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Biomedical subjects

A Singer

Publications and source records attributed to A Singer.

At least 127 records · Page 7Linked to original sources
PubMed1994

Quantitative differences in cell surface expression of class I MHC antigens on murine epidermal Langerhans cells.

Epidermal Langerhans cells are derived from cells of bone marrow origin and, as the primary APC population in the skin, are responsible for initiation of many immune responses. Consequently, cell surface expression of MHC Ags by Langerhans cells is central to their function. Although murine Langerhans cells express class II MHC Ags at high levels, their level of expression of class I MHC has been controversial. In this study, cell surface expression of multiple individual class I MHC Ags on murine epidermal Langerhans cells was analyzed using quantitative immunofluorescence. It was found that Langerhans cells differentially express products of distinct class I genes. Langerhans cells expressed low cell surface amounts of H-2K and Qa-2, whereas expression of surface H-2D and H-2L by the same cells was high. Murine epidermal Langerhans cells therefore express low cell surface amounts of some but not all class I MHC Ags. Differential surface expression of products of distinct class I MHC genes by Langerhans cells may have a profound effect on cutaneous immune responses.

Animals↗
PubMed1994

Expression of the homotypic adhesion molecule E-cadherin by immature murine thymocytes and thymic epithelial cells.

Cadherins mediate homotypic adhesion between lineage-related cells in epithelia and other tissues. One cadherin, E-cadherin, is also responsible for adhesion of murine epidermal Langerhans cells to keratinocytes in vitro, and may play a role in the localization of Langerhans cells in epidermis. The thymus is another tissue in which important adhesive interactions between bone marrow-derived cells and keratinizing epithelia occur. To determine whether cadherins might be involved in interactions between thymocytes and thymic epithelial cells, we examined thymocytes from C57BL/6 mice of various gestational ages for cadherin expression. Most day 14 (D14) and essentially all D16 isolated fetal thymocytes expressed cell surface E-cadherin. After D16, the proportion of fetal thymocytes expressing E-cadherin and the level of E-cadherin expressed by individual thymocytes decreased with increasing gestational age. A minority of neonatal thymocytes and very few adult thymocytes expressed E-cadherin. E-cadherin was maximally expressed by the least mature (CD4-CD8-, HSA (J11d)high, CD5 (Ly-1)low, CD25 (IL-2R alpha)+) thymocytes. P-cadherin, another epithelial cadherin, was not detected on thymocytes at any stage of development. Immunohistologic studies revealed that thymic epithelial cells also expressed E-cadherin. Similar levels of E-cadherin were expressed by neonatal and adult thymic epithelial cells in situ, and E-cadherin was easily demonstrable on the thymic epithelial cell line, TE-71. In contrast, P-cadherin was transiently expressed by thymic epithelial cells in situ, and only small amounts of P-cadherin were detected on TE-71 cells. These studies demonstrate that thymocytes and thymic epithelial cells each have the capacity to express the homotypic adhesion molecule E-cadherin. E-cadherin may play a role in developmentally regulated interactions between early thymocytes and thymic stromal cells.

Animals↗
PubMed1994

Differential effects of zeta and eta transgenes on early alpha/beta T cell development.

The zeta-family dimers (zeta, eta, and gamma) are a group of structurally and functionally related proteins that are expressed in developing thymocytes and function as signal transducing subunits of the T cell antigen receptor (TCR) and certain Ig Fc receptors. Zeta, eta, and gamma each contain one or more copies of a conserved tyrosine-based activation motif (TAM) that is known to be required for signal transduction. To examine the developmental importance of multiple or individual TAM elements we generated transgenic mice that express: (a) full-length (FL) zeta-chain (3 TAMs); (b) eta-chain, a naturally occurring variant of zeta that is derived from alternative splicing (2 TAMs); or (c) truncated zeta-chain (CT108; 1 TAM), under the control of the human CD2 promoter and regulatory elements. Unexpectedly, we found that overexpression of the FL zeta chain caused premature termination of RAG-1 and RAG-2 expression, prevented productive rearrangement of the TCR-alpha and TCR-beta genes and blocked entry of thymocytes into the CD4/CD8 developmental pathway. In contrast, we found that overexpression of eta or CT108 had no effect on normal thymocyte maturation. These results suggest that an early signaling pathway exists in precursor TCR- thymocytes that can regulate RAG-1 and RAG-2 expression and is differentially responsive to individual members of the zeta-family dimers.

Animals↗
PubMed1994

Early progression of thymocytes along the CD4/CD8 developmental pathway is regulated by a subset of thymic epithelial cells expressing transforming growth factor beta.

Precursor cells differentiate into mature CD4+ and CD8+ T cells in the inductive environment of the thymus by undergoing a series of distinct developmental steps marked by expression of the coreceptor molecules CD4 and CD8. Among the earliest cells to enter the CD4/CD8 developmental pathway are CD4-CD8lo precursors cells that differentiate into CD4+CD8+ thymocytes. Here we show that differentiation of precursor cells into CD4+CD8+ thymocytes requires at least one cell division and that their progression through a cell cycle is specifically retarded in the thymus by interaction with thymic epithelial cells that express transforming growth factor beta (TGF-beta) proteins. We also demonstrate that TGF-beta proteins, either in solution or bound to cell membranes, can regulate cell cycle progression and differentiation of CD4-CD8lo precursor cells into CD4+CD8+ thymocytes. The regulatory effect of TGF-beta is specific for CD4-CD8lo precursor cells as TGF-beta proteins do not regulate the earlier generation of CD4-CD8lo precursor cells from CD4-CD8- thymocytes. Finally, we demonstrate that TGF-beta proteins are expressed in vivo in the intact thymus on subcapsular and cortical thymic epithelium where they can contact developing CD4-CD8lo precursor cells. Thus, thymic epithelial cells expressing TGF-beta proteins can actively regulate the rate at which CD4+CD8+ thymocytes are generated from CD4-CD8lo precursor cells.

Animals↗
PubMed1994

Negative selection of CD4+CD8+ thymocytes by T cell receptor-induced apoptosis requires a costimulatory signal that can be provided by CD28.

CD4+CD8+ thymocytes expressing self-reactive T cell antigen receptors (TCR) are deleted in the thymus as a consequence of TCR/self-antigen/major histocompatibility complex interactions. However, the signals that are necessary to initiate clonal deletion have not yet been clarified. Here we demonstrate that TCR engagement does not efficiently induce apoptosis of CD4+CD8+ thymocytes, although it generates signals that increase expression of CD5, a thymocyte differentiation marker. In fact, TCR signals fail to induce thymocyte apoptosis even when augmented by simultaneous engagement with CD4 or lymphocyte function 1-associated molecules. In marked contrast, signals generated by engagement of both TCR and the costimulatory molecule CD28 potently induce apoptosis of CD4+CD8+ thymocytes. Thus, the present results define a requirement for both TCR and costimulatory signals for thymocyte apoptosis and identify CD28 as one molecule that is capable of providing the necessary costimulus. These results provide a molecular basis for differences among cell types in their ability to mediate negative selection of developing thymocytes.

Animals↗
PubMed1994

Isolation of immature and mature T cell receptor complexes by lectin affinity chromatography.

The antigen-specific T cell receptor (TCR) is a multisubunit complex composed of at least six different polypeptide chains (alpha, beta, gamma, delta, epsilon, zeta), several of which are glycoproteins. Assembly of the TCR occurs in the endoplasmic reticulum (ER) and involves intermediary complexes of CD3-gamma, delta, epsilon proteins and TCR alpha and -beta molecules. Egress of TCR from the ER and transport through the Golgi apparatus is most often monitored by the sensitivity of TCR glycoproteins to endoglycosidase H (Endo H), an enzyme specific for immature oligosaccharides which have not yet been processed by Golgi glycosidases and glycosyltransferases. Because they are not glycosylated, the subcellular localization of CD3-epsilon and TCR zeta chains cannot be directly determined by Endo H treatment, and therefore must be inferred by association with glycoprotein members of the TCR complex. Thus, when both immature and mature TCR glycoproteins are present within a given sample, this becomes extremely difficult. In this report, we describe a method for the physical separation of immature and mature murine TCR complexes based on processing of N-linked carbohydrate side chains. Specifically, we report the use of wheat germ agglutinin-affinity matrices to separate TCR complexes which have reached the trans Golgi compartment of the cell from those that have not. This technique is rapid, sensitive, and does not affect the integrity of assembled TCR complexes.

Animals↗
PubMed1994

Detection of DNA and E7 transcripts of human papillomavirus types 16, 18, 31 and 33, TGF beta and GM-CSF transcripts in cervical cancers and precancers.

The association of human papillomavirus (HPV) with a high proportion of cervical cancers should allow the efficiency of cytological screening methods to be improved. We report here that quantitative detection of HPV types 16, 18, 31 and 33 DNA and the corresponding E7 transcripts by polymerase chain reaction (PCR) may be of value in identifying precancers and cancers. In clinical specimens with major cervical lesions, the level of E7 transcription does not appear to be related to concomitant transcription of either transformation growth factor-beta (TGF beta) or granulocyte-macrophage colony stimulating factor (GM-CSF) genes.

Base Sequence↗
PubMed1994

Crosstalk in the mouse thymus.

The development of mature T cells within the thymus is dependent upon intact cortical and medullary microenvironments. In turn, thymic microenvironments themselves are dependent on lymphoid cells to maintain their integrity. Here, Willem van Ewijk and colleagues discuss experiments that have established the phenomenon of 'crosstalk' within the mouse thymus and suggest a mechanism whereby lymphoid and stromal cells influence each other in a consecutive manner during T-cell development.

Animals↗
PubMed1994

Maturation of medullary thymic epithelium requires thymocytes expressing fully assembled CD3-TCR complexes.

Unlike medullary thymic epithelial cells (TEC) of normal mice, medullary TEC of TCR- SCID mice are immature and disorganized. In order to assess directly the role of TCR+ cells in the development of medullary TEC, we bred mice which co-expressed the SCID genetic defect and transgenes encoding clonotypic TCR chains. Immunohistologic examination revealed that medullary thymic epithelial cells from TCR beta transgenic SCID mice, whose thymocytes only express TCR beta chains that inefficiently associate with CD3 and zeta components, remained immature and disorganized. In contrast, medullary TEC from TCR alpha beta transgenic SCID mice, whose thymocytes express fully assembled CD3-zeta-TCR alpha beta complexes were mature and organized. Interestingly, the ability of TCR alpha beta(+)-zeta(+)-CD3+ thymocytes to induce maturation of medullary TEC appeared not to be related to the antigen specificity of the TCR as thymi from positively selecting, negatively selecting and non-selecting TCR alpha beta transgenic SCID mice all possessed induced medullary thymic epithelial cells. In addition, we found that induction of medullary TEC cells was associated with the presence of medullary thymocytes, including those of the CD4-CD8- TCR alpha beta+ phenotype. The present findings demonstrate that fully assembled CD3-zeta-TCR complexes are required to induce maturation of medullary thymic epithelial cells and indicate that thymocyte induction of medullary thymic epithelial cells may result from signaling independently of their clonotypic TCR chains.

Animals↗
PubMed1994

Hypersensitivity to therapeutic murine monoclonal antibodies.

OBJECTIVE: To determine the predictive value of pre-treatment skin tests and in vitro IgE/IgG4 anti-murine monoclonal antibodies in patients treated with murine monoclonal antibodies. DESIGN: Patients treated at two cancer institutions were evaluated by skin testing and solid phase immunoassays to detect IgE and IgG4 specific anti-murine monoclonal antibodies. Skin testing by scratch and intradermal skin testing was done on patients before treatment with murine monoclonal antibodies. IgE & IgG4 specific anti-murine monoclonal antibodies were determined before treatment in all patients and at 1, 7, 14 and 21 days post-treatment in 1 patient. SETTING: Cancer patients undergoing murine monoclonal antibody treatment in two university medical centers were recruited for the study. PARTICIPANTS: Twelve patients, aged 41-75 years with gastrointestinal cancers (colon, stomach, pancreas or liver) with metastatic disease, who had relapses or conventional therapy were enrolled. Some patients had previous exposure to rodents, either as laboratory personnel or had kept them as pets. INTERVENTION: One patient who experienced an anaphylactic reaction to murine monoclonal antibody infusion was desensitized so therapy could continue. MAIN OUTCOME MEASURES: Skin tests, immunoassays, and patient history were correlated with adverse reactions to infusions of murine monoclonal antibodies. MAIN RESULTS: Skin tests (scratch method) and/or in vitro immunoassays may predict allergic outcomes in patients receiving infusions of murine monoclonal antibodies. Intradermal skin testing with murine monoclonal antibodies may result in false positive reactions and have less predictive value. Specific IgE or IgG4 were elevated in the two patients who experienced severe adverse reactions to murine monoclonal antibodies but not in those patients with no reactions and therefore, may have some predictive value. A history of past exposure to mice may also increase the risk of adverse reactions. In one patient, intravenous desensitization enabled treatment to proceed. CONCLUSION: Scratch skin tests, in vitro IgE and/or IgG4 immunoassays together with a past history of previous exposure to murine antigen(s) may predict potential allergic reaction to therapy with murine monoclonal antibodies.

Adult↗
PubMed1994

Integrin expression and function in HPV 16-immortalised human keratinocytes in the presence or absence of v-Ha-ras. Comparison with cervical intraepithelial neoplasia.

Keratinocytes express several receptors of the integrin family which regulate both adhesion and differentiation. We have investigated whether HPV immortalisation, which changes the growth and differentiation properties of keratinocytes, is associated with altered integrin expression or function. We compared two HPV 16-immortalised lines of human keratinocytes, up and vp, with the normal keratinocyte strains, u and v, from which they were derived and with upr, obtained by transfection of up with viral Harvey ras. Immunofluorescence, immunoprecipitation and flow cytometry demonstrated that up and vp had lower levels of integrins than u and v, the reduction in up being greater than in vp. Up and vp also had reduced levels of mRNA encoding the beta 1 integrin subunit. Reduced expression of the alpha 5 beta 1 and alpha 2 beta 1 integrins was correlated with reduced adhesion to fibronectin and collagen in up but not in vp and there were no significant differences between the normal and immortalised cells in adhesion to laminin. Reduced integrin expression was correlated with decreased motility, up showing a greater reduction in motility than vp. Introduction of activated ras into up had no effect on integrin levels, cell motility or tumorigenicity in nude mice; the only difference between up and upr was that upr showed increased adhesion to fibronectin. Examination of eight biopsies of cervical intraepithelial neoplasias with evidence of HPV infection revealed reduced or discontinuous integrin expression in the most severe lesions. We conclude that both in vivo and in culture keratinocytes the impaired differentiation that is associated with the presence of HPV is correlated with reduced integrin expression.

Cell Adhesion↗
PubMed1994

Ciprofloxacin enhances hematopoiesis and the peritoneal neutrophil function in lethally irradiated, bone marrow-transplanted mice.

We analyzed the effect of in vivo ciprofloxacin and ceftazidime treatment on the development of myeloid progenitors and on the survival of lethally irradiated mice rescued with syngeneic bone marrow transplantation (BMT). Ciprofloxacin treatment (15 mg/kg per dose three times daily for 5 days) enhanced myeloid progenitor (colony-forming cell [CFU-C]) number in the bone marrow and the survival of mice transplanted with suboptimal doses (1 x 10(5) of s[Ngeneic bone marrow cells (BMC). Twenty days postirradiation, 50% (38 of 76) of saline-treated mice transplanted with 1 x 10(5) cells died compared with 25% (19 of 76) of ciprofloxacin-treated mice (p < 0.05). Similarly, ciprofloxacin treatment enhanced survival of mice transplanted with 1 x 10(6) syngeneic bone marrow cells: 50% (38 of 76) of saline-treated mice died within 20 days vs. 15% (12 of 80) of ciprofloxacin-treated mice. In contrast, treatment with ceftazidime did not affect progenitor cell number or survival. On day 8 postirradiation, although lethally irradiated mice transplanted with 1 x 10(5) BMC treated with ciprofloxacin demonstrated similar white blood cell (WBC) and red blood cell (RBC) counts as saline-treated mice, a (1.9 +/- 0.2)-fold increase in the percentage of polymorphonuclear cells (PMN) was observed in the peripheral blood of ciprofloxacin-treated mice. On day 5 postirradiation, ciprofloxacin-treated mice showed a (1.6 +/- 0.2)-fold increase in the number of peritoneal PMN and a 6.5-fold increase in their antibacterial activity towards Salmonella typhimurium in comparison with saline-treated mice.

Animals↗
PubMed1994

Preliminary clinical experience with a thermal balloon endometrial ablation method to treat menorrhagia.

OBJECTIVE: To evaluate the clinical effectiveness and safety of a thermal balloon system to ablate the endometrium. METHODS: All 18 patients were candidates for hysteroscopic endometrial ablation or hysterectomy for menorrhagia and consented to a trial of the balloon technique of ablation. All procedures were done in the operating room under general anesthesia, except in one patient who had regional and another who had local anesthesia with analgesia. Follow-up of 6-34 months is reported. RESULTS: Fifteen subjects (83%) reported significant reduction in bleeding or amenorrhea. Two patients underwent subsequent hysterectomy and one a follow-up hysteroscopic examination with biopsy. Histology in these three cases showed areas of scar as well as areas of normal endometrial histology. In one uterus, the entire cavity and the endometrium were normal. The others had endometrial bands of scar and some contraction of the cavity. CONCLUSIONS: Based on follow-up results, the frequency of successful reduction of bleeding and/or amenorrhea in this small series is comparable to hysteroscopic methods of endometrial ablation. There were no complications. A larger trial is warranted to compare this method to hysteroscopic endometrial ablation.

Catheter Ablation↗
PubMed1994

The endometrial ablator: a new instrument.

A thermal balloon and control system designed to produce endometrial ablation blindly is described. A latex balloon on a plastic catheter is inserted into the uterus and connected to a control unit. The unit monitors the pressure and temperature of 5% dextrose in water, which has been injected into the balloon to make it conform to the size and shape of the endometrial cavity. The balloon contains a shielded heating element that is activated to heat the liquid in the balloon to a temperature of 92C. The pressure control deactivates the heating element if the pressure falls below 45 mmHg or rises above 165 mmHg. A timer controls and measures the elapsed interval of heating. The device was tested in human uterine specimens for the potential for uterine perforation, uterine rupture, and thermal effects. Subsequently, the device was tested in six patients in Mexico and four patients in London during hysterectomy just after the abdomen was opened. Thermistor probes were placed at various loci in the uterus to monitor temperature during activation of the thermal balloon. Serosal temperatures were unchanged and endometrial temperatures rose to about 90C. The extent of uterine tissue damage was determined in Mexico City by the zone of visible coagulation of the cut wall of the uterus following removal. In London, tissue diaphorase was measured to determine the depth of destruction of the cellular oxidative enzymes. These measurements varied from 3.3-10 mm under the conditions of time and temperature used. The safety features and the potential for clinical application are discussed.

Catheter Ablation↗