Exploring Plasma-Level Gut Microbiota Mediators and Pro-Inflammatory Markers in Pregnant Women with Short Cervix and Gestational Diabetes Mellitus.

The composition of the gut microbiota (GM) undergoes significant changes during pregnancy, influenced by metabolic status, energy homeostasis, fat storage, and hormonal and immunological modifications. Moreover, dysbiosis during pregnancy has been associated with preterm birth, which is influenced by factors such as cervical shortening, infection, inflammation, and oxidative stress. However, dysbiosis also affects the levels of lipopolysaccharide-binding protein (LBP), short-chain fatty acids (SCFAs), and free fatty acids (FFA) in other tissues and the bloodstream. In this study, we investigated the plasmatic levels of some pro-inflammatory cytokines, such as matrix metalloproteinases-8 (MMP-8), interleukin-8 (IL-8), heat shock protein 70 (Hsp70), and microbial markers in pregnant women with a short cervix (≤25 mm) compared to those with normal cervical length (>25 mm). We examined the differences in the concentration of these markers between the two groups, also assessing the impact of gestational diabetes mellitus. Understanding the relationship between GM dysbiosis, inflammatory mediators, and cervical changes during pregnancy may contribute to the identification of potential biomarkers and therapeutic targets for the prevention and management of adverse pregnancy outcomes, including preterm birth.

Vaginal Microbiome in Pregnant Women with and without Short Cervix.

Cervical shortening is a recognised risk factor for pre-term birth. The vaginal microbiome plays an essential role in pregnancy and in maternal and foetal outcomes. We studied the vaginal microbiome in 68 women with singleton gestation and a cervical length ≤25 mm and in 29 pregnant women with a cervix >25 mm in the second or early third trimester. Illumina protocol 16S Metagenomic Sequencing Library Preparation was used to detail amplified 16SrRNA gene. Statistical analyses were performed in R environment. Firmicutes was the phylum most represented in all pregnant women. The mean relative abundance of Proteobacteria and Actinobacteriota was higher in women with a short cervix. Bacterial abundance was higher in women with a normal length cervix compared to the group of women with a short cervix. Nonetheless, a significant enrichment in bacterial taxa poorly represented in vaginal microbiome was observed in the group of women with a short cervix. Staphylococcus and Pseudomonas, taxa usually found in aerobic vaginitis, were more common in women with a short cervix compared with the control group, while Lactobacillus iners and Bifidobacterium were associated with a normal cervical length. Lactobacillus jensenii and Gardenerella vaginalis were associated with a short cervix.

Tryptophan degradation enzymes expression in the placenta and the Kynurenine/Tryptophan ratio in maternal plasma after elective cesarean section.

Indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO) metabolize tryptophan in the kynurenine pathway. We evaluated these enzymes' mRNA expression in maternal and fetal sides of the placenta of uncomplicated, unlabored full-term pregnancies after elective cesarean section and compared it with that of placentas obtained from vaginal delivery. Tryptophan and kynurenine plasmatic levels after cesarean section were measured, to investigate their possible correlation with IDO1 and TDO mRNA (TDO2) expression. The results suggested that IDO1 and TDO2 expression was higher in the maternal side of the placenta and that labor significantly affects TDO2 expression and the plasma Kynurenine/Tryptophan ratio.

Tryptophan degradation enzymes and Angiotensin (1-7) expression in human placenta.

Indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO) are key enzymes for tryptophan degradation, regulating immune tolerance during pregnancy. The intrauterine renin-angiotensin system is also involved in the progression of a healthy pregnancy. Angiotensin(1-7) maintains the integrity of fetal membranes via counteracting the pro-inflammatory actions of Angiotensin II. No data are available on placental Angiotensin(1-7) co-expression with TDO. We aimed to characterize TDO mRNA expression and its localization in different areas of the placenta of physiological pregnancies delivered at term; its co-expression with Angiotensin(1-7) and its correlation with the plasma kynurenine/tryptophan (Kyn/Trp) ratio was investigated. This prospective observational study included a nonconsecutive series of 20 singleton uncomplicated pregnancies delivered vaginally. TDO mRNA was expressed in both maternal and fetal sides of the placentas and TDO protein also in the villi and it was co-expressed with IDO1 in almost half of the placental cells at these sites. The percentage of TDO+ and IDO1+ cells appeared to be influenced by maternal pre-gestational smoking and newborn weight. A strong correlation was found between the percentage of TDO+ and IDO1+ cells in the villi. TDO+ cells also expressed Angiotensin(1-7), with a higher percentage on the fetal side and in the villi compared to the maternal one. Kyn/Trp plasma ratio was not correlated with IDO and TDO expression nor with the patient's characteristics. Collectively, our data indicate that TDO is detectable in placental tissue and is co-expressed with IDO and with Angiotensin(1-7)+ on the fetal side and in the villi.

Dexamethasone Promotes a Stem-Like Phenotype in Human Melanoma Cells via Tryptophan 2,3 Dioxygenase.

In addition to its well-established immunosuppressive actions, tryptophan 2,3-dioxygenase (TDO) appears to elicit direct effects on tumor cell function. Although TDO has been associated with cancer stemness, its involvement in melanoma stem cell biology remains largely unknown. Since we showed that by upregulating TDO, dexamethasone (dex) promotes proliferation and migration of SK-Mel-28 human melanoma cells, we sought to investigate dex effects on melanoma spherogenesis and stemness, and whether these events are mediated by TDO. We demonstrate here that dex significantly upregulates TDO in A375, a more aggressive melanoma cell line, confirming that dex effects are not limited to SK-Mel-28 cells. Moreover, dex stimulates spherogenesis of both cell lines, which is mediated by TDO, evident by its suppression with 680C91, a TDO inhibitor. The formed melanospheres appear to be enriched with embryonic stem cell marker mRNAs, the expression of which is potentiated by dex. Expression of cancer stem cell markers (CD133, CD44, ganglioside GD2) was significantly increased in A375 spheres, as detected by flow cytometry. Taken together, our results suggest that TDO could represent a promising target in the management of melanoma and that dex, routinely used as a co-medication also in advanced melanoma, may stimulate melanoma cell function/tumor-supporting properties, a rather debilitating and undesired side effect.

Lactate Maintains BCR/Abl Expression and Signaling in Chronic Myeloid Leukemia Cells Under Nutrient Restriction.

This study was directed to deepen the effects of nutrient shortage on BCR/Ablprotein expression and signaling in chronic myeloid leukemia (CML) cells. The backbone of the study was cell culture in medium lacking glucose, the consumption of which we had previously shown to drive BCR/Ablprotein suppression, and glutamine, the other main nutrient besides glucose. In this context, we focused on the role of lactate, the main by-product of glucose metabolism under conditions of rapid cell growth, in particular as a modulator of the maintenance of CML stem/progenitor cell potential, a crucial determinant of disease course and relapse of disease. The results obtained indicated that lactate is a powerful surrogate of glucose to prevent the suppression of BCR/Abl signaling and is therefore capable to maintain BCR/Abl-dependent CML stem/progenitor cell potential. A number of metabolism-related functional and phenotypical features of CML cells were also determined. Among these, we focused on the effect of lactate on oxygen consumption rate, the dependence of this effect on the cell surface lactate carrier MCT-1, and the relationship of the lactate effect to pyruvate and to the activity of mitochondrial pyruvate carrier.

Glutamine Availability Controls BCR/Abl Protein Expression and Functional Phenotype of Chronic Myeloid Leukemia Cells Endowed with Stem/Progenitor Cell Potential.

This study was directed to characterize the role of glutamine in the modulation of the response of chronic myeloid leukemia (CML) cells to low oxygen, a main condition of hematopoietic stem cell niches of bone marrow. Cells were incubated in atmosphere at 0.2% oxygen in the absence or the presence of glutamine. The absence of glutamine markedly delayed glucose consumption, which had previously been shown to drive the suppression of BCR/Abl oncoprotein (but not of the fusion oncogene BCR/abl) in low oxygen. Glutamine availability thus emerged as a key regulator of the balance between the pools of BCR/Abl protein-expressing and -negative CML cells endowed with stem/progenitor cell potential and capable to stand extremely low oxygen. These findings were confirmed by the effects of the inhibitors of glucose or glutamine metabolism. The BCR/Abl-negative cell phenotype is the best candidate to sustain the treatment-resistant minimal residual disease (MRD) of CML because these cells are devoid of the molecular target of the BCR/Abl-active tyrosine kinase inhibitors (TKi) used for CML therapy. Therefore, the treatments capable of interfering with glutamine action may result in the reduction in the BCR/Abl-negative cell subset sustaining MRD and in the concomitant rescue of the TKi sensitivity of CML stem cell potential. The data obtained with glutaminase inhibitors seem to confirm this perspective.

Tryptophan metabolism and immune regulation in the human placenta.

The placenta represents the maternal-fetal vascular interface. It is capable of supplying the bioenergetic needs of the developing conceptus. It is composed of different cell types that engage in highly varied functions, ranging from attachment, invasion and vascular remodeling to cell fusion, hormone production, and nutrient transport. A deep knowledge of the immunological mechanisms responsible for maintaining an active tolerance towards an allogeneic fetus and the anti-inflammatory properties of the placenta can be useful to clarify the pathogenesis of adverse events in pregnancy. While the systemic mechanisms of this immunological regulation in pregnancy have been well studied, the metabolic processes involved in the placental immune response are still poorly understood. The aim of this review is to summarize the most important information concerning the immune regulation in pregnancy, focusing on the role of tryptophan (Trp) catabolism performed by indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) in the placenta.

Dexamethasone Induces the Expression and Function of Tryptophan-2-3-Dioxygenase in SK-MEL-28 Melanoma Cells.

Tryptophan-2,3-dioxygenase (TDO) is one of the key tryptophan-catabolizing enzymes with immunoregulatory properties in cancer. Contrary to expectation, clinical trials showed that inhibitors of the ubiquitously expressed enzyme, indoleamine-2,3-dioxygenase-1 (IDO1), do not provide benefits in melanoma patients. This prompted the hypothesis that TDO may be a more attractive target. Because the promoter of TDO harbors glucocorticoid response elements (GREs), we aimed to assess whether dexamethasone (dex), a commonly used glucocorticoid, modulates TDO expression by means of RT-PCR and immunofluorescence and function by assessing cell proliferation and migration as well as metalloproteinase activity. Our results show that, in SK-Mel-28 melanoma cells, dex up-regulated TDO and its downstream effector aryl hydrocarbon receptor (AHR) but not IDO1. Furthermore, dex stimulated cellular proliferation and migration and potentiated MMP2 activity. These effects were inhibited by the selective TDO inhibitor 680C91 and enhanced by IDO1 inhibitors. Taken together, our results demonstrate that the metastatic melanoma cell line SK-Mel-28 possesses a functional TDO which can also modulate cancer cell phenotype directly rather than through immune suppression. Thus, TDO appears to be a promising, tractable target in the management or the treatment of melanoma progression.

Insulin resistance and obesity affect monocyte-derived dendritic cell phenotype and function.

AIM: Cardiovascular disease (CVD) is prevalent in women after menopause, which may be associated with obesity, insulin resistance and metaflammation. Despite the recognized role of immunological mechanisms in vascular remodeling, the role of dendritic cells (DCs) is still unclear. The aim was to characterize monocyte-derived DCs (Mo-DC) in post-menopausal patients with type 2 diabetes (T2DM) and obese woman, without clinical manifestations of atherosclerosis. METHODS: Obese post-menopausal women with or without T2DM were enrolled and were compared to age-matched healthy women. DCs obtained from patients were phenotypically and functionally characterized by flow cytometry and mixed lymphocyte reaction. MRNA integrins expression was assessed by real time RT-PCR; circulating fetuin-A and adiponectin levels were measured by ELISA. RESULTS: Phenotypic dysregulation of Mo-DC reported was related to a defective allogenic lymphocyte stimulation and to an increased mRNA of CD11c, CD18 and DC-SIGN/CD209 which regulate their adhesion to vascular wall cells. Fetuin-A and adiponectin levels were significantly altered and negatively correlated. Hyperglycaemia significantly impaired CD14+ transdifferentiation into Mo-DC. CONCLUSIONS: These data show a dysfunction of Mo-DCs obtained from precursors isolated from T2DM obese post-menopausal woman without any documented clinical CV event. Association of obesity to diabetes seems to worsen DC's phenotype and function and increase vascular inflammation.

In Vitro Comparison of the Effects of Imatinib and Ponatinib on Chronic Myeloid Leukemia Progenitor/Stem Cell Features.

BACKGROUND: The development of molecularly tailored therapeutic agents such as the BCR/ABL-active tyrosine kinase inhibitors (TKi) resulted in an excellent treatment option for chronic myeloid leukemia (CML) patients. However, following TKi discontinuation, disease relapses in 40-60% of patients, an occurrence very likely due to the persistence of leukemic stem cells that are scarcely sensitive to TKi. Nevertheless, TKi are still the only current treatment option for CML patients. OBJECTIVE: The aim of this study was to compare the effects of TKi belonging to different generations, imatinib and ponatinib (first and third generation, respectively), on progenitor/stem cell expansion potential and markers. PATIENTS AND METHODS: We used stabilized CML cell lines (KCL22, K562 and LAMA-84 cells), taking advantage of the previous demonstration of ours that cell lines contain cell subsets endowed with progenitor/stem cell properties. Primary cells explanted from CML patients were also used. The effects of TKi on the expression of stem cell related genes were compared by quantitative PCR. Flow cytometry was performed to evaluate aldehyde-dehydrogenase (ALDH) activity and the expression of cluster of differentiation (CD) cell surface hematopoietic stem cell markers. Progenitor/stem cell potential was estimated by serial colony formation ability (CFA) assay. RESULTS: Ponatinib was more effective than imatinib for the reduction of cells with ALDH activity and progenitor/stem cell potential of CML patient-derived cells and cell lines. Furthermore, ponatinib was more effective than imatinib in reducing the percentage of CD26-expressing cells in primary CML cells, whereas imatinib and ponatinib showed similar efficacy on KCL22 cells. Both drugs strongly upregulated NANOG and SOX2 in CML cell lines, but in KCL22 cells this upregulation was significantly lower with ponatinib than with imatinib, an outcome compatible with a lower level of enrichment of the stem cell compartment upon ponatinib treatment. CONCLUSION: Ponatinib seems to target CML progenitor/stem cells better than imatinib.

Different effects of tryptophan 2,3-dioxygenase inhibition on SK-Mel-28 and HCT-8 cancer cell lines.

PURPOSE: Indoleamine 2,3-dioxygenase-1 (IDO1) and more recently, tryptophan 2,3-dioxygenase (TDO), are tryptophan-catabolizing enzymes with immunoregulatory properties in cancer. IDO1 is more expressed than TDO in many tumours including melanomas; however, IDO inhibitors did not give expected results in clinical trials, highlighting the need to consider TDO. We aimed to characterize both TDO expression and function in a melanoma cell line, named SK-Mel-28, with the purpose to compare it with a colon cancer cell line, HCT-8, and with a human endothelial cell line (HUVEC). METHODS: TDO expression was assessed as real time-PCR and western blot, for mRNA and protein expression, respectively. While cell proliferation was assessed as cell duplication, cell apoptosis and cell cycle were analysed by means of flow cytometry. RESULTS: SK-Mel-28 cells showed higher TDO levels compared to HCT-8 and to HUVEC cells. A selective TDO inhibitor, 680C91, significantly impaired cell proliferation in a concentration-dependent manner, by inducing cell arrest during the G2 phase for SK-Mel-28 and HUVEC cells, while an early apoptosis was increasing in HCT-8 cells. No toxic effects were observed. These data demonstrated that TDO is highly expressed in SK-Mel-28 cells and may be involved in the regulation of their proliferation. CONCLUSION: TDO may directly modulate cancer cell function rather than immune suppression and can be considered as a target for melanoma progression together with IDO1.

Enhancement of Soft Tissue Sarcoma Cell Radiosensitivity by Poly(ADP-ribose) Polymerase-1 Inhibitors.

Soft tissue sarcomas (STS) are aggressive tumors with a poor prognosis. Poly(ADP-ribose) polymerase (PARP)-1 inhibitors (PARPi) enhance the cytotoxic effects of radiation. In this study, we evaluated the effect of PARPi on survival and DNA damage of irradiated STS cells. For clonogenic assays, STS cell lines were irradiated with or without olaparib, iniparib or veliparib pretreatment. The effect of PARP inhibition on γ-H2AX and Rad51 foci formation, on PARP-1, phospho-ERK and cleaved caspase-3 protein expression and on cell cycle progression was evaluated on irradiated rhabdomyosarcoma cells pretreated with olaparib. The results from this work showed that PARPi induced significant radiosensitization in STS cells. Rhabdomyosarcoma cells showed the highest increase in radiosensitivity, with a radiosensitization enhancement ratio at 50% survival (ER50) of 3.41 with veliparib. All PARPi exerted a synergistic effect when combined with radiation. Fibrosarcoma cells showed an ER50 of 2.29 with olaparib. Leiomyosarcoma and liposarcoma cells showed their highest ER50 with veliparib (1.71 and 1.84, respectively). In rhabdomyosarcoma, olaparib enhanced the formation of radiation-induced γ-H2AX/Rad51 foci and PARP-1 cleavage, induced slightly increased expression of cleaved caspase-3 and reduced phospho-ERK expression. Moreover, the combination of olaparib and radiation resulted in a significantly enhanced cell cycle arrest in the G2/M phase compared to the two treatments alone. In conclusion, we have shown that PARPi are potent radiosensitizers of human STS cells. These results support the pursuit of further investigations into the effects of PARPi combined with radiation on STS.

Inorganic nanoparticles as potential regulators of immune response in dendritic cells.

AIM: The spontaneous adsorption of proteins on nanoparticles (NPs) in biological media is exploited to prepare complexes of NPs and proteins from cancer cells' lysates for application in cancer immunotherapy. MATERIALS & METHODS: Gold (Au) and silica NPs were synthesized, incubated with cancer cells' lysates and characterized. Dendritic cells (DCs) were challenged with protein-coated NPs, their maturation, viability and morphology were evaluated and lymphocytes T proliferation was determined. RESULTS: Silica and Au NPs bound different pools of biomolecules from lysates, and are therefore promising selective carriers for antigens. When incubated with immature DCs, NPs were efficiently endocytosed without cytotoxicity. Finally, protein-coated AuNPs promoted DC maturation and DC-mediated lymphocyte proliferation, at variance with lysate alone and protein-coated silica NPs, that did not promote DCs maturation. CONCLUSION: These results demonstrate that the spontaneous formation of protein coronas on NPs represents a possible approach to fast, easy, cost-effective DCs stimulation.

Dendritic cells: phenotypic and functional heterogeneity.

Dendritic cells are specialized to capture antigens, process them and present them to T cells to initiate, regulate and fine tune immune responses towards pathogens and tumours. The story of these cells began more than forty years ago and the interest for them is ever growing because of their central role in immunobiology. Dendritic cells are heterogeneous for origin, anatomical localization, phenotype and function: several subsets of myeloid dendritic cells and plasmacytoid dendritic cells have been recognized until now. Dendritic cells differentiate from haematopoietic stem cells-derived precursors, migrate from sites of antigen uptake to lymphoid organs and during this process mature to antigen presenting cells capable of interacting with lymphocytes and stimulate both immune response and peripheral tolerance to self. Since dendritic cells play crucial roles in infection, cancer, allergy, autoimmunity and graft rejection, thorough knowledge of their subsets and behaviour will open the path to tools allowing better control of many clinical conditions.