Requirement for lipopolysaccharide-responsive macrophages in galactosamine-induced sensitization to endotoxin.

Injection of D-galactosamine sensitizes mice many thousand-fold to the lethal action of endotoxin (lipopolysaccharide [LPS]). Comparable sensitization was practically absent in LPS-resistant C3H/HeJ mice, which after D-galactosamine treatment were about 500,000 times less sensitive to LPS lethality than histocompatible LPS-sensitive C3H/HeN mice. D-Galactosamine induces changes in the hepatocytes of treated animals, such as depletion of UTP and alterations in the pattern of UDP sugars. These early biochemical changes, which are necessary for development of sensitization, were similar in both mouse strains which we examined. High sensitivity to the lethal effects of LPS was achieved in C3H/HeJ mice after D-galactosamine treatment by transfer of C3H/HeN macrophages obtained in culture from bone marrow precursor cells.     

Increasing return compliance in a tuberculosis detection drive

This study explored the role of subject commitment as a variable for increasing compliance rates in a university-sponsored tuberculosis (TB) detection drive. Return rates for reaction readings were compared between those subjects who had made an overt commitment to return (either a verbal or a verbal plus written agreement) and those subjects who were exposed to a standardized, no commitment procedure. Return rates under both commitment conditions significantly increased. Contrary to expectation, subjects with a known family history of TB were found to be a high-risk group for noncompliance. For this group, obtaining both verbal and written agreement from subjects appeared to be the most efficacious procedure to increase compliance.The authors wish to thank Mr. Joe Hunt, James Leeper, Ph.D., and members of AED (Premedicai Honorary Society) for their assistance in this experiment.Currently a student in the College of Community Health Sciences, University of Alabama, University, Alabama 35486.     

Induction of hyperreactivity to endotoxin in mice by Coxiella burnetii

Intraperitoneal inoculation of mice with live or killed Coxiella burnetii phase I or phase II cells induced a marked hyperreactivity to the lethal effect of bacterial endotoxin and was accompanied by a marked hepatosplenomegaly. The degree and duration of hyperreactivity depended on the dose of C. burnetii administered and were higher with phase I than with phase II cells. Sensitization to the lethal effects of endotoxin and induction of splenomegaly by phase I C. burnetii cells also proceeded in the endotoxin-resistant C3H/HeJ strain of mice. Preincubation of C. burnetii cells with the corresponding immune serum significantly diminished the ability of phase I but not phase II cells to induce hyperreactivity to endotoxin.     

Toll-like receptor 4 expression levels determine the degree of LPS-susceptibility in mice

C57BL/10ScCr (Cr) mice carry a deletion of the Toll-like receptor 4 (tlr4) gene (i.e. they are tlr4(0/0)) and are thus refractory to LPS effects. Insertion of wild-type tlr4 transgene into the tlr4(0/0) Cr germ line endowed LPS susceptibility in the two transgenic lines created, indicating that TLR4 is the only limiting factor for LPS responsiveness in Cr mice. The absolute levels of tlr4 mRNA expressed by the heterozygous transgenic (tlr4(Tr/0)), wild-type C57BL/10ScSn (Sn) (tlr4(+/+)) and heterozygous F1 (Sn x Cr) (tlr4(+/0)) mice varied markedly. However, the pattern of distribution of expression in the different organs was the same in all strains. In different biological assays (B cell mitogenicity, cytokine induction and lethal toxicity) the degree of LPS response obtained in the different strains of mice correlated with the levels of tlr4 mRNA expression. In macrophages, investigation of the LPS-induced cytokine (IL-6) response revealed a linear relationship between the response and the logarithm of TLR4-MD-2 levels.     

Interaction of lipopolysaccharides with plasma high-density lipoprotein in rats.

The method of crossed immunoelectrophoresis was used to investigate early changes in plasma proteins of rats treated with lipopolysaccharide (LPS). Intravenous injection of a smooth (S)- and a rough (R)-form preparation led to alterations in the high-density lipoprotein (HDL) precipitation peak. The changes were dose dependent and characteristic for each LPS. The changes were identified as being due to the formation of a complex of LPS with HDL, the complex of the S-form LPS with HDL migrating slower and that of the R-form LPS with HDL migrating faster than free HDL. The fate of the complex was followed in the plasma of injected rats, and it was shown that the R-form LPS complex disappeared after several hours, whereas the S-form LPS complex was still partly present after 2 days. Plasma clearance studies, carried out with 14C-labeled LPS, revealed similar differences in the rate of elimination of the two LPSs. In both cases the time of clearance resembled that of the disappearance of LPS-HDL complex. These results may indicate that HDL represents a transport protein for LPS in plasma to organs of clearance or to other cellular targets.     

Post‐traumatic growth in advanced cancer patients receiving palliative care

Goals of work To develop the Greek version of the Post‐traumatic Growth Inventory (PTGI‐Gr), and assess its psychometric properties in a palliative care patient sample. Patients and methods The scale was translated with the forward–backward procedure to Greek. It was administered twice, with a 3‐day interval, to 131 eligible patients with advanced cancer. Together with the PTGI, the patients also completed the Greek version of the Impact of Events Scale‐Revised scale (IES‐R‐Gr). The reliability was assessed by the internal consistency (Cronbach's alpha coefficients), test/retest (Spearman's r value), and inter‐item correlations. Validity was demonstrated by factor analysis, inter‐scale correlations, construct validity with the IES‐R‐Gr, and combined with the Eastern Cooperative Oncology Group (ECOG) performance status. Main results The PTGI‐Gr yielded a five‐factor structure, explaining 73.5% of the variance. Cronbach's alphas for the five factors ranged from .66 to .87, respectively. Overall test–retest reliability was satisfactory with a range between .85 and .92 (p<.0005), and inter‐item correlations ranged between .47 and .63. Inter‐scale correlations were found satisfactory (p<.0005, p<.005, and p<.05). Validity as performed using combined validity analysis showed good results. Satisfactory construct validity was supported by the correlation analysis between the PTGI‐Gr and the IES‐R‐Gr scales. Conclusions PTGI‐Gr is an instrument with satisfactory psychometric properties, and is a valid research tool for the post‐traumatic growth of advanced cancer patients.     

Toll-like receptor 2- and 6-mediated stimulation by macrophage-activating lipopeptide 2 induces lipopolysaccharide (LPS) cross tolerance in mice,which results in protection from tumor necrosis factor alpha but in only partial protection from lethal LPS doses

Patients or experimental animals previously exposed to lipopolysaccharide (LPS) become tolerant to further LPS challenge. We investigated the potential of the macrophage-activating lipopeptide 2 (MALP-2) to induce in vivo cross tolerance to tumor necrosis factor alpha (TNF-alpha) and LPS. MALP-2-induced tolerance could be of practical interest, as MALP-2 proved much less pyrogenic in rabbits than LPS. Whereas LPS signals via Toll-like receptor 4 (TLR4), MALP-2 uses TLR2 and TLR6. LPS-mediated cytokine release was studied in mice pretreated with intraperitoneal injections of MALP-2. No biologically active TNF-alpha could be detected in the serum of MALP-2-treated animals when challenged with LPS 24 or 72 h later, whereas suppression of LPS-dependent interleukin (IL)-6 lasted for only 24 h. Protection from lethal TNF-alpha shock was studied in galactosamine-treated mice. Dose dependently, MALP-2 prevented death from lethal TNF-alpha doses in TLR4(-/-) but not in TLR2(-/-) mice, with protection lasting from 5 to 24 h. To assay protection from LPS, mice were pretreated with MALP-2 doses of up to 10 micro g. Five and 24 h later, the animals were simultaneously sensitized and challenged by intravenous coinjection of galactosamine and a lethal dose of 50 ng of LPS. There was only limited protection (four of seven mice survived) when mice were challenged 5 h after MALP-2 pretreatment, and no protection when mice were challenged at later times. The high effectiveness of MALP-2 in suppressing TNF-alpha, the known ways of biological inactivation, and low pyrogenicity make MALP-2 a potential candidate for clinical use.     

ELISA for antibodies to lipid A,lipopolysaccharides and other hydrophobic antigens

Summary A simple, sensitive and rapid ELISA method for the quantification and characterization of antibodies to lipid A has been developed, which can also be applied to other hydrophobic antigens. For coating, antigens were applied to the wells of ELISA plates as solutions in a mixture of chloroform and ethanol 1:9 (v/v), and the solvent evaporated in a stream of warm air. Under these conditions a high coating efficiency was achieved, which made the assay highly sensitive. The use of the above organic solvent considerably reduced the nonspecific adsorption of immunoglobulins to the solid phase, making the usual blocking of unspecific binding sites with BSA or gelatine unnecessary. For screening of lipid A antibodies in the sera of immunized animals, coating with 0.2 µg of the corresponding antigen per well was found to be suitable. For optimal measurement of antibodies in pre-immune sera, sera of healthy human donors, and of monoclonal antibodies, higher amounts of antigen (1–2 µg/well) had to be used. The coating method described here proved excellent also for other antigens directly soluble in organic solvents, such as Re-lipopolysaccharide (LPS) or gangliosides. In addition, the method was successfully applied to less hydrophobic antigens, such as LPS of the classes Ra to Rd and S forms, and lipoteichoic acid. These could be brought into solution in chloroform/ethanol by diluting their aqueous solutions with a large volume of the organic mixture.

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ELISA-Methode zur Bestimmung von Anti-Lipoid-A-Antikörpern, Lipopolysacchariden und anderen hydrophoben Antigenen

Zusammenfassung Eine einfache, empfindliche und schnelle ELISA-Methode zur Bestimmung und Charakterisierung von Anti-Lipoid-A-Antikörpern wurde ausgearbeitet. Zum Beschichten der ELISA-Platten wird eine Lösung des Antigens in Chloroform/Äthanol (1:9 [v/v]) in die Löcher eingetragen und das Lösungsmittel dann mit einem Strom warmer Luft verdampft. Damit wird ein hoher Beschichtungsgrad erreicht, wodurch die Bestimmung sehr empfindlich wird. Gleichzeitig wird die unspezifische Adsorptionskapazität der Platten für Immunglobuline erniedrigt, so daß sich eine Blockierung unspezifischer Bindungsstellen erübrigt, wie sie üblicherweise mit BSA oder Gelatine durchgeführt wird. Zum Auffinden von Lipoid-A-Antikörpern sind 0,2 µg des entsprechenden Antigens pro Plattenloch bei Immunseren, und 1–2 µg bei Nicht-Immunseren und monoklonalen Antikörpern optimal. Die hier beschriebene Beschichtungsmethode kann generell bei Antigenen angewandt werden, die in organischen Lösungen direkt lösbar sind, wie zum Beispiel bei Re-LPS und Gangliosiden. Des weiteren ist diese Methode für Antigene geeignet, die zwar primär in Chloroform/Äthanol unlöslich sind, die aber nach Lösen in einem geeigneten wäßrigen Medium in die Chloroform/Äthanol-Mischung überführt werden können. Dies wird an mehreren Beispielen gezeigt (LPS der Klassen Ra-Rd und S-formen, sowie Lipoteichonsäure).

     

Protective properties of a human IgG preparation rich in antibodies to a wide spectrum of lipopolysaccharides

Naturally occurring human IgG, rich in antibodies to different lipopolysaccharides was investigated for possible protective effects against lethal endotoxin shock and lethal gram-negative infection in mice. The IgG preparation was obtained from pooled serum of selected blood donors with high concentrations of antibodies to 11 different LPS as measured by ELISA. The human IgG (5 mg/mouse) protected C3H/TifF mice against an otherwise lethal infection with Salmonella typhimurium. The human IgG also inhibited the lethality induced by purified LPS in D-galactosamine sensitized C57B1/6 mice. The protection was dependent on the IgG dose given. However, protection was not obtained against all the LPS preparations tested. Absorption of the IgG with different LPS, showed the protection to be caused by serotype-specific anti-LPS antibodies. Protection against a given LPS was not related directly to the corresponding anti-LPS titer as measured by ELISA and passive hemolysis. The interpretation of these results is discussed.     

Endogenous and exogenous glucocorticoids have different roles in modulating endotoxin lethality in D-galactosamine-sensitized mice.

Endotoxin sensitivity and dexamethasone protection have been assessed in mice that were adrenalectomized and also treated with D-galactosamine at the time of endotoxin challenge. Our data establish that adrenalectomy did not detectably alter the magnitude of the increased sensitivity induced by D-galactosamine alone. Furthermore, protection provided by acute exogenous glucocorticoid treatment was still demonstrable in these mice and was not influenced by chronic experimentally induced glucocorticoid deficiency. Our data confirm that the adrenalectomized mouse model of endotoxin lethality is characterized by increased sensitivity to endotoxin and establish that the magnitude of this sensitizing effect is more than 100-fold. We also show for the first time that adrenalectomy causes an appreciable kinetic shift in the endotoxic crisis and that dexamethasone, given at the time of endotoxin challenge, will significantly reverse the increased sensitivity to lethality. Our results indicate that the protective effects of corticosteroids may involve important chronic as well as acute responses. In particular, we conclude that endogenous glucocorticoid need not always increase host resistance to endotoxin, nor does such a circumstance eliminate the possibility for exogenous glucocorticoid-mediated protective effects.