Lifelong dynamics of human CD4+CD25+ regulatory T cells: insights from in vivo data and mathematical modeling.

Despite their limited proliferation capacity, regulatory T cells (T(regs)) constitute a population maintained over the entire lifetime of a human organism. The means by which T(regs) sustain a stable pool in vivo are controversial. Using a mathematical model, we address this issue by evaluating several biological scenarios of the origins and the proliferation capacity of two subsets of T(regs): precursor CD4(+)CD25(+)CD45RO(-) and mature CD4(+)CD25(+)CD45RO(+) cells. The lifelong dynamics of T(regs) are described by a set of ordinary differential equations, driven by a stochastic process representing the major immune reactions involving these cells. The model dynamics are validated using data from human donors of different ages. Analysis of the data led to the identification of two properties of the dynamics: (1) the equilibrium in the CD4(+)CD25(+)FoxP3(+)T(regs) population is maintained over both precursor and mature T(regs) pools together, and (2) the ratio between precursor and mature T(regs) is inverted in the early years of adulthood. Then, using the model, we identified three biologically relevant scenarios that have the above properties: (1) the unique source of mature T(regs) is the antigen-driven differentiation of precursors that acquire the mature profile in the periphery and the proliferation of T(regs) is essential for the development and the maintenance of the pool; there exist other sources of mature T(regs), such as (2) a homeostatic density-dependent regulation or (3) thymus- or effector-derived T(regs), and in both cases, antigen-induced proliferation is not necessary for the development of a stable pool of T(regs). This is the first time that a mathematical model built to describe the in vivo dynamics of regulatory T cells is validated using human data. The application of this model provides an invaluable tool in estimating the amount of regulatory T cells as a function of time in the blood of patients that received a solid organ transplant or are suffering from an autoimmune disease.

Insulated retroviral vectors towards safe and efficient genetic modification of stem cells

In otherwise successful gene therapy trials for the treatment of SCID patients and others, insertional mutagenesis has resulted in leukemia development. Besides the integration of vectors that including strong enhancers, more recently, SIN-vectors have been shown to partially retain oncogenic potential. The identification of genetic elements which would both prevent such activation effects and shield the transgene from silencing, is a main challenge. Previous attempts met with difficulties in producing the vectors and poor efficacy of the insulators (GIE). The improvement of integrating vectors safety has been investigated using new candidate synthetic GIEs. The latter have been introduced in retroviral and lentiviral vectors. Native LTRs, SIN-LTRs, and SIN-insulated constructs have been designed and compared, using two sets of internal promoter, i.e. strong and housekeeping. We could establish that a specific insulator translates at best into functional activity and boundary effect in both vector types. We could also determine that other genetic elements are key determinants in order to achieve accurate expression and viral titre, from these insulated vectors. A dramatic shift in the expression profile is observed in target cells, with a homogenous pattern including data on both cell-lines and primary HSCs from cord blood. The assessment of potential genotoxicity will be presented, based on the comparison of the integration patterns ingenuity in human target cells sampled over a three months period with both reference LTRs and SIN versus test insulated vectors, using high-throughput pyro-sequencing.

Characterization of cardiac-resident progenitor cells expressing high aldehyde dehydrogenase activity.

High aldehyde dehydrogenase (ALDH) activity has been associated with stem and progenitor cells in various tissues. Human cord blood and bone marrow ALDH-bright (ALDH(br)) cells have displayed angiogenic activity in preclinical studies and have been shown to be safe in clinical trials in patients with ischemic cardiovascular disease. The presence of ALDH(br) cells in the heart has not been evaluated so far. We have characterized ALDH(br) cells isolated from mouse hearts. One percent of nonmyocytic cells from neonatal and adult hearts were ALDH(br). ALDH(very-br) cells were more frequent in neonatal hearts than adult. ALDH(br) cells were more frequent in atria than ventricles. Expression of ALDH1A1 isozyme transcripts was highest in ALDH(very-br) cells, intermediate in ALDH(br) cells, and lowest in ALDH(dim) cells. ALDH1A2 expression was highest in ALDH(very-br) cells, intermediate in ALDH(dim) cells, and lowest in ALDH(br) cells. ALDH1A3 and ALDH2 expression was detectable in ALDH(very-br) and ALDH(br) cells, unlike ALDH(dim) cells, albeit at lower levels compared with ALDH1A1 and ALDH1A2. Freshly isolated ALDH(br) cells were enriched for cells expressing stem cell antigen-1, CD34, CD90, CD44, and CD106. ALDH(br) cells, unlike ALDH(dim) cells, could be grown in culture for more than 40 passages. They expressed sarcomeric α -actinin and could be differentiated along multiple mesenchymal lineages. However, the proportion of ALDH(br) cells declined with cell passage. In conclusion, the cardiac-derived ALDH(br) population is enriched for progenitor cells that exhibit mesenchymal progenitor-like characteristics and can be expanded in culture. The regenerative potential of cardiac-derived ALDH(br) cells remains to be evaluated.

A longitudinal study of environmental tobacco smoke exposure in children: parental self reports versus age dependent biomarkers

Background: Awareness of the negative effects of smoking on children's health prompted a decrease in the self-reporting of parental tobacco use in periodic surveys from most industrialized countries. Our aim is to assess changes between ETS exposure at the end of pregnancy and at 4 years of age determined by the parents' self-report and measurement of cotinine in age related biological matrices.Methods: The prospective birth cohort included 487 infants from Barcelona city (Spain). Mothers were asked about maternal and household smoking habit. Cord serum and children's urinary cotinine were analyzed in duplicate using a double antibody radioimmunoassay. Results: At 4 years of age, the median urinary cotinine level in children increased 1.4 or 3.5 times when father or mother smoked, respectively. Cotinine levels in children's urine statistically differentiated children from smoking mothers (Geometric Mean (GM) 19.7 ng/ml; 95% CI 16.83–23.01) and exposed homes (GM 7.1 ng/ml; 95% CI 5.61–8.99) compared with non-exposed homes (GM 4.5 ng/ml; 95% CI 3.71–5.48). Maternal self-reported ETS exposure in homes declined in the four year span between the two time periods from 42.2% to 31.0% (p < 0.01). Nevertheless, most of the children considered non-exposed by their mothers had detectable levels of cotinine above 1 ng/mL in their urine.Conclusion: We concluded that cotinine levels determined in cord blood and urine, respectively, were useful for categorizing the children exposed to smoking and showed that a certain increase in ETS exposure during the 4-year follow-up period occurred.

Adverse obstetrical and neonatal outcomes in elective and medically indicated inductions of labor at term.

OBJECTIVE: To compare the adverse neonatal and maternal outcomes after medically indicated and elective labor induction. Both induction groups were also compared to women with spontaneous onset of labor. METHOD: Retrospective cohort study of 13 971 women with live, cephalic singleton pregnancies who delivered at term (from 1997 to 2007). Adverse maternal and neonatal outcomes were compared between women who underwent an induction of labor in the presence and absence of standard medical indications. RESULTS: Among 5090 patients with induced labor, 2059 (40.5%) underwent elective labor inductions, defined as inductions without any medical or obstetrical indication. Risks of cesarean or instrumental delivery, postpartum hemorrhage >500 ml, prolonged maternal hospitalization >6 days, Apgar<7 at 5 min of life, arterial umbilical cord pH<7.1, admission in neonatal intensive care unit (NICU) and prolonged NICU hospitalization >7 days were similar between nulliparous who underwent elective and medical labor induction. Similar results were obtained for multiparous. All the above mentioned risks, but the Apgar<7 at 5 min of life, were significantly increased after induction in comparison to spontaneous labor. CONCLUSION: Elective induction of labor carries similar obstetrical and neonatal risks as a medically indicated labor induction. Thus, elective induction of labor should be strongly discouraged.

Human natural Treg microRNA signature: role of microRNA-31 and microRNA-21 in FOXP3 expression.

Treg are the main mediators of dominant tolerance. Their mechanisms of action and applications are subjects of considerable debate currently. However, a human microRNA (miR) Treg signature has not been described yet. We investigated human natural Treg and identified a signature composed of five miR (21, 31, 125a, 181c and 374). Among those, two were considerably under-expressed (miR-31 and miR-125a). We identified a functional target sequence for miR-31 in the 3' untranslated region (3' UTR) of FOXP3 mRNA. Using lentiviral transduction of fresh cord blood T cells, we demonstrated that miR-31 and miR-21 had an effect on FOXP3 expression levels. We showed that miR-31 negatively regulates FOXP3 expression by binding directly to its potential target site in the 3' UTR of FOXP3 mRNA. We next demonstrated that miR-21 acted as a positive, though indirect, regulator of FOXP3 expression. Transduction of the remaining three miR had no direct effect on FOXP3 expression or on the phenotype and will remain the subject of future investigations. In conclusion, not only have we identified and validated a miR signature for human natural Treg, but also unveiled some of the mechanisms by which this signature was related to the control of FOXP3 expression in these cells.

Recovery of paraplegia after type B dissection due to spinal collateral recruitment.

Acute paraplegia could be a symptom of aortic dissection due to sudden compromise of arterial spinal cord blood supply. Complete spontaneous neurologic recovery is possible and was observed in the present case 3 hours after symptom onset. Spontaneous spinal cord reperfusion after acute type B dissection was probably due to two main mechanisms. Reperfusion of false lumen and collateral vascular network recruitment, recently confirmed by anatomic animal studies, serve as potential explanations. Favorable evolution of acute paraplegia after aortic dissection exists, but prognosis is uncertain, probably due to individual variable anatomic distribution of spinal cord blood supply.

Haematopoietic stem cell transplantation: activity in Switzerland compared with surrounding European countries.

Haematopoietic stem cell transplantation (HSCT) is a highly specialised procedure used to treat malignancies of the lymphohaematopoietic system as well as some acquired and inherited disorders of the blood. This analysis by the Swiss Blood Stem Cell Transplantation Group, based on data from 2008-2011, describes, treatment rates in Switzerland for specific indications and compares this with data from Germany, France, Italy and the Netherlands, corrected for the size of the population. Differences in transplant rates, in rates for particular indications, and in the use of specific transplant technologies such as use of unrelated donors, use of cord blood or mismatched family donors are described. These data are put in correlation with donor availability from international registries and with number of transplant teams and number of procedures per team all corrected for population size.

Free and total plasma levels of lopinavir during pregnancy, at delivery and postpartum: implications for dosage adjustments in pregnant women.

BACKGROUND: Physiological changes associated with pregnancy may alter antiretroviral plasma concentrations and might jeopardize prevention of mother-to-child HIV transmission. Lopinavir is one of the protease inhibitors more frequently prescribed during pregnancy in Europe. We described the free and total pharmacokinetics of lopinavir in HIV-infected pregnant and non-pregnant women, and evaluated whether significant alterations in its disposition and protein binding warrant systematic dosage adjustment. METHODS: Plasma samples were collected at first, second and third trimester of pregnancy, at delivery, in umbilical cord and postpartum. Lopinavir free and total plasma concentrations were measured by HPLC-MS/MS. Bayesian calculations were used to extrapolate total concentrations to trough (Cmin). RESULTS: A total of 42 HIV-positive pregnant women and 37 non-pregnant women on lopinavir/ritonavir were included in the study. Compared to postpartum and control values, total lopinavir Cmin was decreased moderately (31-39%) during pregnancy, and free Cmin minimally, showing significant alteration only at delivery (-35%). However, total and free Cmin remained in all patients above the target concentrations for wild-type virus of 1,000 ng/ml, and above the unbound IC50(WT) of 0.64-0.77 ng/ml of lopinavir, respectively. Lopinavir free fractions remained higher during pregnancy compared to postpartum and controls, and were influenced by α-1-acid-glycoprotein and albumin decrease. Free cord-to-mother ratio (0.43) was 2.7-fold higher than total cord-to-mother ratio (0.16), suggesting higher fetal exposure. CONCLUSIONS: The moderate decrease of total lopinavir concentrations during pregnancy is not associated with proportional decrease in free concentrations. Both reach a nadir at delivery, albeit not to an extent that would put treatment-naive women at risk of insufficient exposure to the free, pharmacologically active concentrations of lopinavir. No dosage adjustment is therefore needed during pregnancy as it is unlikely to further enhance treatment efficacy but could potentially increase the risk of maternal and fetal toxicity. Nonetheless, in case of viral resistance in treatment-experienced pregnant women, loss of virological control or questionable adherence, it is justified to consider lopinavir dosage adjustment based on total plasma concentration measurement.

Bone marrow-derived cells are implicated as a source of lymphatic endothelial progenitors in human breast cancer.

Bone marrow-derived endothelial progenitor cells (EPCs) infiltrate into sites of neovascularization in adult tissues and mature into functional blood endothelial cells (BECs) during a process called vasculogenesis. Human marrow-derived EPCs have recently been reported to display a mixed myeloid and lymphatic endothelial cell (LEC) phenotype during inflammation-induced angiogenesis; however, their role in cancer remains poorly understood. We report the in vitro differentiation of human cord blood CD133(+)CD34(+) progenitors into podoplanin(+) cells expressing both myeloid markers (CD11b, CD14) and the canonical LEC markers vascular endothelium growth factor receptor 3 (VEGFR-3), lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), and prospero homeobox 1 (PROX-1). These podoplanin(+) cells displayed sprouting behavior comparable to that of LECs in vitro and a dual hemangiogenic and lymphangiogenic activity in vivo in an endothelial cell sprouting assay and corneal vascularization assay, respectively. Furthermore, these cells expressed vascular endothelium growth factor (VEGF) family members A, -C, and -D. Thus, bone-marrow derived EPCs stimulate hemangiogenesis and lymphangiogenesis through their ability to differentiate into LECs and to produce angiogenic factors. Importantly, plasma from patients with breast cancer induced differentiation of CD34(+) cord blood progenitors into hemangiogenic and lymphangiogenic CD11b(+) myeloid cells, whereas plasma from healthy women did not have this effect. Consistent with these findings, circulating CD11b(+) cells from breast cancer patients, but not from healthy women, displayed a similar dual angiogenic activity. Taken together, our results show that marrow-derived EPCs become hemangiogenic and lymphangiogenic upon exposure to cancer plasma. These newly identified functions of bone-marrow derived EPCs are expected to influence the diagnosis and treatment of breast cancer.

Novel insulated gamma and lentis retroviral vectors towards safer genetic modification of stem cells

In otherwise successful gene therapy trials insertional mutagenesis has resulted in leukemia. The identification of new short synthetic genetic insulator elements (GIE) which would both prevent such activation effects and shield the transgene from silencing, is a main challenge. Previous attempts with e.g. b-globin HS4, have met with poor efficacy and genetic instability. We have investigated potential improvement with two new candidate synthetic GIEs in SIN-gamma and lentiviral vectors. With each constructs two internal promoters have been tested: either the strong Fr- MuLV-U3 or the housekeeping hPGK.We could identify a specific combination of insulator 2 repeats which translates into best functional activity, high titers and boundary effect in both gammaretro and lentivectors. In target cells a dramatic shift of expression is observed with an homogenous profile the level of which strictly depends on the promoter strength. These data remain stable in both HeLa cells over three months and cord blood HSCs for two months, irrespective of the multiplicity of infection (MOI). In comparison, control native and SIN vectors expression levels show heterogeneous, depend on the MOI and prove unstable. We have undertaken genotoxicity assessment in comparing integration patterns ingenuity in human target cells sampled over three months using high-throughput pyro-sequencing. Data will be presented. Further genotoxicity assessment will include in vivo studies. We have established insulated vectors which harbour both boundary and enhancer-blocking effect and show stable in prolonged in vitro culture conditions. Work performed with support of EC-DG research FP6-NoE, CLINIGENE: LSHB-CT-2006-018933

Self-Renewing Human Bone Marrow Mesenspheres Promote Hematopoietic Stem Cell Expansion.

Strategies for expanding hematopoietic stem cells (HSCs) include coculture with cells that recapitulate their natural microenvironment, such as bone marrow stromal stem/progenitor cells (BMSCs). Plastic-adherent BMSCs may be insufficient to preserve primitive HSCs. Here, we describe a method of isolating and culturing human BMSCs as nonadherent mesenchymal spheres. Human mesenspheres were derived from CD45- CD31- CD71- CD146+ CD105+ nestin+ cells but could also be simply grown from fetal and adult BM CD45--enriched cells. Human mesenspheres robustly differentiated into mesenchymal lineages. In culture conditions where they displayed a relatively undifferentiated phenotype, with decreased adherence to plastic and increased self-renewal, they promoted enhanced expansion of cord blood CD34+ cells through secreted soluble factors. Expanded HSCs were serially transplantable in immunodeficient mice and significantly increased long-term human hematopoietic engraftment. These results pave the way for culture techniques that preserve the self-renewal of human BMSCs and their ability to support functional HSCs.

Quantitative proteomics in the characterization of T helper lymphocyte differentiation

The term proteome is used to define the complete set of proteins expressed in cells or tissues of an organism at a certain timepoint. Respectively, proteomics is used to describe the methods, which are used to study such proteomes. These methods include chromatographic and electrophoretic techniques for protein or peptide fractionation, mass spectrometry for their identification, and use of computational methods to assist the complicated data analysis. A primary aim in this Ph.D. thesis was to set-up, optimize, and develop proteomics methods for analysing proteins extracted from T-helper (Th) lymphocytes. First, high-throughput LC-MS/MS and ICAT labeling methods were set-up and optimized for analysing the microsomal fraction proteins extracted from Th lymphocytes. Later, iTRAQ method was optimized to study cytokine regulated protein expression in the nuclei of Th lymphocytes. High-throughput LC-MS/MS analyses, like ICAT and iTRAQ, produce large quantities of data and robust software and data analysis pipelines are needed. Therefore, different software programs used for analysing such data were evaluated. Moreover, a pre-filtering algorithm was developed to classify good-quality and bad-quality spectra prior to the database searches. Th-lymphocytes can differentiate into Th1 or Th2 cells based on surrounding antigens, co-stimulatory molecules, and cytokines. Both subsets have individual cytokine secretion profiles and specific functions. Th1 cells participate in the cellular immunity against intracellular pathogens, while Th2 cells have important role in the humoral immunity against extracellular parasites. An abnormal response of Th1 and Th2 cells and imbalance between the subsets are charasteristic of several diseases. Th1 specific reactions and cytokines have been detected in autoimmune diseases, while Th2 specific response and cytokine profile is common in allergy and asthma. In this Ph. D. thesis mass spectrometry-based proteomics was used to study the effects of Th1 and Th2 promoting cytokines IL-12 and IL-4 on the proteome of Th lymphocytes. Characterization of microsomal fraction proteome extracted from IL-12 treated lymphobasts and IL-4 stimulated cord blood CD4+ cells resulted in finding of cytokine regulated proteins. Galectin-1 and CD7 were down-regulated in IL-12 treated cells, while IL-4 stimulation decreased the expression of STAT1, MXA, GIMAP1, and GIMAP4. Interestingly, the transcription of both GIMAP genes was up-regulated in Th1 polarized cells and down-regulated in Th2 promoting conditions.

MHC/Peptide-Specific Interaction of the Humoral Immune System: A New Category of Antibodies.

Abs bind to unprocessed Ags, whereas cytotoxic CD8(+) T cells recognize peptides derived from endogenously processed Ags presented in the context of class I MHC complexes. We screened, by ELISA, human sera for Abs reacting specifically with the influenza matrix protein (IMP)-derived peptide58-66 displayed by HLA-A*0201 complexes. Among 653 healthy volunteers, blood donors, and women on delivery, high-titered HLA-A*0201/IMP58-66 complex-specific IgG Abs were detected in 11 females with a history of pregnancies and in 1 male, all HLA-A*0201(-). These Abs had the same specificity as HLA-A*0201/IMP58-66-specific cytotoxic T cells and bound neither to HLA-A*0201 nor the peptide alone. No such Abs were detected in HLA-A*0201(+) volunteers. These Abs were not cross-reactive to other self-MHC class I alleles displaying IMP58-66, but bound to MHC class I complexes of an HLA nonidentical offspring. HLA-A*0201/IMP58-66 Abs were also detected in the cord blood of newborns, indicating that HLA-A*0201/IMP58-66 Abs are produced in HLA-A*0201(-) mothers and enter the fetal blood system. That Abs can bind to peptides derived from endogenous Ags presented by MHC complexes opens new perspectives on interactions between the cellular and humoral immune system.