Biomedical subjects

A M Friedlander

Publications and source records attributed to A M Friedlander.

At least 55 records · Page 3Linked to original sources

PubMed1995

Pathology of experimental inhalation anthrax in the rhesus monkey.

BACKGROUND: The inhalation form of anthrax, although rare, is nearly always fatal because of the rapid progression of the disease with little host response until the terminal stages of the disease. The Gulf War heightened the concern that anthrax could be used as a biologic weapon. Past studies modeling pathologic changes in human inhalation anthrax have used the rhesus monkey. EXPERIMENTAL DESIGN: We studied pathologic changes in the rhesus monkey model of inhalation anthrax. Gross examination as well as light and electron microscopy were used to define pathologic alterations. Immunolabeling techniques were used to identify the anthrax bacillus by light and electron microscopy. RESULTS: Gross changes included hemorrhage in mesenteric (54%) and tracheobronchial (46%) lymph nodes, meninges (38%), lungs (31%), and small intestinal serosa (31%). Histopathologic changes included suppurative meningitis (77%); hemorrhages in the meninges (54%), neuropil (31%), and pulmonary alveoli (31%); and pneumonia (15%). Spleens and various lymph nodes from all monkeys had one or more of the following changes: hemorrhage, acute inflammation, extracellular bacilli, lymphocytic depletion, and histiocytosis. Spleens of two monkeys were devoid of extracellular bacilli, but degraded intrahistiocytic bacilli reacted with Ab to Bacillus anthracis cell wall polysaccharide. CONCLUSIONS: In our study, compared with previous reports, meningitis and mesenteric lymph node hemorrhages were more common, whereas mediastinal and tracheobronchial lymph node hemorrhages were less common. Immunostaining highlighted intracellular bacilli that would have been otherwise missed by light microscopic examination.

Animals↗

PubMed1994

Structure-function analysis of Bacillus anthracis edema factor by using monoclonal antibodies.

Edema toxin of Bacillus anthracis is composed of protective antigen (PA) and edema factor (EF), a calcium- and calmodulin-dependent adenylate cyclase. At least five different antigenic regions of EF were identified using a competitive-binding, enzyme-linked immunosorbent assay of paired monoclonal antibodies (mAbs). Two mAbs, 9F5 and 7G10, inhibited the binding of 125I-EF to cell-bound PA. However, only 9F5 inhibited the elongation response of Chinese hamster ovary cells in the presence of edema toxin. Cleavage of EF at the two aspartic acid-proline residues by acid hydrolysis resulted in three fragments: a C-terminal 17 kDa fragment, a central 53 kDa fragment, and an N-terminal 18 kDa fragment. Immunoblots of EF cleaved by formic acid mapped mAbs 9F5 and 7G10 to the N-terminal 18 kDa fragment, mAb 1E6 to the C-terminal 17 kDa fragment, and the remaining 7 mAbs to the central 53 kDa fragment. mAbs 7G10 and 9F5 defined an antigenic region within amino acids 1-156 of EF which is involved in interaction with PA in forming edema toxin.

Adenylyl Cyclase Inhibitors↗

PubMed1994

Anthrax edema toxin differentially regulates lipopolysaccharide-induced monocyte production of tumor necrosis factor alpha and interleukin-6 by increasing intracellular cyclic AMP.

Bacillus anthracis exotoxins mediate most of the symptomatology of severe anthrax. In addition to a clinical syndrome reminiscent of septic shock, which may be mediated by cytokines produced by macrophages stimulated with lethal toxin, infected patients show profound edema at sites of infection. Edema is mediated by edema toxin (ET), which comprises of a binding molecule, protective antigen, and an active moiety, edema factor, which possesses intrinsic adenylyl cyclase activity. Intracellular cyclic AMP (cAMP) regulates the production of several cytokines that modulate edema formation and play important roles in host defense against invading bacteria. To determine whether ET enhanced the accumulation of cAMP in monocytes and thereby influenced cytokine production, we cultured human monocytes with endotoxin (lipopolysaccharide [LPS]) and dilutions of ET and determined the levels of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) in culture supernatant fluids. We further estimated cytokine-specific mRNA accumulation in monocytes by reverse transcription PCR and examined intracellular cAMP concentrations following treatment with ET. ET and LPS each induced monocytes to secrete comparable amounts of IL-6. ET did not inhibit and in most experiments modestly enhanced LPS-induced IL-6 production. In contrast to this stimulatory effect on IL-6 production, ET induced little or no TNF-alpha production. Moreover, ET profoundly inhibited LPS-induced TNF-alpha synthesis. These regulatory phenomena were also observed at the mRNA level in association with dose-related enhancement of intracellular cAMP in ET-treated monocytes. Monocytes treated with dibutyryl cAMP, an active analog of cAMP, produced cytokines in a pattern identical to that of cells treated with ET. The disruption of cytokine networks as a consequence of unregulated, ET-induced cAMP accumulation in human monocytes may impair cellular antimicrobial responses and contribute to clinical signs and symptoms.

Antigens, Bacterial↗

PubMed1994

Protein synthesis is required for expression of anthrax lethal toxin cytotoxicity.

Anthrax lethal toxin, which is composed of two proteins, i.e., protective antigen and lethal factor, is cytolytic to mouse peritoneal macrophages and the macrophage-like cell line J774A.1. After exposure of cells to lethal toxin, inhibition of protein synthesis occurred only slightly before the onset of cytolysis. Thus, cell death did not appear to be due to inhibition of protein synthesis. However, prior treatment of J774A.1 cells with cycloheximide or puromycin, which inhibited protein synthesis, protected them completely against lethal toxin-induced cytolysis, which suggested that continuous protein synthesis is required for the expression of lethal toxin activity. Inhibition of protein synthesis had no appreciable effect on the binding of protective antigen to the cell surface receptor or on proteolytic cleavage of surface-bound protective antigen. Furthermore, inhibition of protein synthesis did not alter the uptake of toxin, which suggested that protein synthesis is required at a later stage of the intoxication process. The protection provided by inhibition of protein synthesis was effective, even up to 1 h after exposure to anthrax lethal toxin. The increased uptake of calcium observed in cells exposed to lethal toxin did not occur when they were protected by blocking protein synthesis. Identifying the protein(s) synthesized during the intoxication process may help to understand the mechanism of cell death produced by anthrax lethal toxin.

Animals↗

PubMed1993

Postexposure prophylaxis against experimental inhalation anthrax.

Inhalation anthrax is a rare disease that is almost invariably fatal. This study determined whether a prolonged course of postexposure antibiotics with or without vaccination would protect monkeys exposed to a lethal aerosol dose of Bacillus anthracis when the antibiotic was discontinued. Beginning 1 day after exposure, groups of 10 animals were given penicillin, ciprofloxacin, doxycycline, doxycycline plus vaccination, vaccination alone, or saline. Antibiotics were administered for 30 days and then discontinued. Vaccine was given on days 1 and 15. Two animals died of causes other than anthrax and were not included in the statistical analysis. Nine of 10 controls and 8 of 10 animals given only vaccine died. Each antibiotic regimen completely protected animals while on therapy and provided significant long-term protection upon discontinuance of the drug (penicillin, 7 of 10 survived, P < .02; ciprofloxacin, 8 of 9 survived, P < .002; doxycycline, 9 of 10 survived, P < .002; doxycycline plus vaccination, 9 of 9 survived, P < .0002). Protection against rechallenge was provided by combining postexposure antibiotic treatment with vaccination.

Aerosols↗

PubMed1993

Characterization of macrophage sensitivity and resistance to anthrax lethal toxin.

Anthrax lethal toxin, which consists of two proteins, protective antigen and lethal factor, is cytolytic for macrophages. Macrophages from different mouse strains were found to vary in their sensitivities to toxin. C3H mouse macrophages lysed by lethal factor concentrations of 0.001 micrograms/ml were 100,000 times more sensitive than those from resistant A/J mice. We analyzed various stages of the intoxication process to determine the basis for this resistance. Direct binding studies with radioiodinated protective antigen revealed that the affinity (Kd, approximately 0.5 nM) and number of receptors per cell (25,000 to 33,000) were the same in sensitive and resistant cells. Proteolytic activation of protective antigen by a cell surface protease and subsequent binding of lethal factor were also the same in both sensitive and resistant macrophages. Resistant A/J macrophages were not cross-resistant to other toxins and a virus which, like lethal toxin, require vesicular acidification for activity, implying that resistance is not due to a defect in vesicular acidification. When introduced into the cytosol by osmotic lysis of pinosomes, lethal factor in the absence of protective antigen was cytolytic for the sensitive macrophages while resistant cells were unaffected. Thus, lethal factor by itself possesses the toxic activity of lethal toxin. These results suggest that macrophage resistance is due to a defect at a stage occurring after toxin internalization. A/J macrophages may lack the putative lethal factor target in the cytosol or be defective in the further processing or activation of lethal factor in the cytosol or in endocytic vesicles.

Animals↗

PubMed1992

Functional characterization of protease-treated Bacillus anthracis protective antigen.

Characterization of the functional domains of Bacillus anthracis protective antigen (PA, 83-kDa), the common cellular binding molecule for both anthrax edema toxin and anthrax lethal toxin, is important for understanding the mechanism of entry and action of the anthrax toxins. In this study, we generated both biologically active (facilitates killing of J774A.1 cells in combination with lethal factor, LF) and inactive preparations of PA by protease treatment. Limited proteolytic digestion of PA in vitro with trypsin generated a 20-kDa fragment and a biologically active 63-kDa fragment. In contrast, limited digestion of PA with chymotrypsin yielded a preparation containing 37- and 47-kDa fragments defective for biological activity. Treatment with both chymotrypsin and trypsin generated three major fragments, 20, "17," and 47 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This PA preparation was also biologically inactive. To investigate the nature of the defect resulting from chymotrypsin treatment, we assayed PA preparations for the ability to bind to the cellular receptor and to bind and internalize 125I-LF. All radiolabeled PA preparations bound with specificity to J774A.1 cells and exhibited affinities similar to native 83-kDa PA. Once bound to the cell surface receptor, both trypsin-treated PA and chymotrypsin/trypsin-treated PA specifically bound 125I-LF with high affinity. Finally, these PA preparations delivered 125I-LF to a Pronase-resistant cellular compartment in a time- and temperature-dependent fashion. Thus, the biological defect exhibited by chymotrypsin-treated PA is not at the level of cell binding or internalization but at a step later, such as toxin routing or processing by J774A.1 cells. These protease-treated preparations of PA should prove useful in both elucidating the intracellular processing of anthrax lethal toxin and determining the structure-function relationship of PA and LF.

Animals↗

PubMed1992

Serum concentrations of penicillin, doxycycline, and ciprofloxacin during prolonged therapy in rhesus monkeys.

Concentrations of penicillin, doxycycline, and ciprofloxacin were measured by bioassay in sera of rhesus monkeys treated with these drugs for inhalation anthrax. Antibiotic doses were determined on the basis of published serum concentration data from humans and comparative body surface area calculations for humans and rhesus monkeys. The antibiotics were well tolerated. Serum peak and trough concentrations of penicillin, doxycycline, and ciprofloxacin, respectively, averaged 2.7 and 0.8, 1.31 and 0.26, and 1.22 and 0.14 microgram/mL. These were within the range usually observed with standard oral doses in humans, and peak concentrations in all monkeys exceeded the MICs for 90% of Bacillus anthracis strains.

Animals↗

PubMed1990

Production and characterization of monoclonal antibodies against the lethal factor component of Bacillus anthracis lethal toxin.

The lethal toxin of Bacillus anthracis consists of two components, protective antigen and lethal factor. Protective antigen is cleaved after binding to cell receptors, yielding a receptor-bound fragment that binds lethal factor. Sixty-one monoclonal antibodies to the lethal factor protein have been characterized for specificity, antibody subtype, and ability to neutralize lethal toxin. Three monoclonal antibodies (10G3, 2E7, and 3F6) neutralized lethal toxin in Fisher 344 rats. However, in a macrophage cytolysis assay, monoclonal antibodies 10G3, 2E7, 10G4, 10D4, 13D10, and 1D8, but not 3F6, were found to neutralize lethal toxin. Binding studies showed that five of the monoclonal antibodies that neutralized lethal toxin in the macrophage assay (10G3, 2E7, 10G4, 10D4, and 13D10) did so by inhibiting the binding of lethal factor to the protective antigen fragment bound to cells. Monoclonal antibody 1D8, which was also able to neutralize lethal toxin activity after lethal factor was prebound to cell-bound protective antigen, only partially inhibited binding of lethal factor to protective antigen. Monoclonal antibody 3F6 did not inhibit the binding of lethal factor to protective antigen. A competitive-binding enzyme-linked immunosorbent assay showed that at least four different antigenic regions on lethal factor were recognized by these seven neutralizing hybridomas. The anomalous behavior of 3F6 suggests that it may induce a conformational change in lethal factor. Differences in neutralizing activity of monoclonal antibodies were related to their relative affinity and epitope specificity and the type of assay.

Animals↗

PubMed1989

Internalization and processing of Bacillus anthracis lethal toxin by toxin-sensitive and -resistant cells.

Anthrax lethal toxin consists of two separate proteins, protective antigen and lethal factor (LF). Certain macrophages and a mouse macrophage-like cell line, J774A.1, are lysed by low concentrations of lethal toxin. In contrast, another macrophage cell line, IC-21, and all other cell types tested were resistant to this toxin. To discover the basis for this difference, each step in the intoxication process was examined. No differences between sensitive and resistant cells were found in receptor binding or proteolytic activation of protective antigen, steps that are required prior to LF binding. To determine whether resistance results from a defect in translocation to the cytosol, we introduced LF into J774A.1 and IC-21 cells and a nonmacrophage cell line (L6 myoblast) by osmotic lysis of pinocytic vesicles. Only J774A.1 cells were lysed; no effect was observed in IC-21 and L6 cells. These results suggest that resistant cells either lack the intracellular target of LF or fail to process LF to an active form. The relatively low potency of LF introduced into J774A.1 cells by osmotic lysis suggests that protective antigen may also be required at a stage subsequent to endocytosis.

Animals↗

PubMed1989

Calcium is required for the expression of anthrax lethal toxin activity in the macrophagelike cell line J774A.1.

Anthrax lethal toxin, which consists of two separate proteins, protective antigen (Mr, 82,700) and lethal factor (Mr, approximately 83,000), is cytotoxic to the macrophagelike cell line J774A.1. Removal of calcium from the culture medium protected cells against the action of lethal toxin. Calcium depletion during the binding phase of intoxication afforded only partial protection. Further analysis showed that calcium removal caused some inhibition of protective antigen binding but that it had minimal effect on proteolytic conversion of protective antigen to the active 63-kilodalton fragment and that it had no effect on lethal factor binding. Cells to which lethal toxin had bound in the presence of calcium were protected when transferred to calcium-depleted culture medium, indicating a role for calcium at a postbinding stage. When ammonium chloride is present with lethal toxin, toxin accumulates in intracellular vesicles. Calcium-free medium protected these cells upon removal of the amine block, suggesting that calcium is also required at a step after internalization of lethal toxin. Calcium channel blockers inhibited 45Ca2+ uptake and protected cells against cytotoxicity. Calmodulin inhibitors also protected against the action of lethal toxin, suggesting involvement of calmodulin at a step during intoxication. We conclude that calcium is required at several steps in the intoxication of cells by anthrax lethal toxin.

Animals↗

PubMed1988

Comparative safety and efficacy against Bacillus anthracis of protective antigen and live vaccines in mice.

The efficacy and mechanisms of protection of two live vaccines and of a protective antigen (PA) vaccine against Bacillus anthracis were studied in inbred mice. Mice that differed in their natural resistance to killing by Sterne, a non-encapsulated, toxigenic vaccine strain of B. anthracis, were used. Vaccination with live Sterne spores protected Sterne-resistant mice against challenge with the virulent Vollum 1B (V1B) strain of B. anthracis, but only at doses of Sterne greater than or equal to 0.1 50% lethal dose. The live B. subtilis recombinant strain PA2, which produces the PA component of anthrax toxin, fully protected (CBA/J) or partially protected (BALB/cJ) Sterne-resistant mice against V1B. Neither immunization with the cell-free PA vaccine nor passive administration of anti-PA antiserum protected Sterne-resistant mice against V1B. Sterne-susceptible A/J mice were not protected against V1B by either live vaccine or by the PA vaccine. However, immunization with strain PA2 induced anti-PA antibody and protected A/J mice against Sterne. A/J mice passively treated with antitoxin antibodies also survived Sterne, and survivors were then partially protected against V1B. Thus, immunity to Sterne correlated with an effective anti-PA response. Immunity to fully virulent V1B also required PA but may involve mechanisms in addition to humoral immunity.

Animals↗

PubMed1988

Pathogenesis and genetic control of resistance to the Sterne strain of Bacillus anthracis.

The pathogenesis of lethal infection by the nonecapsulated, toxigenic Sterne strain of Bacillus anthracis and the genetic basis of resistance were characterized in mice. Lethal doses of Sterne spores produced disease in susceptible mice similar to that caused by toxigenic and encapsulated B. anthracis. At the inoculation site, the mice developed an edematous exudate with large concentrations of bacilli and toxin. In the susceptible A/J strain, lethal infection was accompanied by systemic invasion and serum anthrax toxin levels increased in parallel with systemic bacterial concentrations and with the mortality rate. Host resistance to Sterne infection was associated with the ability to synthesize the complement component 5 (C5). All Sterne-resistant mouse strains had a functional gene (Hc) encoding C5, whereas susceptible mice were deficient in C5. A/J mice could be passively protected from lethal challenge by C5-positive serum but not by serum from C5-negative congenic mice. Also resistance was linked to production of C5 in individual backcross (97%) and F2 (98%) mice. The distribution pattern for recombinant inbred mice was consistent with a major role in host resistance of Hc or a closely linked locus, although other genes probably contribute. This mouse model will be useful in characterizing the pathogenesis of anthrax and testing the safety and efficacy of new anthrax vaccines.

Animals↗

PubMed1986

Macrophages are sensitive to anthrax lethal toxin through an acid-dependent process.

Anthrax lethal toxin, which consists of two proteins, protective antigen and lethal factor, is lethal for experimental animals. This study describes the first in vitro system demonstrating lethality of the toxin. Mouse peritoneal macrophages are killed within 1 h of exposure to the toxin. Neither protein component alone shows any toxic activity. The minimal effective concentration of protective antigen and lethal factor was approximately equal to 10(-2) and approximately equal to 10(-3) micrograms/ml, respectively. None of the several established cell lines examined was killed. Cells could be completely protected from the toxin by pretreatment with agents, such as amines or monensin, which dissipate intracellular proton gradients and raise the pH of intracellular vesicles. This protection was reversible and could be overcome by lowering the intravesicular pH. Antitoxin added after preincubation with amines was unable to protect cells subsequently exposed to low pH treatment. These results suggest that anthrax lethal toxin requires passage through an acidic endocytic vesicle in order to exert its toxic effect within the cytosol.

Amines↗

PubMed1984

Inhibition of mouse peritoneal macrophage DNA synthesis by infection with the arenavirus Pichinde.

Macrophage DNA synthesis and proliferation occur during the development of cell-mediated immunity and in the early nonspecific reaction to infection. Arenaviruses have a predilection for infection of cells of the reticuloendothelial system, and in this study we have examined the effect of the arenavirus Pichinde on macrophage DNA synthesis. We have found that infection of mouse peritoneal macrophages with Pichinde caused a profound dose-dependent inhibition of the DNA synthesis induced by macrophage growth factor-colony stimulating factor. At a multiplicity of inoculum of 5, there is a 75 to 95% inhibition of DNA synthesis. Viable virus is necessary for inhibition since Pichinde inactivated by heat or cobalt irradiation had no effect. Similarly, virus pretreated with an antiserum to Pichinde was without inhibitory effect. Inhibition was demonstrated by measuring DNA synthesis spectrofluorometrically as well as by [3H]thymidine incorporation. The inhibition of DNA synthesis was not associated with any cytopathology. There was no evidence that the inhibition was due to soluble factors, such as prostaglandins or interferon, released by infected cells. These studies demonstrate, for the first time in vitro, a significant alteration in macrophage function caused by infection with an arenavirus. It is possible that inhibition of macrophage proliferation represents a mechanism by which some microorganisms interfere with host resistance.

Animals↗

PubMed1978

DNA release as a direct measure of microbial killing by phagocytes.

A new assay for the precise measurement of microbial killing by leukocytes is presented. The method assumes that release of radioactively labeled DNA from the microbe is direct evidence of cell death. Human peripheral blood leukocytes incubated with [14C]thymidine-labeled Salmonella typhimurium released 32 to 59% of the radioactivity after 4 h and 63 to 75% after 18 h. Inactivated leukocytes released less than 5% of the radioactivity. None of the released radioactivity is retained within the leukocyte, and 60% remains precipitable with trichloroacetic acid. Leukocytes released substantial radioactivity from labeled Escherichia coli but only a slight amount from staphylococci. Mouse peritoneal macrophages were also shown to release radioactivity from Salmonella. The DNA release assay avoids the errors inherent in prior killing methods which measure viability by growth inhibition. It is rapid, reproducible, and highly specific.

Blood Bactericidal Activity↗

PubMed1977

Medical therapy of experimental infection stones.

Struvite bladder calculi were induced in rats with an intrarenal injection of urease-producing human T mycoplasma strain T960. Acetohydroxamic acid was effective in inhibiting calculous formation. Methylene blue, tetracycline, orthophosphate, diphosphonate, and hydrochlorothiazide had no inhibitory effect.

Animals↗

PubMed1976

Cellular immunity in pyelonephritis: identification of suppressor cell activity of spleen cells in response to concanavalin A and inhibition of lymphocyte-mediated L cell cytotoxicity.

A population of fast sedimenting spleen cells was identified as the cause of impaired in vitro response of spleen cells to concanavalin A (Con A) in acute pyelonephritis in rats. These fast-sedimenting cells responded to Con A by suppressing the DNA synthetic response of normal spleen cells to Con A. In addition, spleen cells from acute pyelonephritis rats were significantly less able to mediate in vitro cell destruction in xenogeneic aggressor lymphocyte: target L cell mixtures. On the basis of these findings, the hypothesis is proposed that a suppressor cell can protect the pyelonephritic kidney against immunologically mediated tissue damage.

Animals↗