Michael Bruce Whitlow, M.D., Ph.D, Ph.D.

Proliferating trichilemmal cyst with focal calcification
Anolik, Robert; Firoz, Bahar; Walters, Ruth F; Meehan, Shane A; Tsou, Hui C; Whitlow, Michael; Wainwright, Brent
2008 ;14(10):25-25, Dermatology online journal
A 64-year-old man presented with a superficial, well-demarcated, skin-colored tumor on the left posterior scalp that measured 4 x 5 x 6 cm. The tumor was nearly hairless, rubbery, non-tender, and mobile over the underlying subcutaneous tissues. The lesion had grown slowly since arising approximately 30 years ago. Treatment options were declined in the past. However, with relatively more rapid growth over the past five years, the nodule began to cause intermittent pain and interfere with the patient's ability to lie on his back. The patient denied a history of similar lesions in himself or his family. A biopsy specimen showed a ruptured proliferating trichilemmal cyst with focal calcification. Complete excision is recommended for all benign proliferating variants owing to their potential for locally aggressive behavior and malignant transformation
— id: 95417, year: 2008, vol: 14, page: 25, stat: Journal Article,

Pyoderma gangrenosum
Kim L; Whitlow M
Current dermatologic diagnosis & treatment Philadelphia : Lippincott Williams & Wilkins, 2001,
— id: 3753, year: 2001, vol: , page: 184, stat: Chapter,

Aquatic dermatology
Whitlow M
Current dermatologic diagnosis & treatment Philadelphia : Lippincott Williams & Wilkins, 2001,
— id: 3678, year: 2001, vol: , page: 16, stat: Chapter,

Interaction between terminal complement proteins C5b-7 and anionic phospholipids
Liu C; Marshall P; Schreibman I; Vu A; Gai W; Whitlow M
1999 Apr 1;93(7):2297-2301, Blood
We have recently shown that C5b-6 binds to the erythrocyte membrane via an ionic interaction with sialic acid before the addition of C7 and subsequent membrane insertion. In this study we assessed the role of anionic lipids in the binding of the terminal complement proteins to the membrane and the efficiency of subsequent hemolysis. Human erythrocytes were modified by insertion of dipalmitoyl phosphatidylcholine (DPPC), dipalmitoyl phosphatidylserine (DPPS), dipalmitoyl phosphatidylethanolamine (DPPE), or dipalmitoyl phosphatidic acid (DPPA). Lipid incorporation and the hemolytic assays were done in the presence of 100 micromol/L sodium orthovanadate to prevent enzymatic redistribution of lipid. We found that the neutral lipids, DPPC and DPPE, did not affect C5b-7 uptake or hemolysis by C5b-9. In contrast, the two acidic phospholipids, DPPS and DPPA, caused a dose-dependent increase in both lysis and C5b-7 uptake. We conclude that the presence of anionic lipids on the exterior face of the membrane increases C5b-7 uptake and subsequent hemolysis. It is known that sickle cell erythrocytes have increased exposure of phosphatidylserine on their external face and are abnormally sensitive to lysis by C5b-9. The data presented here provide a plausible mechanism for this increased sensitivity
— id: 56412, year: 1999, vol: 93, page: 2297, stat: Journal Article,

Response of SCC-12F, a human squamous cell carcinoma cell line, to complement attack
Whitlow MB; Klein LM
1997 Jul;109(1):39-45, Journal of investigative dermatology
We studied the response of a human squamous cell carcinoma cell line, SCC-12F, to human complement attack and found that the cells were completely resistant to complement lysis. In the absence of lysis, there was significant C3 deposition and C5b-9 deposition on the cells. Removal of the lipid-linked complement regulatory proteins CD59 and decay-accelerating factor (DAF) by treatment of the cells with phosphatidylinositol-specific phospholipase C (PIPLC) resulted in increased C3b and C5b-9 deposition on the cells and a slight increase in cell death. Treatment of the cells with complement caused them to release membrane vesicles containing the terminal complement proteins. In addition, complement induced SCC-12F to produce significant amounts of prostaglandin F2alpha (PGF2alpha). We conclude that CD59 and DAF are important in the resistance of SCC-12F to complement and that these cells produce membrane vesicles and PGF2alpha in response to complement attack. These responses, in the absence of cell death, may be important in the pathogenesis of inflammatory skin disease in which complement is deposited
— id: 7280, year: 1997, vol: 109, page: 39, stat: Journal Article,

Interaction between complement proteins C5b-7 and erythrocyte membrane sialic acid
Marshall P; Hasegawa A; Davidson EA; Nussenzweig V; Whitlow M
1996 Oct 1;184(4):1225-1232, Journal of experimental medicine
The initial phase of membrane attack by complement is the interaction between C5b6, C7, and the cell membrane that leads to the insertion of C5b-7. Here we investigate the role of sialic acid residues in the assembly of C5b-7 intermediates on erythrocyte cell membranes. We find that C5b6 binds to glycophorin, whereas C5 or C6 does not bind, and desialylation of the glycophorin abolishes C5b6 binding. Complement lysis is inhibited by either masking glycophorin sialic acid with F(ab) fragments of an mAb, or by removal of the sialylated region of glycophorin by mild trypsinization. Gangliosides inhibit C5b-7 deposition when added to the aqueous phase. Asialogangliosides and synthetic gangliosides lacking the carboxylic acid residue have no inhibitory activity. We conclude that C5b6 binds to sialylated molecules on the erythrocyte surface. We propose a new model of membrane attack in which C5b6 initially binds to membranes via ionic forces. C7 then binds to C5b6, disrupting the ionic interaction and leading to the exposure of hydrophobic domains. Sialic acid is known to inhibit complement activation. Thus, these findings reveal a paradoxical role for sialic acid in complement attack; the presence of sialic acid inhibits the generation of C5b6, but once the membrane attack pathway is initiated, sialic acid enhances complement lysis
— id: 61953, year: 1996, vol: 184, page: 1225, stat: Journal Article,

Identification of C1q as the heat-labile serum cofactor required for immune complexes to stimulate endothelial expression of the adhesion molecules E-selectin and intercellular and vascular cell adhesion molecules 1
Lozada C; Levin RI; Huie M; Hirschhorn R; Naime D; Whitlow M; Recht PA; Golden B; Cronstein BN
1995 Aug 29;92(18):8378-8382, Proceedings of the National Academy of Sciences of the United States of America
To examine the role of complement components as regulators of the expression of endothelial adhesive molecules in response to immune complexes (ICs), we determined whether ICs stimulate both endothelial adhesiveness for leukocytes and expression of E-selectin and intercellular and vascular cell adhesion molecules 1 (ICAM-1 and VCAM-1). We found that ICs [bovine serum albumin (BSA)-anti-BSA] stimulated endothelial cell adhesiveness for added leukocytes in the presence of complement-sufficient normal human serum (NHS) but not in the presence of heat-inactivated serum (HIS) or in tissue culture medium alone. Depletion of complement component C3 or C8 from serum did not prevent enhanced endothelial adhesiveness stimulated by ICs. In contrast, depletion of complement component C1q markedly inhibited IC-stimulated endothelial adhesiveness for leukocytes. When the heat-labile complement component C1q was added to HIS, the capacity of ICs to stimulate endothelial adhesiveness for leukocytes was completely restored. Further evidence for the possible role of C1q in mediating the effect of ICs on endothelial cells was the discovery of the presence of the 100- to 126-kDa C1q-binding protein on the surface of endothelial cells (by cytofluorography) and of message for the 33-kDa C1q receptor in resting endothelial cells (by reverse transcription-PCR). Inhibition of protein synthesis by cycloheximide blocked endothelial adhesiveness for leukocytes stimulated by either interleukin 1 or ICs in the presence of NHS. After stimulation with ICs in the presence of NHS, endothelial cells expressed increased numbers of adhesion molecules (E-selectin, ICAM-1, and VCAM-1). Endothelial expression of adhesion molecules mediated, at least in part, endothelial adhesiveness for leukocytes, since leukocyte adhesion was blocked by monoclonal antibodies directed against E-selectin. These studies show that ICs stimulate endothelial cells to express adhesive proteins for leukocytes in the presence of a heat-labile serum factor. That factor appears to be C1q
— id: 7927, year: 1995, vol: 92, page: 8378, stat: Journal Article,

INTERACTION OF THE TERMINAL COMPLEMENT PROTEINS WITH SIALIC-ACID CONTAINING MOLECULES OF THE ERYTHROCYTE
WHITLOW, M; MARSHALL, P
1995 DEC ;105(6):873-873, Journal of investigative dermatology
— id: 52661, year: 1995, vol: 105, page: 873, stat: Journal Article,

New York University therapeutic roundtable: a panel of experts answer questions on the treatment of challenging cases
Shupack JL; Kanof N; Stolman LP; Vogel L; Whitlow M; Cohen DE; Washenik K; Lee MP; Stiller MJ
1994 Jul;54(1):29-33, Cutis
— id: 6750, year: 1994, vol: 54, page: 29, stat: Journal Article,

A synthetic peptide from complement protein C9 binds to CD59 and enhances lysis of human erythrocytes by C5b-9
Tomlinson S; Whitlow MB; Nussenzweig V
1994 Feb 15;152(4):1927-1934, Journal of immunology
The membrane glycoprotein CD59 protects host cells from homologous complement attack by inhibiting the assembly of the membrane attack complex. CD59 binds to C8 and C9 in the nascent membrane attack complex and interferes with C9 membrane insertion and polymerization. We show here that a synthetic peptide from the putative C9 hinge region, postulated to be involved in the rearrangement of C9 globular domains during membrane insertion, binds specifically to CD59 and enhances lysis of human erythrocytes by the terminal complement C5b-9 complex. The peptide, C9H, caused a dose-dependent increase in the sensitivity of human erythrocytes to C5b-9-mediated lysis by interfering with the final C9 binding and/or membrane insertion step. C9H exhibited species-specificity, since it had no activity against guinea pig C8 and C9 or on the putative functional homologues of CD59 in guinea pig erythrocytes. A direct association between CD59 and C9H was suggested by two different binding experiments: C9H inhibited the binding of 125I-labeled CD59 to immobilized C9, and C9H immobilized to microtiter plates bound purified CD59 and selectively recognized CD59 from extracts of detergent-solubilized human erythrocyte membranes. These data indicate that the peptide C9H corresponds to a region of the CD59 binding site of C9
— id: 7888, year: 1994, vol: 152, page: 1927, stat: Journal Article,

RESPONSE OF HUMAN KERATINOCYTES TO COMPLEMENT ATTACK
WHITLOW, MB; KLEIN, LM
1994 OCT ;42(3):A454-A454, Clinical research
— id: 52382, year: 1994, vol: 42, page: A454, stat: Journal Article,

RESPONSE OF HUMAN KERATINOCYTES TO COMPLEMENT ATTACK
KLEIN, LM; WHITLOW, MB
1993 APR ;100(4):574-574, Journal of investigative dermatology
— id: 54250, year: 1993, vol: 100, page: 574, stat: Journal Article,

RESPONSE OF HUMAN KERATINOCYTES TO COMPLEMENT ATTACK
KLEIN, M; WHITLOW, MB
1993 APR ;41(2):A470-A470, Clinical research
— id: 54296, year: 1993, vol: 41, page: A470, stat: Journal Article,

Cells lacking glycan phosphatidylinositol-linked proteins have impaired ability to vesiculate
Whitlow M; Iida K; Marshall P; Silber R; Nussenzweig V
1993 Jan 15;81(2):510-516, Blood
Erythrocytes shed membrane vesicles in response to many stimuli. It has been previously demonstrated that glycan phosphatidylinositol-linked (GPI-linked) proteins such as decay accelerating factor and acetylcholinesterase are concentrated in these vesicles relative to the erythrocyte membrane. We have examined the requirement for GPI-linked proteins for the process of vesiculation. Erythrocytes that do not express GPI-linked proteins, obtained from patients with paroxysmal nocturnal hemoglobinuria (PNH), release between 10% and 50% of the quantity of vesicles as normal cells in response to the Ca2+ ionophore A23187. Platelets from the same patients produced 10% to 20% of the amount of vesicles as normal platelets. In addition, a mutant B-lymphoblastoid cell line that lacks GPI-linked molecules produces about half of the number of vesicles as compared with the wild-type cell line in response to the Ca2+ ionophore. Prior findings indicate that vesiculation is one of the mechanisms that the cell uses to remodel the plasma membrane, as well as protect itself from membrane-damaging agents such as the terminal complement components C5b-9. On the basis of the present results, we conclude that GPI-linked proteins play an important role in membrane vesiculation
— id: 61955, year: 1993, vol: 81, page: 510, stat: Journal Article,

CELLS LACKING GLYCAN PHOSPHATIDYLINOSITOL-LINKED PROTEINS HAVE IMPAIRED ABILITY TO VESICULATE
WHITLOW, M; IIDA, K; MARSHALL, P; SILBER, R; ROSSE, W; NUSSENZWEIG, V
1992 APR ;40(2):A242-A242, Clinical research
— id: 51986, year: 1992, vol: 40, page: A242, stat: Journal Article,

Preferential expression of human Fc gamma RIIIPMN (CD16) in paroxysmal nocturnal hemoglobinuria. Discordant expression of glycosyl phosphatidylinositol-linked proteins
Edberg JC; Salmon JE; Whitlow M; Kimberly RP
1991 Jan;87(1):58-67, Journal of clinical investigation
The isoform of Fc gamma RIII (CD16) expressed on PMN has a GPI membrane anchor, and in paroxysmal nocturnal hemoglobinuria (PNH) there is a deficiency in Fc gamma RIII expression on PMN. Contrary to expectation, however, CD16 expression is preserved (albeit at reduced levels) in all affected PNH PMN that completely lack the GPI-anchored proteins DAF (CD55) and CD59. Fc gamma RIII negative PMN are not observed in any of the six PNH patients examined in this study. Analysis of the molecular weight of both glycosylated and deglycosylated Fc gamma RIII from PMN with reduced Fc gamma RIII expression indicates no variations in size relative to normal donor Fc gamma RIIIPMN. Indeed, the Fc gamma RIII expressed at intermediate levels is phosphatidylinositol-specific phospholipase C (PI-PLC)-sensitive. Thus, there is no evidence suggestive of expression of a transmembrane isoform and all data indicate that Fc gamma RIIIPMN on affected cells in PNH is a GPI-linked isoform. With Fc gamma RIIIPMN expression preserved at reduced levels on affected cells in PNH, PMN from PNH patients retain the capacity to internalize the Fc gamma RIIIPMN-specific probe E-ConA (at reduced levels) as well as IgG-opsonized erythrocytes. Reduced expression of GPI-anchored molecules on PNH PMN is not restricted to Fc gamma RIIIPMN since intermediate levels of CD59 were observed in the PNH PMN that were decay-accelerating factor (DAF)-negative and Fc gamma RIIIPMN intermediate. In addition, discordant expression of GPI-linked molecules in individual cells is not restricted to PMN since DAF+/CD14- monocytes were observed in one PNH patient. These data suggest that, when analyzed on an individual cell level, the GPI anchor defect in PNH is not absolute and must involve either a hierarchy of access of different protein molecules to available GPI anchors, distinct anchor biochemistries for the different proteins, or differential regulation of protein-anchor assembly
— id: 57475, year: 1991, vol: 87, page: 58, stat: Journal Article,

Membrane vesiculation protects erythrocytes from destruction by complement
Iida K; Whitlow MB; Nussenzweig V
1991 Oct 15;147(8):2638-2642, Journal of immunology
Nucleated cells can resist attack by C by exocytosis or endocytosis of the terminal C components C5b-9 (membrane attack complex) (MAC), but it is generally accepted that formation of a single MAC channel on E leads to lysis (one-hit theory). We find that human and guinea pig E, but not SRBC, can eliminate the MAC from the membrane in the form of microvesicles and escape destruction. When guinea pig or human E are incubated with C5b-9, vesiculation proceeds without a lag and is detected at nonlytic doses of C9. Continuous Ca2+ influx is required for vesiculation. The amount of released vesicles is in direct relation to Ca2+ concentration, and the increase in vesiculation is associated with a parallel decrease in lysis. SRBC, which do not vesiculate when Ca2+ loaded, are lysed by C5b-9 with the same efficiency in the presence or absence of Ca2+. Vesicles released from guinea pig RBC under C5b-9 attack are enriched in C9 by a factor of 10, compared with the unlysed cells, and by a factor of 3 to 4, compared with ghosts. We conclude that E are protected from lysis not only by CD59 and C8bp/HRF, which prevent MAC assembly, but also by selective elimination of the MAC
— id: 8319, year: 1991, vol: 147, page: 2638, stat: Journal Article,

H19, a surface membrane molecule involved in T-cell activation, inhibits channel formation by human complement
Whitlow MB; Iida K; Stefanova I; Bernard A; Nussenzweig V
1990 Mar;126(1):176-184, Cellular immunology
Here we compare the properties of leukocyte antigens H19 and CD59 with those of the PI-linked 18,000-20,000 Mr molecules which inhibit lysis of human cells by the autologous terminal complement components C5b-9. H19, a 19,000 Mr protein found on human erythrocytes, monocytes, neutrophils, T-lymphocytes and other cells, is one of the ligands involved in the spontaneous rosette formation between human T-lymphocytes and erythrocytes. Recent evidence indicates that H19 also participates in T-cell activation. CD59 is a widely distributed 18,000-25,000 Mr protein anchored to the cell membrane by phosphatidylinositol (PI). The function of CD59 is unknown. Affinity-purified H19 incorporates into cell membranes and inhibits channel formation by human C5b-9 on guinea pig erythrocytes. Significant inhibition is achieved with picogram quantities of H19, corresponding to approximately 600 molecules per erythrocyte. H19 is most effective when C9 is limiting but quite active when C5b-7 or C8 are limiting, indicating that it may interact with several of the structurally related terminal complement components. The inhibitory activity is blocked by mAbs to either CD59 or to H19. H19 is PI-anchored: it is released from the cell membrane by treatment with PI-specific phospholipase C, and it is absent from cells from a patient with paroxysmal nocturnal hemoglobinuria (PNH). Analysis of PNH erythrocytes after treatment with terminal complement proteins shows that the H19-negative erythrocytes are more susceptible to C5b-9-mediated lysis. Treatment of normal human erythrocytes with either anti-H19 or anti-CD59 renders them more susceptible to lysis by human C5b-9. We conclude that H19 and CD59 are probably the same molecule and are identical or closely related to the recently described inhibitors of C5b-9 channel formation
— id: 8318, year: 1990, vol: 126, page: 176, stat: Journal Article,

Secretion by Trypanosoma cruzi of a hemolysin active at low pH
Andrews NW; Whitlow MB
1989 Mar 15;33(3):249-256, Molecular & biochemical parasitology
Trypanosoma cruzi releases into the culture medium heat-labile, trypsin-sensitive molecules which lyse erythrocytes from various animal species. Production of the hemolysin is abolished by removal of glucose from the medium or by addition of the metabolic inhibitors sodium azide, 2-deoxy-D-glucose or puromycin. Sieving experiments with erythrocyte ghosts indicate that large channels are formed on the target membranes. The activity of the hemolysin is maximal at pH 5.5 and undetectable at neutral pH, indicating that it functions in acidic intracellular compartments. This agent could be involved in promoting the escape of T. cruzi into the cytoplasm of the host cell, by mediating the lysis of the membrane of the phagosome in which the parasite resides at early times after invasion
— id: 10698, year: 1989, vol: 33, page: 249, stat: Journal Article,

Amastigotes of Trypanosoma cruzi escape destruction by the terminal complement components
Iida K; Whitlow MB; Nussenzweig V
1989 Mar 1;169(3):881-891, Journal of experimental medicine
We studied the effect of complement on two life cycle stages of the protozoan parasite Trypanosoma cruzi: epimastigotes, found in the insect vector, and amastigotes, found in the mammalian host. We found that while both stages activate vigorously the alternative pathway, only epimastigotes are destroyed. The amounts of C3 and C5b-7 deposited on the amastigotes were similar to those bound to the much larger epimastigotes. Binding of C9 to amastigotes was four to six times less than binding to epimastigotes, resulting in a lower C9/C5b-7 ratio. Although a fairly large amount of C9 bound stably to amastigotes, no functional channels were formed as measured by release of incorporated 86Rb. The bound C9 had the characteristic properties of poly-C9, that is, it expressed a neo-antigen unique to poly-C9, and migrated in SDS-PAGE with an apparent Mr greater than 10(5). The poly-C9 was removed from the surface of amastigotes by treatment with trypsin, indicating that it was not inserted in the lipid bilayer. Modification of amastigote surface by pronase treatment rendered the parasites susceptible to complement attack. These results suggest that amastigotes have a surface protein that binds to the C5b-9 complex and inhibits membrane insertion, thus protecting the parasites from complement-mediated lysis
— id: 10718, year: 1989, vol: 169, page: 881, stat: Journal Article,

Trypanosoma cruzi: mechanisms of cell-invasion and intracellular survival
Andrews NW; Schenkman S; Ley V; Whitlow MB; Robbins ES; Nussenzweig V
1988 Nov;83 Suppl 1:452-455, Memorias do Instituto Oswaldo Cruz
— id: 10908, year: 1988, vol: 83 Suppl 1, page: 452, stat: Journal Article,

EFFICIENCY OF CHANNEL FORMATION BY COMPLEMENT AS MEASURED BY PAROXYSMAL-NOCTURNAL HEMOGLOBINURIA ERYTHROCYTES
Whitlow, MB; Iida, K; Nussenzweig, V
1988 Apr;36(3):A704-A704, Clinical research
— id: 31525, year: 1988, vol: 36, page: A704, stat: Journal Article,

EFFICIENCY OF CHANNEL FORMATION BY COMPLEMENT AS MEASURED ON PAROXYSMAL-NOCTURNAL HEMOGLOBINURIA ERYTHROCYTES
Whitlow, MB; Iida, K; Nussenzweig, V
1988 Apr;90(4):617-617, Journal of investigative dermatology
— id: 31535, year: 1988, vol: 90, page: 617, stat: Journal Article,

EFFICIENCY OF CHANNEL FORMATION BY COMPLEMENT ON PAROXYSMAL- NOCTURNAL HEMOGLOBINURIA ERYTHROCYTES
Whitlow, MB; Iida, K; Nussenzweig, V
1988 Mar 25;2(6):A1643-A1643, FASEB journal
— id: 31527, year: 1988, vol: 2, page: A1643, stat: Journal Article,

The relationship between channel size and the number of C9 molecules in the C5b-9 complex
Ramm LE; Whitlow MB; Mayer MM
1985 Apr;134(4):2594-2599, Journal of immunology
We have recently shown by dose-response analyses with resealed erythrocyte ghosts that the channel formed by complement is a monomer of C5b-9 of the composition C5b61C71C81C9n, in which n = 1 for channels permitting passage of sucrose (0.9 nm molecular diameter) and n = 2 for channels allowing transit of inulin (3 nm molecular diameter) (1). We have now continued these experiments and expanded them by including ribonuclease A (molecular diameter, 3.8 nm) as a marker to assess whether additional C9 molecules enlarge the functional C5b-9 channel. Our results show that formation of C5b-9 channels displays one-hit characteristics with respect to C5b6 when tested by transmembrane passage of inulin or ribonuclease A. By contrast, analysis of dose-response curves of C9 indicate that n = 2-3 for channels allowing transit of inulin and n = 4 for channels allowing transit of ribonuclease A. We have also performed sieving experiments with ghosts carrying C5b-7 and containing two small markers, inositol and sucrose. Dose-response curves for C8 were performed in the presence of excess C9 to ensure conversion of all C5b-8 to C5b-9 channels. The results indicate that small channels (approximately 0.8 nm effective diameter) are not formed at high C9 multiplicity, thus confirming the results obtained with the larger markers, i.e., increase of C9 input leads to formation of larger channels
— id: 17056, year: 1985, vol: 134, page: 2594, stat: Journal Article,

Penetration of C8 and C9 in the C5b-9 complex across the erythrocyte membrane into the cytoplasmic space
Whitlow MB; Ramm LE; Mayer MM
1985 Jan 25;260(2):998-1005, Journal of biological chemistry
We have developed a technique in which transglutaminase is used to measure the penetration of terminal complement proteins across the erythrocyte membrane into the cytoplasmic space. Penetration of a given terminal complement protein into the cytoplasmic space was assessed by labeling the protein of interest with radioactive iodine, forming the complement channel using the labeled protein, adding transglutaminase to only one side of the membrane, and allowing the enzyme to cross-link the susceptible proteins on that side of the membrane. Cross-linking was assessed by measuring the increase in molecular weight of the appropriate molecule on sodium dodecyl sulfate gels under reducing conditions. The results of these experiments indicate that C8 and C9 are rapidly cross-linked to high molecular weight from either the interior or the exterior of the membrane. In order to determine whether the cross-linking mediated by enzyme on the interior was occurring from within the ghosts and not via enzyme that had leaked into the extracellular medium, experiments were performed with dimethylcasein in the extracellular medium. In the presence of this protein, cross-linking of C8 and C9 from outside was negligible. Hence, if cross-linking occurs when transglutaminase is trapped inside the ghosts, it cannot be due to leakage of enzyme, but must be attributable to cross-linking from the inside. The results show that C9 definitely penetrated across the membrane into the intracellular space. With respect to C8, statistical evaluation indicates that C8 probably penetrated into the intracellular space
— id: 17057, year: 1985, vol: 260, page: 998, stat: Journal Article,

Complement lysis of nucleated cells: effect of temperature and puromycin on the number of channels required for cytolysis
Ramm LE; Whitlow MB; Mayer MM
1984 Nov;21(11):1015-1021, Molecular immunology
We have previously shown that lysis of a nucleated mammalian cell requires several complement channels unlike lysis of erythrocytes and that this difference is due primarily to rapid elimination of channels from the plasma membrane. We have now investigated this problem further by studying the rate of channel elimination at low temp, the osmotic fragility of the cells, and the effectiveness of the membrane-associated ion pumps. When complement channels were formed for 3 min at 37 degrees C, followed by prolonged incubation at 2 degrees C, the C6 lytic dose-response curves indicated that a single channel was required for lysis of a cell, whereas multiple channels were required when the entire process was carried out at 30 degrees C. The shift from multi- to one-hit lytic behavior can be explained by the drastic reduction in the rate of channel elimination at low temp. C6 lytic dose-response curves with puromycin-treated cells were also found to display one-hit behavior, but, in this case, the rate of channel elimination was reduced only about 35-40% (which would not suffice to explain the one-hit lytic characteristics). However, cell death was more extensive for puromycin-treated cells than normal cells after incubation in buffers of low ionic strength, suggesting that an increase in osmotic fragility may be a contributing factor in the shift from multi- to one-hit behavior. Blocking of the membrane-associated Na+/K+-ATPases with ouabain did not affect the multi-channel requirement. Presumably, this means that the ion pumping rate does not significantly influence the number of channels required for lysis
— id: 17058, year: 1984, vol: 21, page: 1015, stat: Journal Article,

Elimination of complement channels from the plasma membranes of U937, a nucleated mammalian cell line: temperature dependence of the elimination rate
Ramm LE; Whitlow MB; Koski CL; Shin ML; Mayer MM
1983 Sep;131(3):1411-1415, Journal of immunology
We have studied the release of radiolabeled small markers from nucleated cells carrying complement channels in order to determine the life-span of these channels at various temperatures. U937 cells, a human histiocytic cell line, were labeled with 14C-aminoisobutyric acid or 86RbCl, and treated with sublytic doses of C to form transmembrane channels. The cells were then incubated at various temperatures, and the persistence of channels was evaluated by measuring the release of the intracellular markers through the remaining channels. The results indicate that the life-span of the C channels in the plasma membranes of these cells varies markedly with temperature. Thus, at 2 degrees C, the half-life of the channels was about 2 hr, whereas at 37 degrees C, the half-life was estimated to be approximately 1 min. The rapid elimination of the transmembrane channels from the plasma membranes of these nucleated cells contrasts sharply with the long persistence of C channels in the membranes of erythrocytes or erythrocyte ghosts. It is likely that the multi-hit requirement recently reported for lysis of nucleated mammalian cells by C is due, at least in part, to the rapid disappearance of channels
— id: 17059, year: 1983, vol: 131, page: 1411, stat: Journal Article,

Size distribution and stability of the trans-membrane channels formed by complement complex C5b-9
Ramm LE; Whitlow MB; Mayer MM
1983 Feb;20(2):155-160, Molecular immunology
We have performed double marker sieving experiments with molecules ranging from ca. 0.5-3 nm dia. in order to evaluate the size distribution of the channels formed by complement in resealed sheep erythrocyte ghosts. Evidence is presented that marker release through the channels reached equilibrium between the ghosts and the extracellular fluid in a period of 3 hr and that the channels are stable at 37 degrees C for this period of time. Under these experimental conditions we have observed a differential in the endpoint release of inositol and sucrose, which indicates that some of the ghosts carried channels measuring between 0.7 and 0.9 nm dia. No differential was observed between release of sucrose and raffinose (0.9 and 1.1 nm mol. dia., respectively). Comparisons between sucrose and inulin (0.9 and 3 nm mol. dia, respectively) showed a difference in marker release. Also, there was substantial release of inulin, indicating the presence of channels above 3 nm in dia. Hence, the present data indicate formation of channels in three size ranges, namely, 0.7-0.9 nm, 0.9-3 nm and greater than 3 nm
— id: 17060, year: 1983, vol: 20, page: 155, stat: Journal Article,

Size of the transmembrane channels produced by complement proteins C5b-8
Ramm LE; Whitlow MB; Mayer MM
1982 Sep;129(3):1143-1146, Journal of immunology
It has been shown previously that erythrocytes can be lysed by complement proteins C5b-8, albeit at a much lower rate than C5b-9. We have now performed kinetic sieving experiments with resealed erythrocyte ghosts using sucrose (0.9 nm molecular diameter) and inulin (3.0 nm molecular diameter) as markers. We found that treatment of the ghosts with C5b-8 released sucrose, but not inulin. Addition of C9 to ghosts carrying C5b-8 dramatically increased the rate of sucrose flux and, in addition, caused release of inulin. Hence, unlike C5b-9 channels, those formed by C5b-8 measure less than 3 nm in diameter. Formation of C5b-8 channels was very slow compared with that of C5b-9 channels. Also, we found that about two-thirds of the C5b-8 ghosts did not have sucrose-releasing channels, but such channels were formed on reaction with C9
— id: 17061, year: 1982, vol: 129, page: 1143, stat: Journal Article,

Transmembrane channel formation by complement: functional analysis of the number of C5b6, C7, C8, and C9 molecules required for a single channel
Ramm LE; Whitlow MB; Mayer MM
1982 Aug;79(15):4751-4755, Proceedings of the National Academy of Sciences of the United States of America
Earlier studies have shown that sequential treatment of resealed erythrocyte ghosts with C5b6, C7, C8, and C9 leads to insertion of hydrophobic peptides from these complement proteins into the membrane and assembly of transmembrane channels. The number of molecules of each of the proteins required for assembly of the membrane-associated channel structure was evaluated by measuring the quantitative relationship between the doses of the individual proteins and the release of two trapped markers, sucrose and inulin, from ghosts after channel formation. The incubation period was sufficient to attain equilibrium of marker distribution between the ghosts and the extracellular fluid. Two markers of different size (sucrose and inulin, 0.9 and 3 nm molecular diameter, respectively) were used in order to develop information on the molecular composition of small and large channels, respectively. We found that participation of C5b6, C7, and C8 in channel formation displayed one-hit characteristics, regardless of marker size. By contrast, the participation of C9 was one-hit with respect to the sucrose marker, whereas with respect to the inulin marker the C9 reaction was multi-hit. Our results are compatible with the view that these markers are released through a channel structure in the membrane that is a monomer of C5b--9 of the composition C5b61 C71C81C9n, in which n = 1 for channels permitting passage of sucrose and n = 2 for channels allowing transit of inulin
— id: 17062, year: 1982, vol: 79, page: 4751, stat: Journal Article,