Flow cytometry data mining by cytoChain identifies determinants of exhaustion and stemness in TCR-engineered T cells

The phenotype of infused cells is a major determinant of Adoptive T-cell therapy (ACT) efficacy. Yet, the difficulty in deciphering multiparametric cytometry data limited the fine characterization of cellular products. To allow the analysis of dynamic and complex flow cytometry samples, we developed cytoChain, a novel dataset mining tool and a new analytical workflow. CytoChain was challenged to compare state-of-the-art and innovative culture conditions to generate stem-like memory cells (T_SCM) suitable for ACT. Noticeably, the combination of IL-7/15 and superoxides scavenging sustained the emergence of a previously unidentified nonexhausted Fit-T_SCM signature, overlooked by manual gating and endowed with superior expansion potential. CytoChain proficiently traced back this population in independent datasets, and in T-cell receptor engineered lymphocytes. CytoChain flexibility and function were then further validated on a published dataset from circulating T cells in COVID-19 patients. Collectively, our results support the use of cytoChain to identify novel, functionally critical immunophenotypes for ACT and patients immunomonitoring.     

Antimicrobial and physicochemical characterization of endodontic sealers after exposure to chlorhexidine digluconate

ObjectivesAssess the antibacterial, physical and chemical properties of AH Plus, BioRoot RCS and Pulp Canal Sealer (PCS) in contact with 2% chlorhexidine digluconate (CHX) used as final irrigant prior to root canal obturation.MethodsThe antimicrobial properties were investigated by direct contact tests for planktonic and biofilm growth of E. faecalis, S. mutans, S.epidermidis and S.aureus in vitro. The setting time, wettability, microhardness and surface roughness were also assessed. The sealers were studied in no contact, 1-minute (short-term) and continuous contact (long-term) with CHX. Chemical characterization of sealers was performed by scanning electron microscopy, X-ray diffraction analysis and Fourier-transform infrared spectroscopy after CHX or saline used as the last irrigant in an ex vivo tooth model and in endo training blocks.ResultsCHX increased the antibacterial activity of all the sealers investigated against planktonic bacteria and biofilms with PCS exerting the highest antimicrobial activity with and without the presence of CHX. The setting of AH Plus and BioRoot RCS was retarded, while for PCS accelerated in the presence of CHX. AH Plus and PCS were more hydrophilic after contact with CHX, whilst BioRoot RCS was hydrophobic in a time-dependent manner. The microhardness of sealers was compromised and the surface roughness increased after CHX exposure for AH Plus and BioRoot RCS, and decreased for PCS. CHX did not affect the sealers’ chemistry, but PCS that exhibited two extra phases.SignificanceCHX improved the antibacterial efficacy of endodontic sealers but further evidence is needed to confirm its suitability as a final irrigant prior to root canal obturation.     

FLT4/VEGFR3 and Milroy Disease: Novel Mutations,a Review of Published Variants and Database Update

Milroy disease (MD) is an autosomal dominantly inherited primary lymphedema. In 1998, the gene locus for MD was mapped to 5q35.3 and variants in the VEGFR3 (FLT4) gene, encoding vascular endothelial growth factor receptor 3 (VEGFR3), were identified as being responsible for the majority of MD cases. Several reports have since been published detailing pathogenic FLT4 mutations. To date, a total of 58 different variants in FLT4, 20 of which are unpublished, have been observed in 95 families with MD. A review of published mutations is presented in this update. Furthermore, the unpublished variants are presented including clinical data. Comparison of clinical features in patients and their families with the same mutations reveals incomplete penetrance and variable expression, making genotype–phenotype correlations difficult. Most mutations are missense, but a few deletions and one splicing variant have also been reported. Several animal models have confirmed the role of VEGFR3 in lymphangiogenesis and studies show mutant VEGFR3 receptors are not phosphorylated. Here, an MD patient with the same p.Ile1053Phe change as seen in the Chy mouse is presented for the first time. This finding confirms that this mouse lineage is an excellent model for MD. All the data reviewed here has been submitted to a database based on the Leiden Open (source) Variation Database (LOVD) and is accessible online at www.lovd.nl/flt4.     

Molecular survey of parasites in introduced Pelophylax perezi (Ranidae) water frogs in the Azores

Water frogs, Pelophylax perezi, that are introduced in the Azores, were screened for parasites using PCR primers known to amplify Apicomplexa parasites, and using nematode-specific primers. With the former, three different organisms were detected: Hepatozoon, a trichodinid protozoan ciliate and a possible Stramenopile. Using the latter set of primers, a single unknown spirurid nematode was also detected. Phylogenetic analyses indicate that Hepatozoon detected within amphibian hosts appear to form a clade, although relationships of these parasites do not match the vertebrate intermediate host phylogeny. Regarding the possible Stramenopile, it is unclear whether this organism was actually present on the amphibian or in the water on the surface of the tissue sample. Our findings highlight that many different organisms can be detected with these primers and that they can be used to screen introduced host populations to detect parasites that have been brought with them.     

The protease inhibitor ritonavir inhibits the functional activity of the multidrug resistance related-protein 1 (MRP-1)

BACKGROUND: Efflux pumps situated on the plasma membrane, such as P-glycoprotein (Pgp) and the multidrug resistance related-protein 1 (MRP-1), have been shown to extrude HIV protease inhibitors from the cell. MRP-1 is present on many barrier sites throughout the body, such as the blood-brain and blood-testis interfaces and could reduce the concentration of protease inhibitors in these sanctuary sites for HIV-1 replication. Factors that modulate efflux pump function in vivo are poorly defined. OBJECTIVE: To analyze the inhibitory potential of the anti-retroviral drugs indinavir, amprenavir, ritonavir, lamivudine or zidovudine to modulate MRP-1 function. METHODS: Effect of anti-HIV drugs on the efflux pump activity of MRP-1 was evaluated in the presence of increasing concentrations of human plasma, using UMCC-1/VP cells which stably over-express MRP-1. MRP-1 activity was abrogated by probenecid. The potential of blocking MRP-1 function for an extended (3 day) time period, was also examined in MRP-1 over-expressing cells cultured with either probenecid or the anti-retroviral drugs and a cytotoxic compound (etoposide) that is transported by MRP-1. RESULTS: Ritonavir inhibited the functional activity of MRP-1 similarly to probenecid, as demonstrated by re-sensitization of MRP-1 over-expressing cells to cytotoxic effects of etoposide. Inhibition by ritonavir was inversely related to the concentration of human plasma added to the cells (r2 = 0.89). Other anti-HIV drugs didn't affect the MRP-1 mediated efflux of etoposide. CONCLUSIONS: These data may be exploitable to further improve sanctuary site concentrations of anti-HIV or anti-cancer drugs by using ritonavir as a lead compound to develop more potent MRP-1 inhibitors.     

Survival and cause-specific mortality in ulcerative colitis: follow-up of a population-based cohort in Copenhagen County

BACKGROUND & AIMS: A population-based cohort from Copenhagen County comprising 1160 patients diagnosed with ulcerative colitis between 1962 and 1987 was followed-up until 1997 to describe survival and cause-specific mortality. METHODS: Observed vs. expected deaths were presented as standardized mortality ratio (SMR) with exact 95% confidence intervals (CI) calculated by using individually registered person-years at risk and Danish 1995 mortality rates. Cumulative survival curves were calculated. RESULTS: A total of 261 deaths occurred, not significantly different from the expected number of 249 (SMR, 1.05; 95% CI, 0.92-1.19). The median age at death among men was 70 years (range, 6-96 years) and among women 74 years (range, 25-96 years). Twenty-five deaths (9.6%) were caused by complications to ulcerative colitis, mostly infectious and cardiovascular postoperative complications. Patients older than 50 years of age at diagnosis and with extensive colitis showed an increased mortality within the first 2 years because of ulcerative colitis-associated causes. The mortality from colorectal cancer was not increased and that of cancer in general was significantly lower than expected: 50 vs. 71 (SMR, 0.70; 95% CI, 0.52-0.93). A significantly increased mortality from pulmonary embolism and pneumonia was found. Among women only, death from genitourinary tract diseases and suicide was significantly increased. CONCLUSIONS: Despite an overall normal life expectancy for patients with ulcerative colitis, patients >50 years of age and with extensive colitis at diagnosis had increased mortality within the first 2 years after diagnosis, owing to colitis-associated postoperative complications and comorbidity.     

Perception of sexuality and fertility in women living with HIV: a questionnaire study from two Nordic countries

Introduction

As the human immunodeficiency virus (HIV)-positive population ages, issues concerning sexuality and fertility, among others, are becoming relevant. HIV is still surrounded by stigma and taboos, and there have been few studies conducted in industrialized settings concerning these questions. We therefore wanted to investigate the perception of sexuality and fertility in women living with HIV (WLWH) in an industrialized setting, using a questionnaire.

Methods

WLWH were recruited at their regular outpatient clinic visits, at the major Departments of Infectious Diseases in Denmark and Finland, from January 2012 to October 2013. A questionnaire was developed, study participants were informed of the nature of study and, if they agreed to participate and signed a consent form, they filled in the questionnaire. Demographic information on the participants was obtained from patient files (in Finland) or from a national HIV cohort (in Denmark). Statistical analysis was performed using STATA^®, version 11.

Results

In total, 560 women were included in the study. The median age was 44 years. The majority were of white European origin, with fully suppressed HIV viral load, CD4 cell count >350 µL and mild or no symptoms of their HIV infection. A total of 62% were sexually active, stating condom use as their sole form of contraception. Of the sexually inactive women, one-third were in steady relationships. Eighty percent reported prior pregnancies, of which the majority had one or more children. Most children were born prior to the women''s HIV diagnosis and the mode of conception was predominantly natural. One-quarter of the participating women desired pregnancy, while more than half did not. The remaining quarter either stated that they already had the desired number of children or chose not to answer the question. Fourteen percent stated that their HIV diagnosis ended their wish for children; of these women, the median time of diagnosis was between 1995 and 1996. Pregnancy had been attempted unsuccessfully in one-quarter of study participants. The final question inquired what the risk of mother-to-child transmission was, with all precautions taken. Fifteen percent estimated the risk to be above two percent.

Conclusions

In conclusion, the majority of WLWH in industrialized settings in Denmark and Finland have few HIV-related symptoms, are sexually active and have a strong desire for children.