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Biomedical subjects

M M Frank

Publications and source records attributed to M M Frank.

At least 127 records · Page 7Linked to original sources
PubMed1985

Studies of human C5a as a mediator of inflammation in normal human skin.

C5a is an 11,000-D fragment of the fifth component of complement (C5) with potent anaphylatoxic and leukocyte chemotactic activities. C5a is believed to play an important role in the pathophysiology of certain skin disorders and systemic diseases with cutaneous manifestations. However, there is very little known about the in vivo reactivity of C5a in man. In this study we examined the effects of intradermal injections of human C5a in 17 normal volunteers. C5a was prepared by interacting highly purified human C5 with zymosan bound alternative pathway C5 convertase under conditions resulting in consumption of approximately 90% of the C5 substrate. C5a produced in this manner was chemotactic for human neutrophils and monocytes (0.5 X 10(-7) to 10(-9) M) and caused neutrophil aggregation and myeloperoxidase release (concentrations greater than or equal to 10(-10) M) in vitro. In vivo, C5a produced immediate wheal and flare reactions in all volunteers, and was active in doses as low as 1 ng (10(-13) mol). Intradermal testing with 20 ng of C5a in eight volunteers produced a maximal mean wheal of 11.75 mm (+/- 0.80 mm SEM) 20 min after anaphylatoxin injection, and a maximal mean erythema of 62.50 mm (+/- 3.27 mm SEM) 10 min after C5a administration. Reactions at C5a test sites were dose-related, associated with marked pruritus in some subjects, resolved without lesion formation, and were not associated with late phase reactions. In vivo testing revealed that human C5a was a more potent mediator of wheal and flare reactions than histamine, 48/80, human C3a, or morphine sulfate. Skin biopsies from eight volunteers 20 min after intradermal injection of 20 ng of C5a revealed a neutrophil-predominant perivascular infiltrate, endothelial cell edema, and sites of leukocytoclasis. Mast cell degranulation was observed on both light and electron microscopy of biopsies from C5a test sites. Although erythema at C5a injection sites was reduced by pretreating volunteers with hydroxyzine, whealing reactions and cellular infiltrates in biopsies were unaffected by this H1-antihistamine. Moderate doses of systemic corticosteroids did not alter clinical or histologic reactions at C5a injection sites in two volunteers. This study, using doses within the potential physiologic range of the anaphylatoxin, provides a comprehensive assessment of the effect of human C5a on normal human skin.

Adult↗
PubMed1985

Mechanism of action of blocking immunoglobulin G for Neisseria gonorrhoeae.

Blocking immunoglobulin G (IgG) inhibits complement-mediated killing of serum-resistant Neisseria gonorrhoeae (GC) in immune human serum. We examined the mechanism of action of blocking IgG. Presensitization of GC with increasing concentrations of blocking IgG or F(ab')2 before incubation with bactericidal antibody and absorbed pooled normal human serum increased consumption and deposition of the third component of human complement (C3) and the ninth component of human complement (C9) but inhibited killing in dose-related fashion. We next showed that blocking IgG or F(ab')2 partially inhibited binding of bactericidal IgG to GC. Also, binding of a monoclonal antibody recognizing GC outer membrane protein PIII was almost completely inhibited by blocking F(ab')2, confirming other work (Rice, P. A., M. R. Tam, and M. S. Blake, manuscript submitted for publication) showing that PIII is a target for blocking antibody. Studies of the C3 deposition site showed that one quarter of the C3 deposited on GC in the presence of blocking IgG bound covalently to the antibody molecule. Finally, 125I-GC constituents with covalently bound C3 were affinity purified on Sepharose bearing antibodies to C3 and identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis. C3 deposition on a 40,000-mol wt surface protein was enhanced six- to ninefold by blocking IgG, which indicates that the site of complement deposition was altered by blocking antibody. These studies show that blocking IgG competes with bactericidal antibody for binding to GC, but enhances rather than blocks complement activation, and leads to complement deposition at new sites that do not result in serum killing.

Antibodies, Bacterial↗
PubMed1985

Activation of the C3b receptor: effect of diacylglycerols and calcium mobilization.

The plasma membrane expression and the phagocytic function of the C3b receptor (CR1) on human neutrophils (PMN) are under the control of cellular regulatory mechanisms, and phorbol esters are one class of agents that modulate both membrane expression and function. Phorbol esters also activate protein kinase C; however, the physiologic activation of protein kinase C is thought to be mediated by diacylglycerol. Diacylglycerols are generated during phosphatidyl inositol turnover, which is associated with a rise in intracellular calcium due to another product of polyphosphoinositide metabolism, inositol trisphosphate. We therefore studied the effects of synthetic diacylglycerols and calcium mobilization on CR1 function. In our experiments, treatment of neutrophils with two synthetic diacylglycerols, 1-oleoyl-2-acetoyl-sn-3-glycerol (OAG) and sn-1,2-dioctanoylglycerol, like phorbol esters, induced ligand-independent internalization of CR1. In contrast, the addition of exogenous phospholipase C had no effect on receptor internalization over the time course studied. OAG treatment also enabled neutrophils to specifically phagocytose via CR1. Calcium mobilization with the calcium ionophore A23187 (1 microM) had a synergistic effect on phorbol ester-induced internalization of CR1, but abrogated the phorbol ester enhancement of CR1-dependent phagocytosis. Both trimethoxybenzoate, the intracellular calcium antagonist, and chlorpromazine inhibited phorbol ester-induced internalization of CR1, whereas chelation of extracellular calcium did not. We conclude that activation of protein kinase C modulates the expression and function of CR1, and that calcium mobilization also influences these processes. We speculate that polyphosphoinositide turnover may be involved in the physiologic regulation of CR1.

Calcimycin↗
PubMed1985

Evidence for distinct intracellular pools of receptors for C3b and C3bi in human neutrophils.

In this report, the modulation and localization of complement receptors CR1 and CR3 in neutrophils were examined with the use of monoclonal antibodies (mab) directed against these membrane proteins. We first studied complement receptor modulation in a patient with neutrophil-specific granule deficiency. With flow cytometric analysis, we determined that, while N-formyl-methionyl-leucyl-phenylalanine (f-met-leu-phe) (10(-6) M) caused an increase in the binding of both anti-CR1 and anti-CR3 mab to normal neutrophils, the fmet-leu-phe-stimulated neutrophils from our patient increased anti-CR1 binding but decreased anti-CR3 binding. This suggested that CR3, but not CR1, might be associated with specific granules. We next studied receptor modulation in organelle-depleted neutrophil cytoplasts obtained from normal donors. Unlike the specific granule-deficient neutrophils, the normal cytoplasts failed to augment expression of either receptor after stimulation. Immunofluorescence studies of permeabilized polymorphonuclear leukocytes (PMN) revealed considerable internal binding of both anti-CR1 and anti-CR3. In additional studies, phorbol myristate acetate (PMA) was used as a stimulus for receptor modulation in normal neutrophils. Unlike fmet-leu-phe and C5a, PMA elicited a biphasic dose-response curve. High doses of PMA (greater than 0.5 ng/ml) caused a reduction in the magnitude of membrane expression of both CR1 and CR3. In studies designed to localize the internal pool of receptors, we evaluated the binding of 125I-anti-receptor mab to plasma membrane-, specific granule, and azurophilic granule-enriched fractions obtained from sucrose gradient fractionation of disrupted neutrophils. 125I-anti-CR1 mab bound to the membrane-enriched fraction but bound little to either granule-enriched fraction. In contrast, 125I-anti-CR3 mab bound more to the specific granule-enriched fraction than to the plasma membrane-enriched fraction. Azurophilic granules showed no increased anti-CR3 binding. Immunoprecipitation of radiolabeled solubilized subcellular fractions with anti-receptor mab confirmed these findings. CR3 was present in the plasma membrane-, and specific granule-enriched fraction but not in the azurophilic granule-enriched fraction. CR1, however, was present only in the plasma membrane-enriched fraction. These data indicate that there are intracellular pools for both the CR1 and CR3, but the intracellular locations for these pools are distinct. The pool for CR3 co-sediments with specific granules, while the pool for CR1 does not. Nonetheless, a variety of stimulatory agents increase and decrease the membrane expression of both receptors in parallel.

Acetone↗
PubMed1985

Human C5a modulates monocyte Fc and C3 receptor expression.

FcIgG and C3 (CR1 and CR3) receptors are responsible for binding opsonized particles, phagocytosis, and immune adherence reactions by circulating and tissue-fixed mononuclear phagocytes. Alterations in the expression of these receptors may thus significantly influence the function of these cells. Because chemoattractants have been shown to both recruit and modulate the function of monocytes, this study specifically examines the effects of human C5a and N-formyl-methionyl-leucyl-phenyl-alanine (FMLP) on human peripheral blood monocyte FcIgG and C3 receptor expression in vitro. Adherent, elutriator-purified monocytes were incubated with C5a (10(-7) to 10(-10) M) or FMLP (10(-5) to 10(-10) M) for 30 min at 37 degrees C, and FcIgG receptor expression was assessed by rosetting with sheep erythrocytes sensitized with limiting dilutions of IgG. Human C5a caused dose-related increases in Fc rosettes of 28% at 10(-9) M, 63% at 10(-8) M, and 167% at 10(-7) M (p less than 0.01). In contrast, no significant increases in monocyte Fc receptor expression were induced by FMLP. Similar rosetting experiments were performed with sheep erythrocytes opsonized with limiting amounts of human C3b to assess C3b receptor expression on adherent human monocytes stimulated with C5a (10(-7) to 10(-10) M) or FMLP (10(-6) to 10(-9) M) for 30 min at 37 degrees C. Again, human C5a caused dose-related increases in monocyte C3b rosette formation; at 10(-8) M and 10(-7) M concentrations of C5a, these increases equaled 119% and 196%, respectively (p less than 0.05). In these experiments, 10(-6) M FMLP also caused a significant increase of 110% in monocyte C3b rosette formation (p less than 0.05). Modulation of monocyte cell surface receptors by human C5a or FMLP was also examined by measuring cell fluorescence and side scatter by dual channel flow cytometry after staining normal leukocytes in citrated venous blood with receptor-specific monoclonal antibodies. These flow cytometric studies demonstrated that both C5a and FMLP induce dose-related increases in CR1 (C3b receptor) and CR3 (iC3b receptor) expression in both monocytes and neutrophils.(ABSTRACT TRUNCATED AT 400 WORDS)

Complement C5↗
PubMed1985

Tumor-promoting phorbol esters induce rapid internalization of the C3b receptor via a cytoskeleton-dependent mechanism.

Plasma membrane expression as well as phagocytic capability of the C3b receptor (CR1) are under regulatory control. Phorbol esters are one class of agents which have been shown to influence both of these events. In this study, by using radiolabeled Fab fragments of a monoclonal anti-CR1 antibody to tag the receptor and acid elution of surface-bound Fab, we showed that both phorbol myristate acetate and phorbol dibutyrate induced internalization of the C3b receptor; this occurred in a dose- and time-dependent manner in the absence of occupancy of the receptor by ligand. This was shown to occur in neutrophils, monocytes, and macrophages. We also showed that phorbol esters enhanced CR1-dependent phagocytosis despite the presence of two-thirds fewer receptors present on the plasma membrane. However, fibronectin, another agent that influences phagocytosis, had no effect on receptor internalization. Phorbol ester internalization was temperature-dependent and was inhibitable by cytochalasins B and D. Inhibition of internalization was reversible when cytochalasin B was removed. Phorbol esters also induced increased detergent insolubility of CR1 with kinetics similar to those of receptor internalization. It is possible that association of CR1 with the cytoskeleton is important to the process of "activation" of CR1 in phagocytosis.

Antibodies, Monoclonal↗
PubMed1985

Factor I co-factor activity of CR1 overcomes the protective effect of IgG on covalently bound C3b residues.

We have shown previously that C3b resides in a protected site when it is covalently bound to IgG (C3b-IgG). Such C3b displays a reduced affinity for factor H, with consequent enhanced survival in the presence of factors H and I and increased capacity for promoting alternative pathway consumption of C3. Because erythrocyte CR1 may be a major co-factor for factor I-mediated inactivation of immune complex-borne C3b in blood, we have examined the effect of covalently bound IgG on the C3b-CR1 interaction. Binding of monomeric C3b and C3b-IgG to human erythrocyte CR1 demonstrates identical ionic strength dependence for both species. Identical numbers of binding sites with indistinguishable affinities are detected by both ligands. Cleavage of the alpha'-chain of C3b and the alpha'-heavy chain of C3b-IgG proceeds at the same rate when erythrocyte CR1 serves as co-factor for factor I. Unlike factor H, CR1 supports a second cleavage of fluid-phase iC3b alpha'1 chain (free or bound to IgG) that generates C3c and a 33,000 m.w. fragment, which bears antigenic markers characteristic of C3g. Inactivation of C3b and C3b-IgG by CR1 and factor I also occurs at physiologic ionic strength, but proceeds very slowly relative to rates attainable with sub-physiologic inputs of factor H. CR1 does not recognize IgG-bound C3b as being in a protected site but, because of low binding affinity at physiologic ionic strength, is probably highly dependent on multivalent ligand-receptor interactions to efficiently exert its co-factor functions. Thus, inactivation of C3b-IgG heterodimers or small immune complexes bearing limited numbers of C3b residues may remain largely factor H-dependent in vivo, with resultant enhanced C3b survival.

Antigen-Antibody Complex↗
PubMed1985

Bactericidal but not nonbactericidal C5b-9 is associated with distinctive outer membrane proteins in Neisseria gonorrhoeae.

In this study, we examined the bacterial constituents associated with the complement C5b-9 complex in detergent extracts from serum-treated Neisseria gonorrhoeae (GC). 125I surface-labeled GC were incubated in 10% serum, were washed, and were solubilized in the zwitterionic sulfobetaine detergent SB12. Immunoprecipitation of 125I-GC from the extract with anti-C5 Sepharose was followed by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography of immunoprecipitated material. Polyacrylamide gel analysis of surface-labeled 125I-GC showed prominent bands for proteins I and III for both serum-resistant GC strain 6305 and serum-sensitive GC strain 7189. These same bands were visible with similar intensity in the SB12 extracts from presensitized and non-presensitized 6305 and 7189 after serum incubation. For those organisms bearing bactericidal C5b-9 (6305 + IgG and 7189 +/- IgG), additional distinctive bands immunoprecipitated with antibody to C5 Sepharose. These components were of 93,000, 44,000 40,000, and 15,000 daltons for 6305 + IgG, and were of 90,000, 50,000, 44,000, and 19,000 daltons for 7189 +/- IgG. Nonbactericidal C5b-9 extracted from the surface of 6305 incubated in serum, but not sensitized with antibody, was not associated with these distinctive proteins. However, this nonbactericidal C5b-9 did have a different pattern of associated bacterial surface constituents from that observed in control samples incubated with antibody to human serum albumin, which were similar to those with nonserum-incubated organisms. These studies support our earlier experiments which demonstrated that C5b-9 is in a different molecular configuration on the surface of serum-resistant GC from that on the surface of serum-sensitive GC or resistant GC rendered sensitive with bactericidal antibody.

Animals↗
PubMed1985

Monoclonal antibodies directed against gonococcal protein I vary in bactericidal activity.

Monoclonal antibodies (Mab) with specificity for protein I (PI) from Neisseria gonorrhoeae (GC) were examined for bactericidal activity. Mab 4G5 (gamma 3), ID3 (gamma 2a), and 1G6 (gamma 2a) bound to surface-exposed epitopes on PI of GC strain R11 (IA serotype) as assessed by co-agglutination and 125I protein A uptake. Mab 2H1 (gamma 3) that were directed against IB serotype strains and Mab 2E9 (gamma 2a) were negative in co-agglutination and protein A uptake assays and served as controls for some experiments. Only 4G5 and 1D3 were bactericidal for R11 when presensitized organisms were incubated in 10% absorbed, pooled normal human serum (PNHS) or 10% hypogammaglobulinemic serum (H gamma S) despite binding of nearly equivalent numbers of 4G5, 1D3, and 1G6 to R11 during presensitization, as assessed by 125I-protein A uptake. These Mab activated complement to a similar extent on GC R11, leading to deposition of 56.4 X 10(3), 61.9 X 1093), and 47.1 X 10(3) molecules of C3/organism during incubation in 10% C8-deficient serum. Deposition occurred almost exclusively via the classical complement pathway. Measurement of complement component C9 binding to R11 during incubation in H gamma S showed 35,700 molecules of C9/organism with 4G5, 32,600 C9/organism with 1D3, and surprisingly, 29,600 C9/organism with 1G6. Eight thousand four hundred molecules of C9/organism bound to 2E9-coated organisms, 6000 C9/organism to 2H1-coated bacteria, and 3600 C9/organism to nonpresensitized organisms. The C5b-9 complex deposited by 4G5 had a different sedimentation profile by sucrose density gradient analysis from the C5b-9 complex deposited by 1G6, consistent with a different molecular configuration of the bound complex. Mab 1G6 and 1D3, but not 2E9 or 2H1, were able to compete with 125I-4G5 for binding to GC R11. A Mab (2E6) directed against protein III of GC competed weakly with 125I-4G5 for binding to GC R11. Mab 1G6, but not 1D3, blocked 4G5-dependent killing in a dose-related fashion. Both 4G5 and IG6 reacted weakly with native PI of GC R11 by immunoblotting, but neither Mab recognized the 34,800 m.w. fragment of PI generated by trypsin and chymotrypsin treatment of outer membranes. In contrast, 2E9 reacted strongly by immunoblot with both native and cleaved PI of GC R11, suggesting binding to buried determinants of PI. These experiments show that Mab directed against identical or closely associated, surface-exposed epitopes on gonococcal PI differ markedly in bactericidal activity, despite leading to deposition of nearly equivalent numbers of C3 and C9 molecules per organism.

Anti-Bacterial Agents↗
PubMed1984

C3b covalently bound to IgG demonstrates a reduced rate of inactivation by factors H and I.

We have prepared C3b covalently linked to IgG via a hydroxylamine-sensitive bond between the C3b alpha' chain and sites predominantly, but not exclusively, located in the IgG heavy chain. This C3b species displays relative resistance to inactivation by factors H and I when compared with free C3b. This resistance appears to be due entirely to reduced affinity of C3b-IgG for factor H. Resistance to inactivation is not conferred on C3b by binding to another serum glycoprotein of similar size, ceruloplasmin, and may be a special property of IgG. C3b-IgG demonstrates an enhanced capacity to consume serum C3 relative to C3b. These alterations of the behavior of C3b when bound to IgG may in part explain the augmentation of alternative pathway activity by IgG. In addition, IgG-induced protection of C3b might influence both complement-mediated killing and phagocytosis of bacteria, as well as modify the in vivo handling of IgG-containing soluble immune complexes.

Ceruloplasmin↗
PubMed1984

A prospective clinical and immunologic analysis of patients with serum sickness.

We prospectively evaluated the clinical and immunologic features of serum sickness in 12 patients with bone-marrow failure treated for 10 days with intravenous infusions of horse antithymocyte globulin. Eleven of the 12 patients had signs and symptoms of serum sickness 8 to 13 days after beginning therapy with antithymocyte globulin. Eleven patients (including 10 of 11 with signs and symptoms of serum sickness) acquired circulating immune complexes, with peak levels occurring at 10 to 12 days. Serum C4 and C3 levels fell precipitously, with nadirs on the 10th day. Plasma levels of C3a anaphylatoxin were elevated in the four patients in whom it was measured. Eight of the 11 patients with signs and symptoms of serum sickness had a characteristic serpiginous erythematous and purpuric eruption on the hands and feet at the junction of palmar and plantar skin. Direct immunofluorescence of skin lesions revealed immune deposits (IgM, IgE, IgA, or C3) in the blood vessels of three of five patients. This study documents the immunopathology of serum sickness in human beings and describes a cutaneous marker for the disease.

Adolescent↗
PubMed1984

Studies on the fibronectin receptors of human peripheral blood leukocytes. Morphologic and functional characterization.

We have investigated the interactions between plasma fibronectin (Fn) and human peripheral blood phagocytic cells. As shown by studies of the binding of Fn-coated fluorescent microspheres (Fn-ms), both polymorphonuclear leukocytes (PMN) and monocytes had specific binding sites for Fn at the plasma membrane. However, as purified from blood, only monocytes were stimulated by Fn to become more actively phagocytic. This increase in phagocytosis was reflected by an Fn-induced increase in the ingestion of IgG-coated erythrocytes and, more dramatically by an Fn-dependent initiation of phagocytosis of C3b-coated erythrocytes. Despite this difference between PMN and monocytes in the functional consequences of Fn binding, the cell surface molecules responsible for Fn binding on the two cell types shared many characteristics. On both cells, binding of Fn-ms was inhibited by sufficient concentrations of fluid-phase Fn; both PMN and monocytes bound fewer Fn-ms at 4 degrees C than at 37 degrees C; both achieved maximal binding at similar Fn-ms/cell ratios; and phenylmethylsulfonyl fluoride did not inhibit Fn-ms binding to either cell type. Most dramatically, monoclonal anti-Fn antibodies that inhibited binding of Fn-ms to one cell type inhibited binding to both; conversely, monoclonal anti-Fn antibodies that did not inhibit Fn-ms binding to either cell type did not inhibit binding to the other. Fn will stimulate PMN to a more actively phagocytic state, like that induced in monocytes, if the PMN are first exposed to C5a or N-formyl-methionyl-leucylphenylalanine. This effect occurs without apparent change in the number of Fn receptors. We conclude that the PMN and monocyte receptors for Fn are very similar, but that their milieu is very different in the two cells as purified from peripheral blood. Whereas Fn induces increased phagocytosis in monocytes, PMN must be activated before the Fn can be effective.

Animals↗
PubMed1984

Deficiency in C3b receptors on neutrophils of patients with chronic granulomatous disease and hyperimmunoglobulin-E recurrent infection (Job's) syndrome.

C3b receptor (CR1) expression by neutrophils (PMNs) and erythrocytes (Es) from patients with chronic granulomatous disease (CGD) or with hyper-IgE, frequent infection (Job's) syndrome was compared with that of control subjects. The control subjects consisted of one group of patients with infections and a second group of normal, healthy individuals. Three quantitative assays were used: rosette formation with C3b-coated cellular intermediates (EAC43b), binding of radiolabeled monoclonal anti-CR1 ([125I]anti-CR1) to PMN surfaces, and binding of the antibody to nonidet P-40 (NP-40) extracts of PMNs and Es in an immunoradiometric assay. Rosette formation by the PMNs of five male CGD patients was about 50% of that of paired normal control subjects, whereas the rosette formation of three female CGD patients was similar to that of the control subjects. Surface binding of [125I]anti-CR1 to PMNs of 10 CGD patients was about half that of the normal subjects (mean percent binding was 2.33% for the CGD patients vs. 3.86% for the normal subjects, giving a difference of -1.53 +/- 0.22%, P less than 0.001 by the paired-sample t test). The degree of PMN binding was similarly low for both the male and the female CGD patients. Conversely, the binding of anti-CR1 to the PMNs of 11 infected control patients appeared to be similar to that of the normal subjects (4.51% for the patient vs. 4.21% for the paired normal subjects). The infected control group originally included four Job's syndrome patients, and when this subgroup was analyzed separately, their PMNs were shown to bind significantly less anti-CR1 than did the PMNs of the normal subjects (P less than 0.01 by the paired-sample t test). In contrast, the other infected control patients showed higher-than-normal levels of anti-CR1 binding (P less than 0.05). When compared to that of the normal subjects, the total CR1 quantitated in PMN extracts was also lower than normal in CGD patients (P less than 0.01 and in the PMN extracts of eight Job's syndrome patients tested (P less than 0.01). The PMNs of the other infected control subjects were not significantly different from those of the normal subjects in total CR1 expression. Extracts of Es from Job's syndrome patients also had fewer than normal CR1 (P less than 0.02). On the other hand, CR1 levels in E extracts from the CGD patients and the other control patients were similar to those in the normal control subjects. Quantitations of C3, C4, and factor B were normal in CGD.(ABSTRACT TRUNCATED AT 400 WORDS)

Antigen-Antibody Complex↗
PubMed1984

Mechanism of bacterial resistance to complement-mediated killing: inserted C5b-9 correlates with killing for Escherichia coli O111B4 varying in O-antigen capsule and O-polysaccharide coverage of lipid A core oligosaccharide.

The interaction of C3 and terminal complement components with three isogenic strains of Escherichia coli O111B4 varying in outer membrane and capsule composition was examined. Strains CL99 and 1-1, which possess O-antigen capsule and 74 to 77% coverage of lipid A core oligosaccharide, were sensitive to killing in pooled normal human serum (PNHS) or magnesium ethylene glycoltetraacetic acid PNHS in the presence but not the absence of antibody, although 1-1 contained 35% more lipopolysaccharide than CL99 and was slightly less sensitive to alternative pathway killing. In contrast, strain 1-2 lacks O-antigen capsule but contains 84% coverage and resists serum killing in the presence and absence of antibody in both PNHS and magnesium ethylene glycoltetraacetic acid PNHS. All three strains consumed C3 and C9 when incubated in PNHS, but consumption was most rapid with 1-2, which also bound the largest number of C3 molecules per CFU. Between 15 X 10(3) and 24 X 10(3) molecules of C9 per CFU bound to CL99 and 1-1 during incubation in 10% PNHS or 10% magnesium ethylene glycoltetraacetic acid PNHS, and binding was relatively stable. Binding and release of 3 X 10(3) to 8 X 10(3) molecules of C9 per CFU was observed for strain 1-2. The majority of C9 bound to CL99 and 1-1 in the presence of antibody distributed with the outer membrane after lysis of the organisms in a French press, whereas only 16.1 to 20.1% of C9 was deposited on these organisms in the absence of antibody, and 31.5 to 39.8% of C9 on strain 1-2 with or without antibody sedimented with the outer membrane. Between 4.6 X 10(3) and 5.5 X 10(3) molecules of C9 per CFU remained bound in a salt- and trypsin-resistant form to the outer membrane of organisms that were killed, whereas fewer than 1.4 X 10(3) molecules of C9 per CFU were bound to the outer membrane of organisms not killed by serum. These results indicate that C5b-9 that is bound to the outer membrane of E. coli O111B4 in a form resistant to salt or protease elution correlates with bacterial killing.

Antigens, Bacterial↗
PubMed1984

Reduction of chronic hypoxic pulmonary hypertension in the rat by beta-aminopropionitrile.

We administered antifibrotic agent beta-aminopropionitrile (BAPN) to rats exposed to 10% O2-90% N2 for 3 wk to prevent excess vascular collagen accumulation. Groups of Sprague-Dawley rats studied were air breathing, hypoxic, and hypoxic treated with BAPN, 150 mg/kg twice daily intraperitoneally. After the 3-wk period, we measured mean right ventricular pressure (RVP), the ratio of weight of right ventricle to left ventricle plus septum (RV/LV + S), and hydroxyproline content of the main pulmonary artery (PA) trunk. Hypoxia increased RVP from 14 to 29 mmHg; RVP was 21 mmHg in hypoxic BAPN-treated animals. Hypoxia increased the RV/LV + S ratio from 0.28 to 0.41; the ratio was 0.32 in hypoxic BAPN-treated animals. Hypoxia increased PA hydroxyproline from 20 to 239 micrograms/artery; hydroxyproline was 179 micrograms/artery in hypoxic BAPN-treated animals. Thus BAPN prevented pulmonary hypertension, right ventricular hypertrophy, and excess vascular collagen produced by hypoxia. We conclude that vascular collagen contributes to the maintenance of chronic hypoxic pulmonary hypertension.

Aminopropionitrile↗