Intestinal alkaline phosphatase preserves the normal homeostasis of gut microbiota.

BACKGROUND AND AIMS: The intestinal microbiota plays a critical role in maintaining human health; however, the mechanisms governing the normal homeostatic number and composition of these microbes are largely unknown. Previously it was shown that intestinal alkaline phosphatase (IAP), a small intestinal brush border enzyme, functions as a gut mucosal defence factor limiting the translocation of gut bacteria to mesenteric lymph nodes. In this study the role of IAP in the preservation of the normal homeostasis of the gut microbiota was investigated. METHODS: Bacterial culture was performed in aerobic and anaerobic conditions to quantify the number of bacteria in the stools of wild-type (WT) and IAP knockout (IAP-KO) C57BL/6 mice. Terminal restriction fragment length polymorphism, phylogenetic analyses and quantitative real-time PCR of subphylum-specific bacterial 16S rRNA genes were used to determine the compositional profiles of microbiotas. Oral supplementation of calf IAP (cIAP) was used to determine its effects on the recovery of commensal gut microbiota after antibiotic treatment and also on the colonisation of pathogenic bacteria. RESULTS: IAP-KO mice had dramatically fewer and also different types of aerobic and anaerobic microbes in their stools compared with WT mice. Oral supplementation of IAP favoured the growth of commensal bacteria, enhanced restoration of gut microbiota lost due to antibiotic treatment and inhibited the growth of a pathogenic bacterium (Salmonella typhimurium). CONCLUSIONS: IAP is involved in the maintenance of normal gut microbial homeostasis and may have therapeutic potential against dysbiosis and pathogenic infections.

Assessment of ability of murine and human anti-lipid A monoclonal antibodies to bind and neutralize lipopolysaccharide.

The use of monoclonal antibodies (mAbs) directed to lipid A for the therapy of gram-negative sepsis is controversial. In an attempt to understand their biologic basis of action, we used a fluid-phase radioimmunoassay to measure binding between bacterial lipopolysaccharide (LPS) and two IgM mAbs directed to lipid A that are being evaluated for the treatment of gram-negative bacterial sepsis. Both antibodies bound 3H-LPS prepared from multiple strains of gram-negative bacteria when large excesses of antibody were used, although binding was modest and only slightly greater than control preparations. We also studied the ability of each anti-lipid A antibody to neutralize some of the biological effects of LPS in vitro. Despite large molar excesses, neither antibody neutralized LPS as assessed by the limulus lysate test, by a mitogenic assay for murine splenocytes, or by the production of cytokines interleukin (IL)-1, IL-6, or tumor necrosis factor from human monocytes in culture medium or in whole blood. Our experiments do not support the hypothesis that either of these anti-lipid A mAbs function by neutralizing the toxic effects of LPS.

Binding of a dimeric derivative of vancomycin to L-Lys-D-Ala-D-lactate in solution and at a surface.

BACKGROUND: The emergence of bacteria that are resistant to vancomycin (V), a glycopeptide antibiotic, results from the replacement of the carboxy-terminal D-Ala-D-Ala of bacterial cell wall precursors by D-Ala-D-lactate. Recently, it has been demonstrated that covalent dimeric variants of V are active against vancomycin-resistant enterococci (VRE). To study the contribution of divalency to the activities of these variants, we modeled the interactions of V and a dimeric V with L-Lys-D-Ala-D-lactate, an analog of the cell-wall precursors of the vancomycin-resistant bacteria. RESULTS: A dimeric derivative of V (V-Rd-V) was found to be much more effective than V in inhibiting the growth of VRE. The interactions of V and V-Rd-V with a monomeric lactate ligand - diacetyl-L-Lys-D-Ala-D-lactate (Ac2KDADLac) - and a dimeric derivative of L-Lys-D-Ala-D-lactate (Lac-R'd-Lac) in solution have been examined using isothermal titration calorimetry and UV spectroscopy titrations; the results reveal that V-Rd-V binds Lac-R'd-Lac approximately 40 times more tightly than V binds Ac2KDADLac. Binding of V and of V-Rd-V to Nalpha-Ac-L-Lys-D-Ala-D-lactate presented on the surface of mixed self-assembled monolayers (SAMs) of alkanethiolates on gold indicates that the apparent off-rate for dissociation of V-Rd-V from the surface is much slower than that of V from the same surface. CONCLUSIONS: The results are compatible with the hypothesis that divalency is responsible for tight binding, which correlates with small values of minimum inhibitory concentrations of V and V-Rd-V.

Future prospects for vaccine adjuvants.

Since the landmark experiments of Ramon 60 years ago, attempts have been made to augment the humoral and cellular responses to administered antigens in order to develop more potent and less toxic vaccines. The need for an acceptable adjuvant suitable for clinical use has been underscored by recent advances in recombinant biotechnology and synthetic chemistry which have made it possible to create antigens that are smaller and better characterized, yet less immunogenic, than before. It is likely that these antigens will require an adjuvant to achieve protective immunity. Some of these same technological advances, together with a better understanding of the immune system in general, have permitted the study of adjuvants to evolve from an empirical field to a developmental one. This article discusses the currently known agents capable of immunopotentiation and possible strategies for their use in future vaccines.

Distinct granzyme expression in human CD3- CD56+ large granular- and CD3- CD56+ small high density-lymphocytes displaying non-MHC-restricted cytolytic activity.

Cultured natural killer (NK) cells derived from CD3- CD56+ high-density small lymphocytes (HDLs) exhibit similar morphology and high levels of non-major histocompatibility complex-restricted (NK) cytotoxicity equivalent to those of cultured NK cells from CD3- CD56+ low-density large granular lymphocytes (LGLs). To examine the similarities and differences between NK cells from HDLs and NK cells from LGLs, we investigated the expression of three distinct members of the granule serine protease (granzyme) family within cultured CD3- CD56+ LGLs and HDLs. CD3- subpopulations of nonadherent peripheral blood mononuclear cells, LGLs (density < 1.063 g/ml), and HDLs (density > 1.063 g/ml) were stimulated to proliferate in culture. The cultured cells from each population were entirely CD3- CD56+ and were indistinguishable in terms of their increased granularity and size once activated. All cultured CD3- CD56+ LGLs and HDLs displayed cytolytic activity against K562 and immunoglobulin-coated P815. Western analysis detected perforin in both cultured LGL and HDL populations. Cultured HDLs and LGLs both expressed BLT-esterase activity and human granzyme A mRNA. Granzyme B mRNA and protein and Asp-ase activity were detected in unstimulated and cultured LGLs and cultured HDLs. By contrast, unstimulated HDLs did not express significant levels of granzyme B. High levels of Hu-Met-1 granzyme mRNA and Met-ase activity were detected only in cultured LGLs. Thus, despite the development of large granular morphology during proliferation, interleukin-2 cultured CD3- CD56+ HDLs display a different pattern of granzyme expression from CD3- CD56+ LGLs. These data also further suggest an unusually restricted expression of the Hu-Met-1 granzyme in LGLs.

Preparation and characterization of human bone marrow-derived macrophages.

Bone marrow-derived macrophages were prepared from human bone marrow mononuclear cells following cultivation in GCT-conditioned medium (GCT-CM) and purification by adherence to fibronectin-coated flasks. The growth of bone marrow mononuclear cells in GCT-CM was dependent on the shape of the culture vessels, being increased in round-bottomed versus flat-bottomed wells. Proliferation was confined to nonadherent cells; like blood monocytes, bone marrow-derived macrophages did not incorporate [3H]thymidine in response to GCT-CM or human serum. Purified macrophages from this source expressed nonspecific esterase and OKM1, OKla, FMC 17, 32, and 34 and 25F9 antigens but lacked Mo2. They expressed high levels of an inactivator of plasminogen activator, minactivin, and gave a substantial metabolic burst in response to phorbol myristate acetate or opsonized (but not unopsonized) zymosan. Bone marrow-derived macrophages acted as accessory cells in the response of T lymphocytes to phytohemagglutinin. The results suggest that liquid bone marrow cultures are useful in the study of the differentiation of human mononuclear phagocytes.

Outer membrane protein A (OmpA), peptidoglycan-associated lipoprotein (PAL), and murein lipoprotein (MLP) are released in experimental Gram-negative sepsis.

We previously showed that Escherichia coli bacteria incubated in normal human serum release complexes that contain three conserved Gram-negative bacterial outer membrane proteins (OMPs) and LPS. We have identified the OMPs as outer membrane protein A (OmpA), peptidoglycan-associated lipoprotein (PAL), and murein lipoprotein (MLP). These OMPs are conserved among enteric Gram-negative bacteria and are bound by IgG in antisera raised to heat-killed rough bacteria such as E. coli J5 (J5 IgG). The present experiments were performed to further analyze the release of these OMPs in a rat wound infection model of sepsis. Plasma was collected from thermally injured rats with E. coli O18 sepsis and filtered. LPS was affinity-purified from plasma filtrates using monoclonal antibody specific for the O-polysaccharide side chain of E. coli O18 LPS. Plasma filtrates were also incubated with J5 IgG conjugated to magnetic beads. Affinity-purified samples were analyzed for the OMPs by immunoblotting. OmpA, PAL, and MLP were released into septic rat blood in complexes with LPS. PAL was consistently present in samples affinity-purified using J5 IgG. The results indicate that OmpA, PAL, and MLP are released and circulate in experimental Gram-negative sepsis and suggest that a proportion of released OMPs are tightly associated with LPS.

A method for the production and quantitative assay of human lymphokine preparations.

A procedure for the preparation and assay of a human lymphokine supernatant is described. Tonsillar lymphocytes at a concentration of 2 X 10(7)/ml in a serum-free medium are incubated for 2 h with PHA-P, washed free of unbound mitogen and incubated for a further 17 h. The supernatant is harvested and concentrated, and the active protein fraction is absorbed to and eluted from a column of hydroxylapatite. The eluate is desalted and sterile filtered. The preparations contain active material released from 10(8) cells in 1 ml and do not contain residual PHA-P. The most active preparations are obtained using PHA-P as the mitogen and at concentrations of 25-50 microgram/ml during the 2 h pulsing period. Activity is assessed by a quantitative assay based on the maintenance of mitogen activated blast cells in culture for 2 days.

Rejection of cultured thyroid allografts by the transfer of sensitized LYT 2+ T cells.

Cultured BALB/c (H-2d) thyroid allografts, which are accepted permanently in fully immunocompetent allogeneic CBA/H (H-2k) recipients, are rejected following the transfer of immune spleen cells generated by immunization of recipient-strain mice with P815 (H-2d) which, like the cultured thyroid, expresses only class I major histocompatibility (MHC) antigens. Depletion of Lyt 2+ T cells from the transferred population abolished the capacity of immune spleen cells to trigger rejection, whereas depletion of L3T4+ T cells was without effect. Transfer of in-vitro-activated H-2d specific Lyt 2+ T cells also triggered rejection of cultured thyroid allografts. The results are consistent with previous studies showing that sensitized Lyt 2+ T cells are required for rejection of cultured pancreatic islet allografts, which also express only class I MHC antigens. These results are in accord with the notion that Lyt 2+ T cells are activated by and are active against class I MHC antigens.

Induction of class II major histocompatibility antigens on thyroid, adult pancreatic islet, and fetal proislet allografts.

Cultured BALB/c (H-2d) thyroid, adult pancreatic islet and fetal proislet tissues can be accepted permanently in CBA/H (H-2k) recipients until rejection is triggered by the transfer of antidonor strain activated immune T cells. Class II MHC antigens are not expressed on the accepted grafts and are induced following immune cell transfer, but expression is quantitatively and qualitatively different for the different tissues. Thyroid allografts show intense class II MHC antigen expression early after immune cell transfer and before extensive tissue destruction has occurred. In contrast, adult islet and fetal proislet allografts show patchy and cytoplasmic staining usually associated with areas of tissue destruction. Gamma-interferon treatment of tissues in vitro showed that proislet tissue has a greater capacity for class II MHC antigen induction than adult islet tissue. The differences in the capacity for class II MHC induction by fetal and adult islet tissue may be important in relation to the pathogenesis of autoimmune disease.

Cyclosporin inhibits a two-signal mechanism for the generation of cytotoxic NK-like cells, from small lymphocyte precursors.

Cytotoxic cells resembling NK cells are generated from CD 3- small, lymphokine (LK) nonresponsive precursor cells during an 8 day culture period with mitomycin C-treated autologous T cell blasts and LK. Recombinant interleukin 2 (rIL2) can replace LK in this coculture system. Both stimuli are required for the generation of the cytotoxic cells, which can then be maintained in short term cultures by LK alone. The generation of the cytotoxic cells was inhibited in cultures containing 0.1 micrograms/ml cyclosporin (Cys). Cys did not inhibit the LK dependent growth of the cytotoxic cells. Cys also inhibited the mitogen and LK dependent stimulation of purified T cells, but did not inhibit the LK dependent proliferation of T cell blasts. In contrast to the results for the generation of the cytotoxic cells, Cys did not inhibit the activation/growth of peripheral blood NK cells by LK or rIL2. These studies support the notion that the precursors of the NK-like cytotoxic cells are at an earlier stage of differentiation than peripheral blood NK cells responsive to LK alone, and that the precursor cells are triggered into a differentiation pathway leading to the NK-like cytotoxic cells by a two-signal mechanism.

Protective effects of anti-O polysaccharide and anti-lipid A monoclonal antibodies on pulmonary hemodynamics.

Monoclonal antibodies (MAbs) directed to endotoxin can protect in some animal models against the pathophysiological effects of endotoxin infusion. When 0.02 microgram/kg of lipopolysaccharide (LPS) derived from Escherichia coli O111:B4 was incubated in vitro for 2 h with the murine immunoglobulin G MAb, 5B10, directed against the O-polysaccharide antigenic domain of E. coli O111:B4 and then the mixture was infused into sheep, we noted significant protection. The second temperature peak was decreased (P < 0.05 vs. LPS control). The acute pulmonary arterial pressure elevation was diminished (mean peak pulmonary arterial pressure 23.2 +/- 2.5 mmHg, P < 0.05 vs. LPS control), and the peak plasma thromboxane B2 level was reduced (mean peak thromboxane B2 level 0.50 +/- 0.15 ng/ml, P < 0.05 vs. LPS control). In contrast, preincubation of the LPS with a human immunoglobulin M MAb, HA-1A, directed against the core glycolipid of the LPS molecule provided no protective effects in this sheep model. This finding is in agreement with recent studies reporting HA-1A may bind to antibiotic-treated bacteria but not to purified smooth LPS.

Tolerance to low-dose endotoxin in awake sheep.

Dose response and tolerance to a small intravenous dose of Serratia marcescens lipopolysaccharide (LPS) were studied in awake sheep. Core temperature significantly increased after a dose of 0.002 micrograms/kg; changes in pulmonary arterial pressure, pulmonary vascular resistance, plasma thromboxane B2, and circulating leukocyte concentration occurred after 0.02 micrograms/kg; plasma 6-keto-prostaglandin F1 alpha increased after 0.2 micrograms/kg. Development of acute tolerance was studied by injection of S. marcescens LPS (0.02 micrograms/kg iv) on 3 consecutive days: pulmonary arterial pressure and thromboxane B2 levels were significantly lower than controls after the second dose, whereas fever and the degree of leukopenia were not diminished until the third dose. After intravenous administration of LPS given in increasing doses from 0.1 to 3.2 micrograms/kg three times weekly over 7 wk, there were no measurable changes in any of the above parameters after challenge with S. marcescens LPS (0.02 micrograms/kg) after a 1-wk rest period. In awake sheep, small intravenous doses of LPS can cause physiologically important changes of the pulmonary circulation and can alter the hemodynamic and eicosanoid mediator responses to subsequent challenges with LPS. Large intravenous doses of LPS can ablate the physiological responses to subsequent small doses of LPS.

Antiendotoxin strategies.

Endotoxin is a potent stimulator of the inflammatory response and is believed to initiate the pathology in Gram-negative sepsis. Agents are being developed that bind and neutralize or block the effects of endotoxin, with the goal of improving outcome in the treatment of sepsis. Strategies discussed in this article include anti-LPS antibodies, LPS binding proteins and lipoproteins, polymyxin B conjugates, lipid A analogues, and extracorporeal techniques for endotoxin removal.