Avian antigen-binding cells. II. Enrichment of antigen-binding B and T cells.

Antigen-binding B and T cells from chicken spleens were selected on plates of antigen-derived gelatin. Hapten-specific B cells from DNP-immune normal chicken spleens were selected on DNP-gelatin. As much as 165-fold functional enrichment of precursors of anti-DNP antibody-producing cells, as measured in an adoptive transfer system, was achieved. However, the enrichment of DNP-binding cells assessed by rosette formation and autoradiography was no more than 25-fold. HGG-specific T cells from bursectomized agammaglobulinemic chickens immunized with deaggregated HGG were selected on HGG-gelatin. In the fraction adherent to HGG-gelatin, at least a 20-fold enrichment of suppressors of the antibody response to TNP-HGG, as measured by adoptive transfer, was accomplished. In contrast, no more than 6-fold enrichment of HGG-binding cells was detected by autoradiography. Antigen-specific depletion and enrichment of suppressor T cells and of HGG-binding cells occurred in parallel, suggesting that suppressor cells can bind soluble antigen and can be isolated on antigen coupled to a solid support.     

Gastrointestinal stem cells. II. Intestinal stem cells

Current views of the identity, distribution, and regulation of small intestinal epithelial stem cells and their immediate progeny are discussed. Recent works implicating Wnt signaling in stem and progenitor proliferation, the involvement of Notch signaling in epithelial lineage specification, and the role of hedgehog and bone morphogenetic protein families in crypt formation are integrated. We had the good fortune that many of these papers came in pairs from independent groups. We attempt to identify points of agreement, reinterpret each in the context of the other, and indicate directions for continued progress.     

Gastrointestinal stem cells. I. Pancreatic stem cells

The transplantation of islets isolated from donor pancreas has renewed the interest in cell therapy for the treatment of diabetes. In addition, the capacity that stem cells have to differentiate into a wide variety of cell types makes their use ideal to generate beta-cells for transplantation therapies. Several studies have reported the generation of insulin-secreting cells from embryonic and adult stem cells that normalized blood glucose values when transplanted into diabetic animal models. Finally, although much work remains to be done, there is sufficient evidence to warrant continued efforts on stem cell research to cure diabetes.     

Characterization of T and B antigen-binding cells for beta-galactosidase. II. T antigen-binding cells.

The results reported in this paper suggest that the specific Z-binding cells of the normal mouse include a large portion of T lymphocytes. Depletion of T cells with anti-theta serum and cortisone indicates that the majority of the ZBC of the thymus, which occur at frequencies of about 150/10(6), are indeed T cells. Similar treatment of spleen cells suggests that approximately half the binding cells in that organ are contributed by the T lymphocyte population. T-and B-enriched populations obtained from the spleen by using differential adherence to nylon wool contained equal numbers of ZBC and bound equivalent amounts of the antigen. Hence, there appears to be a high frequency of T lymphocytes that can be shown to bind beta-galactosidase specifically in both the thymus and spleen of normal mice.     

Specific, transient suppression of the immune response by HGG tolerant spleen cells. II. Effector cells and target cells.

In a previous report, it was shown that spleen cells from mice made tolerant to human gamma-globulin (HGG)5 could specifically inhibit the immune response of normal spleen cells after adoptive transfer to lethally irradiated recipients. However, that report also showed that the suppressive activity was only transiently associated with tolerant spleen cell populations. It was concluded from those experiments that while suppressive activity could be demonstrated in tolerant spleen cells under certain conditions, such activity was not obligatory for the maintainance of the tolerant state. The experiments presented here were performed to determine the nature of the effector cell(s) and the target cell(s) involved in this system of suppression of the immune response. Treatment of cells from tolerant animals with anti-thymocyte serum and complement to remove thymus-derived (T) cells completely abrogated suppresive activity. Removal of adherent cells from tolerant spleen cells by passage over glass wool columns resulted in partial loss of the suppression. The inhibitory activity of the suppressor cells was resistant to 900 R irradiation regardless of whether the tolerant spleen cells were irradiated before or after adoptive transfer. The cellular target(s) for the supprssor cells was examined by using lipopolysaccharide (LPS) as an alternative source of helper activity for the response to HGG. LPS, injected at the time of the initial antigenic challenge of mice that had been reconstituted with tolerant and normal spleen cells, prevented the expression of suppression against bone marrow-derived (B) cells. However, when LPS was presented only at the time of secondary antigenic challenge, it was unable to overcome suppression of the immune response of reconstituted recipients. Thus, LPS could produce a state where the B cells were resistant to suppression, but LPS could not rescue the responsiveness of B cells once the cells in the reconstituted recipient had been suppressed. In addition, the immune response to both the hapten dinitrophenol (DNP) and the carrier (HGG) were suppressed when recipients of tolerant and normal spleen cells were challenged with DNP6HGG. This indicates that T helper cells are also a target for suppression. The results presented in this paper are discussed in relation to a possible mechanism of suppression which proposes that suppressive activity represents the induction of tolerance in immunologically competent cells by HCG which is closely associated with the tolerant spleen cells.     

Epithelial cells and their neighbors. III. Interactions between intraepithelial lymphocytes and neighboring epithelial cells

Intraepithelial gammadelta-T cells are present in all epithelial tissues, where they reside in close contact with neighboring epithelial cells. Our data support the idea that the role of these cells is to monitor neighboring cells for signs of damage or disease. Once a problem is detected, the intraepithelial gammadelta-T cells can lyse damaged or malignant epithelial cells, directly participate in tissue repair through production of epithelial growth factors, and play a unique role in the recruitment of inflammatory cells to the site of damage. Intraepithelial gammadelta-T cells play unique roles in homeostasis and disease.     

Antigen-presenting cells and anti-Salmonella immunity.

The present article summarizes studies aimed at addressing the role of antigen-presenting cell populations, particularly dendritic cells (DC), in the immune response to Salmonella typhimurium. Data from in vitro studies shed light on presentation of antigens expressed in Salmonella on major histocompatibility complex class I and class II molecules by infected DC and macrophages, and the activation state of DC following infection. Finally, data from in vivo studies addressing the role of DC and defined DC subsets during the host response to Salmonella using a murine infection model are discussed.     

Guidepost cells.

Guidepost cells, as classically defined in the grasshopper embryo have only rarely been found in other systems. If the concept of guidepost cells is expanded, recognizing that any special role of specific cells in axon guidance is a function of the entire landscape in which axons are growing, and that growth cone--guidepost interactions may share mechanisms with many other cell--cell interactions, then numerous examples are found in both the peripheral and central nervous systems of many species.     

Mucosal mast cells. I. Isolation and functional characteristics of rat intestinal mast cells

We have developed a procedure for the dispersion of mast cells from the intestinal lamina propria (LP) and epithelium of rats infected with the intestinal nematode, Nippostrongylus brasiliensis. The dispersed cells are morphologically and histochemically similar to intestinal mucosal mast cells (MMC) in situ and are distinguishable from peritoneal mast cells (PMC). MMC derived from the LP or epithelium of parasitized animals secrete histamine in response to the specific parasite antigens as well as anti-IgE. Unlike PMC, these cells are unresponsive to the basic secretagogues 48/80 and bee venom peptide 401. Similarly, bee venom peptide 401 conjugated with dansyl chloride binds to PMC and mast cells in the thymus and intestinal serosa, but not to mast cells in or derived from the intestinal LP and epithelium. Studies on PMC treated by the intestinal cell isolation procedure show that the functional characteristics of the MMC cannot be solely attributed to the isolation procedure. Thus, MMC have been isolated and shown to be morphologically, histochemically, and functionally different from PMC, as suggested by previous in vivo studies of the normal intestine.     

Primary anti-trinitrophenyl memory cells investigated by plaque-forming cells and ELISA.

Primary anti-trinitrophenyl antibody production was investigated from spleen cells of mice immunized with trinitrophenylated-keyhole limpet hemocyanin, using the plaque-forming cell method and ELISA. Cells taken 5 days after antigen injection do not produce IgE, but do produce IgM and IgG1 anti-trinitrophenyl antibodies as demonstrated by plaque-forming cells. Substantial increase of IgM, IgG1, and IgE antibody production was seen from cells taken 7 days after immunization, followed by a rapid decline. By ELISA it was seen that cells taken 3 days after immunization already produce small amounts of anti-trinitrophenyl antibodies. Presence of antigen from the start of the cultures did not increase antibody production from cells taken 3 days after immunization, but potentiated antibody secretions from cells taken 5 days or later after immunization. This potentiation was interpreted as recruitment of antibody-forming cells from early memory B cells. The presence of IL-4 from the start of the cultures had no appreciable effect. Cell sorting with specific antibody-coated magnetic beads showed that plaque-forming cells from nonsorted cells, membrane IgE+ or membrane IgE- cells secreted similar amounts of anti-trinitrophenyl IgG1 and IgE antibodies. No difference in anti-trinitrophenyl IgM, IgG1, or IgE production was found in controls; cells sorted negatively or positively for CD23. The data show that memory B cells can be demonstrated already on Day 5 after immunization, and their antigen-induced antibody secretion is IL-4 dependent.     

Time domain dielectric spectroscopy study of human cells. II. Normal and malignant white blood cells.

The dielectric properties of human lymphocyte suspensions were studied by time domain dielectric spectroscopy (TDDS). Nine populations of malignant and normal lymphocytes were investigated. Analysis of the dielectric parameters of cell structural parts were performed in the framework of Maxwell-Wagner mixture formula and the double-shell model of cell. The specific capacitance of the cell membranes was estimated by the Hanai-Asami-Koisumi formula. It was shown that the dielectric permittivity, capacitance and conductivity values of cell membranes are higher for normal lymphocytes than for the malignant ones. The difference of the same parameters for normal B- and T-cells is also discussed.     

Role of NKT cells in the digestive system. II. NKT cells and diabetes

Natural killer T (NKT) cells are a subset of regulatory T lymphocytes that recognize glycolipid antigens presented by the major histocompatibility complex class I-related glycoprotein CD1d. NKT cells have been implicated in regulating the progression of Type 1 diabetes (T1D) in human patients and in an animal model for T1D. In addition, glycolipid agonists of NKT cells have been successful in preventing diabetes in mice, raising enthusiasm for the development of NKT cell-based therapies for T1D.     

Development of rat mast cells in vitro. I. Differentiation of mast cells from thymus cells.

Mast cells were differentiated by long-term culture of rat thymus cells on rat embryonic fibroblasts monolayers. Mature mast cells obtained in the culture were morphologically similar to normal peritoneal and thoracic mast cells and possessed specific receptors for IgE on their surface. In culture, blast cells appeared on the monolayer several days after seeding of thymus cells. These cells developed into young mast cells in the monolayer and became free in the culture medium with maturation. Receptors for IgE were detected on the surface of mastoblasts which contained a small amount of metachromatic granules. Evidence was obtained which suggested that the number and/or affinity of the receptors for IgE increases with maturation of mast cells. It was found that some mast cells differentiated from monolayers of embryo cells without seeding thymus cells. The present experiments, however, clearly showed that mast cells can be differentiated from thymus cell culture without monolayer. It appears that both thymus and embryo tissues contain precursors of mast cells.     

Lysis of tumor cells by antibody and complement. VI. Enhanced killing of enzyme-pretreated tumor cells.

The ascites form of a chemically induced guinea pig hepatoma, line-10, was resistant to killing in vitro by xenogeneic antibody and guinea pig complement. Pretreatment of line-10 cells with certain proteolytic enzymes rendered tham susceptible to the killing action of antibody and guinea pig complement. The effects of enzyme pretreatment were dependent on enzyme concentration, temperature, and could be blocked by addition of competitive or non-competitive inhibitors. The effect of the enzyme treatment could reversed by incubating the treated cells at 37 degrees C (but not at 0 degrees C), in the absence of the enzyme. Effective enzymes included ficin, bromelain, pronase, elastase, papain, trypsin, collagenase, lipases type I and type VI, and the neuraminidase preparation isolated from Clostridium perfringens. The activity of the lipase preparations and the neuraminidase preparation isolated from Clostridium perfringens appeared to be caused by proteolytic enzyme contamination. Enzyme preparations that proved ineffecitve in rendering the line-10 cells sensitive to killing by antibody and guinea pig complement included DNase, RNase, beta-glucuronidase type 6A or type B10, hyaluronidase type V or type VI, and pectinesterase.     

Enucleation of mammalian cells by cytochalasin B. II. Formation of hybrid cells and heterokaryons by fusion of anucleate and nucleated cells

The development of methods for the formation of hybrid cells and heterokaryons by virus-induced fusion of chemically-enucleated cells and nucleated cells has been described. Heterokaryons and hybrid cells formed by fusion of anucleate mouse peritoneal macrophages (MPM) and nucleated mouse L and human HEp-2 cells were identified by mixed haemadsorption, by their sensitivity to trypsin and by their capacity to ingest antibody-coated sheep red blood cells. The expression of macrophage markers in these cells declined rapidly after fusion. Hybrid cell and heterokaryon formation was identified in mixed cultures of anucleate L cells and nucleated MPM, and was accompanied by the reactivation of DNA synthesis in the macrophage nuclei. Other hybrids and heterokaryons were formed by virus-induced fusion of anucleate MPM and nucleated chick embryo erythrocytes and anucleate L cells and nucleated HEp-2 cells. The value of anucleate-nucleate cell hybrids in the study of metabolic and genetic regulation in mammalian cells is discussed.