Peripheral Blood Of Subjects Research Articles

Mitochondrial DNA: a proinflammatory ‘enemy from within’ during HIV infection?

Mitochondria are crucial in cell life, as they are the main intracellular source of energy, and have a main role in cell death as they contain molecules able to trigger apoptosis. However, mitochondria can also release molecules called ‘damage-associated molecular patterns' (DAMPs) that trigger a potent innate immune response and cause inflammation through the engagement of the Toll-like receptors (TLR). During human immunodeficiency virus (HIV) infection, a proinflammatory status is present that is not completely explained by the activity of the virus per se, or by the effective immune response against the virus. However, the presence of significant amounts of DAMPs of mitochondrial origin, such as extracellular plasmatic mtDNA, in the peripheral blood of subjects with HIV infection suggests that part of the inflammation typical of such infection could be because of the activity of these molecules, and thus opens new therapeutic perspectives. Mitochondria are intracellular organelles involved in a wide range of processes, as they are the main source of cellular energy, in the form of ATP, are deeply involved in the regulation of programmed cell death, in the homeostasis of intracellular calcium, and are the site of several biosynthesis pathways. Furthermore, they are the main source of reactive oxygen species. Mitochondria derive from ancestral endosymbiont bacteria; as a reminiscence of such origin, they contain several copies of a circular genome, the mtDNA, which encodes key proteins of the oxidative phosphorylation system. Like bacterial proteins, those synthetized within mitochondria contains formyl-methionin, and their degradation origin chemotactic formyl peptides,1 which behave as danger signals and can activate inflammasome.2 When cells are coping with a massive insult that is potentially harmful for the entire organism, mitochondria release into the cytoplasm molecules able to trigger cell death, such as cytochrome-c or apoptosis-inducing factor.

Effect of occupational combined exposure of chromium and iron on erythrocyte metabolism

To investigate the effect of combined occupational exposure of chromium and iron on erythrocyte metabolism, and the possible mechanism. A total of 115 chromate production workers were selected in a chemical factory of Jinan as exposure group, Dec, 2008, and 60 healthy residents from a community which was far away from the factory were enrolled as control group. Environmental concentrations of chromium and iron were collected by filter membrane sampling and determined. The peripheral blood of subjects were collected for determination of chromium, iron, copper in whole blood and folate, vitamin B₁₂ in serum, mean corpuscular hemoglobin (MCH) and mean corpuscular volume (MCV) and correlation analysis was conducted. The median (quartile interval) concentration of air-chromium and air-iron in workplace were 9.0 (10.5) and 11.2 (10.1) µg/m³, respectively, which were significantly higher than that of the control (0.1 (0.1) and 7.2 (2.5) µg/m³) (all P values < 0.01). Blood-chromium and blood-iron of the exposed group were 15.5 (14.1) µg/L and (895.1 ± 90.2) mg/L, which were significantly higher than the counterpart of the control (3.6(2.0) µg/L, (563.7 ± 49.3) mg/L) (all P values < 0.01). Serum folate ((6.9 ± 2.5) µg/L), serum vitamin B₁₂ ((396.4 ± 177.0) µg/L) and blood copper ((777.6 ± 103.5) µg/L) of the exposed group were all significantly lower comparing to the control group ((558.0 ± 330.8), (8.1 ± 3.8), (812.1 ± 94.6) µg/L) (all P values < 0.05). The relationships between blood chromium and serum folate, serum vitamin B₁₂ were statistical significant (r = -0.319 and -0.293, P < 0.01). Both serum vitamin B₁₂ and blood copper correlated with mean corpuscular hemoglobin (MCH) and mean corpuscular volume (MCV) (r = -0.223, -0.242, -0.261, -0.292, all P values < 0.01). Combined chromium and iron exposure existed in the workplace. Adverse effect of Chromium on human erythrocyte may via folate and vitamin B₁₂ metabolism, while iron may via copper metabolism.

The Role of Fibrocytes in Sickle Cell Lung Disease

BackgroundInterstitial lung disease is a frequent complication in sickle cell disease and is characterized by vascular remodeling and interstitial fibrosis. Bone marrow-derived fibrocytes have been shown to contribute to the pathogenesis of other interstitial lung diseases. The goal of this study was to define the contribution of fibrocytes to the pathogenesis of sickle cell lung disease.Methodology/Principal FindingsFibrocytes were quantified and characterized in subjects with sickle cell disease or healthy controls, and in a model of sickle cell disease, the NY1DD mouse. The role of the chemokine ligand CXCL12 in trafficking of fibrocytes and phenotype of lung disease was examined in the animal model. We found elevated concentration of activated fibrocytes in the peripheral blood of subjects with sickle cell disease, which increased further during vaso-occlusive crises. There was a similar elevations in the numbers and activation phenotype of fibrocytes in the bone marrow, blood, and lungs of the NY1DD mouse, both at baseline and under conditions of hypoxia/re-oxygenation. In both subjects with sickle cell disease and the mouse model, fibrocytes expressed a hierarchy of chemokine receptors, with CXCR4 expressed on most fibrocytes, and CCR2 and CCR7 expressed on a smaller subset of cells. Depletion of the CXCR4 ligand, CXCL12, in the mouse model resulted in a marked reduction of fibrocyte trafficking into the lungs, reduced lung collagen content and improved lung compliance and histology.ConclusionsThese data support the notion that activated fibrocytes play a significant role in the pathogenesis of sickle cell lung disease.

Estrogen and progesterone play pivotal roles in endothelial progenitor cell proliferation

BackgroundIt has been previously suggested that angiogenesis occurs during the menstrual cycle. Moreover, a rise in uterine blood flow is largely maintained by vasodilatation and substantial increases in angiogenesis. It is known that estradiol (E2) and progesterone (P4) are involved in angiogenesis. Recently, endothelial progenitor cells (EPCs) were found to be involved in neovascularization; however, their roles in uterine neovascularization have not been well characterized. We hypothesized that E2- or P4-mediated EPC proliferation plays important roles in uterine neovascularization during the menstrual cycle.MethodsThe number of EPCs in peripheral blood from subjects in the menstrual phase (n = 12), follicular phase (n = 8), and luteal phase (n = 16), was measured using flow cytometry. Peripheral blood mononuclear cells (PBMCs) were cultured for seven days with or without 17beta-estradiol (E2beta) or P4, followed by assessment of EPC proliferation based upon the uptake of acetylated low density lipoprotein (LDL) and lectin. The expression of estrogen receptor (ER) or progesterone receptor (PR) in EPCs was also evaluated using real-time PCR.ResultsE2beta and P4 significantly increased the proliferation of EPCs derived from the peripheral blood of subjects in menstrual phase, but not subjects in the luteal phase. In addition, the expression level of ERalpha was markedly higher than ERbeta in EPCs derived from women in menstrual phase.ConclusionsEPC proliferation is induced during the menstrual phase and proliferation can be affected by estrogen through ERalpha activation.

Ara h 1–reactive T cells in individuals with peanut allergy

Effective immunotherapy for peanut allergy is hampered by a lack of understanding of peanut-reactive CD4(+) T cells. To identify, characterize, and track Ara h 1-reactive cells in subjects with peanut allergy by using Ara h 1-specific class II tetramers. Tetramer-guided epitope mapping was used to identify the antigenic peptides within the peanut allergen Ara h1. Subsequently, HLA class II/Ara h 1-specific tetramers were used to determine the frequency and phenotype of Ara h 1-reactive T cells in subjects with peanut allergy. Cytokine profiles of Ara h 1-reactive T cells were also determined. Multiple Ara h 1 epitopes with defined HLA restriction were identified. Ara h 1-specific CD4(+) T cells were detected in all of the subjects with peanut allergy tested. Ara h 1-reactive T cells in subjects with allergy expressed CCR4 but did not express CRTH2. The percentage of Ara h1-reactive cells that expressed the β7 integrin was low compared with total CD4(+) T cells. Ara h 1- reactive cells that secreted IFN-γ, IL-4, IL-5, IL-10, and IL-17 were detected. In individuals with peanut allergy, Ara h 1-reactive T cells occurred at moderate frequencies, were predominantly CCR4(+) memory cells, and produced IL-4. Class II tetramers can be readily used to detect Ara h 1-reactive T cells in the peripheral blood of subjects with peanut allergy without in vitro expansion and would be effective for tracking peanut-reactive CD4(+) T cells during immunotherapy.

TGF-β signaling is altered in the peripheral blood of subjects with multiple sclerosis

Multiple sclerosis (MS) is a central nervous system inflammatory disorder with evidence of peripheral immune dysregulation. Abnormalities of the immune suppressive cytokine TGF-β have been reported, but not fully defined, in MS. Through a pathway-focused expression profiling of the peripheral blood, we found abnormalities of TGF-βRII, SMAD4 and SMAD7 expression in subjects with MS, and reduction in the levels of TGF-β regulated genes, indicating an overall reduction in TGF-β signaling in MS. The response to exogenous TGF-β was intact, however, indicating an extrinsic defect of TGF-β signaling in MS. These results indicate that TGF-β control is diminished in MS.

Ara h 2 peptides containing dominant CD4+ T-cell epitopes: Candidates for a peanut allergy therapeutic

Peanut allergy is a life-threatening condition; there is currently no cure. Although whole allergen extracts are used for specific immunotherapy for many allergies, they can cause severe reactions, and even fatalities, in peanut allergy. This study aimed to identify short, T-cell epitope-based peptides that target allergen-specific CD4(+) T cells but do not bind IgE as candidates for safe peanut-specific immunotherapy. Multiple CD4(+) T-cell lines specific for the major peanut allergen Ara h 2 were generated from PBMCs of 16 HLA-diverse subjects with peanut allergy by using 5,6-carboxyfluorescein diacetate succinimidylester-based methodology. Proliferation and ELISPOT assays were used to identify dominant epitopes recognized by T-cell lines and to confirm recognition by peripheral blood T cells of epitope-based peptides modified for therapeutic production. HLA restriction of core epitope recognition was investigated by using anti-HLA blocking antibodies and HLA genotyping. Serum-IgE peptide-binding was assessed by dot-blot. Five dominant CD4(+) T-cell epitopes were identified in Ara h 2. In combination, these were presented by HLA-DR, HLA-DP, and HLA-DQ molecules and recognized by T cells from all 16 subjects. Three short peptide variants containing these T-cell epitopes were designed with cysteine-to-serine substitutions to facilitate stability and therapeutic production. Variant peptides showed HLA-binding degeneracy, did not bind peanut-specific serum IgE, and could directly target T(H)2-type T cells in peripheral blood of subjects with allergy. Short CD4(+) T-cell epitope-based Ara h 2 peptides were identified as novel candidates for a T-cell-targeted peanut-specific immunotherapy for an HLA-diverse population.

PD‐1 modulates regulatory T cells and suppresses T‐cell responses in HCV‐associated lymphoma

T regulatory (TR) cells suppress T cell responses that are critical in the development of chronic viral infection and associated malignancies. Programmed death-1 (PD-1) also plays a pivotal role in regulation of T cell functions during chronic viral infection. To examine the role of PD-1 pathway in regulating TR cell functions that inhibit T cell responses during virus-associated malignancy, TR cells were investigated in the setting of hepatitis C virus-associated lymphoma (HCV-L), non-HCV-associated lymphoma (non-HCV-L), HCV infection alone, and healthy subjects (HS). Relatively high numbers of CD4+CD25+ and CD8+CD25+ TR cells as well as high levels of PD-1 expressions on these TR cells were found in the peripheral blood of subjects with HCV-L compared to those from non-HCV-L or HCV alone or HS. TR cells from the HCV-L subjects were capable of suppressing the autogeneic lymphocyte response, and depletion of TR cells in PBMC from HCV-L improved T cell proliferation. Additionally, the suppressed T cell activation and proliferation in HCV-L was partially restored by blocking the PD-1 pathway ex vivo, resulting in both a reduction in TR cell number and the ability of TR to suppress the activity of effector T cells. This study suggests that the PD-1 pathway is involved in regulating TR cells that suppress T cell functions in the setting of HCV-associated B cell lymphoma.

The Percentages of Peripheral Blood CD4+ CD25+ FoxP3+ T regulatory (Treg) Cells are Lower in Subjects with Chronic Rhinosinusitis Compared to Healthy Controls

RATIONALE: Chronic rhinosinusitis (CRS) is a burdensome public health problem. The hallmark of CRS is extensive sinus mucosal inflammation. A number of studies have shown the presence of T cells, eosinophils, and Th1 and Th2 cytokines in the sinus tissue of patients with CRS. The main objective of this study was to determine whether the fraction of regulatory T cells (Tregs) differs in the peripheral blood of subjects with CRS compared to healthy individuals.

Lymphocytes T régulateurs et maladies auto-immunes systémiques : lupus érythémateux systémique, polyarthrite rhumatoïde et syndrome de Gougerot-Sjögren primaire

Regulatory/suppressor T cells (Tregs) maintain immunologic homeostasis and prevent autoimmunity. They are the guardians of dominant tolerance. Recent research reveals quantitative and/or functional defect of Tregs in systemic autoimmune diseases. In this article, past and recent studies of Tregs in human systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and primary Sjögren's syndrome (pGSS) are reviewed. Most studies report that Tregs are decreased in peripheral blood of subjects with active SLE. A population of CD4+CD25−Foxp3+ is specifically described in SLE. Tregs functions are still discussed. Tregs counts in peripheral blood of RA patients vary across studies. Enrichment of synovial fluid in Tregs contrasts with inflammation. Tregs suppressive effects are altered in vivo in RA secondary to proinflammatory cytokines environment and resistance of effector T cells to Tregs. In pGSS, the conflicting place of Tregs in the balance prevention of autoimmunity/antitumor immunity is unspecified. Immunosuppressive treatments, like corticosteroids and anti-TNF, modulate Tregs cells population. There is increasing interest in the use of Tregs as a biological therapy to preserve and restore tolerance to self-antigen. However, difficulties to characterize these lymphocytes and controversies in the results of studies refrain their use in current clinical practice.

A Comparison of Serum Hepcidin and GDF15 Expression in Cases of Iron Depletion Due to Blood Loss.

Abstract 4043Poster Board III-978Iron depletion affects billions worldwide, and anemia develops when iron stores become insufficient to maintain normal erythropoiesis. Hepcidin is a negative regulator of iron absorption in the body. Low serum hepcidin levels provide a physiological response to iron demand in patients with iron deficiency. Based on a recent discovery of elevated growth differentiation factor 15 (GDF15) as a pathological suppressor of hepcidin in cases of thalassemia (Tanno et al. 2007), it was hypothesized that GDF15 may be elevated under physiological circumstances such as iron deficiency, but this area of research remains controversial. Here GDF15 and hepcidin levels were measured in the peripheral blood of subjects with iron-depleted erythropoiesis, and monitored in patients receiving iron replacement therapy. Initially, clinical studies were performed to measure GDF15 in the serum of iron-depleted blood donors (n = 10) and healthy volunteers (n = 10). Iron depletion was defined for this study as serum ferritin level (<10 ng/ml) and transferrin saturation (<15%). When compared to the control group, hemoglobin and RBC counts were also significantly reduced (p< 0.01) among the iron-depleted group. Serum hepcidin levels were also significantly reduced in the iron-depleted group (<5 ng/ml) compared to healthy volunteers (46 ± 41 ng/ml, p < 0.01). However, the GDF15 levels were not elevated in the iron-depleted group. Instead, iron-depletion was associated with a slight decrease in serum GDF15 (304 ± 90 pg/ml) compared to that in the control group (386 ± 104 pg/ml, p = 0.084). In three additional patients with iron depletion due to menstrual blood loss, serum GDF15 and hepcidin levels were monitored during oral iron replacement. In association with normalization of the ferritin and transferrin saturation, hepcidin levels increased in each patient, but GDF15 levels remained stable (202 ± 15 pg/ml versus 215 ± 23 pg/ml). In summary, iron depletion due to blood loss was not associated with increased GDF15, and there was no obvious correlation between GDF15 and hepcidin expression at the time of depletion or during iron replacement therapy. The data support the hypothesis that GDF15 elevation contributes to the pathological suppression of hepcidin and iron overload in cases of ineffective erythropoiesis rather than the physiological suppression of hepcidin in cases of iron depletion. Disclosures:No relevant conflicts of interest to declare.

Successful Use of CD154 Expression and Intracellular Cytokine Staining to Monitor Changes in Allergen-Specific Th2 Cells in Peripheral Blood of Subjects in a Placebo-Controlled Study of Tolamba™, a Novel Immunotherapeutic Vaccine for Ragweed Allergies

RATIONALE: Sensitive and accurate measurement of allergen-specific TH cells in PBMC is possible using CD154 expression to identify antigen-reactive T cells (Frentsch et al., Nat. Med. 11:1118). To explore the potential of this method for monitoring allergen therapy, we examined changes in ragweed allergen-specific Th2 cell frequency in Tolamba™ or placebo-treated subjects undergoing controlled ragweed pollen exposure. METHODS: PBMC were collected at the last pre-treatment and post-treatment ragweed exposures from a subset of Tolamba™ (n = 17) and placebo-treated (n = 15) ragweed-allergic rhinitis subjects in an environmental ragweed-exposure chamber study. In vitro allergen-stimulated CD3+CD4+CD154+ T cell intracellular IL-4, IL-5, IL-13 and IFN-γ was evaluated by flow cytometry. RESULTS: Placebo-treated subjects responded to multiple ragweed exposures with significant increases in Amb a 1-specific T cell IL-4 (P = 0.049) and IL-13 (P = 0.048) expression and ragweed-specific T cell IL-5 (P = 0.0006) and IL-13 (P = 0.018) expression. In contrast, Tolamba™-treated subjects demonstrated no significant differences in allergen-specific Th2 cytokine expression from pre- to post-treatment. In comparing pre- to post-treatment changes between Tolamba™ and placebo groups, there was a significant difference in Amb a 1-specific T cell IL-5 expression (P = 0.029) and a trend to significance for changes in Amb a 1-specific T cell IL-4 (P = 0.057) and IL-13 expression (P = 0.061). Allergen-specific T cell IFN-γ expression remained low and un-changed. CONCLUSIONS: Ragweed exposure increased placebo-treated subjects' allergen-specific CD154+ T cell IL-4, IL-5 and IL-13 expression. Tolamba™ treatment prevented these increases. Combined CD154 and intracellular cytokine staining appears to be a sensitive biomarker assay for monitoring changes in specific Th subset frequencies in studies of new clinical interventions.

HIV-1/AIDS vaccine development: are we in the darkness before the dawn?

HIV-1/AIDS vaccine development: are we in the darkness before the dawn?

BCG-Induced Dendritic Cell Responses and Suppression of Interleukin-5 Production from T Cells in Atopic Asthmatics

Bacille Calmette-Guérin (BCG) induces potent Th1 responses with the help of interleukin (IL)-10 and IL-12 released from dendritic cells (DCs), and suppresses Th2-associated allergic reactions. However, there are still some controversies on therapeutic effects of BCG in asthmatics. This study investigated whether BCG administration to DCs suppresses IL-5 production from T cells in atopic asthmatics. DCs derived from peripheral blood of subjects were cultured with or without BCG and Dermatophagoides farinae extract. Some DCs were co-cultured with T cells in the presence of BCG or the above culture supernatants. In the atopic asthmatics, BCG significantly increased IL-10 and IL-12 production from DCs. In the presence of D. farinae extract, BCG further increased IL-10 production. BCG-induced IL-10 production was significantly higher in the atopics (n=14) than in the non-atopics (n=9). Both BCG and the BCG-treated DCs culture supernatant significantly increased IFN-γ production from T cells. Both BCG and the supernatant from DCs+BCG+D. farinae co-cultures significantly decreased IL-5 production (all p<0.05), but the supernatant from DCs+BCG co-cultures did not. In conclusion, administration of BCG together with D. farinae extract effectively decreased IL-5 production from T cells, probably through the action of IL-10 and IL-12 released from DCs in D. farinae-sensitive asthmatics.

A Constitutive Increase in Gamma Globin Gene Production during the Erythroid Differentiation of CD34+ Cells from Sickle Cell Disease Patients and Alterations in Cyclic Nucleotide Levels during This Differentiation.

Studies have demonstrated that the activation of soluble guanylate cyclase (sGC) in erythrocytic cell lines and in primary erythroid precursor culture causes an increase in gamma globin gene transcription and, consequently, an augmented production of fetal hemoglobin (HbF). The cyclic nucleotides, cGMP and cAMP, are second messengers that are responsible for mediating the signaling of various extracellular signals, including those of nitric oxide (NO), hormones and neurotransmitters and, probably, have an important role in SCD by regulating the production of HbF. The aim of this study was to compare gamma-globin gene production, in association with cGMP- and cAMP-dependent signaling pathways, during the erythroid differentiation of CD34+ cells from steady-state SCD patients and healthy controls. Hematopoietic CD34+ cells were separated from the peripheral blood of subjects by CD34+-positive cell magnetic separation and were then cultured on semi-solid medium for 7 days until reaching the erythroid colony forming unit (CFU-e) stage; these colonies were then differentiated in liquid-phase culture until day 13 when cells reached the orthochromatic erythroblast/ reticulocyte stage. Cell samples were collected at days 7, 10 and 13 of culture for flow cytometry, citospin, measurement of intracellular cGMP/cAMP levels and evaluation of the gene expressions of gamma globin and the sGC alpha and beta subunits by real-time RT-PCR. Genes for endothelial nitric oxide synthase (eNOS) and phosphodiesterase 3B (PDE3B) were also studied, since these genes are important for NO/cyclic nucleotide signaling pathways. Gamma globin gene expression was observed to be significantly higher in the cultures of SCD cells on the 13th day of erythroid differentiation compared to the same time point in control cells (2.95 ± 0.30 vs. 1.45 ± 0.17 rel expression, respectively, p<0.01, Mann-Whitney, n=6). The expressions of the sGC alpha and beta subunits, eNOS and PDE3B did not present any significant differences during control and SCD erythroid differentiation. Levels of cGMP were significantly decreased during erythropoiesis in the SCD cultures, compared to control cultures (day 7: 277 ± 53 vs. 639 ± 122 fmol/5x106cells (p<0.05); day 10: 117 ± 22 vs. 268 ± 118 fmol/5x106cells; day 13: 37 ± 6 vs. 93 ± 24 fmol/5x106cells (p<0.02), respectively). In contrast, levels of cAMP were significantly increased in the SCD cultures compared to the control cultures (day 7: 72.02 ± 14.85 vs. 24.99 ± 13.50 pmol/5x106cells (p<0.05); day 10: 36.34 ± 16.71 vs. 8.66 ± 4.59 pmol/5x106cells; day 13: 1.74 ± 1.09 vs. 0.68 ± 0.37 pmol/5x106cells, respectively). Results suggest that the hematopoietic stem cells of SCD patients are significantly and constitutively different to those of control stem cells, since under the same in vitro culture conditions, consistently higher levels of gamma globin were observed during sickle cell erythroid differentiation. Intracellular cGMP and cAMP levels were also significantly altered during SCD erythroid differentiation, providing further support to the theory that cyclic nucleotide signaling may regulate HbF production. The precise role of these signaling pathways in the mechanism studied and whether any cross talk between these pathways occurs remains to be determined.

Anti-inflammatory effects of probiotic yogurt in inflammatory bowel disease patients

Our aim was to assess anti-inflammatory effects on the peripheral blood of subjects with inflammatory bowel disease (IBD) who consumed probiotic yogurt for 1 month. We studied 20 healthy controls and 20 subjects with IBD, 15 of whom had Crohn's disease and five with ulcerative colitis. All the subjects consumed Lactobacillus rhamnosus GR-1 and L. reuteri RC-14 supplemented yogurt for 30 days. The presence of putative regulatory T (T(reg)) cells (CD4(+) CD25(high)) and cytokines in T cells, monocytes and dendritic cells (DC) was determined by flow cytometry from peripheral blood before and after treatment, with or without ex vivo stimulation. Serum and faecal cytokine concentrations were determined by enzyme-linked immunosorbent assays. The proportion of CD4(+) CD25(high) T cells increased significantly (P = 0.007) in IBD patients, mean (95% confidence interval: CI) 0.84% (95% CI 0.55-1.12) before and 1.25% (95% CI 0.97-1.54) after treatment, but non-significantly in controls. The basal proportion of tumour necrosis factor (TNF)-alpha(+)/interleukin (IL)-12(+) monocytes and myeloid DC decreased in both subject groups, but of stimulated cells only in IBD patients. Also serum IL-12 concentrations and proportions of IL-2(+) and CD69(+) T cells from stimulated cells decreased in IBD patients. The increase in CD4(+) CD25(high) T cells correlated with the decrease in the percentage of TNF-alpha- or IL-12-producing monocytes and DC. The effect of the probiotic yogurt was confirmed by a follow-up study in which subjects consumed the yogurt without the probiotic organisms. Probiotic yogurt intake was associated with significant anti-inflammatory effects that paralleled the expansion of peripheral pool of putative T(reg) cells in IBD patients and with few effects in controls.

Plasma obestatin and ghrelin levels in subjects with Prader–Willi syndrome

Prader-Willi syndrome (PWS) is an obesity syndrome characterized by rapid weight gain and excessive food intake. Food intake is regulated by the hypothalamus but directly influenced by gastrointestinal peptides responding to the nutritional status and body composition of an individual. Ghrelin, derived from preproghrelin, is secreted by the stomach and increases appetite while obestatin, a recently identified peptide derived post-translationally from preproghrelin, works in opposition to ghrelin by decreasing appetite. The objective of this study was to measure fasting obestatin and ghrelin levels in peripheral blood of subjects with PWS and compare to age and gender matched control subjects. Plasma obestatin and ghrelin levels were measured in subjects with PWS (n = 16, mean age = 16.0 +/- 13.3 years; age range 1-44 years) and age and gender matched control subjects (n = 16). Significantly higher obestatin levels were seen in the 16 PWS subjects (398 +/- 102 pg/ml) compared with 16 controls (325 +/- 109 pg/ml; matched t-test, P = 0.04), particularly in 5 young (< or =3 years old) PWS subjects (460 +/- 49 pg/ml) compared with 5 young controls (369 +/- 96 pg/ml; matched t-test, P = 0.03). No significant difference in ghrelin levels was seen between the PWS and comparison groups. No significant correlation was observed for either peptide when compared with body mass index but a significant negative correlation was seen for ghrelin and age in PWS subjects. Our observations suggest that obestatin may be higher in infants with PWS compared to comparison infants. The possibility that obestatin may contribute to the failure to thrive which is common in infants with PWS warrants further investigation.

Activated T cell subsets in human type 1 diabetes: evidence for expansion of the DR+ CD30+ subpopulation in new-onset disease

An important limitation in T cell studies of human autoimmune (type 1) diabetes is lack of direct access to cells infiltrating the pancreas. We hypothesized that cells recently released from the pancreas into the blood might express a characteristic combination of markers of activation. We therefore examined the recently activated circulating T cell population [CD3+, human leucocyte antigen D-related (HLA-DR+)] using cytokine production and 10 additional subset markers [CD69, CD25, CD122, CD30, CD44v6, CD57, CD71, CCR3 (CD193), CCR5 (CD195) or CXCR3 (CD183)], comparing newly diagnosed adult (ND) (age 18-40 years) patients (n=19) to patients with diabetes for >10 years [long-standing (LS), n=19] and HLA-matched controls (C, n=16). CD3+ DR+ cells were enriched by two-step immunomagnetic separation. No differences in basal or stimulated production of interleukin (IL)-4, IL-10, IL-13 or interferon (IFN)-gamma by CD3+ DR+ enriched cells were observed between the different groups of subjects. However, among the CD3+ DR+ population, significant expansions appeared to be present in the very small CD30+, CD69+ and CD122+ subpopulations. A confirmatory study was then performed using new subjects (ND=26, LS=15), three-colour flow cytometry, unseparated cells and three additional subset markers (CD38, CD134, CD4/CD25). This confirmed the expansion of the CD3+ DR+ CD30+ subpopulation in ND subjects. We conclude that a relative expansion in the T cell subpopulation with the activated phenotype CD3+ DR+ CD30+ is seen in the peripheral blood of subjects with newly diagnosed type 1 diabetes. This subpopulation represents less than 0 x 7% of circulating T cells and may provide a rich source of disease-specific T cells that can be isolated from blood.

Four novel single nucleotide polymorphisms within the promoter region of p53 gene and their associations with uterine leiomyoma

Dysregulated p53 expression has been implicated as a major contributor to numerous tumorigenesis. Single nucleotide polymorphisms (SNPs) within the functional consequence of the novel p53 promoter region remains obscure. Herein, we aimed to establish the extent of genetic variability within the promoter region of p53 gene as well as their association with leiomyoma susceptibility. Women were divided into two groups, leiomyoma (n = 160) and nonleiomyoma controls (n = 200). Total DNA was isolated from the peripheral blood of subjects. The DNA fragment containing p53 promoter regions (+64 approximately -404 bp) were obtained by amplification of polymerase chain reaction. The variations of DNA fragments were detected by DNA sequencing or restriction fragment length polymorphism (RFLP). Sequence alignment was used to identify sequence variations in p53 promoter regions. Genotypes were analyzed by method of RFLP. Genotypes/allelic frequencies in the leiomyoma and control groups were compared. A total of 15 sequence variations within p53 promoter region were identified, including -408 T/C, -382 A/G, -359 A/G, -325 T/C, -250 A/G, -216 T/C, -205 G/A, -198 G/A, -177 T/C, -103 A/G, -81 G/A, -71 G/A, -51 T/A, -33 A/G, and -17 T/C. Among these variations, four SNPs (-250 A/G, -216 T/C, -103 A/G, and -33 A/G) were established. Allele frequencies of -250*G/-216*C/-103*G/-33*G in the leiomyoma group and control group 6.9/5.0/5.9/3.8% and 3.8/1.8/2.3/4.0%, respectively. Two of them (-216*C and -103*G) are associated with higher leiomyoma susceptibility. We concluded that some sequence variations were observed within the promoter region of p53 gene. The SNPs of -216*C and -103*G among the identical sequence variations are associated with leiomyoma development.

Warfarin Resistance Due to a Val29Leu Mutation in the VKORC1 Protein.

Warfarin resistance is a common clinical problem encountered by physicians of all subspecialities. Medical noncompliance and dietary indiscretions are felt to be the usual causes of a subject's “resistance” to anticoagulation with coumarin derivatives. Warfarin's target is the vitamin K epoxide reductase complex (VKOR). This complex functions within the vitamin K dependent γ-carboxylase system to recycle oxidized vitamin K. Until recently, the genetic sequence of components of this complex remained a mystery (Li et al., Nature 2004). Mutations in a subunit of this complex named vitamin K epoxide reductase complex subunit 1 (VKORC1) were isolated in human subjects with warfarin resistance (Rost et al., Nature 2004). Knowledge of this coding sequence has enabled us to study individual subjects suspected to have a genetic source for their warfarin resistance. Our study subject is a 24-year-old Caucasian female who received warfarin after developing bilateral pulmonary emboli. She required 30 mg of warfarin daily to achieve an international normalized ratio greater than 2.0. After institutional review board approval and with the patient's informed consent, genomic DNA was extracted from the subject's peripheral blood. The three exons comprising the VKORC1 gene were amplified by polymerase chain reaction (PCR). PCR primers were designed using the published sequence and online Primer3 software. Prolonged extension times were required to obtain good amplification of all three exons. After purification, the PCR products were sequenced and analyzed. The sequence analysis demonstrated a heterozygous G→T mutation in exon 1 at base pair 85. This results in a Val29Leu substitution, which has been described in a warfarin resistant patient by Rost et al. The second and third exons matched completely with the published sequence. The gene frequency of the mutation was then determined by examining 400 random, discarded DNA samples. The HypCH4 IV restriction enzyme cleaves at 2 positions in exon 1 in the wild-type genotype, but at just one position if our known mutation is present. The mutation was not identified in any sample.In conclusion, we have identified a second patient with a Val29Leu substitution in vitamin K epoxide reductase resulting in warfarin resistance. Our analysis of 400 random samples has not identified any other example of this mutation, indicating that this is a rare occurrence. Genetic analysis of patients requiring more than 20 mg of warfarin daily would likely reveal more mutations in the VKORC1 protein. The identification of such mutations would provide valuable insight into the structure-function relationships of this protein complex.