31P NMR study of intracellular pH during the respiratory burst of macrophages

The metabolic events occurring during the respiratory burst of macrophages previously primed in vivo with lipopolysaccharide were studied by 31P nuclear magnetic resonance, using the P388D1 cell line as a model of the mature macrophages. Using perchloric acid extracts, the presence of phosphocreatine was shown in the primed cells, indicating that in control P388 D1 macrophages, in which no phosphocreatine was seen, in vivo maturation was incomplete. The cells primed in vivo exhibited greater maturation than the control cells, as well as greater creatine kinase activity. Perfusion of gel-embedded macrophages allowed the monitoring of phosphorylated metabolite peak intensities and of the intracellular pH. After the respiratory burst of the primed macrophages had been triggered by concanavalin A, these intensities did not alter significantly, but the intracellular pH decreased. 31P NMR spectra reflected transient acidification in the primed cells, possibly due to the formation of endocytic vesicles and their fusion with lysosomes.     

Mycobacterium bovis BCG decreases MHC-II expression in vivo on murine lung macrophages and dendritic cells during aerosol infection

Mycobacterium tuberculosis and M. bovis BCG infect APCs. In vitro, mycobacteria inhibit IFN-gamma-induced MHC-II expression by macrophages, but the effects of mycobacteria on lung APCs in vivo remain unclear. To assess MHC-II expression on APCs infected in vivo, mice were aerosol-infected with GFP-expressing BCG. At 28 d, ∼1% of lung APCs were GFP+ by flow cytometry and CFU data. Most GFP+ cells were CD11b^high/CD11c^neg-mid lung macrophages (58-68%) or CD11b^high/CD11c^high DCs (28-31%). Lung APC MHC-II expression was higher in infected mice than naïve mice. Within infected lungs, however, MHC-II expression was lower in GFP+ cells than GFP− cells for both macrophages and DCs. MHC-II expression was also inhibited on purified lung macrophages and DCs that were infected with BCG in vitro. Thus, lung APCs that harbor mycobacteria in vivo have decreased MHC-II expression relative to uninfected APCs from the same lung, possibly contributing to evasion of T cell responses.     

13C and 31P NMR studies on sugar metabolism in Bombyx mori and Philosamia cynthia ricini larvae

Studies were made on ¹³C and ³¹P NMR in larvae of two species of silkworm, Bombyx mori and Philosamia cynthia ricini, in vivo as well as in vitro to determine the pathways of glucose utilization, especially those to amino acids as components of silk fibroin. Results showed that the ¹³C of [1-¹³C]glucose administered orally into 5th instar larvae of both species was incorporated into glucose-1-phosphate, glucose-6-phosphate and trehalose. Serine, glutamate, glutamine, citrate, malate, trehalose and sorbitol-6-phosphate were detected in the hemolymphs of these larvae as metabolites of [1-¹³C]glucose. Two days after [1-¹³C]glucose administration, labeled alanine, glycine, serine, urea, glycogen, trehalose and glycerol were clearly detected in Bombyx larvae. Starvation caused rapid consumption of administered [1-¹³C]glucose with very little accumulation of ¹³C in glycogen or trehalose. In the in vivo³¹P NMR spectra of Bombyx larvae, ATP, arginine phosphate, sorbitol-6-phosphate, uridine diphosphoglucose, phosphoenolpyruvate and inorganic phosphate were detected with some sugar phosphates, such as glucose-1-phosphate and glucose-6-phosphate. During starvation, the intensity of the signal of inorganic phosphate increased and those of sugar phosphate other than sorbitol-6-phosphate decreased, but these changes were reversed by oral administration of glucose.     

Accessory cells in the in vitro generation of type C virus-specific T-killer lymphocytes. III. Two methods of antigen presentation of cytolytic T-lymphocyte precursors

F₁ hybrid mice primed in vivo with tumor cells bearing the virus-induced FMR antigen and the H-2 specificities of each parent are able to produce in vitro in secondary response cytolytic T lymphocytes (CTL) reacting with FMR in the context of the H-2 antigens of both parents. This suggests that the processing in vivo of the immunizing cells by f₁ macrophages results in the presentation of FMR antigens in the context of both H-2 specificities. It has also been suggested that FMR antigens are recognized by cytolytic T-lymphocyte precursors (CTL-P) at the surface of tumor cells and not of macrophages (4). The results reported here show that there are two methods of CTL-P priming: (a) in most cases, FMR antigens are presented directly by the tumor cells; (b) however, in the absence of antigen-presenting tumor cells in vivo or in vitro, macrophages present FMR to CTL-P. The presentation by macrophages appears less efficient but is probably sufficient to explain the priming of memory cells corresponding to both parental H-2.     

High strength binding of P815 mastocytoma cells is not necessary for their lysis by macrophages which have been primed and triggered in vitro

We have examined the hypothesis that binding of P815 mastocytoma cells is a necessary step in lysis of these cells by macrophages which are both "primed" and "triggered" in vitro, Macrophages "primed" by conditioned media containing IFN-gamma, or by rIFN-gamma have an increased ability to bind P815. However, adding either heat-killed Listeria or endotoxin to trigger the primed macrophages has opposite effects on lysis and binding of P815. Lysis is increased. Binding is dramatically decreased. This is true when centrifugal forces of 200 x g, 400 x g, and 800 x g are used to disrupt P815-macrophage binding. Although 100% of P815 cells bound by cytotoxic macrophages are lysed, a large additional population of unbound P815 is also lysed. Detailed kinetic studies indicate that macrophages do not rapidly bind and lyse several cycles of P815. There is an initial lag period of 4 to 6 h before P815 lysis can be detected, and completion of lytic events then occurs within 12 to 14 h. Lysis of P815 bound to cytotoxic macrophages is slightly slower than lysis of the total population of bound and unbound P815. In contrast, D3.1, a cloned CD4+ T cell line, is tightly bound to macrophages but not lysed efficiently. When macrophages are simultaneously confronted with P815 and macrophage-bound D3.1, only the former are lysed. Altogether, the data indicate that P815-macrophage binding, as operationally defined by our assay, is not a necessary step for lysis. These results, by use of macrophages primed and triggered in vitro, are in contrast to previously reported experiments examining P815 binding and lysis by macrophages activated in vivo by infection with bacillus Calmette-Guérin.     

Migration of Myeloid Cells during Inflammation Is Differentially Regulated by the Cell Surface Receptors Slamf1 and Slamf8

Previous studies have demonstrated that the cell surface receptor Slamf1 (CD150) is requisite for optimal NADPH-oxidase (Nox2) dependent reactive oxygen species (ROS) production by phagocytes in response to Gram- bacteria. By contrast, Slamf8 (CD353) is a negative regulator of ROS in response to Gram+ and Gram- bacteria. Employing in vivo migration after skin sensitization, induction of peritonitis, and repopulation of the small intestine demonstrates that in vivo migration of Slamf1^-/- dendritic cells and macrophages is reduced, as compared to wt mice. By contrast, in vivo migration of Slamf8^-/- dendritic cells, macrophages and neutrophils is accelerated. These opposing effects of Slamf1 and Slamf8 are cell-intrinsic as judged by in vitro migration in transwell chambers in response to CCL19, CCL21 or CSF-1. Importantly, inhibiting ROS production of Slamf8^-/- macrophages by diphenyleneiodonium chloride blocks this in vitro migration. We conclude that Slamf1 and Slamf8 govern ROS–dependent innate immune responses of myeloid cells, thus modulating migration of these cells during inflammation in an opposing manner.     

Phosphorus-31 NMR of rat brain in vivo with bloodless perfluorocarbon perfused rat

Rat brain in vivo has been examined by ³¹p NMR under conditions of normal blood perfusion (hematocrit 38%) and under conditions in which a perfluorocarbon blood substitute, devoid of any phosphorus containing compounds, largely replaced the animal's normal blood supply (hematocrit 7%). These studies demonstate that 2,3-diphosphoglycerate does not — as has been suggested — contribute to, and thus does not interfere with, the ³¹p NMR analysis of rat brain in vivo. However, low intensity ³¹P resonances assigned to choline phosphate, glycerol 3-phosphorylethanolamine, and glycerol 3-phosphorylcholine are observed. “High energy phosphorus” metabolite levels show no marked change over two hours with perfluorocarbon blood substitution from those of the normal blood perfused animal. This supports use of perfluorocarbon media for tissue perfusion in vitro and for ¹⁹F NMR vascular imaging in vivo.     

Development of matrix metalloproteinase-13 inhibitors – A structure-activity/structure-property relationship study

A structure-activity/structure-property relationship study based on the physicochemical as well as in vitro pharmacokinetic properties of a first generation matrix metalloproteinase (MMP)-13 inhibitor (2) was undertaken. After systematic variation of inhibitor 2, compound 31 was identified which exhibited microsomal half-life higher than 20?min, kinetic solubility higher than 20?μM, and a permeability coefficient greater than 20?×?10^?6?cm/s. Compound 31 also showed excellent in vivo PK properties after IV dosing (C_max?=?56.8?μM, T_1/2 (plasma)?=?3.0?h, Cl?=?0.23?mL/min/kg) and thus is a suitable candidate for in vivo efficacy studies in an OA animal model.     

Haplotype preference in lymphocyte differentiation. II. F1 hybrid helper T cells generated with antigen-bearing parental macrophages can cooperate with B lymphocytes of either parent.

Carrier-specific helper T cells were generated in F₁ hybrid mice by either conventional immunization procedures or by repeated immunizations with antigen-bearing macrophages derived from either F₁ or parental donors. The F₁ helper T cells generated in these various ways were then analyzed for their capacities to help hapten-primed B lymphocytes derived from each of the two parental strains as well as from F₁ donors in the development of secondary anti-hapten antibody responses. These analyses were conducted using two different types of in vivo assay systems as well as a totally in vitro system. Under all circumstances, helper T cells from F₁ mice, primed either in conventional fashion or with antigen bearing parental or F₁ macrophages, were capable of interacting effectively with B lymphocytes of each parent and of F₁ origin. Moreover, in the case of F₁ helper cells primed with antigen-bearing parental macrophages, there was no evidence of preferential helper activity for parental B lymphocytes corresponding to the type of macrophage used for sensitization; this was true irrespective of whether in vivo or in vitro assay systems were employed. The relevance of these findings and others which are either similar to, or discordant with, them to the general question of genetic restrictions in macrophage-T lymphocyte interactions is discussed.     

Antigen presentation by macrophage-enriched cells from the mouse Peyer's patch

Macrophage-enriched cells derived from Peyer's patches were prepared with or without preincubation of whole patches in collagenase-containing medium. The cells thus obtained were pulsed with ovalbumin (OVA), added to OVA-primed whole lymph node (LN) cells or LN T cells and proliferative responses stimulated in the latter cells were assessed after 5 days by thymidine incorporation. Macrophage-enriched cells obtained from Peyer's patches (PP) preincubated with collagenase presented antigen as well or better than macrophage-enriched cells obtained from spleens. In contrast, macrophage-enriched cells obtained from PP which were not preincubated with collagenase were frequently unable to function as antigen-presenting cells. In further studies, macrophage-enriched cells obtained from mice which had been fed OVA were found to be capable of stimulating antigen-primed LN T cells in an antigen-specific manner without further exposure to antigen in vitro. These results indicate PP macrophage-enriched cells are fully capable of supporting the induction of immune responses in vitro and that macrophages from PP of orally primed mice may present antigen without further antigen exposure. Thus macrophages in PP may participate in the induction of immune responses to gastrointestinal antigens in vivo.     

Bioactive microbial metabolites from glycyrrhetinic acid

Biotransformation of 18β-glycyrrhetinic acid, using Absidia pseudocylinderospora ATCC 24169, Gliocladium viride ATCC 10097 and Cunninghamella echinulata ATCC 8688a afforded seven metabolites, which were identified by different spectroscopic techniques (¹H, ¹³C NMR, DEPT, ¹H-¹H COSY, HMBC and HMQC). Three of these metabolites, viz. 15α-hydroxy-18α-glycyrrhetinic acid, 13β-hydroxy-7α,27-oxy-12-dihydro-18β-glycyrrhetinic acid and 1α-hydroxy-18β-glycyrrhetinic acid are new. The ¹³C NMR data and full assignment for the known metabolite 7β, 15α-dihydroxy-18β-glycyrrhetinic acid are described here for the first time. The major metabolites were evaluated for their hepatoprotective activity using different in vitro and in vivo models. These included protection against FeCl₃/ascorbic acid-induced lipid peroxidation of normal mice liver homogenate, induction of nitric oxide (NO) production in rat macrophages and in vivo hepatoprotection against CCl₄-induced hepatotoxicity in albino mice.     

Control of mitochondrial respiration in the heart in vivo

The role of the hydrolysis products of adenosine triphosphate (ATP), adenosine diphosphate (ADP) and inorganic phosphate (Pi), in the control of myocardial respiration was evaluated in vivo using ³¹P NMR. These studies were conducted to evaluate whether increases in the ATP hydrolysis products can be detected through the cardiac cycle or during increases in cardiac work. ³¹P NMR data acquisitions gated to various portions of the cardiac cycle (50 msec time resolution) revealed that cytosolic ATP, ADP and Pi did not change over the course of the cardiac cycle. These metabolites were also monitored during steady-state increases in cardiac work in conjunction with measurements of coronary blood flow and oxygen consumption. No changes were observed during 2 to 3 fold increases in myocardial oxygen consumption induced by various methods. These results demonstrate that the cytosolic ATP, ADP, and Pi concentrations remain relatively constant throughout the cardiac cycle and during physiological increases in cardiac work and oxygen consumption. Furthermore, it is shown that ADP and Pi cannot be solely responsible for the regulation of cardiac respiration in vivo based on the in vitro Km values of these compounds for oxidative phosphorylation. It is concluded that other mechanisms, working in concert with the simple kinetic feedback of ATP hydrolysis products, must be present in the cytosol to provide control of myocardial respiration in vivo.     

Prospects of in vivo 31P NMR method in glyphosate degradation studies in whole cell system

The degradation of the phosphonate herbicide glyphosate (N-phosphonomethylglycine) by four taxonomically distinct microorganisms was studied in vivo in whole cell system using phosphorus nuclear magnetic spectroscopy (³¹P NMR). The time-course of glyphosate metabolization in dense cell cultures was followed by means of ³¹P NMR up to 21 days after the addition. The results obtained by this non-invasive way confirmed that the cells of Spirulina platensis and Streptomyces lusitanus biodegrade herbicide. Moreover, phosphorus starvation influenced the rate of glyphosate degradation by S. platensis. On the other hand, the results of similar measurements in the cultures of green algae Chlorella vulgaris showed that this aquatic plant, however growing in the medium containing 1 mM of N-phosphonomethylglycine, did not seem to posses the ability of its biodegradation. Additionally, the use of this method allowed us to find the new fungal strain Fusarium dimerum, which is able to biodegrade and utilize the glyphosate as the sole source of phosphorus. The results of our studies on usefulness of in vivo ³¹P NMR for tracing glyphosate degradation in whole cell systems revealed that this non-invasive, one-step method, might be considered as a valuable tool in environmental biotechnology of organophosphonate xenobiotics.     

In VivoP NMR Spectroscopic Studies of Soybean Bradyrhizobium Symbiosis: I. Optimization of Parameters

³¹P NMR spectroscopy was used to study in vivo the symbiotic state established between soybean (Glycine max [L.] Merr. cv Williams) and Bradyrhizobium japonicum (USDA 110 and 138). Different experimental conditions were used to maintain perfused, respiring detached or attached nodules in an NMR magnet. The pH of the perfusion medium affected the cytoplasmic pH and the resolution of the spectra. The internal Pi content and distribution were assessed as a function of nodule age and green-house growth conditions and the rate of glucose and 2-deoxyglucose uptake into nodules in split and intact states. The major metabolites (glucose-6-P, fructose-1,6-diP, P-choline, Pi, NTP, UDP-glc, and NAD) were readily identified from ³¹P NMR spectra of perchloric acid extracts of nodules with the exception of one unknown phosphorus metabolite. Nodules stressed by glucose deprivation demonstrated movement of Pi between the vacuole and cytoplasmic compartments not previously observed in ³¹P NMR studies.     

Characterization of the 31P NMR spectrum of Manduca sexta and effects of antimetabolites

High resolution ³¹P nuclear magnetic resonance spectroscopy (NMR) was successfully applied to 5th instar larvae of Manduca sexta. Conditions for in vivo analysis under non-saturating conditions are described. The ³¹P NMR spectrum of intact larvae was composed of six peaks. Their resonance frequencies are reported relative to orthophosphoric acid. Analysis of tissue extracts demonstrated the in vivo peaks to be composed of the β phosphorus resonance of nucleotide triphosphates (NTP) at −19.36 ppm; α phosphorus of NTP and nucleotide diphosphates (NDP) at −10.51 ppm; β and γ phosphorus of NDP and NTP, respectively, at −5.42 ppm; phosphoarginine (PA) at −3.45 ppm; inorganic phosphate (Pi) at +2.76 ppm and sugar phosphates at +3.34 ppm. The major sugar phosphate present in fat body extracts was trehalose-6-phosphate and this was the major phosphorus component of the spectrum of hemolymph. The spin-lattice relaxation times for each in vivo peak were determined.Titration of aqueous fat body and hemolymph extracts was carried out and the relationship between the chemical shift of Pi and pH determined. On this basis the pH of the hemolymph was estimated at approx. 6.7.The metabolic inhibitors, iodoacetate and dinitrophenol, had significant effects on the ³¹P NMR spectrum of intact larvae. Administration of iodoacetate caused a rapid increase in the levels of sugar phosphates together with decreases in NTP and PA. Dinitrophenol also caused declines in the relative levels of NTP and PA but sugar phosphates decreased as well. The experiments demonstrated the potential of in vivo NMR analysis for metabolic studies on high energy phosphate metabolites in M. sexta.     

Studies on the regulation of lymphocyte reactivity by normal and activated macrophages.

To determine the potential role of macrophages as regulators of the immune response, the effect of mouse peritoneal macrophages on transforming mouse spleen lymphocytes was investigated. Mitogen and antigen stimulated lymphocyte transformation, as measured by DNA synthesis, was enhanced by all concentrations of normal macrophages tested, but only by low concentrations of activated macrophages. High concentrations of activated macrophages markedly inhibited lymphocyte transformation. This inhibition occurred whether lymphocyte DNA synthesis was measured by incorporation of [³H]TdR or of ³²P. Activated macrophages cultured with lymphocytes within 4 hr of being removed from the peritoneal cavity inhibited lymphocyte transformation. When activated macrophages were cultured alone for 24 or more hours before addition of lymphocytes, enhancement of transformation was noted. Once lymphocytes were exposed to activated macrophages, they could not be induced to undergo transformation in the presence of Con A. Whereas heat-killed activated macrophages, which appeared intact morphologically, lost their capacity to inhibit lymphocyte transformation, macrophages treated with mitomycin C to inhibit DNA synthesis retained this capacity. Syngeneic and allogeneic macrophages had similar inhibitory ability. Supernatants from cultures of many cell types (including normal or activated macrophages, lymphocytes, lymphocytes plus macrophages, and L cells) inhibited [³H]TdR incorporation by both mitogen stimulated lymphocytes and tumor cells. These studies demonstrate the capacity of macrophages to regulate lymphocyte transformation in vitro and suggest a role for these cells as regulators of cell-mediated immunity in vivo.     

Formation of heterophile antibodies by human tonsilar lymphocytes: II. In vitro stimulation with allogeneic cells

Short-term cultures of human tonsilar lymphocytes (HTL), 5 × 10⁶ cells/culture, in medium RPMI 1640 supplemented with human group AB serum were studied for the production of plaque-forming cells (PFC) against sheep (SRBC) and bovine (BRBC) red blood cells following in vitro stimulation by various allogeneic lymphoid cells. Of 55 HTL specimens examined, 48 produced a significant number (50–300/culture) of PFC against SRBC and/or BRBC following the in vitro stimulation. The optimal doses of the stimulator HTL and peripheral blood lymphocytes (PBL) were 10⁷ and 5 × 10⁶/culture, respectively. After the stimulation, PFC appeared in significant numbers on the third day, reached the peak number on the sixth day, and decreased sharply in number thereafter. Removal of E-rosetting cells from both stimulator and responder populations abolished the PFC formation. PFC formation against SRBC was inhibited by solubilized Forssman antigen, while PFC formation against BRBC was inhibited strongly by Hanganutziu-Deicher antigen, hardly by Paul-Bunnell antigen and not at all by Forssman antigen. Supernatants of mixed lymphocyte culture of PBL were shown to enhance PFC formation of HTL cultures stimulated by allogeneic lymphocytes. The results of this study indicated that in vivo primed B cells of the HTL were triggered in vitro by allogeneic stimulation for the heterophile antibody formation. Since these antibodies are apparently directed against Forssman and Hanganutziu-Deicher antigens, the “allo” nature of these antigens as well as their relationship to the previously described heterophile transplantation antigens have to be clarified.     

Synthesis and fungicidal activity of novel pyrazole derivatives containing 5-Phenyl-2-Furan

Pyrazole constitutes an important heterocyclic family covering a broad range of synthetic as well as natural products that exhibit numerous chemical, biological, agrochemical and pharmacological properties. In order to explore compounds with good fungicidal activity, a series of new pyrazole derivatives containing 5-phenyl-2-furan were designed and synthesized. In vitro and in vivo fungicidal activities were evaluated and the compound ethyl-1-(5-phenylfuran-2-carbonyl)-5-propyl-1H-pyrazole-3-carboxylate (I8) displayed significant fungicidal activity against various fungi, especially against P. infestans. The structures of the novel pyrazole derivatives were confirmed by ¹H NMR, ¹³C NMR, MS, elemental analysis and X-ray single crystal diffraction. Further study showed that compound I8 might act on the synthesis of cell walls from morphological and ultrastructural studies by SEM and TEM. The results also revealed that compound I8 could block the nutritional transportation leading to cells senescence and death. These results suggested that the novel pyrazole derivatives proved to be promising lead compounds.     

Modulation of the recognition and lysis of EL4 tumor target cells by cytotoxic T lymphocytes

When the EL4 targets were harvested from the peritoneal cavity (in vivo), they had less than half as much cell-surface sialic acid as EL4 cells harvested from tissue culture (in vitro), apparently due to the presence of a neuraminidase activity in the peritoneal cavity. Both the recognition and the lysis of either EL4 in vivo or EL4 in vitro target cells by allogeneically primed cytotoxic T lymphocytes were enhanced upon removal of cell-surface sialic acid by neuraminidase treatment. However, even after neuraminidase treatment, there still remained a difference in the lytic profile when using EL4 targets that were harvested in vivo versus in vitro. Both conjugate formation between the target and the T cells and anti-H-2D^b adsorption by the target cells were unaffected by the culture conditions of the target line. However, antibody-induced capping and exocytosis of vesicles differed between the differently cultured target cells, suggesting that there was a membrane organizational difference between them that was detected by the cytotoxic T cells. These data are consistent with the idea that cell surface sialic acid as well as the membrane organization can influence T-cell recognition and lysis of target cells.