Deep microbial analysis of multiple placentas shows no evidence for a placental microbiome.

OBJECTIVES: To resolve the controversy regarding the presence of a microbiota in the placenta. DESIGN: Classical and molecular microbiological study. SETTING: All samples were collected during caesarean section. POPULATION: A total of 28 human placentas and six murine placentas. METHODS: All 28 human placentas were checked for 16S rRNA gene amplification products. Three locations from four selected human placentas and three 'environmental controls' for each placenta were placed in seven culture media. The four selected human placentas were further analysed using Gram stain, immunohistochemistry for bacteria, electron microscopy, and TaqMan RT-qPCR. Six placentas from three SPF mice were cut into four pieces each, and further analysed for 16S rRNA gene amplification. MAIN OUTCOME MEASURES: Microbiological and molecular evidence of bacteria. RESULTS: None of the placental cultures used for the full analysis, or their environmental cultures, was positive for bacterial growth. None of the other methods showed any evidence of bacteria. Immunohistochemistry showed negligible bacterial counts. None of the murine placentas showed evidence of 16S rRNA gene amplification. CONCLUSIONS: Our results support that the fetal environment in the womb is sterile. Based on the immunohistochemistry and the limit of detection of the other methods used, if a placental microbiome exists, it is of extreme low biomass, and thus its effect on clinical phenotypes is probably minor, if it exists at all. TWEETABLE ABSTRACT: Using several microbiological and molecular methods in parallel, we found no compelling evidence of bacteria in human and mouse placentas.

Resistance of T suppressor cells to cyclophosphamide administration in total-lymphoid-irradiated mice.

The effect of administration of cyclophosphamide (Cy) on the susceptibility of mice to tolerance induction and its influence on the rate of induced T suppressor cells in antigen-specific and nonspecific systems was assessed using mice that had undergone total lymphoid irradiation (TLI). Tolerance to bovine serum albumin (BSA) was induced in adult mice conditioned by a short course of fractionated total lymphoid irradiation with an injection of nondeaggregated BSA; Cy was administered upon termination of TLI, two days prior to administration of the tolerizing antigen. Susceptibility to tolerance induction and rate of induced T suppressor cells were assessed one month later. Cy-treated TLI mice and mice treated with TLI alone did not respond to a challenge with DNP-BSA in complete Freund's adjuvant, whereas a good response was obtained to an unrelated protein antigen. The induced T suppressor cell rate was measured by inhibition of a secondary anti DNP-BSA response in an adoptive transfer system. The rate of non-antigen-specific T suppressor cells was measured by inhibition of the mixed leukocyte reaction with Concanavalin A-induced T suppressor cells obtained from spleen cells of TLI-treated, or Cy-treated mice, or those given both treatments. The results reveal that Cy treatment of TLI-conditioned mice does not reduce the rate of induced of T suppressor cells in antigen-specific and antigen-nonspecific systems.

Regulation of the immune response in experimental models of autoimmune disorders: resistance of (NZB X NZW)F1 mice to tolerance induction in vivo.

Studies were carried out to test whether tolerance to alloantigens and to heterologous proteins could be induced in (NZB X NZW)F1 (B/W) female mice, compared with females of various other mouse strains, including BALB/c, C3H/eb, C57Bl/Ka and (BALB/c X C57Bl/6)F1. Untreated BALB/c and B/W mice were resistant to tolerance induction by deaggregated BSA, while all other strains were susceptible, as indicated by their lack of response to antigen challenge. Tolerance induction to BSA was further potentiated in all mouse strains including BALB/c with the exception of B/W, following prior conditioning of the mice with total lymphoid irradiation (TLI). Similarly, specific and permanent tolerance to H-2 incompatible alloantigens was successfully induced in TLI conditioned BALB/c, C3H/eb, (BALB/c X C57Bl/6)F1 injected with bone marrow cells, however, B/W mice were resistant. Stable chimeras could be established in TLI treated B/W mice only across a semi-allogeneic combination (BALB/c--greater than B/W). No graft vs host disease (GVHD) was observed in any of the chimeras including B/W mice. We conclude that B/W mice are resistant to tolerance induction to heterologous proteins and alloantigens, even after TLI conditioning. We postulate that this phenomenon is a function of both the intrinsic properties of the haemopoietic stem cells, including their differentiated progeny, as well as characteristics of their cellular microenvironment.

Sugar-specific endocytosis of glycoproteins by Lewis lung carcinoma cells.

Lewis lung carcinoma cells from tumors, metastasis nodules, or from culture bind fluorescent derivatives of neoglycoproteins containing alpha-D-glucose residues: This binding is competitively inhibited by neoglycoproteins containing alpha-D-glucose, by mannan, and by several other neoglycoproteins. Cell binding and uptake of the fluorescent derivatives of the neoglycoproteins was quantified by lysing the cells with an alkylpolyol (MAC 19 or MAC 18) and measuring the fluorescence intensity of the supernatant. The amount of cell-associated neoglycoprotein was higher at 37 degrees C than at 4 degrees C with LLC from tumor. The binding and uptake were inhibited by glycoconjugates containing alpha-D-glucose. These results suggest the presence of sugar specific receptors in Lewis lung carcinoma cells which are involved in a sugar-specific binding and endocytosis phenomenon. The implication of the existence of a carbohydrate-binding protein on the surface of Lewis lung carcinoma cells are discussed with regard to the in vivo behaviour of these cells, especially in relation to their metastatic properties and to the possibility of using neoglycoproteins as specific carriers of cytotoxic drugs. Hybrid molecules of gelonin and neoglycoprotein containing alpha-D-glucose were used as targetted toxin: The targetted toxin was found to bind to and to enter the intact cells and was 100 times more toxic than free drug.

The relationship between surface immunoglobulin isotype and immune function of murine B lymphocytes. V. IgD-bearing cells in immunological tolerance.

The role of IgD (delta) in the induction of tolerance in murine B lymphocytes was explored in the in vivo adoptive antibody response to bovine serum albumin (BSA). Delta+ or delta- B cells purified on the fluorescence-activated cell sorter were injected, with or without purified T cells, into lethally irradiated intermediate hosts, which were rendered tolerant by an injection of deaggregated BSA one day after the cell transfer. Spleen cells from the intermediate hosts were treated with anti-Thy-1.2 antiserum and complement mixed with normal T cells and were transferred to final hosts. The ability of the B cells of the final hosts to respond to the tolerogen BSA and to dinitrophenylated human gamma globulin antigen was examined. This approach enabled examination of the function of the B cell subpopulations in an environment free of tolerogen and of induced T suppressor cells. The results revealed that in the presence of T cells the delta+ subpopulation was highly resistant to tolerance induction, whereas the delta- subpopulation was highly susceptible to tolerance induction. However, there was no difference in susceptibility to tolerance induction between the two subpopulations when tolerance was induced in the absence of T cells.

Immunoregulatory cells in aging mice. I. Concanavalin A-induced and naturally occurring suppressor cells.

The status of concanavalin A (Con A)-triggered suppressor cells in aging mice was assessed in relation to untreated cells. Spleen cells of (C3H/ebJ X C57BL/6J)F1 and (BALB/cJ X C57BL/6J)F1 aging (20-41 months' old) and young (2-3 months' old) mice were triggered with Con A and examined for their capacity to suppress lymphocyte proliferative responses elicited by mitogens (Con A and phytohemagglutinin), by mixed lymphocyte reactions or by human gamma-globulin. Con A-triggered cells of the aging mice were found to be less efficient suppressors than those of the young, yet the aging mouse spleen cells exerted a more potent suppressive effect than the young, a priori, without pretreatment. Hence, the immune system in aging is characterized by a change in suppressor cell types; the Con A-triggered suppressors diminish and the naturally occurring, untreated ones become abundant.

Anion transport in red blood cells. I. Chemical properties of anion recognition sites as revealed by structure-activity relationships of aromatic sulfonic acids.

The present study is concerned with the chemical factors that determine the inhibitory properties of reversible aromatic sulfonic acids on sulfate exchange system of human red blood cells. Two series of compounds were tested for inhibitory potencies: benzene sulfonic acid (BS) and 2,2'-disulfonic stilbene (DS) derivatives, each series with substituent groups such as Cl, OH, NH2, NO2, NNN, N-acetamido, and N-benzoamido. As judged by various kinetic criteria, all congeners of BS and DS appear to have common sites of action in the anion transport system. The range of inhibitory potencies, as defined by the concentration required to produce 50% inhibition (ID50), varied over a 10(4) range (ID50:2-50,000 microM). The degree of inhibition was correlated with two physicochemical properties of the substituent groups: (a) lipophilicity, as judged by the pi values (Hansch factor) of the groups; and (b) the electronic character, as judged by sigma values (Hammett factor) of the groups. Optimal correlations were obtained with a linear combination of the two factors. Based on the above structure-activity relationships and on a comparison between the inhibitory properties of congeners of BS and DS, we suggest that the microenvironment of substrate recognition sites bears a positive multipolar character and possesses functionally essential groups with electron donor capacity embedded in a hydrophobic area.

Anion transport in red blood cells. II. Kinetics of reversible inhibition by nitroaromatic sulfonic acids.

The anion exchange system of human red blood cells is highly inhibited and specifically labeled by isothiocyano derivatives of benzene sulfonate (BS) or stilbene disulfonate (DS). To learn about the site of action of these irreversibly binding probes we studied the mechanism of inhibition of anion exchange by the reversibly binding analogs p-nitrobenzene sulfonic acid (pNBS) and 4,4'-dinitrostilbene-disulfonic acid (DNDS). In the absence of inhibitor, the self-exchange flux of sulfate (pH 7.4, 25 degrees C) at high substrate concentration displayed self-inhibitory properties, indicating the existence of two anion binding sites: one a high-affinity transport site and the other a low-affinity modifier site whose occupancy by anions results in a noncompetitive inhibition of transport. The maximal sulfate exchange flux per unit area was JA = (0.69 +/- 0.11) X 10(-10) moles . min-1 . cm-2 and the Michaelis-Menten constants were for the transport site KS = 41 +/- 14 mM and for the modifier site Ks' = 653 +/- 242 mM. The addition to cells of either pNBS at millimolar concentrations or DNDS at micromolar concentrations led to reversible inhibition of sulfate exchange (pH 7.4, 25 degrees C). The relationship between inhibitor concentration and fractional inhibition was linear over the full range of pNBS or DNDS concentrations (Hill coefficient n approximately equal to 1), indicating a single site of inhibition for the two probes. The kinetics of sulfate exchange in the presence of either inhibitor was compatible with that of competitive inhibition. Using various analytical techniques it was possible to determine that the sulfate transport site was the target for the action of the inhibitors. The inhibitory constants (Ki) for the transport sites were 0.45 +/- 0.10 microM for DNDS and 0.21 +/- 0.07 mM for pNBS. From the similarities between reversibly and irreversibly binding BS and DS inhibitors in structures, chemical properties, modus operandi, stoichiometry of interaction with inhibitory sites, and relative inhibitory potencies, we concluded that the anion transport sites are also the sites of inhibition and of labeling of covalent binding analogs of BS and DS.

Anion transport in red blood cells. III. Sites and sidedness of inhibition by high-affinity reversible binding probes.

Studies of binding of the reversible inhibitor DNDS (for abbreviations, see Nomenclature) and red blood cell membranes revealed 8.6 +/- 0.7 x 10(5) high-affinity binding sites per cell (KD = 0.8 +/- 0.4 muM). Under conditions of "mutual depletion," inhibition studies of anion exchange revealed 8.0 +/- 0.7 x 10(5) DNDS inhibitory sites per cell (KD = 0.87 +/- 0.04 muM). Binding and kinetics studies with DNDS indicate that there are 0.8 -- 0.9 x 10(6) functional anion transport sites per blood cell. The transport of DNDS displayed high temperature and concentration dependencies, chemical specificity, susceptibility to inhibition by DIDS, and differences between egress and ingress properties. Under conditions of no DNDS penetration (e.g., 0 degrees C), inhibition of anion exchange by DNDS showed marked sidedness from the outside inhibitions and were demonstrable at micromolar concentrations, whereas from the inside no inhibition occurred even at millimolar concentrations. The asymmetry of DNDS transport properties and the sidedness of binding and inhibition suggest that anion transport sites have a very low affinity for or are inaccessible to DNDS at the inner membrane face. The site of DNDS permeation, although susceptible to DIDS, is apparently not the site of anion exchange.

Recombination of integral and peripheral protein fractions from human red cell membrane with homologous lipids.

Integral and peripheral protein fractions from human red cells membranes were recombined with a total red cell lipid extract and with homologous lipids in varying mixtures, by dialysis from 2-chlorethanol solutions. The 2 protein fractions were compared for lipid binding capacity and for selectivity towards individual lipids.