PubMed Health⌕ Search

Biomedical subjects

M M Frank

Publications and source records attributed to M M Frank.

At least 163 records · Page 9Linked to original sources
PubMed1983

Studies of the mechanism of bacterial resistance to complement-mediated killing. V. IgG and F(ab')2 mediate killing of E. coli 0111B4 by the alternative complement pathway without increasing C5b-9 deposition.

The mechanism of antibody-dependent complement-(C) mediated killing of Escherichia coli 0111B4, strain 12015 (12015), was examined. 12015 was resistant to serum killing when incubated in hypogammaglobulinemic serum (H gamma S) or pooled normal human serum (NHS) that had been previously adsorbed to remove specific antibody (Abs NHS). Presensitization with immune rabbit serum or purified immune rabbit IgG resulted in 1 to 3 log killing when 5 X 10(8) colony forming units (CFU)/ml were incubated in 10 to 40% Abs NHS. Binding of 125I-C3 and 131I-C9 to the bacterial surface of the presensitized and the nonpresensitized strain was quantitated when these organisms were incubated in 10, 20, and 40% Abs NHS. Stable binding of up to 3.0 X 10(5) molecules of C3 and 8.0 X 10(4) molecules of C9 to presensitized and nonpresensitized isolates occurred in the highest concentration of serum, but there was no killing without presensitization. Similar results were found when Abs NHS was chelated with ethylene bis glycoltetraacetic acid containing 2 mM MgCl2 (Mg EGTA) to block classical pathway activation, indicating that antibody mediated the bactericidal reaction through the alternative pathway. Deposition of C3 and C9 and killing of 120 15 in 10% Abs NHS or 10% H gamma S was measured after presensitization with increasing amounts of IgG, F(ab')2, or Fab'. There was a dose-dependent increase in C3 deposition and killing, but only minimal change in C9 binding when 1.0 X 10(3) to 3.2 X 10(4) IgG or F(ab')2/CFU were bound to the bacterial surface. In contrast, there was no increase in C3 or C9 binding and no bacterial killing when 1 X 10(3) to 3.4 X 10(4) molecules Fab'/CFU were bound to the bacterial surface. These experiments show that immune IgG and F(ab')2 can mediate killing of E. Coli 0111B4 by the alternative pathway without changing the extent of terminal C component attachment to the bacterial surface.

Animals↗
PubMed1983

Studies on the mechanism of bacterial resistance to complement-mediated killing. VI. IgG increases the bactericidal efficiency of C5b-9 for E. coli 0111B4 by acting at a step before C5 cleavage.

Our previous experiments showed that immune IgG and F(ab')2, but not Fab', mediated serum killing of Escherichia coli 0111B4, strain 12015 (12015), without significantly increasing the extent of terminal complement (C) component attachment to the bacterial surface. We concluded that bactericidal antibody must change either the site or the nature of C5b-9 bacterial attachment. To pursue this possibility, conditions necessary for elution of C5b-9 from the bacterial surface were examined. Forty-two to 44% of 125I-C9 was released from the serum-resistant nonpresensitized 12015 by 1 M NaCl or 0.1% trypsin, compared with the 21 to 24% release from the serum-sensitive presensitized isolate under the same condition. When strain 12015 bearing 125I-C9 was lysed in a French pressure cell, 73.1% of 125I-C9 was released with the capsular fraction if the organisms had not been presensitized. In contrast, on presensitized 12015, 70.2% of 125I-C9 remained associated with the outer membrane after such lysis. These results suggested that C5b-9 was trapped within or underneath the capsule of 12015 in the absence of bactericidal antibody, but that addition of antibody led to C5b-9 insertion into the outer membrane with bacterial killing. The requirement of C components preceding C5 for bacterial killing was next examined. Minimal killing of presensitized 12015 occurred when a terminal C complex was formed by acid activation from purified C5, C6, C7, C8, and C9 in the absence of C3 or earlier components. In contrast, between 1.2 and 3 log killing of nonpresensitized rough Salmonella minnesota and rough E. coli was observed in the same system. Killing of 12015 was examined with bacteria incubated in C5-deficient serum (C5D), followed by washing and the addition of purified C5, C6, C7, C8, and C9 to permit C5b-9 formation. Antibody was added before or after incubation in C5D serum, or after the addition of purified C5-C9. Under conditions of equivalent C3 and C9 binding, significant killing occurred only when antibody was added before incubation in C5D serum. These results show that antibody must be present at or before the time of C5 convertase formation to mediate killing of 12015 by C5b-9. Therefore, antibody is unlikely to be functioning primarily to alter the bacterial surface to expose sites for C5b-9 insertion, nor is the effect of antibody simply to increase C3 and terminal component binding. We postulate that antibody mediates killing of 12015 by localizing C5b-9 around antibody-clustered sites of C3 and C5 convertase formation.

Animals↗
PubMed1983

Surface modulation of classical pathway activation: C2 and C3 convertase formation and regulation on sheep, guinea pig, and human erythrocytes.

We examined the formation of the early classical complement (C) pathway enzymes on sheep (Es), guinea pig (Egp), and human (Eh) erythrocytes (E). Each species' E were sensitized with sufficient IgM or IgG anti-E Ab to establish equal numbers of C1-fixing sites on all E. After sensitization with 100 C1-fixing sites of Ab and excess C1, uptake of C4 was equivalent on all three cell types, judged by anti-C4 binding (for guinea pig C4) or by direct uptake of radiolabeled protein (for human C4). With equal numbers of cell-bound C1 and C4, however, there were marked differences in C2 convertase activity on Es, Egp, and Eh. Sheep EAC14 utilized C2 at least 20 times faster than Egp and Eh bearing the same number of C1 and C4 molecules. C3 cleavage was even further depressed on Egp and Eh, and was not changed by the substitution of oxyC2 for normal human C2. In whole guinea pig serum (GPS), 300 times more C1-fixing sites were required on Egp than on Es to achieve similar amounts of lysis; however, equivalent C3 uptake on Egp and Es was associated with equal extents of lysis, demonstrating that GPS lysis of these cells was regulated by early steps in classical pathway (CP) activation. Incubation of E bearing radiolabeled C4b with Factor I demonstrated that C4b on Egp was highly resistant to cleavage compared to the same protein bound to Es or Eh. Studies with partially purified C3 convertase decay-accelerating factors from Eh stroma demonstrated that these membrane proteins could not account for the observed surface regulation of CP activity because these proteins do not affect the rate of C2 cleavage by EAC14. We conclude that E surface molecules have an important role in modulation of CP activation. This surface-associated CP regulation occurs at the level of cell-bound C4b.

Animals↗
PubMed1983

Reticuloendothelial system fc receptor function in rheumatoid arthritis.

Patients with systemic lupus erythematosus (SLE) have abnormal reticuloendothelial system (RES) IgG Fc receptor function which is correlated with disease activity and levels of immune complexes (IC). We studied RES function, IC levels, clinical and laboratory manifestations in 43 patients with RA. The articular index was abnormal in 42 patients (range of 6 to 153). Thirty were seropositive; 31 had an elevated erythrocyte sedimentation rate. Extraarticular manifestations included nodules (25), anemia (19), Sjögren's syndrome (6), vasculitis (4), and Felty's syndrome (2). Medication included corticosteroids (32), nonsteroidal antiinflammatory drugs (33), slow acting antirheumatic drugs (18), and cytotoxics (2). Thirty-three patients had IC (30 by the Clq binding assay, 21 by the Raji cell assay); 13 patients had normal RES function (t 1/2-24-60 min; upper 95% confidence limit in normals = 62 min). Thirty had prolonged clearance times (65-210 min, mean 106 min). In contrast to SLE, clearance times were not found to be correlated with IC levels, or clinical or laboratory measures of disease activity. Defective RES immune clearance may not be an important determinant in the pathogenesis of immunologic tissue damage in rheumatoid arthritis.

Adult↗
PubMed1982

Studies on the mechanism of bacterial resistance to complement-mediated killing. I. Terminal complement components are deposited and released from Salmonella minnesota S218 without causing bacterial death.

The mechanism of resistance of gram-negative bacteria to killing by complement was investigated. Complement consumption and uptake of purified, radiolabeled complement components on bacteria was studied using a serum- sensitive and a serum-resistant strain of Salmonella minnesota. Twice as many molecules of (125)I C3 were bound per colony-forming unit (CFU) of the smooth, serum-resistant S. minnesota S218 as were bound per CFU of the rough, serum-sensitive S. minnesota Re595 in 10 percent pooled normal human serum (PNHS), although 75-80 percent of C3 was consumed by both organisms. Hemolytic titrations documented total consumption of C9 by 5 min and more than 95 percent consumption of C5 and C7 by 15 min in the reaction with S218 with 10 percent PNHS. In contrast, negligible C5 depletion, 10 percent C7 consumption, and only a 26 percent decrease in C9 titer occurred with the serum-sensitive Re595. Binding of (125)I C5, (125)I C7, and (125)I C9 to S218 and Re595 was measured in 10 percent PNHS. A total of 6,600 molecules C5/CFU, 5,200 molecules C7/CFU, and 3,100 molecules C9/CFU bound to S218 after 5-10 min of incubation at 37 degrees C, but 50-70 percent of the C5, C7, and C9 bound to S218 was released from the organism during incubation at 37 degrees C for 60 min. Binding of 2,000 molecules C5/CFU, 1,900 molecules C7/CFU, and 9,000 molecules C9/CFU to Re595 was achieved by 20 min and was stable. The ratio of bound C9 molecules to bound C7 molecules, measured using (131)I C9 and (125)I C7, was constant for both organisms after 15 min and was 4.3:1 on Re595 and 0.65:1 on S218 in 10 percent PNHS. With addition of increasing amounts of purified, unlabeled (29 to 10 percent PNHS, there was no change in the C9:C7 ratio on Re595. However, with S218 there was a linear increase of the C9:C7 ratio, which approached the ratio on Re595. There was no (14)C release from S218 incubated in PNHS, nor was there evidence by electron microscopy of outer membrane damage to S218. Therefore, S. minnesota S218 is resistant to killing by PNHS, despite the fact that the organism consumes terminal complement components efficiently and that terminal components are deposited on the surface in significant amounts. The C5b-9 complex is released from the surface of S218 without causing lethal outer membrane damage.

Adsorption↗
PubMed1982

Studies on the mechanism of bacterial resistance to complement-mediated killing. II. C8 and C9 release C5b67 from the surface of Salmonella minnesota S218 because the terminal complex does not insert into the bacterial outer membrane.

The mechanism for consumption of terminal complement components and release of bound components from the surface of serum-resistant salmonella minnesota S218 was studied. Consumption of C8 and C9 by S218 occurred through interaction with C5b67 on the bacterial surface because C8 and C9 were consumed when added to S218 organisms previously incubated in C8-deficient serum and washed to remove all C5b67 on the bacterial surface because C8 and C9 were consumed when added to S218 organisms previously incubated in C8- deficient serum and washed to remove al but cell bound C5b67. Rapid release of (125)I C5 and (125)I C7 from the membrane of S218 was dependent on binding of C8 because (125)I C5 and (125)I C7 deposition in C8D serum was stable and was twofold higher in C8D than in PNHA, and addition of purified C8 or C8 and C9 to S218 previously incubated in C8D serum caused rapid release of (125)I C5 and (125)I C7 from the organism. Analysis by sucrose density gradient ultracentrifugation of the fluid phase from the reaction of S218 and 10 percent PNHS revealed a peak consistent with SC5b-9, in which the C9:C7 ratio was 3.3:1, but the NaDOC extracted bound C5b-9 complex sedimented as a broad peak with C9:C7 of less than 1.2:1. Progressive elution of C5b67 and C5b-9 from S218 but not serum-sensitive S. minnesota Re595 was observed with incubation in buffers of increasing ionic strength. Greater than 90 percent of the bound counts of (125)I C5 or (125)I C9 were released from S218 by incubation in 0.1 percent trypsin, but only 57 percent of (125)I C9 were released by treatment of Re595 with trypsin. These results are consistent with the concept that C5b-9 forms on the surface of the serum-sensitive S. minnesota S218 in normal human serum, but the formed complex is released and is not bactericidal for S218 because it fails to insert into hydrophobic outer membrane domains.

Blood Bactericidal Activity↗
PubMed1982

A serial study of splenic reticuloendothelial system Fc receptor functional activity in systemic lupus erythematosus.

A serial study of reticuloendothelial system splenic Fc receptor function was undertaken in 13 patients with systemic lupus erythematosus (SLE). Fc receptor function was assessed by measuring the clearance half times of IgG-sensitized 51Cr-labeled autologous erythrocytes from the circulation. Prolonged clearance half times were initially detected in all patients, and the magnitude of the clearance defect was significantly correlated with disease activity (P less than 0.01, Spearman rank correlation) and immune complex levels (P less than 0.01). Clinical course and changes in receptor functional activity over time were found to be significantly correlated. Clearance half-times tended to return to normal in all 11 patients who clinically improved. The clearance half-time remained unchanged in 1 patient whose disease activity was stable, and it worsened in 1 patient whose illness flared. Levels of immune complexes also changed concordantly with clearance half-times and disease activity. These studies underscore the interrelationship between the presence of circulating immune complexes, defects in Fc receptor function that might lead to prolonged circulation of these complexes, and disease activity in SLE.

Adult↗
PubMed1982

Biotinylation of human C3.

Purified human C3 was biotinylated using the biotinyl-N-hydroxysuccinimide imidoester (BNHS). Depending on the input of BNHS, from three to six molecules of biotin were incorporated per C3 molecule. The biotinyl-C3 retained over 90% of its specific hemolytic activity and when bound to sheep erythrocytes maintained its ability to adhere to human C3b receptors. These functions could be blocked by avidin. The biotinyl-C3 was fragmented normally to C3c and C3d in human serum and adsorption with avidin-Sepharose indicated that biotin moities were present in both fragments. Fluorescein-conjugated avidin reacted well with cell-bound biotinyl-C3b and was useful for quantitating C3 fixation by flow cytometry. Ferritin-conjugated avidin was used as a marker to characterize the distribution of biotinyl-C3b on erythrocytes by electron microscopy. These results suggest that biotinyl-C3 and avidin derivatives may be very useful tools for studies of many of the biological functions of C3.

Avidin↗
PubMed1982

A quantitative analysis of the interactions of antipneumococcal antibody and complement in experimental pneumococcal bacteremia.

The mechanism of protection of type-specific antipneumococcal antibody and complement in bacteremia was investigated with purified rabbit antibody and a guinea pig model of pneumococcal bacteremia. IgG and IgM were isolated from the sera of rabbits immunized with type 7 pneumococci (Pn), and their binding to Pn was quantitated. The number of antibody-binding sites on the pnuemococcal capsule was also determined. Pn were incubated with various amounts of the immunoglobulin preparations before intravenous injection into nonimmune guinea pigs. Whereas 120 molecules of IgM per Pn were sufficient to enhance bloodstream clearance of Pn, 1,400 molecules of IgG per bacterium were required to produce this effect. As the amount of either IgG or IgM added to the Pn was increased, the rate of bloodstream clearance accelerated. In striking contrast, greater than 1,000 molecules of IgM had no effect on the rate of clearance in C4-deficient guinea pigs, which cannot activate complement via the classic pathway. Similarly, 5,000 molecules of IgG had only minimal effect in C4-deficient guinea pigs, and 24,000 molecules of IgG had no effect in guinea pigs depleted of complement by cobra venom factor. Thus, the in vivo opsonic effects of both IgG and IgM anticapsular antibody are mediated via their ability to activate complement. IgG anti-pneumococcal cell wall antibody, raised by intravenous injection of rabbits with unencapsulated Pn, had no effect on the rate of bloodstream clearance of Pn or on the polymorphonuclear leukocyte killing of type 7 Pn in an in vitro bacterial assay. Because the opsonic effects of anticapsular antibody required complement activation, the ability of anticell wall IgG to activate complement was compared with the two classes of anticapsular antibody. As judged by depletion of C3 and C4 from guinea pig serum, as well as by the fixation of radiolabeled C3 to Pn, IgM anticapsular antibody was the best complement activator. However, anticell wall IgG was somewhat more active than anticapsular IgG in each of these tests of complement activation and fixation. When equivalent amounts of C3 were fixed to Pn by each of the three antibodies, Pn sensitized with IgG and IgM anticapsular antibodies caused immune adherence, whereas Pn sensitized with anticell wall IgG did not. This may explain the failure of anticell wall antibody of mediate complement-dependent phagocytosis of Pn in vivo or in vitro. Although anticell wall IgG is capable of activating complement and fixing C3 to Pn, it is not opsonic; the most likely reason is that the nonopsonic antibody mediates C3 deposition in sites on the Pn that cannot interact efficiently with phagocytic cell C3 receptors.

Animals↗
PubMed1982

The requirement of specific anticapsular IgG for killing of Haemophilus influenzae by the alternative pathway of complement activation.

Employing an in vitro bactericidal assay using C4-deficient guinea pig serum (C4D-GPS), we evaluated the ability of the alternative pathway to kill a number of strains of encapsulated and unencapsulated Haemophilus influenzae (HI). Complement activation occurred, but no bactericidal activity was observed against any of the tested HI in nonimmune C4D-GPS without detectable antibody to HI-type b (HIb) capsular polysaccharide (HIb-ps). In the presence of high-titered human anti- (type b) IgG, C4D-GPS killed the encapsulated strains. Restoration of classical pathway activity by the addition of purified C4 to C4D-GPS, which contained antibody to somatic antigens, resulted in killing of some strains of both encapsulated and unencapsulated HI. In nonimmune C4D-GPS the alternative pathway is activated but does not mediate bactericidal activity. The addition of specific high-titered anticapsular IgG results in killing of encapsulated HI by the alternative pathway in guinea pig serum.

Animals↗
PubMed1982

Monocyte receptors for the Fc portion of IgG studies with monomeric human IgG1: normal in vitro expression of Fc gamma receptors in HLA-B8/Drw3 subjects with defective Fc gamma-mediated in vivo clearance.

The Fc gamma receptors of reticuloendothelial cells are presumed to play an important role in the clearance of circulating particles opsonized with IgG. In order to quantify these receptors and assess their contribution to the clearance phenomenon in man, Scatchard analysis has been applied to 125I-IgG1 myeloma protein binding by a model mononuclear phagocyte, the peripheral blood monocyte. Close compliance to the criteria for linear Scatchard plots has been obtained. The kinetics of binding at 37 degrees C were consistent with a simple, reversible, bimolecular reaction. A saturable, single class of high-affinity binding sites was discerned with a Ka of 2.61 +/- 0.13 x 10(8) M-1 and a mean of 35,500 +/- 1700 receptors per monocyte. These receptors expressed approximately equal affinities for IgG subclasses 1 and 3, with progressively lower affinities for IgG subclasses 4 and 2, respectively. Parameters of IgG1 binding to monocytes of patients with dermatitis herpetiformis and normal individuals of HLA-B8/Drw3 haplotype were not significantly different from controls, despite the previous demonstration of retarded IgG-mediated clearance in one-half of such subjects. Receptor number and affinity failed to correlate with T 1/2 for sensitized erythrocyte clearance in vivo. Functional defects in in vivo clearance, even in the absence of circulating immune complexes, are not necessarily related to abnormal expression of Fc gamma receptors by phagocytes.

Antigen-Antibody Complex↗
PubMed1982

The use of conglutinin in a quantitative assay for the presence of cell-bound C3bi and evidence that a single molecule of C3bi is capable of binding conglutinin.

We have developed a quantitative assay for cell surface C3bi using 125I-labeled conglutinin. Conglutinin was purified to homogeneity from bovine serum and radiolabeled with 125I Bolton Hunter reagent. Conditions of time, temperature, ionic strength, and cell concentration that optimized the binding of conglutinin to erythrocytes bearing C3bi were then determined. The interaction between conglutinin and C3bi under these conditions was highly specific, since EAC4b3b, EAC4b3d, EAC4b3b-beta IH, and EAC4b treated with serum did not bind radioconglutinin significance better with EA or EAC4b. Using this assay, was examined the kinetics of inactivation of both human and guinea pig C3b bound to erythrocytes and showed that, for both, maximum conglutinin binding occurred after EAC4b3b had been incubated with a source of beta 1H and C3INA for 10 to 20 min at 37 degrees C.l We showed a linear relationship between the number of molecules of C3bi per erythrocyte and the amount of conglutinin bound for both guinea pig and human C3bi. The affinity of conglutinin for cell-bound C3bi was shown to be independent of C3bi density on the erythrocyte surface, and the Kd for conglutinin binding to erythrocytes bearing human C3bi was determined to be 1.3 X 10(-8) M. The number of conglutinin binding sites per erythrocyte as calculated from Scatchard plots was equal to the number of C3bi molecules on the cell surface as determined by direct assay using 125I-labeled C3. Moreover, for both human and guinea pig C3bi, the plot of log (cell surface C3bi) vs log (conglutinin bound) had a slope of 1. These findings imply that a single molecule of C3bi is capable of binding a molecule of conglutinin under the conditions of our assay.

Animals↗
PubMed1981

Impaired immune response of splenectomised patients to polyvalent pneumococcal vaccine.

Immune response to polyvalent pneumococcal vaccine was measured in normal volunteers and splenectomised patients, by means of a newly developed enzyme-linked immunosorbent assay for pneumococcal antibodies. IgG and IgM responses to eight of the nine polysaccharides tested were markedly impaired in splenectomised patients. The response of splenectomised patients was less than that of the controls in terms of absolute titre of antibody achieved, relative rise in titre (fold increase of post-immunisation titre over pre-immunisation titre), and rate of rise.

Antibodies, Bacterial↗
PubMed1981

Opsonic requirements for intravascular clearance after splenectomy.

We investigated the opsonic requirements for intravascular clearance of pneumococci in guinea pigs and of sensitized erythrocytes in human beings after splenectomy. The impaired clearance of injected pneumococci in splenectomized guinea pigs was corrected by immunization. This improvement in clearance was due to increased hepatic sequestration of organisms. There was a significant delay in antibody-mediated clearance of autologous erythrocytes sensitized with IgG (P < 0.001), although the rate of complement-mediated clearance in splenectomized patients was normal. A fourfold increase in sensitizing antibody resulted in a significant improvement in clearance that was due to increased hepatic sequestration (P < 0.005). One patient who had an intact spleen and who had previously received Thorotrast (thorium oxide) had impaired antibody-mediated clearance despite increased sensitization. These observations suggest that, after splenectomy the remaining macrophages of the reticuloendothelial system require increased amounts of antibody to mediate efficient intravascular clearance of opsonized particles.

Animals↗
PubMed1981

Defective Fc-receptor functions associated with the HLA-B8/DRw3 haplotype: studies in patients with dermatitis herpetiformis and normal subjects.

Dermatitis herpetiformis is associated with the HLA-B8/DRw3 haplotype in over 90 per cent of patients, and various percentages have been reported to have circulating immune complexes. Since removal of immune complexes from the circulation is thought to depend on the Fc-receptor function of tissue macrophages, we studied this function by measuring the clearance of IgG-sensitized autologous erythrocytes in 16 patients with dermatitis herpetiformis, in normal controls with the HLA-B8/DRw3 haplotype, and in randomly selected controls. All patients were HLA-B8 positive, and all of 12 patients tested were HLA-DRw3 positive. Erythrocyte clearance was reduced in eight of the 16 patients, but did not correlate with immune-complex levels. Four of eight controls with HLA-B8/DRw3 also had delayed Fc-receptor-mediated clearance as compared with normal controls. In addition, both patients and HLA-B8/DRw3-positive controls had decreased percentages and total numbers of T cells bearing Fc receptors for IgG. These findings indicate a functional Fc-receptor defect associated with the HLA-B8/DRw3 antigens.

Adult↗
PubMed1981

Binding of immunoglobulin- and complement-coated erythrocytes to human neutrophil subpopulations.

Human neutrophils are separable into two populations on the basis of the presence or absence of Fc receptors as detected by rosette assays with IgG antibody-sensitized human erythrocytes. In the present report it was shown that IgG-sensitized sheep erythrocytes could detect some Fc receptor activity on those cells which are Fc receptor-negative with the human cell assay. There was clearly a difference between Fc receptor expressed on the positive and negative cells. Neutrophils with "high" and "low" density Fc receptor activity were studied for a range of membrane receptors for complement fragments including, C4b, C3b, C3d, and C3bi. No difference was found between the two populations of cells in their expression of complement receptors. Moreover, neither population expressed receptors for C3d, suggesting that neither population was a high proportion of immature neutrophils.

Animals↗