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Biomedical subjects

M M Frank

Publications and source records attributed to M M Frank.

At least 73 records · Page 4Linked to original sources
PubMed1988

Linkage between brain blood flow and respiratory drive during rapid-eye-movement sleep.

The correlation between brain blood flow (BBF) and respiratory neuromotor output, as reflected by diaphragmatic electromyogram (EMG) activity (EMGdi), was studied during wakefulness, rapid-eye-movement (REM) sleep, and non-REM sleep (NREM). Compared with the awake state, mean BBF increased by 4.7% during NREM and by 32.6% during REM (P less than 0.001). Also, surges of BBF during REM occurred during periods of intense phasic activity. EMGdi [peak and peak/inspiratory time (TI)] was highly variable within REM periods but fluctuated as a reciprocal function of simultaneously measured BBf (r = -0.49, P less than 0.001). Furthermore, mean EMGdipeak decreased from NREM to REM in a manner reciprocally related to the corresponding change in BBF (r = -0.77, P = 0.015). These findings suggest that a component of the reduction of respiratory neuromotor output during REM is attributable to increased BBF with consequent relative hypocapnia in the central chemoreceptor environment.

Animals↗
PubMed1988

Interactions of a nonneutralizing IgM antibody and complement in parainfluenza virus neutralization.

While many viruses activate the complement cascade directly, this is generally not a neutralizing event in the absence of antibody. We used a nonneutralizing IgM monoclonal antibody to parainfluenza virus type 3 (PIV3) hemagglutinin-neuraminidase (HN) to explore the role of antibody in complement-dependent neutralization of PIV3. Neither the antibody nor nonimmune guinea pig serum (GPS) neutralized PIV3 significantly, but a more than 100-fold reduction in titer was found when antibody and GPS were combined. Heat-inactivated GPS or GPS lacking either of two different complement proteins were all inactive with or without antibody. Specific repletion of the deficient sera with highly purified complement proteins restored neutralizing activity, indicating an absolute requirement for the classical pathway of complement activation and lytic terminal complement components, and viral lysis was confirmed by electron microscopy. The presence of antibody before complement activation was essential; later addition had no effect. Spontaneous complement activation by PIV3 occurred via the classical pathway in the absence of antibody. Addition of antibody did not alter the overall rate or extent of complement component C3 binding to PIV3 in these experiments. We conclude that certain nonneutralizing antibodies may support complement-dependent PIV3 neutralization by facilitating viral lysis. This process does not, however, involve enhanced activation through the C3 step. Lysis may require antibody-dependent localization of the membrane attack complex or reorganization of the viral envelope structures to facilitate attack complex insertion and lysis.

Antibodies, Monoclonal↗
PubMed1988

CR1-receptor recycling in phorbol ester-activated polymorphonuclear leucocytes.

Complement-receptor type 1, CR1, which recognizes the C3b cleavage fragment of C3, is present on the membranes of human phagocytic cells, but does not mediate phagocytosis or undergo internalization unless activated by one of a variety of stimuli. Among these stimuli low doses of phorbol esters have been shown to induce a consistently increased expression of CR1, despite apparently continuous receptor internalization. We have studied the fate of internalized receptor-ligand complexes in neutrophils activated with low concentrations of phorbol dibutyrate. In our studies, we followed CR1 with either 125I-C3b, the physiologic ligand, or with 125I-Fab fragments of a monoclonal anti-CR1 antibody. We observed rapid internalization of CR1-C3b complexes by PMN treated with 10 ng/ml (1.98 x 10(-8)M) PDBu, consistent in rate and extent with previously reported results using monoclonal antibodies. The fate of the internalized ligand was studied after elution of cell-surface C3b at 0 degrees. Intracellular ligand was externalized in a time- and temperature-dependent fashion, reaching a plateau at 10-15 min. Released C3b was totally TCA precipitable and structurally unaltered, as determined by SDS-PAGE, suggesting that recycling occurs via a prelysosomal predegradative compartment. Loading the cells with chloroquine did not affect this process. A monoclonal anti-CR1 Fab probe behaved in exactly the same manner, suggesting that the recycling of intact ligand-receptor complexes takes place. The possible physiological consequences of this finding are discussed.

Chloroquine↗
PubMed1988

Human serum sickness: a prospective analysis of 35 patients treated with equine anti-thymocyte globulin for bone marrow failure.

We have prospectively evaluated the clinical and immunological features of serum sickness in 35 patients treated for bone marrow failure with anti-thymocyte globulin (ATG 15 mg/kg/day) and methylprednisolone (1 to 1.5 mg/kg/day). Twenty-one patients were treated for 10 days and 14 were treated for 28 days. Clinical evidence of serum sickness developed in 30 patients (86%) and included fever and malaise (100%), cutaneous eruptions (93%), arthralgias (67%), gastrointestinal complaints (67%), cephalgia (57%), blurring of vision (37%), arthritis, (30%) and lymphadenopathy (13%). Clinical serum sickness began on day 7 +/- 1 (X +/- S.E.M.) and lasted for 10 +/- 2 days in the 18 affected patients receiving the shorter course of ATG. In the 12 affected patients receiving the longer course of ATG, serum sickness began on day 9 +/- 1. The earliest manifestations of serum sickness were fever, malaise, and cutaneous eruptions. Cutaneous findings consisted of morbilliform eruptions (n = 19) and urticaria (n = 1) or a combination (n = 8) that lasted 10 to 14 days. Twenty-one patients (75%) developed a highly characteristic serpiginous band of erythema and purpura along the sides of the fingers, toes, palms and soles 12 to 48 hours before other symptoms of serum sickness. Biopsies of lesional skin during the course of serum sickness revealed immune deposits (IgM, IgE, IgA and C3) in dermal vasculature in 7 of 9 patients. Immunological changes that occurred during the course of serum sickness included increased serum levels of IgG, IgM, IgA, and IgE. Circulating immune complexes, as measured by the C1q-binding assay, increased from a mean value of 12% to 45% on day 13 +/- 1. Complement levels (C3, C4, and CH50) decreased 50 to 80% from their baseline levels on day 10 +/- 2. Acute phase reactants increased: erythrocyte sedimentation rate, C-reactive protein and beta-2 microglobulin. Abnormal urinalysis developed in 17 patients (57%) over the course of serum sickness and included proteinuria, hematuria and hemoglobinuria on day 10 +/- 3. Hematopoietic response occurred in 43%. All 5 patients who did not develop serum sickness recovered from bone marrow failure. Our data document the clinical and immunopathological findings in human serum sickness and suggest that the principles of antigen-antibody interaction, complement activation, and resultant inflammatory response as seen in the previous animal studies are directly applicable to studies of patients with serum sickness.

Adolescent↗
PubMed1988

Decline in rates of clearance of IgG-sensitized erythrocytes with increasing age.

Decreased immune functions have been suggested as a cause for the increased incidence of autoimmunity, malignancy, and infection in the elderly population. To assess the possible role of changes in macrophage function in the aging process we studied the Fc receptor-mediated clearance of IgG-coated erythrocytes in 56 healthy normal volunteers by following the removal of radiolabeled autologous erythrocytes. An age-related decrease in Fc-mediated clearance rates in both female and male subjects was found, which suggests a physiological decline of this macrophage function in older individuals.

Adult↗
PubMed1987

C5a induction of human interleukin 1. Synergistic effect with endotoxin or interferon-gamma.

Interleukin-1 (IL-1) is a potent cytokine which possesses the ability to mediate systemic acute phase responses as well as local tissue inflammation. In these studies, we have examined the ability of C5a and C5a des Arg to induce IL-1 production in vitro. Human C5a and C5a des Arg were purified to homogeneity and were found to stimulate IL-1 release from freshly obtained human mononuclear cells into the extracellular medium. Only 2 hr of exposure to the purified complement components were necessary in order to stimulate IL-1 production. The minimal concentration of C5a required was 25 ng/ml, whereas 125 ng/ml of C5a des Arg induced comparable amounts of IL-1. This dose relationship was maintained at higher concentrations (150 ng/ml vs 750 ng/ml, respectively). That the effect was due to the anaphylatoxins themselves, and not endotoxin contamination, was shown by negative Limulus amebocyte lysate tests and employing preincubation of C5a/C5a des Arg with polymyxin B. The latter blocked a wide dose range of endotoxin-stimulated IL-1 production. However, when endotoxin was added to C5a or C5a des Arg, significant synergism in the stimulation of IL-1 production was observed, occurring at various concentrations of either agent. A similar synergism with C5a/C5a des Arg was seen with interferon-gamma. In these studies, IL-1 production was measured by bioassay employing cloned D . 10 . G4 . 1 murine T cells and by radioimmunoassay for human IL-1 beta; using C5a/C5a des Arg as stimulants, there was a high degree of correlation (r = 0.82) between the two assays. Since traumatic, infectious, and inflammatory diseases may result in the simultaneous appearance of these stimuli, the synergism described herein is likely to be clinically relevant.

Biological Assay↗
PubMed1987

C3b generation is affected by the structure of the O-antigen polysaccharide in lipopolysaccharide from salmonellae.

Salmonellae differing in the O-antigen side chain of their lipopolysaccharide were previously shown to activate the alternative pathway of complement to different extents. We now examine the generation of the major cleavage fragment of the complement component C3 (C3b) on these bacteria in a system that contains the purified components C3, B, D, and P but lacks the regulatory proteins H and I. The deposition of C3b in this system reproduces the same pattern obtained earlier with the use of whole serum, with the expected differences among the strains bearing different O-antigen. However, two distinct mechanisms for these differences in C3b generation became apparent. The intermediate activating strain showed 3 to 4 times less initial deposition of C3b than the other two strains. In contrast, the least activating strain showed adequate initial deposition but poor amplification, as shown by 2 to 3.4 lower amplification indexes as compared with those on the other two strains. Binding studies with factor B showed that decreased C3 convertase formation was responsible for the low amplification on this strain. Only 25% of the C3b bound to its surface was able to bind factor B with a high affinity, in comparison with 90% on the other two strains. No differences were found for the binding of factor H among the strains. These studies identify the molecular mechanisms by which these bacteria avoid complement activation.

Antigens, Bacterial↗
PubMed1987

A complement-resistant HeLa cell line (T638) is blocked at the step of C3 deposition.

A complement-resistant line of HeLa cells (T638) was derived by serial passage of complement-susceptible HeLa cells in anti-beta 2-microglobulin (b2m) antiserum and complement. The T638 line maintained stable complement resistance when passed for an additional 1500 generations in the absence of antiserum and complement. T638 cells expressed equivalent levels of cell-associated b2m as did the parent HeLa cell line. Furthermore, T638 cells were resistant to killing by complement and anti-HeLa antiserum with specificity for molecules other than b2m. These results indicate that the resistance of T638 cells does not simply reflect loss of anti-b2m binding antigens. We next investigated the mechanism of resistance of T638 cells to complement-mediated killing. Antibody-sensitized HeLa and T638 cells both consumed CH50 activity completely from normal human serum; cytotoxicity was not mediated via the alternative complement pathway. HeLa and T638 cells caused equivalent utilization of C4 from normal human serum in the presence of antibody. Consumption of C2, greater with T638 than with HeLa cells during incubation in serum, was complete when cells bearing purified C1 and limited C4 were incubated with C2. T638 cells bound more 3H-C4 than HeLa cells during incubation in serum, but binding of 3H-C3 by T638 cells was fourfold to fivefold less than by HeLa cells. Finally, we investigated the rate of decay in the capacity of C142 on HeLa and T638 to cleave and deposit 3H-C3. The T1/2 for decay of C142-mediated binding of 3H-C3 on HeLa was 3.9 min, whereas minimal C3 deposition was detected on T638 cells at all time points. These results show that T638 cells evade complement-mediated lysis despite activating early components of the classical complement pathway. The mechanism of resistance is a failure to form an effective C3 convertase.

Antibodies↗
PubMed1987

Purification of C1 inhibitor. A new approach for the isolation of this biologically important plasma protease inhibitor.

C1 inhibitor (C1-INH) acts to inhibit active enzymes of both the classical complement and Hageman factor-dependent pathways. Previously reported C1-INH purification procedures were multistep and most have been associated with significant loss in specific functional activity. We have developed a simple chromatographic procedure which yields a pure C1-INH protein from normal human plasma with a specific activity equal to or greater than the starting sample. Briefly, protease inhibitor-treated, pooled human citrated plasma was fractionated with polyethylene glycol (PEG 4000); the supernatant fraction that remained soluble at 16% was obtained. The inhibitor was precipitated with 45% PEG. The resulting precipitate was solubilized and chromatographed on DEAE Sephacel using a linear salt gradient. The eluted fractions containing the C1-INH and other contaminants were pooled and dialyzed against the starting buffer of the next chromatographic step. A unique separation procedure using zinc ion chelate-coupled agarose was employed as the second chromatographic step. The eluted C1-INH, after zinc ion chromatography, displayed a significant enhancement in purity and maintained a specific functional activity twice that of plasma. The final procedure utilized immunoadsorption chromatography using an anti-contaminant column. Under reducing conditions on sodium dodecyl sulfate polyacrylamide gel electrophoresis, the purified C1-INH migrated as a single band with an apparent molecular weight of 90,000-105,000, but under non-reducing conditions, a doublet with apparent molecular weights of 94,000-100,000 and 85,000-93,000 was seen. C1-INH antigenic concentrations were measured and shown to be correlated in serum, citrate plasma, and EDTA plasma from 16 normal subjects.

Chelating Agents↗
PubMed1987

Detection of activated terminal complement (C5b-9) in cerebrospinal fluid from patients with central nervous system involvement of primary Sjogren's syndrome or systemic lupus erythematosus.

We have examined cerebrospinal fluid (CSF) and serum from patients with Sjogren's syndrome (SS) and systemic lupus erythematosus (SLE) for evidence of activation of the terminal pathway of complement. Fluid phase terminal complement complexes (SC5b-9), quantitated by ELISA, were detected in the CSF of 14 of 16 patients with SS and focal central nervous system (CNS) disease. Five of six SS patients without focal CNS disease but with psychiatric disease or cognitive dysfunction had detectable CSF SC5b-9, whereas two other SS patients without focal CNS or neuropsychiatric disease had no detectable CSF SC5b-9. Six of seven patients with SLE or SLE overlap syndrome with CNS involvement had CSF SC5b-9, whereas two patients with SLE without CNS involvement had no CSF SC5b-9. A subset of SS and SLE patients with CNS disease had SC5b-9 detected in CSF but not in serum. SC5b-9 was generally absent from the CSF of patients with noninflammatory CNS diseases. These findings demonstrate intrathecal activation of terminal complement in patients with CNS SS or CNS SLE, and suggest a role for terminal complement activation in the pathophysiology of CNS involvement in both SS and SLE.

Complement Membrane Attack Complex↗
PubMed1987

Modulation of FcR function by complement: subcomponent C1q enhances the phagocytosis of IgG-opsonized targets by human monocytes and culture-derived macrophages.

We have investigated the interaction of C1q, a subunit of the first component of complement, with human monocytes and culture-derived macrophages. Adherence of these mononuclear phagocytes to surfaces coated with C1q induced a marked enhancement of the phagocytosis of sheep erythrocytes opsonized with IgG anti-Forssman antibody (EA-IgG). This C1q-mediated enhancement of phagocytosis was dose dependent, and was specifically blocked by pretreatment of the C1q-coated surfaces with F(ab')2 anti-C1q. The augmentation of FcR-mediated phagocytosis by C1q was determined to be a result of the interaction between the C1q and the phagocytic effector cell, and was not due to interaction between the surface-bound C1q and the EA-IgG. Neither resting nor N-formyl-methionyl-leucyl-phenylalanine-stimulated polymorphonuclear leukocytes were induced by C1q to increase FcR-mediated phagocytosis. Experiments conducted with purified fragments of C1q suggest that the C1q phagocytosis enhancement signal resides in the collagen-like tail domain of the molecule. This region is the same portion of the molecule previously shown to interact with the cell surface C1q receptor. Native type I collagen was unable to enhance FcR-mediated phagocytosis by mononuclear phagocytes. It has been demonstrated that C1q can be localized to areas of inflammation, and additionally C1q can be secreted by macrophages in culture. In view of these findings and the results of our present study, we hypothesize that C1q could provide local, direct, and non-opsonic enhancement of phagocytosis by mononuclear phagocytes in areas of infection and inflammation.

Cell Adhesion↗
PubMed1987

Deficiency of the homologous restriction factor in paroxysmal nocturnal hemoglobinuria.

The affected E of two patients with paroxysmal nocturnal hemoglobinuria (PNH) were enriched by lysing the unaffected, normal E with anti-human decay-accelerating factor (DAF) and guinea pig serum. The membranes of the unlysed, DAF-deficient cells (PNH-E) were dissolved and examined by SDS-PAGE and immunoblotting using an antiserum to homologous restriction factor (HRF). Whereas the 65 kD complement regulatory protein was readily detectable in the normal controls, it was completely lacking in both samples of PNH-E membranes. Functional studies likewise indicated the absence of HRF activity from PNH-E. When radiolabeled, isolated HRF protein was offered to PNH-E, it became firmly attached to the cell. Approximately 1,000 molecules of HRF per cell reduced the characteristic susceptibility of these cells to reactive lysis by C5b-9 to nearly normal levels. The results suggest that HRF, which is known to control the action of C8 and C9 on normal human E membranes, is deficient in PNH, as well as acetylcholinesterase and DAF.

Blood Proteins↗
PubMed1987

Multimeric C9 within C5b-9 is required for inner membrane damage to Escherichia coli J5 during complement killing.

We have shown recently that an average of three or more C9 molecules must bind to C5b-8 on Escherichia coli strain J5 to cause direct complement killing in the absence of serum lysozyme. We initially confirmed and extended this observation by showing that deposition of a large number of C5b-9 complexes bearing 1C9 per C5b-8 was not bactericidal for J5. To identify the target site for bactericidal C5b-9 deposition, we measured release of periplasmic and cytoplasmic markers of different size from J5 as the C9:C5b-8 ratio was changed, because the diameter of the C5b-9 channel is known to increase as the C9:C5b-8 ratio increases. To facilitate measurement of release of the periplasmic marker beta-lactamase (BLA), J5 was transformed for high level constitutive TEM-1 BLA production (J5-Amp). Multimeric C9 within C5b-9 (C9:C5b-8 greater than 3) was required to release BLA (m.w. 28,900) from J5-Amp regardless of whether cells bore 310, 560, or 890 C5b-9/organism. Curves of both BLA release and killing vs C9:C5b-8 ratio were sigmoidal and nearly superimposable. Release of the small cytoplasmic marker 86Rb, a potassium analog, also required a minimum C9:C5b-8 ratio of 3:1; specific 86Rb release did not occur in the absence of killing. Release of the large cytoplasmic marker beta-galactosidase (m.w. 505,000) did not occur even at the highest achievable C9:C5b-8 ratio of 11:1, despite greater than 99.9% killing, indicating that there was no dissolution of the peptidoglycan layer due to incomplete removal of serum lysozyme. Complement-mediated killing of J5 requires sufficient damage to the outer membrane or formation of a sufficiently large C5b-9 channel to release the large periplasmic marker BLA. The requirement of multimeric C9 for 86Rb release suggests that at low C9:C5b-8 ratios, either C5b-9 does not have access to the cytoplasmic space or that the J5 K+ transport systems are able to compensate for putative C5b-9 channels.

Blood Bactericidal Activity↗
PubMed1987

Terminal complement components play a role in the expression of C5a.

This study examined the expression of C5a detected antigenically (RIA) and functionally (PMN-myeloperoxidase release) consequent to classical or alternative pathway convertase cleavage. Maximal C5a expression occurred when C5 was cleaved in the presence of the later-acting complement components, C6, C7, and C8. This effect was detected by using both purified components and normal human serum immunochemically depleted of C7 or C8 and reconstituted with the purified component. C6 alone was not sufficient to augment C5a expression. Subsequent incubation of C6 and C7 with C5 cleaved in the absence of the terminal components was not sufficient for C5a release. Repeated freezing and thawing of C5 cleaved in the absence of C6 and C7 produced C5a equivalent to that detected when convertase cleavage occurred in the presence of the terminal components. Mild detergent treatment of convertase-cleaved C5 was not sufficient for C5a release. We believe that these data indicate a role for the terminal complement components in the expression of both C5a antigen and function. The mechanism for this effect is not known, but it may involve conformational changes in the C5 molecule that occur during membrane attack complex formation.

Complement C5↗
PubMed1987

Studies of phagocytosis in chronic granulomatous disease.

Abnormal phagocyte function in chronic granulomatous disease (CGD) is associated with decreased bactericidal activity. Ingestion of serum-opsonized organisms is reported to be normal in these patients. We previously showed that in CGD the expression of C3b receptors (CR1) on polymorphonuclear leukocytes (PMNs) is significantly depressed. In this study, we compared the phagocytic activity of the PMNs from normal healthy controls with that of CGD patients and one individual with myeloperoxidase (MPO) deficiency. The ingestion of sheep erythrocytes (E) by PMNs adherent to a glass surface was examined; the E were coated either with excess IgG (E-IgG) or with C3b plus limited IgG (EAC3b-IgG). The PMNs, both in CGD and in MPO deficiency, ingested E-IgG and EAC3b-IgG at levels markedly above normal. C3b-coated erythrocytes were not phagocytosed. Preincubating the PMNs with sodium azide, which blocks MPO, or catalase, a scavenger of H2O2, caused a marked increase in phagocytosis by normal PMNs. Azide had a variable effect on PMN activity in CGD and no effect on the activity in the subject with MPO deficiency. Even in the presence of azide, the ingestion of EAC3b-IgG by the PMNs from the CGD patients was significantly greater than that seen in paired normals [mean phagocytic index (PI), 2.13 for CGD vs. 1.48 for normals; P less than 0.05 by the paired sample t test]. Similar results were obtained with ingestion of E-IgG. Notably, ingestion of serum-opsonized Candida organisms (relatively nondegradable particles) was markedly above normal with CGD PMNs and, in normal PMNs, azide treatment also evoked an increase. In addition, rosette formation of the adhered PMNs with E-IgG was enhanced with CGD and the azide-treated normal PMNs. We demonstrated that this increased activity was not the result of increased Fc receptor (FcR) number, as determined from the binding of a monoclonal anti-FcR antibody. Both the E-IgG rosette formation and the ingestion by CGD PMNs were abrogated in the presence of an H2O2-generating system. In contrast, the phagocytic activity of MPO-deficient PMNs was not altered by exogenous H2O2. These findings suggest that cellular products generated by the H2O2-MPO-halide system down-regulate the rosette-forming and phagocytic activity of PMNs from normal healthy individuals, but not that from CGD and MPO-deficient patients.

Azides↗