State-of-the-art cytometry in the search of novel biomarkers in digestive cancers.

Despite that colorectal and liver cancer are among the most prevalent tumours in the world, the identification of non-invasive biomarkers to aid on their diagnose and subsequent prognosis is a current unmet need that would diminish both their incidence and mortality rates. In this context, conventional flow cytometry has been widely used in the screening of biomarkers with clinical utility in other malignant processes like leukaemia or lymphoma. Therefore, in this review, we will focus on how advanced cytometry panels covering over 40 parameters can be applied on the study of the immune system from patients with colorectal and hepatocellular carcinoma and how that can be used on the search of novel biomarkers to aid or diagnose, prognosis, and even predict clinical response to different treatments. In addition, these multiparametric and unbiased approaches can also provide novel insights into the specific immunopathogenic mechanisms governing these malignant diseases, hence potentially unravelling novel targets to perform immunotherapy or identify novel mechanisms, rendering the development of novel treatments. As a consequence, computational cytometry approaches are an emerging methodology for the early detection and predicting therapies for gastrointestinal cancers.

Expression profile of microRNAs related with viral infectivity, inflammatory response, and immune activation in people living with HIV.

Objective: To evaluate the serum expression of microRNAs (miRNAs) with ability to modulate the human immunodeficiency (HIV) replication or inflammatory status in people living with HIV (PLWH). Methods: Forty healthy controls and two groups of PLWH were evaluated: (a) Group 1 (n = 30), patients with detectable viral load at inclusion, analyzed before receiving antiretroviral therapy (ART) and 12 months after initiating it; (b) Group 2 (n = 55), PLWH with prolonged undetectable viral load. Intestinal barrier disruption (I-FABP) and bacterial translocation (16S rDNA) markers, inflammatory markers such as interleukin (IL)-6 and sCD163, immune activation and expression of specific miRNAs were evaluated. Results: Serum concentrations of I-FABP, 16S rDNA, IL-6, sCD163 and activated T lymphocytes were increased in PLWH. Serum miR-34a was overexpressed at inclusion and remained elevated after ART. The expression of the remaining miRNAs that modulate HIV infectivity (miR-7, mir-29a, miR-150, and miR-223) was similar in PLWH and controls. Related to miRNAs implicated in inflammation (miR-21, miR-155, and miR-210), significant overexpression were observed in miR-21 and miR-210 levels in untreated PLWH, but levels were restored in those patients treated for a long period. Conclusion: A sustained overexpression of miR-34a was detected even after prolonged HIV controlled replication. miR-21 and miR-210 can be considered new markers of inflammation with high sensitivity to its modifications.

Implication of Neutrophils Extracellular Traps in the Pathogenesis of SARS-CoV-2 pneumonia.

Peripheral blood polymorphonuclear neutrophils (PMNs) forming extracellular traps (NETs), as well as endothelial- and platelet-derived parameters, have been analyzed in patients with SARS-CoV-2 pneumonia, and their prognostic role has been evaluated. Eighty-seven consecutive patients hospitalized with SARS-CoV-2 pneumonia were prospectively selected. A sample of 30 healthy individuals served as the control group. Clinical and oxygenation (oxygen saturation to fraction of inspired oxygen ratio-SpO2/FiO2) characteristics and PMNs forming NETs, serum levels of myeloperoxidase, E-selectin, vascular cell adhesion molecule 1-VCAM1-vascular endothelial growth factor, P-selectin, platelet factor 4 and plasma concentrations of D-dimer were evaluated at hospital admission, at discharge and 14 days after discharge. Intensive care unit admission or death was the primary composite endpoint. Patients showed a higher number of PMNs forming NETs than healthy controls. The absolute number of PMNs forming NETs was inversely correlated with oxygen status (SpO2/FiO2) and positively with inflammatory (C-reactive protein, ferritin) markers and VCAM1. A decrease in, but not a normalization of NETs and endothelial-derived parameters was observed in patients who survived. In conclusion, the formation of NETs runs parallel to that of other inflammatory and endothelial activation markers, and is inverse to the oxygenation parameters, supporting a pathogenic role for PMNs in this entity.

Hepatitis C: Problems to extinction and residual hepatic and extrahepatic lesions after sustained virological response.

Loss of follow-up or reinfections hinder the expectations of hepatitis C eradication despite the existence of highly effective treatments. Moreover, the elimination of the infection does not imply the reversion of those chronic alterations derived from the previous infection by hepatitis C virus (HCV). This review analyzes the risk factors associated with loss to follow-up in diagnosis or treatment, and the possibility of reinfection. Likewise, it assesses the residual alterations induced by chronic HCV infection considering the liver alterations (inflammation, fibrosis, risk of decompensation, hepatocellular carcinoma, liver transplantation) and, on the other hand, the comorbidities and extrahepatic manifestations (cryoglobulinemia, non-Hodgkin lymphoma, peripheral insulin resistance, and lipid, bone and cognitive alterations). Peculiarities present in subjects coinfected with human immunodeficiency virus are analyzed in each section.

Modifications of liver stiffness and CXCL4, TGF-ß1 and HGF are similar in HCV- and HIV/HCV-infected patients after DAAs.

The objective of this work was to identify predictive factors of fibrosis regression after direct antiviral agents (DAAs) in HCV-monoinfected and HIV/HCV-coinfected patients. This was a prospective study of HCV-monoinfected (n = 20), HIV/HCV-co-infected (n = 66) patients and healthy controls (n = 15). Patients had started DAAs and achieved sustained virological response. Liver stiffness (LS) and serum concentrations of profibrotic transforming growth factor (TGF)-ß1 and CXC chemokine ligand 4 (CXCL4) and antifibrotic HGF hepatocyte growth factor (HGF) were analyzed at baseline (M0) and 12 months after starting DAAs (M12). A M12 LS achievement of ≤ 9.5 kPa was considered the cutoff point to discharge from a liver clinic. The LS decrease from M0 to M12 was 34%. No significant differences were observed in LS decline between HCV- and HIV/HCV-infected individuals. Changes of serum CXCL4, TGF-ß1 and HGF levels did not correlate with LS improvement. 16 out from 56 patients (28%) with a baseline LS > 9.5 achieved a M12 LS ≤ 9.5. HCV-monoinfected and HIV/HCV coinfected patients experienced a significant reduction of LS after sustained virological response. This improvement did not correlate with changes in serum profibrotic or antifibrotic markers. A 29% of those with a baseline LS > 9.5 achieved a LS under this cutoff point.

Modifications of CD4 T cells, CD4/CD8 ratio and serum levels of soluble CD14 in HIV-HCV-coinfected patients after sustained HCV response induced by direct-acting antiviral agents: influence of liver cirrhosis.

To analyze the modifications of CD4 T cell, CD4/CD8 ratio, and serum levels of soluble CD14 (sCD14) in HIV/HCV-coinfected patients after treatment with direct anti-HCV antiviral agents. Consecutive cases of HIV/HCV-coinfected patients, attended at the University Hospital, who achieved sustained virological responses with interferon-free hepatitis C antiviral drugs, were analyzed. Thirty-five percent of patients (n = 39) had been diagnosed with liver cirrhosis. The evaluation criteria were changes in CD4 T-cell counts and percentages and inflammation (measured by serum sCD14 levels) or immune activation indexes (determined by CD4/CD8 ratio) from beginning anti-HCV therapy to 12 months later. One hundred twelve patients were included (87% male; median age, 54 years; median time from the infection diagnosis, 22 years; previous drug users, 87%). Significant increases in CD4 T cell count and percentage were detected only in individuals without liver cirrhosis. No significant differences in CD4/CD8 ratios or sCD14 levels were observed in patients with or without cirrhosis. The proportion of patients with less than 500 CD4 T cell/mm3 before therapy who achieved more than 500 CD4 T cell/mm3 after it increased only in the group without liver cirrhosis. The finding that CD4 T cell count and percentage were improved only in patients without liver cirrhosis supports the idea that treatment against HCV in HIV/HCV-coinfected patients is needed in the early phases of liver disease.

Similarities and differences between HIV and SARS-CoV-2.

In the last 50 years we have experienced two big pandemics, the HIV pandemic and the pandemic caused by SARS-CoV-2. Both pandemics are caused by RNA viruses and have reached us from animals. These two viruses are different in the transmission mode and in the symptoms they generate. However, they have important similarities: the fear in the population, increase in proinflammatory cytokines that generate intestinal microbiota modifications or NETosis production by polymorphonuclear neutrophils, among others. They have been implicated in the clinical, prognostic and therapeutic attitudes.

Neutrophil Expression of T and B Immunomodulatory Molecules in HIV Infection.

Objective: Evaluate the expression of B and T cell immunomodulatory molecules in polymorphonuclear neutrophils (PMN) in HIV-infected patients. Methods: HIV load, bacterial translocation and neutrophils' expression of T [programmed death ligand, interleukin-10+, arginase 1+] and B [BAFF, APRIL] molecules were analyzed in different cohorts and time points: a control group of 25 healthy individuals and two groups of HIV-infected patients. Group 1 of patients included 35 untreated patients, studied at baseline and after antiretroviral therapy (ART). Group 2 was composed of 25 patients with undetectable viral load after a median of 101 months of ART prior to inclusion in the study. Results: Compared with the control group, group 1 patients showed increased bacterial translocation and their PMN had a significantly higher expression of T and B-cell immunomodulatory molecules, both at baseline and after 12 months of ART. Group 2 patients showed reduced bacterial translocation levels when compared with group 1 patients after 12 months of treatment. PMN expression of B-cell modulators was similar between group 2 patients and healthy controls, although the expression of T-cell modulators remained increased. Conclusion: In HIV-infected patients, the expression of B-cell stimulatory and T-cell suppressive molecules by neutrophils was increased at baseline and after a limited time of therapy. After a prolonged period of ART, only PMNs expression of T-cell immunosuppressive molecules remained elevated.

HIV antibodies level as a marker of HIV persistence: the role of hepatitis C virus coinfection.

Human immunodeficiency virus (HIV) antibodies have been proposed as a measure of the size of the HIV reservoir. The aim of our study is to quantify the anti-HIV antibodies level in a cohort of people living with HIV (PLWH), stratified based on the presence of continuous undetectable HIV viral load and the co-existence of hepatitis C virus infection. A sample of 229 HIV-monoinfected (n = 114) or HIV/HCV-coinfected [either with resolved HCV infection (n = 75) or active HCV coinfection (n = 40)] patients, followed up a median of 34 (IQR 20-44) months, was studied. Anti-HIV index was obtained as the 1:800 dilution of HIV antibodies. CD4+ T cell count, time with undetectable HIV viral load, annual increase of CD4+ T cell count, anti-HCV therapy, and diagnosis of cirrhosis were analyzed. Patients with a continued suppressed HIV viral load had significant lower anti-HIV index compared with those with virologic failure during the follow-up. Significant higher CD4+ T cell increase was observed in those with a lower anti-HIV index. HIV-monoinfected patients showed an anti-HIV index significantly lower than patients with HCV coinfection. Resolved HCV infection after interferon-based therapy, but not with direct acting antivirals, was associated with a lower anti-HIV index. HIV/HCV-coinfected patients showed higher HIV antibodies level when compared with HIV-monoinfected individuals. A decrease in anti-HIV index in HIV/HCV-coinfected patients was detected when a sustained virological HCV response was obtained after interferon-based therapy, in possible relation with the direct or indirect effect of interferon on PLWH CD4 T cells.

Giant Extra-Axial Cavernous Angioma of the Falx: Case Report.

BACKGROUND AND IMPORTANCE: Cavernous angiomas or cavernomas are vascular malformations usually located in the brain parenchyma. However, they rarely present as extra-axial lesions, attached to the dura, and may mimic meningiomas. Most reported cases concern the cavernous sinus region and other locations are very uncommon. CLINICAL PRESENTATION: A 61-yr-old female known for long-standing mental illness presented with progressive gait instability. Imaging studies revealed an extra-axial lesion in relation to the anterior part of the falx cerebri. An interhemispheric approach was used to remove the lesion. Pathological analysis revealed features compatible with an extra-axial cavernoma: structureless vascular channels lacking smooth muscle and elastic lamellae, without intervening brain parenchyma. CONCLUSION: Cavernous angiomas or cavernomas can present as extra-axial lesions. Although progressive growth can be observed, they should not be considered as tumoral lesions, because there is no cellular duplication. Unlike other locations, resection of anterior cranial fossa extra-axial cavernomas seems to be facilitated by minimal bleeding.

Fibersol-2 induces apoptosis of Apc-deficient colorectal Cancer (SW480) cells and decreases polyp formation in Apc MIN mice.

The consumption of dietary fibers has been implicated with a lowered risk of human colorectal cancer. Proposed mechanisms involve alterations in the stool consistency, transit time, and formation of short-chain fatty acid by dietary fiber fermentation, and the reorganization of gut microbiota. Here we show that Fibersol-2, a digest-resistant maltodextrin, not only inhibits proliferation of colorectal SW480 cancer cell lines by increasing reactive oxygen species (ROS), but decreases the numbers of the adenoma count in Multiple Intestinal Neoplasia (MIN) mice carrying a mutation in the Adenomatous Polyposis Coli gene by 84 d of age. These observations provide direct evidence that Fibersol-2 intrinsically contains anti-cancer activity, independent of the intestinal metabolism and any potential interactions with the microbiota.

Tumor suppression by resistant maltodextrin, Fibersol-2.

Resistant maltodextrin Fibersol-2 is a soluble and fermentable dietary fiber that is Generally Recognized As Safe (GRAS) in the United States. We tested whether Fibersol-2 contains anti-tumor activity. Human colorectal cancer cell line, HCT116, and its isogenic cells were treated with FIbersol-2. Tumor growth and tumorigenesis were studied in vitro and in vivo. Apoptotic pathway and generation of reactive oxygen species (ROS) were investigated. We discovered that Fibersol-2 significantly inhibits tumor growth of HCT116 cells by inducing apoptosis. Fibersol-2 strongly induces mitochondrial ROS and Bax-dependent cleavage of caspase 3 and 9, which is shown by isogenic HCT116 variants. Fibersol-2 induces phosphorylation of Akt, mTOR in parental HCT116 cells, but not in HCT116 deficient for Bax or p53. It prevents growth of tumor xenograft without any apparent signs of toxicity in vivo. These results identify Fibersol-2 as a mechanism-based dietary supplement agent that could prevent colorectal cancer development.

Cell Differentiation and Checkpoint.

DNA damage is induced in many types of cells by internal and external cell stress. When DNA is damaged, DNA Damage Response (DDR) programs are activated to repair the DNA lesions in order to preserve genomic integrity and suppress subsequent malignant transformation. Among these programs is cell cycle checkpoint that ensures cell cycle arrest and subsequent repair of the damaged DNA, apoptosis and senescence in various phases of the cell cycle. Moreover, recent studies have established the cell differentiation checkpoint, the other type of the checkpoint that is specifically activated in the course of differentiation. We will discuss the evidences that support the link between DNA damage proteins and C2C12 cell differentiation.

Long-term p110α PI3K inactivation exerts a beneficial effect on metabolism.

The insulin/insulin-like growth factor-1 signalling (IIS) pathway regulates cellular and organismal metabolism and controls the rate of aging. Gain-of-function mutations in p110α, the principal mammalian IIS-responsive isoform of PI 3-kinase (PI3K), promote cancer. In contrast, loss-of-function mutations in p110α impair insulin signalling and cause insulin resistance, inducing a pre-diabetic state. It remains unknown if long-term p110α inactivation induces further metabolic deterioration over time, leading to overt unsustainable pathology. Surprisingly, we find that chronic p110α partial inactivation in mice protects from age-related reduction in insulin sensitivity, glucose tolerance and fat accumulation, and extends the lifespan of male mice. This beneficial effect of p110α inactivation derives in part from a suppressed down-regulation of insulin receptor substrate (IRS) protein levels induced by age-related hyperinsulinemia, and correlates with enhanced insulin-induced Akt signalling in aged p110α-deficient mice. This temporal metabolic plasticity upon p110α inactivation indicates that prolonged PI3K inhibition, as intended in human cancer treatment, might not negatively impact on organismal metabolism.

Physiological and biochemical parameters controlling waterlogging stress tolerance in Prunus before and after drainage.

Waterlogging is associated with poor soil drainage. As a consequence oxygen levels decrease in the root environment inducing root asphyxia and affecting plant growth. Some plants can survive under these conditions triggering complex anatomical and biochemical adaptations, mostly in the roots. Long- and short-term responses to waterlogging stress were compared in two trials using a set of two myrobalans (Prunus cerasifera Erhr), 'P.2175' and 'P.2980', as tolerant rootstocks and two almond × peach [Prunus amygdalus Batsch ×Prunus persica (L.) Batsch] interspecific hybrids, 'Garnem' and 'Felinem', as sensitive ones in two consecutive years. Stomatal conductance and chlorophyll content were measured in the long-term trials to assess survival performance, while the enzyme activities of superoxide dismutase (SOD, EC 1.15.1.1), guaiacol peroxidase (POD, EC 1.11.1.7), and catalase (CAT, EC 1.11.1.6) were measured in the short-term trials to study early antioxidant response. The incidence of the stress in the root environment was different as a result of the different plant development at the moment of the treatment, as a consequence of different environmental conditions both before and during the treatment between the 2 years. The activity of the different enzymes was higher in the sensitive genotype 'Felinem' than in the tolerant 'P.2175'. This result shows an activation of the antioxidant system and has been observed to depend of the different nature of the roots between the 2 years. As the antioxidant enzymes seem to work more efficiently when roots are more aerated, we cannot conclude that they are responsible for the higher tolerance observed in the myrobalan plums.

Tacrolimus modulates liver and pancreas nitric oxide synthetase and heme-oxygenase isoforms and cytokine production after endotoxemia.

Cytoprotective effects of tacrolimus are due to its unspecific anti-inflammatory and anti-oxidant properties. Neither the exact mechanisms nor if there is any organ-specificity or dose-dependent response have not been yet elucidated. Our aim was to evaluate the effect of tacrolimus on oxidative stress and mediator production in liver and pancreatic tissue secondary to endotoxemia. Wistar rats were pretreated with intraperitoneal injection of tacrolimus (0.07, 0.15, and 0.3mg/kg) 24h before Escherichia coli LPS was administrated. Animals were sacrificed 24h after LPS administration and iNOS, eNOS, and nNOS and type 1 and 2 heme-oxygenase (HO) expression were measured. TNF-α and IL-1 tissue expression and plasmatic NO, CO, TNF-α, and IL-1 were also determined. LPS exposure increased iNOS expression in both organs, eNOS did not show variations and liver nNOS expression was significantly lower. Tacrolimus diminished both pancreas and liver iNOS and nNOS expression. Both liver and pancreatic eNOS expression augmented when tacrolimus was administrated. High doses of tacrolimus were correlated with ameliorated liver HO-1 plus HO-2 and pancreas HO-1 expression after LPS stimulation. Tacrolimus treatment diminished TNF-α but not IL-1 expression increase after LPS challenge in hepatic tissue. Pancreatic TNF-α and IL-1 values diminished partially when high doses were employed. Plasmatic NO, CO, TNF-α, and IL-1 concentrations increase after LPS challenge was diminished when highest doses of tacrolimus were given. In conclusion, tacrolimus exerts a protective effect on commonly observed harmful phenomena after LPS stimulation by modulating liver and pancreas oxidative enzyme expression and cytokine production.

Satisfaction with pregnancy and birth services: the quality of maternity care services as experienced by women.

OBJECTIVES: To investigate the opinions of women regarding satisfaction with the quality of maternity care received, and to establish whether health-care technology increases satisfaction or interferes with the construction of personal satisfaction in the process of care. DESIGN AND SETTING: Information was gathered using focus groups. The area of study comprised the postnatal groups run as part of the Sexual and Reproductive Health Programme of the Catalan Health Authority, Spain. PARTICIPANTS: Five focus groups were held between May 2006 and July 2007. FINDINGS: Quality of care is a complex concept in which a number of independent core features can be identified. These core features were grouped into three basic categories: safety (the hospital and its technological facilities, and the technical expertise of health professionals), the relationship between the carers and the service user, and the structural aspects that determine the context in which health care is provided. KEY CONCLUSIONS AND IMPLICATIONS FOR PRACTICE: The mothers in this study were satisfied with health-care technology and viewed it as a source of security. Technology was indispensable to reduce the anxiety provoked by their perceived lack of confidence in their ability as mothers. During pregnancy and, especially, when giving birth, women believe that their feelings and values should be understood by professionals, from whom they seek empathy and personal commitment, not just information.

Increased Susceptibility to Skin Carcinogenesis in TREX2 Knockout Mice.

TREX2 is a proofreading 3'-5' exonuclease that can be involved in genome maintenance; however, its biological role remains undefined. To better understand the function and physiologic relevance of TREX2, we generated mice deficient in TREX2 by targeted disruption of its unique coding exon. The knockout mice are viable and do not show relevant differences in growth, survival, lymphocyte development, or spontaneous tumor incidence compared with their wild-type counterparts over a period of up to 2 years. Also, we did not observe chromosomal instability or defects in cell proliferation and cell cycle upon loss of TREX2. We have observed that TREX2 expression is not ubiquitous, being expressed preferentially in tissues with stratified squamous epithelia, such as the skin or esophagus, and specifically in keratinocytes. Interestingly, TREX2-null mice are more susceptible to skin carcinogenesis induced by 7,12-dimethylbenz(a)anthracene (DMBA) compared with wild-type mice. This phenotype correlates with a reduction of DMBA-induced apoptosis in both the epidermis and keratinocytes of TREX2-null mice. Altogether, our results suggest a tumor suppressor role for TREX2 in skin carcinogenesis through which it contributes to keratinocyte apoptosis under conditions of genotoxic stress.

Tecnología y humanización en la asistencia al nacimiento. La percepción de las mujeres / Technology and humanization in childbirth care. Women’s perception

Objetivos: Conocer la opinión de las mujeres en relación con la tecnología sanitaria aplicada al seguimiento del embarazo y asistencia al parto, así como sus vivencias y sentimientos respecto a la calidad humana de la relación asistencial, y si ésta se adecuó a sus expectativas. Personas y método: Estudio con metodología cualitativa. Los datos se han obtenido mediante la realización de cuatro grupos focales. Resultados: El análisis de los datos ha permitido la construcción de dos categorías básicas: la opinión sobre las diferentes tecnologías aplicadas a lo largo del proceso asistencial, y la adecuación a las expectativas previas de la vivencia de la calidad humana en la relación asistencial. Conclusiones: La mayoría de las mujeres acepta las tecnologías sanitarias a su alcance. El significado del aumento de la dependencia tecnológica, sobre todo para la ecografía obstétrica, permite diferentes lecturas; un sector minoritario de mujeres prefiere una atención menos medicalizada durante el proceso de atención al parto. Todas las mujeres quieren sentirse protagonistas de su embarazo y parto. Confían plenamente en la competencia profesional de los expertos, cuya presencia les confiere seguridad, pero demandan mayor presencia de cualidades personales como el cariño y el trato deferente (AU)

Objective: To know women’s opinion regarding health care technologies applied to pregnancy follow-up and labour care, besides their personal experiences and feelings towards the human quality of the care provided and if this care relationship was meeting patient’s expectations. Participants and method: The study was performed with quality evaluation methods. Data were obtained into four focal groups. Results: Data analysis allowed us to make a classification on two basic categories: the patient perception about the different technologies applied on the care process, and the fit of the care relationship to the human quality standards that the patient expected. Conclusions: Most women accept the available health care technologies. This means there is an increase on the technological dependence, in particular regarding obstetric echography, can have different interpretations; there is a small women’s sector who prefers a less medicalized care in the process of childbirth. But all women want to play a major part in pregnancy and delivery; they are quite confident in the professional competence of experts; their presence alone reassures the patient, but the latter demands more personal qualities in care providers, such as warmth and deferential attitude in their relationship (AU)

Cutting edge: the phosphoinositide 3-kinase p110 delta is critical for the function of CD4+CD25+Foxp3+ regulatory T cells.

CD4+CD25+Foxp3+ regulatory T cells (Tregs) contribute to the maintenance of peripheral tolerance by inhibiting the expansion and function of conventional T cells. Treg development and homeostasis are regulated by the Ag receptor, costimulatory receptors such as CD28 and CTLA-4, and cytokines such as IL-2, IL-10, and TGF-beta. Here we show that the proportions of Tregs in the spleen and lymph nodes of mice with inactive p110delta PI3K (p110deltaD910A/D910A) are reduced despite enhanced Treg selection in the thymus. p110deltaD910A/D910A CD4+CD25+Foxp3+ Tregs showed attenuated suppressor function in vitro and failed to secrete IL-10. In adoptive transfer experiments, p110deltaD910A/D910A T cells failed to protect against experimental colitis. The identification of p110delta as an intracellular signaling protein that regulates the activity of CD4+CD25+Foxp3+ Tregs may facilitate the further elucidation of the molecular mechanisms responsible for Treg-mediated suppression.