more representative of the TME than tissue culture polystyrene (TCPS). These biomaterials could be particularly useful for drug screening applications to make better predictions of efficacy, offering better translation to preclinical in vivo models and clinical trials. However, it can be challenging to compare drug response reports across different platforms and conditions in the current literature. This is, in part, as a result of inconsistent reporting and use of drug response metrics, and vast differences in cell growth rates across a large variety of biomaterial design. This perspective paper attempts to clarify the definitions of drug response measurements used in the field, and presents examples in which these measurements can and cannot be applied. We suggest as best practice to include appropriate controls, always measure the growth rate of cells in the absence of drug, and follow our provided \"decision tree\" matrix when reporting drug response metrics.

bioengineering↗

bioRxiv2018

New genetic variants associated with major adverse cardiovascular events in patients with acute coronary syndromes and treated with clopidogrel and aspirin

ImportanceAlthough a few studies have reported the effects of several polymorphisms on major adverse cardiovascular events (MACE) in patients with acute coronary syndromes (ACS) and those undergoing percutaneous coronary intervention (PCI), these genotypes account for only a small fraction of the variation and evidence is insufficient. This study aims to identify new genetic variants associated with MACE by large-scale sequencing data.\n\nObjectiveTo identify the genetic variants that caused MACE.\n\nDesignAll patients in this study were allocated to dual antiplatelet therapy for up to 12 months and have the follow-up duration of 18 months.\n\nSettingA two-stage association study was performed.\n\nParticipantsWe evaluated the associations of genetic variants and MACE in 1961 patients with ACS undergoing PCI (2009-2012), including high-depth whole exome sequencing of 168 patients in the discovery cohort and high-depth targeted sequencing of 1793 patients in the replication cohort.\n\nMain Outcomes and MeasureThe primary clinical efficacy endpoint was the major adverse cardiovascular events (MACE) composite endpoint, including cardiovascular death, myocardial infarction (MI), stroke (CT or MR scan confirmed) and repeated revascularization (RR).\n\nResultsWe discovered and confirmed six new genotypes associated with MACE in patients with ACS. Of which, rs17064642 at MYOM2 increased the risk of MACE (hazard ratio [HR] 2.76; P = 2.95 x 10-9) and reached genome-wide significance. The other five suggestive variants were KRTAP10-4 (rs201441480), WDR24 (rs11640115), ECHS1 (rs140410716), AGAP3 (rs75750968) and NECAB1 (rs74569896). Notably, the expressions of MYOM2 and ECHS1 are down-regulated in both animal models and patients with phenotypes related to MACE. Importantly, we developed the first superior classifier for predicting MACE and achieved high predictive accuracy (0.809).\n\nConclusions and RelevanceWe identified six new genotypes associated with MACE and developed a superior classifier for predicting MACE. Our findings shed light on the pathogenesis of cardiovascular outcomes and may help clinician to make decision on the therapeutic intervention for ACS patients.\n\nTrial RegistrationThis study has been registered in the Chinese Clinical Trial Registry (http://www.chictr.org.cn, Registration number: ChiCTR-OCH-11001198).

genetics↗

bioRxiv2018

The true size of placebo analgesia: Concordant neural and behavioural measures of placebo analgesia during experimental acute pain

Placebo analgesia refers to the reduction of pain following the administration of an inactive treatment. While most clinical trials compare a drug treatment against a placebo to determine the efficacy of the analgesic, most experimental studies of placebo analgesia do not include a real analgesic condition. A direct comparison of placebo against a real analgesic can inform us about the true size of the placebo effect. To this end, we aimed to provide a robust estimate of placebo analgesia by contrasting the effect of pain relief expectation from an inert cream (vaseline) against a real topical analgesic agent (lidocaine) applied on two different limbs and their respective control conditions. Pain reports and electroencephalography (EEG) responses triggered by laser nociceptive stimulation were collected. Forty typical healthy adults were enrolled in a double-blind randomized within-subject study where a standard placebo induction script of verbal suggestions in a sham medical setting was used to enhance the expectation on treatment outcome. In line with the earliest studies of placebo analgesia, majority (30 of 40) of participants was placebo responders, i.e. they reported lower pain to the placebo treatment. Placebo responders reported low pain and displayed low laser evoked potentials (LEPs) amplitude for both the analgesic and placebo treatment limbs compared to the respective control limbs. Placebo analgesia correlated positively with the amplitude of the LEPs, thus establishing convergent validity of the findings. This study provides a robust estimate of the neural and behavioural measures of placebo analgesia, in comparison to a real analgesic. These estimates can help inform the quantitative criteria for similar neural and behavioural measures in assessing the effectiveness of a real drug in placebo controlled trials.

neuroscience↗

bioRxiv2018

Can pharmacological enhancement of the placebo effect be a novel therapy for working memory impairments?

Working memory is considered as a core aspect of cognitive function and its impairment in a wide range of mental disorders has resulted in it being considered as an important transdiagnostic feature. To date pharmacological and behavioural strategies for augmenting working memory have achieved only moderate success. Here we have taken a different approach by combining expectancy effects with intranasal oxytocin as an adjunct given previous evidence that it may enhance placebo effects. In a randomised controlled clinical trial we demonstrate that while working memory performance is not influenced by expectancy per se when it is given in conjunction with oxytocin performance in terms of accuracy can be significantly enhanced following positive expectancy induction (placebo effect) and impaired following negative expectancy induction (nocebo effect). Thus combining expectancy effects with intranasal oxytocin may represent a radical new approach for improving working memory function in mental disorders.

neuroscience↗

bioRxiv2018

Optimization of Dose Schedules for Chemotherapy of Early Colon Cancer Determined by High Performance Computer Simulations

Cancer chemotherapy dose schedules are conventionally applied intermittently, with dose duration of the order of hours, intervals between doses of days or weeks, and cycles repeated for weeks. The large number of possible combinations of values of duration, interval, and lethality has been an impediment to empirically determine the optimal set of treatment conditions. The purpose of this project was to determine the set of parameters for duration, interval, and lethality that would be most effective for treating early colon cancer. An agent-based computer model that simulated cell proliferation kinetics in normal human colon crypts was calibrated with measurements of human biopsy specimens. Mutant cells were simulated as proliferating and forming an adenoma, or dying if treated with cytotoxic chemotherapy. Using a high performance computer, a total of 28,800 different parameter sets of duration, interval, and lethality were simulated. The effect of each parameter set on the stability of colon crypts, the time to cure a crypt of mutant cells, and the accumulated dose was determined. Of the 28,800 parameter sets, 434 parameter sets were effective in curing the crypts of mutant cells before they could form an adenoma and allowed the crypt normal cell dynamics to recover to pretreatment levels. A group of 14 similar parameter sets produced a minimal time to cure mutant cells. A different group of 9 similar parameter sets produced the least accumulated dose. These parameter sets may be considered as candidate dose schedules to guide clinical trials for early colon cancer.

cancer biology↗

bioRxiv2018

Do mechanical strain magnitude and rate drive bone adaptation in adult women? A 12-month prospective study.

Although there is strong evidence that certain activities can increase bone density and structure in some individuals, it is unclear what specific mechanical factors govern the response. This is important because understanding the effect of mechanical signals on bone could contribute to more effective osteoporosis prevention methods and efficient clinical trial design. The degree to which strain rate and magnitude govern bone adaptation in humans has never been prospectively tested. Here, we studied the effects of a voluntary upper extremity compressive loading task in healthy adult women during a twelve month prospective period. One hundred and two women age 21-40 participated in one of two experiments. (1): low (n=21) and high (n=24) strain magnitude. (2): low (n=21) and high (n=20) strain rate. Control: (n=16): no intervention. Strains were assigned using subject-specific finite element models. Load cycles were recorded digitally. The primary outcome was change in ultradistal integral bone mineral content (iBMC), assessed with QCT. Interim timepoints and secondary outcomes were assessed with high resolution pQCT (HRpQCT). Sixty-six subjects completed the intervention, and interim data were analyzed for 77 subjects. Both the low and high strain rate groups had significant 12-month increases to ultradistal iBMC (change in control: -1.3{+/-}2.7%, low strain rate: 2.7{+/-}2.1%, high strain rate: 3.4{+/-}2.2%), total iBMC, and other measures. \"Loading dose\" was positively related to 12-month change in ultradistal iBMC, and interim changes to total BMD, cortical thickness and inner trabecular BMD. Subjects who gained the most bone completed, on average, 130 loading bouts of (mean strain) 550 {varepsilon} at 1805 {varepsilon}/s. Those with the greatest gains had the highest loading dose. We conclude that signals related to strain magnitude, rate, and number of loading bouts contribute to bone adaptation in healthy adult women, but only explain a small amount of variance in bone changes.

physiology↗

bioRxiv2018

Adopting Literature-based Discovery on Rehabilitation Therapy Repositioning for Stroke

Stroke is a common disabling disease severely affecting the daily life of the patients. There is evidence that rehabilitation therapy can improve the movement function. However, there are no clear guidelines that identify specific, effective rehabilitation therapy schemes, and the development of new rehabilitation techniques has been fairly slow. One informatics translational approach, called ABC model in Literature-based Discovery, was used to mine an existing rehabilitation candidate which is most likely to be repositioned for stroke. As in the classic ABC model originated from Don Swanson, we built the internal links of stroke (A), assessment scales (B), rehabilitation therapies (C) in PubMed relating to upper limb function measurements for stroke patients. In the first step, with E-utility we retrieved both stroke related assessment scales and rehabilitation therapies records, and complied two datasets called Stroke_Scales and Stroke_Therapies, respectively. In the next step, we crawled all rehabilitation therapies co-occurred with the Stroke_Theapies, named as All_Therapies. Therapies that were already included in Stroke_Therapies were deleted from All_Therapies, so that the remaining therapies were the potential rehabilitation therapies, which could be repositioned for stroke after subsequent filtration by manual check. We identified the top ranked repositioning rehabilitation therapy following by subsequent clinical validation. Hand-arm bimanual intensive training (HABIT) ranked the first in our repositioning rehabilitation therapies list, with the most interaction links with Stroke_Scales. HABIT showed a significant improvement in clinical scores on assessment scales of Fugl-Meyer Assessment and Action Research Arm Test in the clinical validation on upper limb function for acute stroke patients. Based on the ABC model and clinical validation of the results, we put forward that HABIT as a promising rehabilitation therapy for stroke, which shows that the ABC model is an effective text mining approach for rehabilitation therapy repositioning. The results seem to be promoted in clinical knowledge discovery.\n\nAuthor SummaryIn the present study, we proposed a text mining approach to mining terms related to disease, rehabilitation therapy, and assessment scale from literature, with a subsequent ABC inference analysis to identify relationships of these terms across publications. The clinical validation demonstrated that our approach can be used to identify potential repositioning rehabilitation therapy strategies for stroke. Specifically, we identified a promising rehabilitation method called HABIT previously used in pediatric congenital hemiplegia. A subsequent clinical trial confirmed this as a highly promising rehabilitation therapy for stroke.

bioinformatics↗

bioRxiv2018

Lovastatin, not simvastatin, corrects core phenotypes in the fragile X mouse model

The cholesterol-lowering drug lovastatin corrects neurological phenotypes in animal models of fragile X syndrome (FX), a commonly identified genetic cause of autism and intellectual disability. The therapeutic efficacy of lovastatin is being tested in clinical trials for FX, however the structurally similar drug simvastatin has been proposed as an alternative due to an increased potency and brain penetrance. Here, we perform a side-by-side comparison of the effects of lovastatin and simvastatin treatment on two core phenotypes in the Fmr1-/y mouse model. We find that while lovastatin normalizes excessive hippocampal protein synthesis and reduces audiogenic seizures (AGS) in the Fmr1-/y mouse, simvastatin does not correct either phenotype. These results caution against the assumption that simvastatin is a valid alternative to lovastatin for the treatment of FX.

neuroscience↗

bioRxiv2018

Ancient origin and complex evolution of porcine endogenous retroviruses

BackgroundXenotransplantation may overcome significant shortage of human allotransplant. Porcine organs are considered favorable for xenotransplantation duo to similar size and function to human organ. However, porcine endogenous retroviruses (PERVs) are potential infectious agents during xenotransplantation as they are able to infect and horizontally transfer among human cells. Furthermore, PERVs can be endogenized in pig genomes and are transmitted genetically in a Mendelian fashion. Here, we depict a complex evolutionary history of modern PERVs.\n\nResultsWe in silico mined 142 mammalian genomes and 14 pig genomes. This led to the documentation of 185 PERVs and a new viral cluster. Large-scale genomic alterations were found in most PERVs including many insertion-deletion events and which are suggestive of ancient origins, and pig genomes have been shaped by PERV-mediated genomic rearrangement during evolution. Notably, we found that lesser Egyptian jerboa and rock hyrax harbor ancestral PERV-related elements indicative of ancient cross-species transmission events from none-porcine species to pigs. A comprehensive analysis of these viral \"fossils\" suggested that recombination among none-porcine endogenous retroviruses led to the origination of PERVs.\n\nConclusionFor the first time, using large scale genomic mining, we decipher a complex evolutionary history for the PERVs. These new findings help us to understand the past of PERVs which pose the potential risk in clinical trials of xenotransplantation and provide novel insights into the origin and evolution of a human-infecting pathogen.

microbiology↗

bioRxiv2018

Single-cell integrative analysis of CAR-T cell activation reveals a predominantly TH1/TH2 mixed response independent of differentiation

We present the first comprehensive portrait of single-cell level transcriptional and cytokine signatures of anti-CD19 4-1BB/CD28/CD3{zeta} CAR-T cells upon antigen-specific stimulation. Both CD4+ helper and CD8+ cytotoxic CAR-T cells are equally effective in directly killing target tumor cells and their cytotoxic activity is associated with the elevation of a range of TH1 and TH2 signature cytokines (e.g., IFN{gamma}, TNF, IL5, and IL13), as confirmed by the expression of master transcription factors TBX21 (T-bet) and GATA3. However, rather than conforming to stringent TH1 or TH2 subtypes, single-cell analysis reveals that the predominant response is a highly mixed TH1/TH2 function in the same cell and the regulatory T cell (Treg) activity, although observed in a small fraction of activated cells, emerges from this hybrid TH1/TH2 population. GM-CSF is produced from the majority of cells regardless of the polarization states, further contrasting CAR-T to classic T cells. Surprisingly, the cytokine response is minimally associated with differentiation status although all major differentiation subsets such as naive, central memory, effector memory and effector are detected. All these suggest that the activation of CAR-engineered T cells is a canonical process that leads to a highly mixed response combining both type 1 and type 2 cytokines together with GMCSF, supporting the notion that polyfunctional CAR-T cells correlate with objective response of patients in clinical trials. This work provides new insights to the mechanism of CAR activation and implies the necessity for cellular function assays to characterize the quality of CAR-T infusion products and monitor therapeutic responses in patients.

bioengineering↗

bioRxiv2018

Vitamin D3 regulates estrogen’s action and affects mammary epithelial organization in 3D cultures

Vitamin D3 (vitD3) and its active metabolite, calcitriol (1,25-(OH)2D3), affect multiple tissue types by interacting with the vitamin D receptor (VDR). Although vitD3 deficiency has been correlated with increased incidence of breast cancer and less favorable outcomes across ethnic groups and latitudes, randomized human clinical trials have yet to provide conclusive evidence on the efficacy of vitD3 in treating and/or preventing breast cancer. When considering that carcinogenesis is \"development gone awry\", it becomes imperative to understand the role of vitD3 during breast development. Mammary gland development in VDR KO mice is altered by increased ductal elongation and lateral branching during puberty, precocious and increased alveologenesis at pregnancy and delayed post-lactational involution. These developmental processes are largely influenced by mammotropic hormones, i.e., ductal elongation by estrogen, branching by progesterone and alveologenesis by prolactin. However, research on vitD3s effects on mammary gland morphogenesis focused on cell proliferation and apoptosis in 2D culture models and utilized supra-physiological doses of vitD3, conditions that spare the microenvironment in which morphogenesis takes place. Here, using two 3D culture models, we investigated the role of vitD3 in mammary epithelial morphogenesis. We found that vitD3 interferes with estrogens actions on T47D human breast cancer cells in 3D differently at different doses, and recapitulates what is observed in vivo. Also, vitD3 can act autonomously and affect the organization of MCF10A cells in 3D collagen matrix by influencing collagen fiber organization. Thus, we uncovered how vitD3 modulates mammary tissue organization independent of its already known effects on cell proliferation.

developmental biology↗

bioRxiv2018

Spheroid culture of mesenchymal stromal cells results in morpho-rheological properties appropriate for improved microcirculation

Human bone marrow mesenchymal stromal cells (MSCs) have been used in clinical trials for the treatment of systemic inflammatory diseases due to their regenerative and immunomodulatory properties. However, intravenous administration of MSCs is hampered by cell trapping within the pulmonary capillary networks. Here, we hypothesize that traditional twodimensional (2D) plastic-adherent cell expansion fails to result in appropriate morphorheological properties required for cell-circulation. To address this issue, we adapted a novel method to culture MSCs in non-adherent three-dimensional (3D) spheroids (mesenspheres). The biological properties of mesensphere-cultured MSCs remained identical to conventional 2D cultures. Morpho-rheological analyses revealed a smaller size and lower cell stiffness of mesensphere-derived MSCs compared to plastic-adherent MSCs, measured using real-time deformability cytometry (RT-DC) and atomic force microscopy, resulting in an increased ability to pass through micro-constrictions in an ex vivo microcirculation assay. This ability was confirmed in vivo by analysis of cell accumulation in various organ capillary networks after intravenous injection of mesensphere-derived MSCs in mouse. Our findings generally identify cellular morpho-rheological properties as attractive targets to improve microcirculation and specifically suggest mesensphere cultures as a promising approach for optimized MSC-based therapies.

biophysics↗

bioRxiv2018

Bacteroides fragilis defense against Cronobacter sakazakii -induced pathogenicity by regulating the intestinal epithelial barrier function and attenuating both apoptotic and pyroptotic cell death

Cronobacter sakazakii (CS), an important pathogen, is associated with the development of necrotizing enterocolitis (NEC), infant sepsis, and meningitis. Several randomized prospective clinical trials demonstrated that oral probiotics could decrease the incidence of NEC. Previously, we isolated and characterized a novel probiotic, B. fragilis strain ZY-312. However, it remains unclear how ZY-312 protects the host from the effects of CS infection. To understand the underlying mechanisms triggering the probiotic effects, we tested the hypothesis that there was a cross-talk between probiotics/probiotics-modulated microbiota and the local immune system, governed by the permeability of the intestinal mucosa using in vitro and in vivo models for the intestinal permeability. The probiotic effects of ZY-312 on intestinal epithelial cells were first examined, which revealed that ZY-312 inhibited CS invasion, CS-induced dual cell death (pyroptosis and apoptosis), and epithelial barrier dysfunction in vitro and in vivo. ZY-312 also decreased the expression of an inflammasome (NOD-like receptor family member pyrin domain-containing protein 3 (NLRP3), caspase-3, and serine protease caspase-1 in a neonatal rat model. Furthermore, ZY-312 significantly modulated the compositions of the intestinal bacterial communities, and decreased the relative abundances of Proteobacteria, Gamma proteobacteria, but increased the relative abundance of Bacteroides and Bacillus in neonatal rats. In conclusion, our findings have shown for the first time that the probiotic, B. fragilis ZY-312, suppresses CS-induced NEC by modulating the pro-inflammatory response and dual cell death (apoptosis and pyroptosis).\n\nAuthor summaryCronobacter sakazakii, a major necrotizing enterocolitis pathogen, is used as a model microorganism for the study of opportunistic bacteria in the pathogenesis of necrotizing enterocolitis. Here, we have now unequivocally demonstrated that both apoptotic and pyroptotic stimuli contribute to the pathogenesis of Cronobacter sakazakii -induced necrotizing enterocolitis. Previously, we isolated and characterized a novel probiotic, B. fragilis strain ZY-312. We found that the ZY-312 defense against Cronobacter sakazakii-induced necrotizing enterocolitis by inhibiting Cronobacter sakazakii invasion, epithelial barrier dysfunction, the expression of inflammatory cytokines and dual cell death (pyroptosis and apoptosis). This study demonstrates the utility of ZY-312 as a promising probiotic agent for the prevention and treatment of various intestinal diseases, including NEC.

microbiology↗