Stepwise Induction Research Articles

Oral tolerance to food protein.

Oral tolerance is the state of local and systemic immune unresponsiveness that is induced by oral administration of innocuous antigen such as food proteins. An analogous but more local process also regulates responses to commensal bacteria in the large intestine and, together, mucosally induced tolerance appears to prevent intestinal disorders such as food allergy, celiac disease, and inflammatory bowel diseases. Here we discuss the anatomical basis of antigen uptake and recognition in oral tolerance and highlight possible mechanisms underlying the immunosuppression. We propose a model of stepwise induction of oral tolerance in which specialized populations of mucosal dendritic cells and the unique microenvironment of draining mesenteric lymph nodes combine to generate regulatory T cells that undergo subsequent expansion in the small intestinal lamina propria. The local and systemic effects of these regulatory T cells prevent potentially dangerous hypersensitivity reactions to harmless antigens derived from the intestine and hence are crucial players in immune homeostasis.

Stepwise renal lineage differentiation of mouse embryonic stem cells tracing in vivo development

The in vitro derivation of renal lineage progenitor cells is essential for renal cell therapy and regeneration. Despite extensive studies in the past, a protocol for renal lineage induction from embryonic stem cells remains unestablished. In this study, we aimed to induce renal lineages from mouse embryonic stem cells (mESC) by following in vivo developmental stages, i.e., the induction of mesoderm (Stage I), intermediate mesoderm (Stage II) and renal lineages (Stage III). For stage I induction, in accordance with known signaling pathways involved in mesoderm development in vivo, i.e., Nodal, bone morphogenic proteins (BMPs) and Wnt, we found that the sequential addition of three factors, i.e., Activin-A (A), a surrogate for Nodal signaling, during days 0–2, A plus BMP-4 (4) during days 2–4, and A4 plus lithium (L), a surrogate for Wnt signaling, during days 4–6, was most effective to induce the mesodermal marker, Brachyury. For stage II induction, the addition of retinoic acid (R) in the continuous presence of A4L during days 6–8 was most effective to induce nephrogenic intermediate mesodermal markers, such as Pax2 and Lim1. Under this condition, more than 30% of cells were stained positive for Pax2, and there was a concomitant decrease in the expression of non-mesodermal markers. For stage III induction, in resemblance to the reciprocal induction between ureteric bud (UB) and metanephric mesenchyme (MM) during kidney development, we found that the exposure to conditioned media derived from UB and MM cells was effective in inducing MM and UB markers, respectively. We also observed the emergence and gradual increase of cell populations expressing progenitor cell marker CD24 from Stage I to Stage III. These CD24+ cells correlated with higher levels of expression of Brachyury at stage I, Pax2 and Lim1 at stage II and MM markers, such as WT1 and Cadherin 11, after exposure to UB-conditioned media at stage III. In conclusion, our results show that stepwise induction by tracing in vivo developmental stages was effective to generate renal lineage progenitor cells from mESC, and CD24 may serve as a useful surface marker for renal lineage cells at stage II and MM cells at stage III.

CD24: A Novel Surface Marker for PDX1-Positive Pancreatic Progenitors Derived from Human Embryonic Stem Cells

Human ESCs provide a promising cell resource for the treatment of type I diabetes mellitus. Although PDX1-positive pancreatic progenitors can be efficiently generated from human ESCs by stepwise induction, further in vitro differentiation into functional, mature beta cells is not efficient or reproducible. Purification of pancreatic progenitor cells could facilitate the identification of signals that regulate beta cell differentiation and maturation. Here, we report the identification of a novel surface marker for PDX1-positive pancreatic progenitors based on an in vitro human ESC differentiation system. By costaining PDX1 and a panel of cell surface antigens at the pancreatic progenitor stage of human ESC differentiation, we discovered a positive marker, CD24. CD24-positive cells coexpressed most of the key transcription factors of pancreatic progenitors, and the expression of important pancreatic genes was greatly enriched in CD24-positive cells compared with the CD24-negative cells. In addition, CD24-positive cells could differentiate into insulin-producing cells but CD24-negative cells could not. These results indicate that CD24 could be a surface marker for PDX1-positive pancreatic progenitors derived from human ESCs. Enrichment of pancreatic progenitors with this marker will facilitate the investigation of beta cell maturation during the human ESC differentiation.

Algebraic Signal Processing Theory: Cooley–Tukey-Type Algorithms for Polynomial Transforms Based on Induction

A polynomial transform is the multiplication of an input vector by a matrix whose th element is defined as for polynomials from a list and sample points from a list . Such transforms find applications in the areas of signal processing, data compression, and function interpolation. An important example includes the discrete Fourier transform. In this paper we introduce a novel technique to derive fast algorithms for polynomial transforms. The technique uses the relationship between polynomial transforms and the representation theory of polynomial algebras. Specifically, we derive algorithms by decomposing the regular modules of these algebras as a stepwise induction. As an application, we derive novel general-radix algorithms for the discrete Fourier transform and the discrete cosine transform of type 4.

Analysis of Electrophysiological State Patterns and Changes During Hypnosis Induction

Hypnosis can be seen as a guided induction of various states of consciousness. This article details a time-series analysis that visualized the electrophysiological state changes during a session as a correlate to the instructions. Sixty-four channels of EEG and peripheral physiological measures were recorded in 1 highly susceptible subject. Significant state changes occurred synchronously with specific induction instructions. Some patterns could be physiologically explained, such as sensorimotor desynchronization over the right hemispheric hand area during left arm levitation. There was a highly significant increase in broadband activity during the stepwise trance induction that may point to a deep hypnotic state. This study provides illustrated proof for the detectability of physiological state changes as correlates to different states of awareness, consciousness, or cognition during hypnosis.

Intestinal Tolerance Requires Gut Homing and Expansion of FoxP3+ Regulatory T Cells in the Lamina Propria

Tolerance to food antigen manifests in the absence and/or suppression of antigen-specific immune responses locally in the gut but also systemically, a phenomenon known as oral tolerance. Oral tolerance is thought to originate in the gut-draining lymph nodes, which support the generation of FoxP3(+) regulatory T (Treg) cells. Here we use several mouse models to show that Treg cells, after their generation in lymph nodes, need to home to the gut to undergo local expansion to install oral tolerance. Proliferation of Treg cells in the intestine and production of interleukin-10 by gut-resident macrophages was blunted in mice deficient in the chemokine (C-X3-C motif) receptor 1 (CX3CR1). We propose a model of stepwise oral tolerance induction comprising the generation of Treg cells in the gut-draining lymph nodes, followed by migration into the gut and subsequent expansion of Treg cells driven by intestinal macrophages.

Abstract 4894: Human endogenous retroviral over expression in endometrial carcinoma is due to hypomethylation of the 5′LTR

Abstract The purpose of this study was to determine the expression of envelope (env) genes from five Human Endogenous Retrovirus (HERV) family members in Endometrial Carcinomas (EnCa) and correlate with CpG-methylation of the 5′LTR HERV-W in EnCa and prestages compared to matched control tissues. Over 85% of all EnCa are classified as endometrioid expressing steroid hormone receptors and involve prior stages, like hyperplasia. HERV families embody over 8% of the human genome and are derived from ancestral germ line retroviral infections. Sixteen different functional HERV-env genes are essential for cytotrophoblast cell-cell fusion during placentogenesis, but are also implicated in tumorigenesis. Aberrant methylation and hypomethylation are hallmarks of tumorigenesis. Hypomethylated 5′ LTRs of HERVs correlating with an increased expression have been shown for HERV-K and HERV-W (ovary) in different human carcinomas. Previously, we demonstrated a significant step-wise increase of the Syncytin-1 HERV-W env gene at both mRNA and protein levels for EnCa and prestages compared to controls. A pivotal role for Syncytin-1 in EnCa cell-cell fusions and cellular proliferation was shown using RNAi technology after treatment of cell lines with cAMP analogs or steroid hormones and tamoxifen, respectively. Tamoxifen also activated phosphorylation of AKT (Thr308) and ERα (Ser118) leading to Syncytin-1 induction via non-genomic ER signalling. We have established methods for absolute quantification real time PCR (qPCR) for five different HERV-env genes and methylation-specific-PCR (MSP) using bisulfite treated genomic DNA. Methylated CpGs were verified by cloning and sequencing. Gene expression results of benign and malignant tissues (EnCa n=44, polyps n=21, hyperplasia n=12) compared to control postmenopausal endometrium (n=33) showed that the HERV-env genes Syncytin-1, HERV-K, ERV-3, HERV-P(b) and Syncytin-2 were significantly up-regulated. We observed that in control endometrium the highest expressed env was ERV3; whereas in hyperplasia and EnCa Syncytin-1 was the most prominent. Methylation studies of the Syncytin-1 5′ LTR identified 20 potential CpGs where many correlated with protein binding sites, e.g. for ERα, AP-1, GATA1-3 and MeCP2. MSP results showed that the 5′LTR region was hypomethylated in tumors compared to matched controls. Cloning and sequencing of PCR fragments showed that 3-4 CpGs corresponding with protein binding sites were hypomethylated in tumors (n=10) compared to matched controls. Studies using 5′Aza-2′Cytidine treatment of EnCa cell lines resulted in a 2.5-fold increase of Syncytin-1. In conclusion, up-regulation of Syncytin-1 in EnCa is due to specific hypomethylation of the 5′ LTR. Understanding epigenetic regulation of HERV-env genes will contribute to the molecular etiology in EnCa and stepwise induction from benign prestages but also other carcinomas. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4894.

Adult Stem Cell Therapy for Injured Solid-Organ Tissue (with Emphasis on Cardiac Tissue Repair)

Adult hematopoietic tissue-derived stem cells are primarily used for hematopoietic reconstitution in patients with malignant lympho-hematopoietic disorders undergoing stem cell transplantation. Their therapeutic use for solid- organ tissue repair such as cardiac tissue is a novel treatment strategy. Besides hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs), endothelial progenitor cells (EPCs) and even solid-organ tissue resident stem cells (e.g. cardiac stem cells (CSC)) are known to contribute to solid-organ tissue repair. Stem cell delivery for cardiac tissue repair is by transvascular or intramyocardial injection. Myocardial tissue engineering takes advantage of biocompatible materials as stem cell carriers (scaffolds). Stem cell concentration at the site of tissue injury and engineering the stem cell microenvironment are additional components that determine treatment outcome. Mechanisms that explain how stem cells generate solid-organ specific cells include nuclear re-programming (transdifferentiation), cell fusion, and paracrine secretion of transplanted cells (e.g., MSCs). One preferred therapeutic concept is the stepwise induction of neovascularization followed by activation of the solid-organ resident stem cell pool. Cardiac tissue repair in patients with acute myocardial infarction is currently at the forefront of clinical stem cell treatment research. The outcome of major clinical trials will be discussed.

Evolution of Changes in Upper Airway Collapsibility during Slow Induction of Anesthesia with Propofol

Upper airway collapsibility is known to increase under anesthesia. This study assessed how this increase in collapsibility evolves during slow Propofol induction and how it relates to anesthesia-induced changes in upper airway muscle activity and conscious state. Nine healthy volunteers were studied. Anesthesia was induced with Propofol in a step-wise manner (effect-site concentration steps of 0.5 microg x ml(-1) from 0 to 3 microg x ml(-1) and thereafter to 4 microg x ml(-1) and 6 microg x ml(-1) [target-controlled infusion]). Airway patency was maintained with continuous positive airway pressure. Pharyngeal collapsibility was assessed at each concentration by measuring critical pressure. Intramuscular genioglossus electromyogram and anesthetic depth (bispectral index score) were monitored throughout. Loss of consciousness was defined as failure to respond to loud verbal command. Loss of consciousness occurred at varying Propofol effect-site concentrations between 1.5 and 4.0 microg x ml(-1). Initially genioglossus electromyographic activity was sustained with increases in Propofol concentration, increasing in some individuals. At or approaching loss of consciousness, it decreased, often abruptly, to minimal values with an accompanying increase in critical pressure. In most subjects, bispectral index score decreased alinearly with increasing Propofol concentration with greatest rate of change coinciding with loss of consciousness. Slow stepwise induction of Propofol anesthesia is associated with an alinear increase in upper airway collapsibility. Disproportionate decreases in genioglossus electromyogram activity and increases in pharyngeal critical closing pressure were observed proximate to loss of consciousness, suggesting that particular vulnerability exists after transition from conscious to unconscious sedation. Such changes may have parallels with upper airway behavior at sleep onset.

The Potted-Plant Microcosm Substantially Reduces Indoor Air VOC Pollution: II. Laboratory Study

Indoor air-borne loads of volatile organic compounds (VOCs) are usually significantly higher than those outdoors, and chronic exposures can cause health problems. Our previous laboratory studies have shown that the potted-plant microcosm, induced by an initial dose, can eliminate high air-borne VOC concentrations, the primary removal agents being potting-mix microorganisms, selected and maintained in the plant/root-zone microcosm. Our office field-study, reported in the preceding paper, showed that, when total VOC (TVOC) loads in reference offices (0 plants) rose above about 100 ppb, levels were generally reduced by up to 75% (to < 100 ppb) in offices with any one of three planting regimes. The results indicate the induction of the VOC removal mechanism at TVOC levels above a threshold of about 100 ppb. The aims of this laboratory dose-response study were to explore and analyse this response. Over from 5 to 9 days, doses of 0.2, 1.0, 10 and 100 ppm toluene and m-xylene were applied and replenished, singly and as mixtures, to potted-plants of the same two species used in the office study. The results confirmed the induction of the VOC removal response at the lowest test dosage, i.e in the middle of the TVOC range found in the offices, and showed that, with subsequent dosage increments, further stepwise induction occurred, with rate increases of several orders of magnitude. At each dosage, with induction, VOC concentrations could be reduced to below GC detection limits (< 20 ppb) within 24 h. A synergistic interaction was found with the binary mixtures, toluene accelerating m-xylene removal, at least at lower dosages. The results of these two studies together demonstrate that the potted-plant microcosm can provide an effective, self-regulating, sustainable bioremediation or phytoremediation system for VOC pollution in indoor air.

Conventional stepwise vs. vital capacity rapid inhalation induction at two concentrations of sevoflurane

Conventional stepwise vs. vital capacity rapid inhalation induction at two concentrations of sevoflurane

Reduction of transglutaminase 2 expression is associated with an induction of drug sensitivity in the PC-14 human lung cancer cell line.

Recently a role for transglutaminase 2 (TGase 2) in the drug resistance of cancer cells has been suggested, although the mechanism is unclear. In the present study, we observed that doxorubicin-resistant PC-14/ADR cells showed a ten-fold higher level of TGase 2 expression than drug-sensitive PC-14 cells. PC-14/ADR cells exhibited the classical multidrug resistance (MDR) phenotype, which was cross-resistant to vincristine, but not to cisplatin. The stepwise induction of resistance to doxorubicin and vincristine in PC-14 cells was accompanied by a gradual increase of TGase 2 expression, but this expression was not increased with induction of cisplatin resistance. To confirm the role of TGase 2 protein in the acquisition of drug resistance in PC-14 cells, the TGase 2 expression in PC-14/ADR cells was reduced by stable transfection with the antisense or ribozyme construct. In the clones showing reduced expression of TGase 2, lactate dehydrogenase released from drug-treated cells was increased in the presence of either MDR-related drugs (doxorubicin and vincristine) or a non-MDR-related drug (cisplatin). These data suggest that TGase 2 can play a role in the acquisition of drug resistance in PC-14 cells through a general cellular defense system other than the MDR-related system.

Single catheter technique of hepatic venous isolation and extracorporeal charcoal hemoperfusion for malignant liver tumors

Background A single catheter technique of hepatic venous isolation and charcoal hemoperfusion (HVI-CHP) using a 4-lumen/2-balloon (4L-2B) catheter was developed to perform high-dose intra-arterial chemotherapy of the liver. Herein we report the technique, safety, and pharmacokinetics of this system in comparison with the original double-balloon technique. Patients and methods Sixteen patients with malignant liver tumors were treated by hepatic arterial infusion (HAI) with adriamycin at a dose of 100 mg/m 2 under HVI-CHP. Seven patients underwent HVI-CHP by the double-balloon technique (group A), in which filtered hepatic effluent and the rest of the inferior vena caval blood were separately drawn and returned to the left axillary vein. The other nine patients were treated by the single catheter technique (group B). In group B, hepatic effluent was isolated by balloon inflations and directed to filters through fenestrations of one major lumen of a 4L-2B catheter. The filtered blood was returned straight to the right atrium through the other major lumen of the catheter. Results All patients in group A had a smooth stepwise induction of HVI-CHP, whereas one of nine patients in group B developed severe hypotension requiring interruption of HVI. The hepatic venous flow rate in group B during HVI-CHP was significantly higher than that in group A ( P < 0.05). Systemic adriamycin exposure, as assessed by the area under the time concentration curve in systemic serum, was significantly higher in group A compared to that in group B ( P < 0.01). Conclusion The single catheter technique is hemodynamically tolerable and feasible in the majority of patients with malignant liver tumors. In view of systemic drug exposure, the single catheter technique is superior to the original doubleballoon technique.

CURRENT STATUS OF PROSTATE-CANCER RESEARCH - DEVELOPMENT OF IN-VITRO MODEL SYSTEMS (REVIEW)

Prostate cancer is a major medical problem which is expected to affect over 200,000 US men in 1994. Despite its widespread prevalence and the difficulties in clinical diagnosis and treatment of the disease, the contribution of environmental factors and the etiological mechanism of prostate cancer are poorly understood. A brief update on recent progress in cellular and molecular research of prostate cancer is provided. Specific areas discussed include oncogenes (vas, myc, c-erbB-2 and bcl-2), tumor suppressor genes (p53, RB, DCC, putative suppressor genes), growth factors (EGF, FGF, IGF) and human papillomavirus (HPV) infections. The elucidation of a multistep mechanism of prostate tumorigenesis has been slowed by a lack of tumor tissue and the limited number of in vitro cell lines available for study. Reviewed here are the newly developed in vitro normal prostate cell systems which supply a base for addition of oncogenes and chemical mutagens and may provide insight into the molecular genetic events which accompany the stepwise induction of prostatic neoplasia.

Feasibility of Pulse Oxymetry to Measure Arterial O2Saturation in Studies on Cochlear Blood Circulation

To understand the characteristics of oxygen transport to the inner ear, the relationship between arterial O2 saturation and cochlear microcirculation was investigated under different respiratory condition in guinea pigs. To monitor arterial O2 saturation a pulse oxymeter instead of an arterial blood gas analyzer was used. When the arterial O2 saturation was measured in the foot pad by a pulse oxymeter under different respiratory conditions, the data showed a close correlation with the results of blood gas analysis. For the measurement of cochlear microcirculation, a pulse oxymeter was found to be a feasible respiratory monitor for animal experiments. With this apparatus our study demonstrated a slower reaction in the decrease of perilymphatic oxygen tension than of cochlear blood flow during stepwise induction of hypoventilation monitored by a pulse oxymeter. Under certain conditions of hyperventilation in which arterial O2 saturation and perilymphatic oxygen tension increased gradually, cochlear blood flow was found to decrease. This decrease of cochlear blood flow could be attributed to chemical controls which are regulated, as in the cerebral blood circulation, by the content of CO2 and H+ in the vascular bed in the cochlea.

The effect of hypoglycaemia on visual function: a clinical and electrophysiological study.

Disturbance of vision commonly accompanies hypoglycaemia. This study was designed to investigate the nature of the visual disturbance, the blood glucose threshold at which the disturbance occurred and the physiological basis. Measurements were made of the corrected visual acuity, colour vision (100 Hue test), visual evoked potentials (VEP), electroencephalography (EEG) frequency analysis and psychometry (digit recall) during stepwise induction of controlled hypoglycaemia produced by an intravenous insulin infusion. Six male volunteers and five insulin-dependent diabetic subjects were studied. During hypoglycaemia corrected visual acuity was unchanged. Colour vision was significantly impaired. Baseline VEP were normal in both groups but significantly prolonged during hypoglycaemia (mean increment 10.8 ms) and increased by greater than 5 ms in nine out of 11 subjects. Quantitative EEG analysis demonstrated slowing with a power density spectral shift from fast alpha to slow alpha, theta and delta which correlated with VEP latency and amplitude changes. The findings have clinical implications. A deterioration in colour vision is likely to impair the ability to read reagent strips by eye. VEP measurements in diabetic patients are likely to be misleading if hypoglycaemia is present; EEG changes are a sensitive index of cortical dysfunction during hypoglycaemia and provide a theoretical basis for developing a portable device to detect early hypoglycaemia.

987 SHOCK: NEONATAL HYPOPERFUSION SYNDROMES AND MUSCLE SURFACE

Low perfusion states are inadequately defined by standard methods in the Neonatal ICU. Using a stepwise induction of hemorrhagic shock in 7 spontaneously breathing nembutal-sedated laboratory cats, pHm was shown to vary independently of routinely monitored parameters and most strikingly, autonomous of blood pH (0-1.21 difference). In 4 critically ill human infants (760-3500 gms) pHm was shown to fall during states known to be associated with tissue underperfusion although compensatory mechanisms maintained the patient at clinically acceptable levels. During hypotensive episodes, acute volume expansion with PRBC or albumin resulted in an average transient rise in pHm of 0.18 pH units (range 0.11-0.27) with no significant change in blood pH. Trends associated with a rising mean aortic blood pressure and general improvement correlated with pHm returning to “healthy” values in HMD patients (range 6.52-7.47). There were no complications in monitoring up to 6 days. This study demonstrates (1) pHm can be used as an independent indicator of tissue perfusion at the end organ; (2) blood volume expansion temporarily increases tissue perfusion in critically ill neonates; (3) newborn and premature infants can regulate blood flow to non-vital organs (muscle); (4) this previously inaccessible information in the newborn nursery may help reduce neonatal morbidity and mortality by permitting earlier diagnosis of shock and institution of appropriate therapy.

Transcription and translation mechanisms in a «permeabilized E. coli system

Summary An E. coli «permeabilized cell system (PCS) is described, resulting from 2 M-sucrose plasmolysis in the presence of 10−4 M EDTA. This treatment induces some interesting properties in the cells: permeability to various phosphorylated derivatives without osmotical fragility, depletion from endogenous metabolites and lack of growth on rich medium. This system appears well suited for the study of uncoupled macromolecular synthesis: o (1) in the presence of the usual RNA polymerase substrates (ATP, CTP, GTP and UTP), PCS prominently synthesizes messenger RNA of exceptional great stability (half-life greater than 60 minutes). The reasons of this stability will be discussed; (2) the mRNAs thus formed are active in protein synthesis as it can be shown by a stepwise induction experiment: RNA synthesized in the presence of the four ribonucleoside triphosphates plus IPTG can direct the synthesis of β-galactosidase, after PCS is transferred in an amino acid medium plus actinomycin D; (3) when PCS is supplemented with a complete amino mixture in addition to the four ribonucleoside triphosphates, it synthesizes proteins and also accumulates large amounts of ribosomal and transfer RNA's.