Combining conventional chemotherapy and γδ T cell-based immunotherapy to target cancer-initiating cells.

Colon cancer comprises a small population of cancer initiating stem cells (CIC) that is responsible for tumor maintenance and resistance to anti-cancer therapies, possibly allowing for tumor recapitulation once treatment stops. Combinations of immune-based therapies with chemotherapy and other anti-tumor agents may be of significant clinical benefit in the treatment of colon cancer. However, cellular immune-based therapies have not been experimented yet in the population of colon CICs. Here, we demonstrate that treatment with low concentrations of commonly used chemotherapeutic agents, 5-fluorouracyl and doxorubicin, sensitize colon CICs to Vγ9Vδ2 T cell cytotoxicity. Vγ9Vδ2 T cell cytotoxicity was largely mediated by TRAIL interaction with DR5, following NKG2D-dependent recognition of colon CIC targets. We conclude that in vivo activation of Vγ9Vδ2 T cells or adoptive administration of ex-vivo expanded Vγ9Vδ2 T cells at suitable intervals after chemotherapy may substantially increase anti-tumor activities and represent a novel strategy for colon cancer immunotherapy

Chemotherapy Sensitizes Colon Cancer Initiating Cells to Vc9Vd2 T Cell-Mediated Cytotoxicity

Colon cancer comprises a small population of cancer initiating stem cells (CIC) that is responsible for tumor maintenance and resistance to anti-cancer therapies, possibly allowing for tumor recapitulation once treatment stops. Combinations of immune-based therapies with chemotherapy and other anti-tumor agents may be of significant clinical benefit in the treatment of colon cancer. However, cellular immune-based therapies have not been experimented yet in the population of colon CICs. Here, we demonstrate that treatment with low concentrations of commonly used chemotherapeutic agents, 5- fluorouracyl and doxorubicin, sensitize colon CICs to Vc9Vd2 T cell cytotoxicity. Vc9Vd2 T cell cytotoxicity was largely mediated by TRAIL interaction with DR5, following NKG2D-dependent recognition of colon CIC targets. We conclude that in vivo activation of Vc9Vd2 T cells or adoptive administration of ex-vivo expanded Vc9Vd2 T cells at suitable intervals after chemotherapy may substantially increase anti-tumor activities and represent a novel strategy for colon cancer immunotherapy

Gene regulation of GPR17, a checkpoint receptor in oligodendroglial differentiation

GPR17 is a G protein-coupled receptor activated by both uracil nucleotides and cysteinyl-leukotrienes. We have previously demonstrated GPR17 is a key regulator of oligodendroglial differentiation and myelination. The receptor starts to be expressed in early oligodendrocyte precursor cells (OPCs), it reaches its maximal expression in immature oligodendrocytes and is then progressively down-regulated. In late OPCs, GPR17 forced expression led to impaired maturation, suggesting that its expression needs to be tightly time regulated. Based on these evidences, this work was aimed at identifying the signaling molecules regulating GPR17 expression during oligodendroglial differentiation. For this purpose, we cloned a putative promoter region of Gpr17 into a reporter vector upstream to a gene encoding for a luciferase. Then, we transfected this construct in Oli-neu cells, an immortalized oligodendroglial cell line, and we set up a reporter assay to evaluate the bioluminescence produced in response to an array of stimuli. Our results showed that treatment with both dibutyryl-cAMP, an analogue of cAMP, and forskolin, an activator of adenylyl cyclase, led to a significant increase of promoter activity, suggesting that cAMP signaling triggers GPR17 expression. To evaluate if GPR17 could be regulated by neuronal factors, we incubated cells with medium conditioned by cortical neurons. After 48h, we observed a significant induction of promoter activity; this effect was enhanced by heating the medium, suggesting neurons release one or more factors promoting oligodendroglial differentiation via Gpr17 gene, but that an inhibitory thermolabile factor is also present in the neuronal-conditioned medium. In line with this hypothesis, we found that insulin, a component of the medium formulation known to activate the mTOR pathway, strongly inhibited GPR17 promoter activity, whereas rapamycin, an inhibitor of the same pathway, significantly increased it. These data are consistent with the hypothesis that, while a neuronal-derived product activating cAMP is involved in turning GPR17 on, the mTOR pathway, likely activated by insulin-like growth factors, may be responsible for its physiological silencing at later stages of oligodendroglial development. These results may be relevant to the identification of new pharmacological strategies to activate/inhibit GPR17 under dysregulated conditions accompanied by myelination defects

Acute Inflammation and Elevated Cardiac Markers in a Two-Month-Old Infant with Severe Acute Respiratory Syndrome Coronavirus 2 Infection Presenting with Cardiac Symptoms

Severe acute respiratory syndrome coronavirus 2 infection in children mainly shows a milder course. In complicated cases, it is unknown whether inflammation is predictive of disease severity, as in adults. Moreover, cardiac involvement is anecdotally described. We report the case of a 2-month-old infant with severe acute respiratory syndrome coronavirus 2 infection presenting with fever, tachycardia and elevated interleukin-6, who was diagnosed with myocarditis and treated with immunoglobulins

Distinctive features of tumor-infiltrating gd T lymphocytes in human colorectal cancer

gd T cells usually infiltrate many different types of cancer, but it is unclear whether they inhibit or promote tumor progression. Moreover, properties of tumor-infiltrating gd T cells and those in the corresponding normal tissue remain largely unknown. Here we have studied features of gd T cells in colorectal cancer, normal colon tissue and peripheral blood, and correlated their levels with clinicopathologic hallmarks. Flow cytometry and transcriptome analyses showed that the tumor comprised a highly variable rate of TILs (5-90%) and 4%gd T cells on average, with the majority expressing Vd1. Most Vd1 and Vd2 T cells showed a predominant effector memory phenotype and had reduced production of IFN-g which was likely due to yet unidentified inhibitory molecules present in cancer stem cell secretome. Transcriptome analyses revealed that patients containing abundant gd T cells had significantly longer 5-year disease free survival rate, suggesting their efficacy in controlling tumor at very early stage

microRNAs and Cardiac Cell Fate

The role of small, non-coding microRNAs (miRNAs) has recently emerged as fundamental in the regulation of the physiology of the cardiovascular system. Several specific miRNAs were found to be expressed in embryonic, postnatal, and adult cardiac tissues. In the present review, we will provide an overview about their role in controlling the different pathways regulating cell identity and fate determination. In particular, we will focus on the involvement of miRNAs in pluripotency determination and reprogramming, and specifically on cardiac lineage commitment and cell direct transdifferentiation into cardiomyocytes. The identification of cardiac-specific miRNAs and their targets provide new promising insights into the mechanisms that regulate cardiac development, function and dysfunction. Furthermore, due to their contribution in reprogramming, they could offer new opportunities for developing safe and efficient cell-based therapies for cardiovascular disorders

Predicting 2-drug antiretroviral regimen efficacy by genotypic susceptibility score: results from a cohort study

Background: HIV drug resistance has a deleterious effect on the virological outcome of antiretroviral therapy (ART). The aim of the study was to evaluate the ability of genotypic susceptibility score (GSS) to predict virological outcome following an ART switch to a 2-drug regimen in virosuppressed HIV-1 infected patients. Material and methods: From the ARCA database we selected HIV-1 infected patients virologically suppressed switching to 2-drug ART (2006-2018, time of switch=baseline), with pre-baseline resistance genotype and at least one HIV-1 RNA determination during follow up. Primary endopoint was virological failure (VF: an HIV-RNA, VL, ≥ 200 cps/mL or 2 consecutive ≥ 50 cps/mL). Survival analysis was used to investigate predictors of VF. The GSS predicted by the latest and the cumulative genotype (CGSS) was calculated using the Stanford hivdb (v.8.5) with respect to the 2-drug regimen started. CD4 changes from baseline at weeks 24, 48 and 96 were assessed using Student’s t-test for paired samples. Results: We included 773 patients: 522 (68%) were males, 186 (24%) heterosexuals, with median age of 50 years (IQR, 43-56), 10 years of HIV (5-20), 7 years of ART (4-15) and 5 (3-8) previous antiretroviral (ARV) lines. At baseline patients had been virologically suppressed for 6.4 years (2.5-14), allowing isolated blips. The median zenith VL was 4.9 log10 (4.4-5.5), CD4 cells count at nadir 222 (108-324) and at baseline 640 (477-860). Median GSS was 2 (1.5-2), with GSS <2 in 213 (28%) pts, median CGSS was 2 (1-2), with CGSS <2 in 250 (33%). The previous ARV classes used were NRTI in 770 patients (99%), NNRTI in 416 (54%), boosted PI in 639 (83%) and INSTI in 218 (28%). Current ARV regimens included: PI+3TC in 455 pts (59%), of which 3TC+ ATV unboosted or ATV/r or ATV/c in 181 (23%) and DRV/r or DRV/c in 274 (36%), DTG+3TC in 260 (34%) and DTG+RPV in 58 (7%). During a median observation time of 75 wks (IQR 37-120) the estimated probability of VF at 48 weeks was 6% (95% CI 5-7) among patients with GSS=2, 4% (3-5) among patients with GSS 1-1.99 and 11% (4-18) among those with GSS <1 (Log Rank p=0.21). According to CGSS, the estimated probability of VF at 48 weeks was 5% (95% CI 1-6) among patients with CGSS =2, 6% (4-8) among patients with CGSS 1-1.99 and 8% (3-13) among those with CGSS <1 (Log Rank p=0.006) (Fig 1). Observed median changes of CD4+ counts from baseline were +24 cells/μL (IQR -67;+132) at 24 weeks, +49 cells/μL (IQR -31;+159) at 48 weeks and +74 cells/μL (IQR -30; +197) at 96 weeks (p<0.001 for all comparisons). At multivariate analysis, adjusting for years of ART, CD4 cell count at nadir and at baseline, CGSS strata, number of previous ARV lines, only longer time since last VL>50 cps/mL was associated with lower risk of VF (+ 1 year, aHR 0.89, 95% CI 0.82-0.98; p=0.01). Conclusions: Despite an effect of CGSS, the duration of virosuppression was the only independent predictor of virological efficacy of switching to 2-drug regimens

Generation of human induced pluripotent stem cells (EURACi001-A, EURACi002-A, EURACi003-A) from peripheral blood mononuclear cells of three patients carrying mutations in the CAV3 gene

Caveolinopathies are a heterogeneous family of genetic pathologies arising from alterations of the caveolin-3 gene (CAV3), encoding for the isoform specifically constituting muscle caveolae. Here, by reprogramming peripheral blood mononuclear cells, we report the generation of induced pluripotent stem cells (iPSCs) from three patients carrying the ΔYTT deletion, T78K and W101C missense mutations in caveolin-3. iPSCs displayed normal karyotypes and all the features of pluripotent stem cells in terms of morphology, specific marker expression and ability to differentiate in vitro into the three germ layers. These lines thus represent a human cellular model to study the molecular basis of caveolinopathies

IL4 primes the dynamics of breast cancer progression via DUSP4 inhibition

The tumor microenvironment supplies proinflammatory cytokines favoring a permissive milieu for cancer cell growth and invasive behavior. Here we show how breast cancer progression is facilitated by IL4 secreted by adipose tissue and estrogen receptor-positive and triple-negative breast cancer cell types. Blocking autocrine and paracrine IL4 signaling with the IL4R\uce\ub1 antagonist IL4DM compromised breast cancer cell proliferation, invasion, and tumor growth by downregulating MAPK pathway activity. IL4DM reduced numbers of CD44+/CD24-cancer stem-like cells and elevated expression of the dual specificity phosphatase DUSP4by inhibiting NF-\uce\ubaB. Enforced expression of DUSP4 drove conversion of metastatic cells to nonmetastatic cells. Mechanistically, RNAi-mediated attenuation of DUSP4activated the ERKand p38 MAPK pathways, increased stem-like properties, and spawned metastatic capacity. Targeting IL4 signaling sensitized breast cancer cells to anticancer therapy and strengthened immune responses by enhancing the number of IFN\uce\ub3-positive CTLs. Our results showed the role of IL4 in promoting breast cancer aggressiveness and how its targeting may improve the efficacy of current therapies