Next generation sequencing (NGS)-based approach to diagnosing Algerian patients with suspected inborn errors of immunity (IEIs).

The advent of next-generation sequencing (NGS) technologies has greatly expanded our understanding of both the clinical spectra and genetic landscape of inborn errors of immunity (IEIs). Endogamous populations may be enriched for unique, ancestry-specific disease-causing variants, a consideration that significantly impacts molecular testing and analysis strategies. Herein, we report on the application of a 2-step NGS-based testing approach beginning with targeted gene panels (TGPs) tailored to specific IEI subtypes and reflexing to whole exome sequencing (WES) if negative for Northwest Algerian patients with suspected IEIs. Our overall diagnostic yield of 57% is comparable to others broadly applying short-read NGS to IEI detection, but data from our localized cohort show some similarities and differences from NGS studies performed on larger regional IEI cohorts. This suggests the importance of tailoring diagnostic strategies to local demographics and needs, but also highlights ongoing concerns inherent to the application of genomics for clinical IEI diagnostics.

miRNA-23b as a biomarker of culture-positive neonatal sepsis.

BACKGROUND: Neonatal sepsis remains an important cause of morbidity and mortality. The ability to quickly and accurately diagnose neonatal sepsis based on clinical assessments and laboratory blood tests remains difficult, where haemoculture is the gold standard for detecting bacterial sepsis in blood culture. It is also very difficult to study because neonatal samples are lacking. METHODS: Forty-eight newborns suspected of sepsis admitted to the Neonatology Department of the Mother-Child Specialized Hospital of Tlemcen. From each newborn, a minimum of 1-2 ml of blood was drawn by standard sterile procedures for blood culture. The miRNA-23b level in haemoculture was evaluated by RT-qPCR. RESULTS: miR-23b levels increased in premature and full-term newborns in early onset sepsis (p < 0.001 and p < 0.005 respectively), but lowered in late onset sepsis in full-term neonates (p < 0.05) compared to the respective negative controls. miR-23b levels also increased in late sepsis in the negative versus early sepsis negative controls (p < 0.05). miR-23b levels significantly lowered in the newborns who died from both sepsis types (p < 0.0001 and p < 0.05 respectively). In early sepsis, miR-23b and death strongly and negatively correlated (correlation coefficient = - 0.96, p = 0.0019). In late sepsis, miRNA-23b and number of survivors (correlation coefficient = 0.70, p = 0.506) positively correlated. CONCLUSIONS: Lowering miR-23b levels is an important factor that favours sepsis development, which would confirm their vital protective role, and strongly suggest that they act as a good marker in molecular diagnosis and patient monitoring.

High-density lipoprotein immunomodulates the functional activities of macrophage and cytokines produced during ex vivo macrophage-CD4+ T cell crosstalk at the recent-onset human type 1 diabetes.

BACKGROUND: Both CD4+ T cells and macrophages are mainly involved in the autoimmune-mediated ß-cells destruction in type 1 diabetes (T1D). The aim of this study was to examine the effect of HDL on functional activities of macrophage and its ability to regulate the production of cytokines in autologous mixed macrophage/CD4+ T cells at the recent-onset human type 1 diabetes. METHODS: Cell samples were isolated from volunteers with recent-onset T1D or healthy controls. RESULTS: The levels of the production of IL-1ß, IL-2, IFN-γ, nitric oxide (NO), and hydrogen peroxide (H2O2) were significantly increased in the co-culture of T1D cells when compared to that of cells from healthy controls. Similarly, those of intracellular free calcium ions (ifCa2+) were slightly, but not significantly increased (p> 0.05). Conversely, macrophage exhibited significantly decreased levels of the relative tyrosine phosphorylation of STAT6 (p-STAT6, Tyr641) in culture of T1D cells than in that of cells from healthy controls; while those of p-STAT4 (Tyr693) were significantly increased. Likewise, the levels of IL-4 and IL-10 were significantly decreased in the co-culture of T1D cells compared to co-culture of cells from healthy controls. Additionally, HDL treatment significantly down-regulated the production of IL-1ß, IL-2, IFN-γ, NO, H2O2, phagocytosis, bacterial killing, the relative tyrosine phosphorylation of macrophage-expressed STAT4 (p-STAT4, Tyr693), as well as the ratio of IL-1ß/IL-10, NO production/arginase activity, p-STAT4/p-STAT6, IFN-γ/IL-4, IFN-γ/IL-10, and the combined proinflammatory (PICs)/anti-inflammatory (AICs) cytokines. Moreover, HDL treatment significantly up-regulated the production of IL-4, IL-10, arginase activity, and p-STAT6 (Tyr641) (for all comparisons, p< 0.001). CONCLUSIONS: We show for the first time that HDL may reverse both the functional activities of macrophages and immunoinflammatory response during reciprocal macrophage-CD4+ T cell crosstalk at the beginning of T1D. These findings should open the way for therapeutic trials in the short- and medium-term.

ERK1/2 activation in human taste bud cells regulates fatty acid signaling and gustatory perception of fat in mice and humans.

Obesity is a major public health problem. An in-depth knowledge of the molecular mechanisms of oro-sensory detection of dietary lipids may help fight it. Humans and rodents can detect fatty acids via lipido-receptors, such as CD36 and GPR120. We studied the implication of the MAPK pathways, in particular, ERK1/2, in the gustatory detection of fatty acids. Linoleic acid, a dietary fatty acid, induced via CD36 the phosphorylation of MEK1/2-ERK1/2-ETS-like transcription factor-1 cascade, which requires Fyn-Src kinase and lipid rafts in human taste bud cells (TBCs). ERK1/2 cascade was activated by Ca2+ signaling via opening of the calcium-homeostasis modulator-1 (CALHM1) channel. Furthermore, fatty acid-evoked Ca2+ signaling and ERK1/2 phosphorylation were decreased in both human TBCs after small interfering RNA knockdown of CALHM1 channel and in TBCs from Calhm1-/- mice. Targeted knockdown of ERK1/2 by small interfering RNA or PD0325901 (MEK1/2 inhibitor) in the tongue and genetic ablation of Erk1 or Calhm1 genes impaired preference for dietary fat in mice. Lingual inhibition of ERK1/2 in healthy volunteers also decreased orogustatory sensitivity for linoleic acid. Our data demonstrate that ERK1/2-MAPK cascade is regulated by the opening of CALHM1 Ca2+ channel in TBCs to modulate orogustatory detection of dietary lipids in mice and humans.-Subramaniam, S., Ozdener, M. H., Abdoul-Azize, S., Saito, K., Malik, B., Maquart, G., Hashimoto, T., Marambaud, P., Aribi, M., Tordoff, M. G., Besnard, P., Khan, N. A. ERK1/2 activation in human taste bud cells regulates fatty acid signaling and gustatory perception of fat in mice and humans.

Facial cutaneo-mucosal venous malformations can develop independently of mutation of TEK gene but may be associated with excessive expression of Src and p-Src.

We aimed to search for mutations in the germline and somatic DNA of the TEK gene and to analyze the expression level of Src and phospho-Src (p-Src) in tumor and healthy tissues from patients with facial cutaneo-mucosal venous malformations (VMCM). Eligible patients from twelve families and thirty healthy controls were recruited respectively at the Departments of Stomatology and Oral Surgery, and Transfusion Medicine of Tlemcen University Medical Centre. Immunoblot analyses of Src and p-Src were performed after direct DNA sequencing. No somatic or germline mutations were found in all the 23 exons and their 5' and 3' intronic flanking regions, except for one case in which a c.3025+20-3025+22 del mutation was highlighted at the intron 15, both in the germline and somatic DNA. Additionally, elevated expression levels of Src and p-Src were observed only in the patient with such mutation. However, when normalized to ß-actin, the overall relative expression levels of both Src and p-Src were significantly increased in VMCM tissues when compared to healthy tissues (for both comparisons, p <0.001). In conclusion, we confirm the outcomes of our previous work suggesting that VMCM can develop independently of mutation of the TEK gene. Additionally, the results for Src activity are of particular interest in the context of specific targeted therapies and biological diagnosis. Nevertheless, such a conclusion should be confirmed through a mechanistic study and/or in a satisfactory number of patients.

Sodium Selenite Modulates Global Activation of Proinflammatory M1-like Macrophages, Necroinflammation and M1-like/M2-like Dichotomy at the Onset of Human Type 1 Diabetes.

AIM: The study aims to show that sodium selenite (Ss) would have an immunomodulatory effect on the functional activity of proinflammatory macrophages (Mφs) during their extended extracellular activation at the onset of human type 1 diabetes (T1D). BACKGROUND: Exacerbated activation of proinflammatory "M1" macrophages (Mφs) can promote chronic local pancreatic islet inflammation and T1D development. OBJECTIVE: We investigated the ex vivo effects of Ss on the immune modulation of global/extended activation of human proinflammatory M1-like Mφs. METHODS: Experiments were carried out on primary monocytes-derived Mφs (MDMs). RESULTS: The levels of IL-1ß, TNF-α, H2O2 and intracellular free calcium ions (ifCa2+), and the ratios of IL-1ß-to-IL-10 and TNF-α-to-IL-10 were markedly increased in T1D Mφs than in healthy control Mφs. Conversely, both IL-10 production and arginase 1 (ARG1) activity were downregulated in T1D Mφs. Additionally, Ss treatment induced a marked downregulation of respiratory burst, ifCa2+ levels, M1-like Mφ-associated inducible nitric oxide (NO) synthase (iNOS) activity, cell necrosis and related necroinflammation biomarkers, including IL-1ß and TNF-α, CD14 expression, and the ratios of iNOS-to-ARG1, IL-1ß-to-IL-10, and TNF-α-to-IL-10. Moreover, Ss upregulated anti-inflammatory "M2-like" Mφ activity as demonstrated by ARG1 activity and IL-10 production, as well as phagocytosis capacity. CONCLUSION: Ss exerts a potent immunomodulatory role on functional activities of human proinflammatory T1D M1-like Mφs subjected to extended activation, as well as on the M1-like/M2-like dichotomy. Additionally, the current study provides a novel therapeutic approach using Ss to promote the anti-inflammatory function of Mφs at the onset of T1D.

Clinical and immunological aspects of microRNAs in neonatal sepsis.

Neonatal sepsis constitutes a highly relevant public health challenge and is the most common cause of infant morbidity and mortality worldwide. Recent studies have demonstrated that during infection epigenetic changes may occur leading to reprogramming of gene expression. Post-transcriptional regulation by short non-coding RNAs (e.g., microRNAs) have recently acquired special relevance because of their role in the regulation of the pathophysiology of sepsis and their potential clinical use as biomarkers. ~22-nucleotide of microRNAs are not only involved in regulating multiple relevant cellular and molecular functions, such as immune cell function and inflammatory response, but have also been proposed as good candidates as biomarkers in sepsis. Nevertheless, establishing clinical practice guidelines based on microRNA patterns as biomarkers for diagnosis and prognosis in neonatal sepsis has yet to be achieved. Given their differential expression across tissues in neonates, the release of specific microRNAs to blood and their expression pattern can differ compared to sepsis in adult patients. Further in-depth research is necessary to fully understand the biological relevance of microRNAs and assess their potential use in clinical settings. This review provides a general overview of microRNAs, their structure, function and biogenesis before exploring their potential clinical interest as diagnostic and prognostic biomarkers of neonatal sepsis. An important part of the review is focused on immune and inflammatory aspects of selected microRNAs that may become biomarkers for clinical use and therapeutic intervention.

Alteration of antioxidant defense status precedes humoral immune response abnormalities in macrosomia.

BACKGROUND: This study aimed to investigate whether the anomalies affecting the antioxidant and humoral immune defenses could start at birth and to check whether the decrease in antioxidant defenses may precede the immune abnormalities in macrosomic newborns. MATERIAL/METHODS: Thirty macrosomic and 30 sex-matched control newborns were recruited for a retrospective case-control study at the Maghnia Maternity Hospital of Tlemcen Department (Algeria). RESULTS: The serum IgG levels were similar in both groups. However, plasma ORAC, albumin, vitamin E, SOD, CAT and GSH-Px levels were significantly decreased in macrosomic as compared to control newborns, yet no difference was observed after adjustment for weight. Additionally, serum concentrations of complement C3, MDA and XO were significantly higher in macrosomic as compared to controls before adjustment for weight. Moreover, macrosomia was significantly associated with high levels of complement C3 (OR=8, p=0.002); whereas no association with those of IgG was observed (OR<1, p>0.05). Furthermore, macrosomia was significantly associated with low levels of ORAC (OR=4.96, p=0.027), vitamin E (OR=4.5, p=0.018), SOD (OR=6.88, p=0.020) and CAT (OR=5.67, p=0.017), and with high levels of MDA (OR=10.29, p=0.005). CONCLUSIONS: Abnormalities of the humoral defense system in excessive weight could be preceded by alterations of the anti-oxidative defense and by inflammatory response and activation of innate immunity at birth. Additionally, excessive weight could be a potential factor contributing to decreased anti-oxidative capacity and increased oxidative stress.

L-Threoascorbic acid treatment promotes S. aureus-infected primary human endothelial cells survival and function, as well as intracellular bacterial killing, and immunomodulates the release of IL-1ß and soluble ICAM-1.

BACKGROUND: Vitamin C (ascorbic acid, AscH2) has been shown to enhance immunity. Here, we studied its immunomodulatory effect on human endothelial cells (ECs) during S. aureus infection. MATERIALS AND METHODS: The ex vivo effects of AscH2 were performed on primary human umbilical vein endothelial cells (HUVECs) infected or not with S. aureus. RESULTS: AscH2 treatment induced a marked downregulation of nitric oxide (NO) production and a moderate upregulation of arginase activity in S. aureus-infected HUVECs (respectively, p < 0.05 and p > 0.05). Although the upregulated release levels of soluble intercellular adhesion molecular 1 (sICAM-1/sCD54) and sE-selectin (sCD62E) molecules were not significantly different between treated and untreated S. aureus-infected HUVECs, AscH2 treatment induced reversing effect on sICAM-1 release when comparing to uninfected control HUVECs. Moreover, AscH2 treatment appears to have a significant effect on preventing HUVEC necrosis induced by S. aureus infection (p < 0.05). Furthermore, AscH2 treatment induced a significant upregulation of cell protective redox biomarker in S. aureus-infected, as shown by superoxide dismutase (SOD) activity (p < 0.05), but not by catalase activity (p > 0.05). Additionally, S. aureus infection markedly downregulated total bound calcium ions (bCa2+) levels as compared to control HUVECs, whereas, AscH2 treatment induced a slight upregulation of bCa2+ levels in infected HUVECs as compared to infected and untreated HUVECs (p > 0.05). On the other hand, AscH2 treatment downregulated increased total cellular cholesterol content (tccCHOL) levels in HUVECs induced by S. aureus infection (p < 0.05). In addition, AscH2 treatment markedly reversed S. aureus effect on upregulation of intracellular glucose (iGLU) levels within infected HUVECs (p < 0.05). Moreover, AscH2 treatment significantly downregulated S. aureus growth (p < 0.05), and significantly upregulated bacterial internalization and intracellular killing by HUVECs (p < 0.05), as well as their cell cycle activation (p < 0.01). Finally, AscH2 treatment has a slight effect on the production of interleukin 6 (IL-6), but induced a marked downregulation of that of IL-1ß in S. aureus-infected HUVECs (respectively, p > 0.05, and p < 0.05). CONCLUSIONS: Our outcomes demonstrated that, during S. aureus infection, AscH2 treatment promotes human ECs survival and function, as well as prevents inflammatory response exacerbation, while inducing bactericidal activity.

Lack of cell movement impairs survival of peripheral blood IL-2-stimulated natural killer cells originating from solid cancer and promotes red blood cells to induce their switch toward a regulatory phenotype.

BACKGROUND: Red blood cells (RBCs) can have a modulatory effect on immune cells; so changes in their dynamism could considerably influence their physiology, and consequently the immune activities of neighbouring cells, like natural killer (NK) cells. Herein, we studied the effect of both RBCs and lack of cell movement on the proliferation, survival and regulation of peripheral IL-2-stimulated NK cells from normal and solid malignant conditions. METHODS: Experiments were conducted on twelve cell culture groups, including NK cells from patients with solid malignant tumor or healthy controls, cultured alone or with autologous or nonautologous RBCs under shaking or no shaking conditions. RESULTS: NK cells from neoplastic patients behaved differently depending on the culture conditions including shaking and/or RBCs presence. Therefore, NK cells survival was downregulated in the absence of shaking; whereas, shaking have not only upregulated cell survival, but also downregulated the levels of p53-related apoptosis. Moreover, RBCs enhanced NK cells proliferation; while, this effect was modulated by shaking. Furthermore, RBCs can generate opposite effects on the production and modulation of protumoral or immunosuppressive cytokines, depending on the origin of NK cells, i.e., whether they derive from healthy or solid malignant tumor conditions. Finally, NK cells become able to express Foxp3 regulatory marker when combining three main conditions that include (i) treatment with high dose of IL-2, (ii) presence of RBCs, and (iii) absence of shaking. CONCLUSIONS: Our outcomes showed for the first time that cell stagnation would be markedly involved in peripheral NK cell apoptosis, as well as in switching toward a regulatory phenotype-induced Foxp3. Cell movement may be one of ex vivo potential approaches in boosting the activities and survival of such cells during solid cancer.

Aspirin enhances regulatory functional activities of monocytes and downregulates CD16 and CD40 expression in myocardial infarction autoinflammatory disease.

BACKGROUND: Exacerbation of CD16 as molecule marker of both intermediate and non-classical monocytes (MOs) has been shown to be involved in the pathogenesis of myocardial infarction (MI). In this study, we have tried to evaluate the aspirin (acetylsalicylic acid, ASA) treatment effect on the CD16-expressed MOs and activation-associated CD40 in MI. METHODS: MOs were isolated from the whole blood of healthy controls and patients with MI. The cells were stimulated and treated with different doses of ASA. RESULTS: ASA significantly decreased nitric oxide (NO) production and inducible NO synthase (iNOS) activity, but significantly increased arginase activity. Levels of interleukin (IL)-1ß, IL-6 and interferon-γ (IFN-γ) were downregulated, whereas those of IL-10 were upregulated. Additionally, ASA induced a markedly increase in both phagocytosis and intracellular pathogen killing activities. Moreover, ASA treatment induced significantly upregulation of intracellular levels of glucose (iGlu), and free calcium ions (ifCa2+), and, covertly, significantly downregulation of total cellular cholesterol content (tccCHOL). Furthermore, the expression levels of CD16 and CD40 were significantly downregulated in ASA-treated MOs. CONCLUSIONS: We show for the first time that ASA immunomodulates the functional activities of MOs during MI and promotes their switching toward a classical phenotype, exhibiting low CD16 expression levels and thereby anti-inflammatory properties.

Metformin partially reverses the inhibitory effect of co-culture with ER-/PR-/HER2+ breast cancer cells on biomarkers of monocyte antitumor activity.

BACKGROUND: Immune activities of monocytes (MOs) can be altered within the microenvironment of solid malignancies, including breast cancer. Metformin (1,1-dimethylbiguanide hydrochloride, MET), has been shown to decrease tumor cell proliferation, but its effects have yet to be explored with respect to MOs (monocytes) activity during their crosstalk with breast cancer cells. Here, we investigated the effects of MET on overall phenotypic functional activities, including cellular immunometabolism and protective redox signaling based-biomarkers, intracellular free calcium ions (ifCa2+), phagocytosis and co-operative cytokines (IFN-γ and IL-10) of autologous MOs before and during their interplay with primary ER-/PR-/HER2+ breast cancer cells. METHODS: Human primary breast cancer cells were either cultured alone or co-cultured with autologous MOs before treatment with MET. RESULTS: MET downregulated breast cancer cell proliferation and phagocytosis, while having no significant effect on the ratio of phosphorylated Akt (p-Akt) to total Akt. Additionally, we observed that, in the absence of MET treatment, the levels of lactate dehydrogenase (LDH)-based cytotoxicity, catalase, ifCa2+, IL-10 and arginase activity were significantly reduced in co-cultures compared to levels in MOs cultured alone whereas levels of inducible nitric oxide synthase (iNOS) activity were significantly increased. In contrast, MET treatment reduced the effects measured in co-culture on the levels of LDH-based cytotoxicity, arginase activity, catalase, ifCa2+, and IFN-γ. MET also induced upregulation of both iNOS and arginase in MO cells, although the increase did not reach significant difference for iNOS activity. Moreover, MET induced a robust increase of superoxide dismutase (SOD) activity in MOs, but not in MOs co-cultured with breast cancer cells. Furthermore, MET markedly upregulated the levels of IFN-γ production and downregulated those of IL-10 in isolated MOs, while inducing a slight opposing up-regulation of IL-10 production in co-cultures. CONCLUSIONS: Our results show that the biomarkers of phenotypic functional activities of MOs are modified after co-culturing with primary human breast cancer cells. Treatment of co-cultures with MET resulted in increased release of antitumor cytokine IFN-γ and ifCa2+, and increased cell necrosis during breast cancer cells-MOs crosstalk.

Rituximab Treatment Modulates the Release of Hydrogen Peroxide and the Production of Proinflammatory Cytokines by Monocyte at the Onset of Type 1 Diabetes.

BACKGROUND: Monocytes are the main blood innate mononuclear phagocyte and one of the most important effector cells expressing Fcγ receptor, which is critical for the interaction with Fc domain of antibodies. OBJECTIVE: To evaluate the effect of Rituximab (RTX, a chimeric human anti-CD20 monoclonal antibody) on the functional activities of Monocytes (MOs) at the onset of human Type 1 Diabetes (T1D). METHODS: MOs were isolated from peripheral blood mononuclear cells (PBMCs) obtained from volunteer patients with recent-onset T1D and healthy control donors. RESULTS: The levels of the production of Interleukin 1ß (IL-1ß) and IL-6 were significantly increased in MOs from patients with T1D when compared to MOs from healthy controls (respectively, p < 0.01 and p < 0.05). Similarly, Interferon γ (IFN-γ), and intracellular free Calcium Ion (ifCa2+) levels were increased in T1D MOs than in control MOs, but the difference did not reach a significant level. Conversely, the production levels of IL-4 and catalase activity, as well as of both phagocytosis and killing capacities were decreased in MOs of T1D patients compared to MOs from healthy controls, but the difference was not significant for catalase activity and killing capacity (respectively, p < 0.01, p > 0.05, p < 0.01, and p > 0.05). Additionally, treatment with RTX significantly upregulated phagocytosis (p < 0.05), markedly downregulated the release of IL-1ß (p < 0.01), ifCa2+, hydrogen peroxide (H2O2), and slightly downregulated the Nitric Oxide Synthase (NOS) activity, NOS activity-to-arginase activity ratio, the levels of Lactate Dehydrogenase (LDH)-based cytotoxicity, and the production of IL-6 and IFN-γ. Moreover, RTX treatment significantly upregulated the production of IL-4 (p < 0.05), IL-10 (p < 0.01) and the catalase activity (p < 0.05). CONCLUSION: Our study has shown for the first time that RTX can reverse the abnormal functional activities of MOs as well as their production of proinflammatory cytokines at the onset of T1D. From a therapeutic point of view, RTX may potentially be suggested at the beginning of T1D to immunomodulate innate immunity and inflammatory conditions.

A comprehensive catalog of LncRNAs expressed in T-cell acute lymphoblastic leukemia.

Several studies have demonstrated that LncRNAs can play major roles in cancer development. The creation of a catalog of LncRNAs expressed in T cell acute lymphoblastic leukemia (T-ALL) is thus of particular importance. However, this task is challenging as LncRNA expression is highly restricted in time and space manner and thus may greatly differ between samples. We performed a systematic transcript discovery in RNA-Seq data obtained from T-ALL primary cells and cell lines. This led to the identification of 2560 novel LncRNAs. After the integration of these transcripts into a large compendium of LncRNAs (n = 30478) containing both known LncRNAs and those previously described in T-ALLs, we then performed a systematic genomic and epigenetic characterization of these transcript models demonstrating that these novel LncRNAs share properties with known LncRNAs. Finally, we provide evidence that these novel transcripts could be enriched in LncRNAs with potential oncogenic effects and identified a subset of LncRNAs coregulated with T-ALL oncogenes. Overall, our study represents a comprehensive resource of LncRNAs expressed in T-ALL and might provide new cues on the role of lncRNAs in this type of leukemia.

A critical regulator of Bcl2 revealed by systematic transcript discovery of lncRNAs associated with T-cell differentiation.

Normal T-cell differentiation requires a complex regulatory network which supports a series of maturation steps, including lineage commitment, T-cell receptor (TCR) gene rearrangement, and thymic positive and negative selection. However, the underlying molecular mechanisms are difficult to assess due to limited T-cell models. Here we explore the use of the pro-T-cell line P5424 to study early T-cell differentiation. Stimulation of P5424 cells by the calcium ionophore ionomycin together with PMA resulted in gene regulation of T-cell differentiation and activation markers, partially mimicking the CD4-CD8- double negative (DN) to double positive (DP) transition and some aspects of subsequent T-cell maturation and activation. Global analysis of gene expression, along with kinetic experiments, revealed a significant association between the dynamic expression of coding genes and neighbor lncRNAs including many newly-discovered transcripts, thus suggesting potential co-regulation. CRISPR/Cas9-mediated genetic deletion of Robnr, an inducible lncRNA located downstream of the anti-apoptotic gene Bcl2, demonstrated a critical role of the Robnr locus in the induction of Bcl2. Thus, the pro-T-cell line P5424 is a powerful model system to characterize regulatory networks involved in early T-cell differentiation and maturation.

Combination of metformin with sodium selenite induces a functional phenotypic switch of human GM-CSF monocyte-derived macrophages.

OBJECTIVES: We evaluated the effects of metformin (Met, 1,1­dimethylbiguanide hydrochloride) combined or not with sodium selenite (Ss, Na2SeO3) on the functional activities of LPS-activated GM-CSF monocyte-derived macrophages (GM-MDM). MATERIALS AND METHODS: Human GM-MDMs from three healthy donors were treated with Met or Ss alone, or with the combination of Met and Ss, and assayed for various biological activities and cytokines expression. RESULTS: Met alone and Ss alone had significantly different effects on phagocytosis and killing capacities and IL-ß production, but had similar effects on the downregulation of inducible nitric oxide synthase (iNOS) activity, relative nicotinamide adenine dinucleotide reduced (NADH) dehydrogenase (Complex I), intracellular free calcium ions (ifCa2+), and on the upregulation of arginase activity. Additionally, iNOS activity-to-arginase activity ratio was downregulated in Met or Ss treated-GM-MDMs, and, conversely, upregulated in GM-MDMs treated with Met + Ss in combination, indicating that arginase activity dominates that of iNOS when the two treatments are associated. Moreover, combination of Met with Ss significantly upregulated hydrogen peroxide (H2O2) production and phagocytic capacity, but significantly downregulated the production of IL-1ß, iNOS activity and killing capacity. On the contrary, we show that Met alone induced significant downregulation of phagocytic capacity and slight upregulation of killing capacity. Nevertheless, Ss seems to accentuate the effect of Met on the downregulation of NO production, as well as to reverse its effect on both phagocytic and killing capacities. On the other hand, all treatments induced a sharp decrease in relative levels of NADH dehydrogenase, and a marked decrease in the levels of ifCa2+. Finally, we found that GM-MDMs treated with Met or Ss, or Met combined with Ss exhibited different functional activation phenotypes, as indicated by the surface expression of co-stimulatory and cell activation and presentation molecules CD14, CD80, CD86 and HLA-DR. CONCLUSIONS: Our results demonstrated that Met/Ss combination can play an important role in the modulation of functional activities of human LPS-activated GM-MDMs. Additionally, the overall effects of Met and the induction of "M2" GM-MDMs-associated arginase could be influenced by its combination with Ss.

Macrophage Bactericidal Assays.

The search for the bactericidal activity of macrophage (Mϕ) is crucial not only during infection, but also to explore its functional activities in normal and pathological conditions, such as autoimmune and inflammatory disorders, allergic inflammation, and cancer. There are several methods exploring the phagocytic and bactericidal activities of Mϕ. This chapter focuses specifically on the technique called antibiotic protection assay and on the methods for the determination of Mϕ production of nitric oxide and hydrogen peroxide as antimicrobial agents and biomarkers of respiratory burst. The protocols presented herein are valid for both Mϕ cell lines and monocyte-derived Mϕs (MDMs).

Thymoquinone Potently Enhances the Activities of Classically Activated Macrophages Pulsed with Necrotic Jurkat Cell Lysates and the Production of Antitumor Th1-/M1-Related Cytokines.

Antitumor activity of classically activated macrophage (Mϕ) may be impaired within the tumors, spleen, and bone marrow. Thus, it is possible to boost its antitumor activity after its pulsing with necrotic tumor cell lysates combined with an adjuvant. We set out to determine the potential adjuvant effects of thymoquinone (TQ; 2-isopropyl-5-methyl-1,4-benzoquinone, C10H12O2) on both functional activities of classically activated Mϕs, pulsed or not with necrotic Jurkat T cell line lysates (NecrJCL), and the balance of antitumor cytokines (ATCs) versus immunosuppressive cytokines (ISCs) during crosstalk with autologous human CD4+ T cells. We found that TQ treatment resulted in a significant upregulation of phagocytic activity, respiratory burst, the production of interleukin-2 (IL-2), IL-6, and IL-17 in NecrJCL-pulsed Mϕ co-culture system, and, conversely, in downregulation of the production of IL-6, IL-17, nitric oxide (NO), and arginase activity in nonpulsed TQ-treated Mϕs co-culture system. In addition, TQ has also shown low upregulation effect on the production of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and IL-1ß, pathogen killing capacity and H2O2 in NecrJCL-pulsed Mϕs co-cultures. Moreover, TQ significantly downregulated arginase activity, and significantly upregulated inducible NO synthase (iNOS) activity-to-arginase activity ratio in NecrJCL-pulsed Mϕ co-cultures. Furthermore, TQ downregulated IL-10-to-IL-17 ratio and total cellular cholesterol content (ttcCHOL), but upregulated the ratios of IL-1ß-to-IL-4, IL-1ß-to-IL-10, IFN-γ-to-IL-4, IFN-γ-to-IL-10, TNF-α-to-IL-4, TNF-α-to-IL-10, and combined proinflammatory cytokines (PICs)-to-anti-inflammatory cytokines (AICs) in NecrJCL-pulsed Mϕs co-culture system, whereas significant differences were highlighted only for IL-10-to-IL-17, IFN-γ-to-IL-10, and PICs-to-AICs ratios. Our outcomes demonstrated that TQ can act as potent adjuvant for enhancing both the functional activities of NecrJCL-pulsed Mϕ and the production of ATCs during their interplay with CD4+ T cells.