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C W Vogel

Publications and source records attributed to C W Vogel.

At least 19 recordsLinked to original sources
PubMed2001

Initial characterization of the apoptosis-inducing receptor for natural human anti-neuroblastoma IgM.

BACKGROUND: Human neuroblastoma (NB) cells contain a 260 kDa surface antigen (NB-p260), which serves as receptor for natural human IgM antibodies (anti-NB IgM). Upon binding to NB-p260, these antibodies induce apoptosis in human NB cells. PROCEDURE AND RESULTS: In this study, we purified NB-p260 to homogeneity from human LA-N-1 NB cells by sequential ion exchange chromatography followed by preparative SDS gel electrophoresis. Purified NB-p260 exhibited rapid autodegradation despite the presence of various protease inhibitors. The autodegradation process precluded extensive N-terminal sequencing. However, from repeat N-terminal sequence analysis, a consensus sequence of seven amino acid residues emerged that exhibited significant homology to the subunit c of the human mitochondrial ATP synthase, a hydrophobic membrane protein of 7.6 kDa. Western blot analyses demonstrated that purified NB-p260 was recognized by polyclonal antibodies raised against both subunit c-containing storage bodies and a synthetic peptide consisting of amino acid residues 32-45 of subunit c. In addition to peptide sequences related to subunit c, NB-p260 also contained epitopes related to the human heat shock protein HSP90. In Western blots, a monoclonal anti-HSP90 antibody reacted with purified NB-p260 as well as with a predominant protein fragment of approximately 90 kDa that appeared during the process of NB-p260 autodegradation. The anti-HSP90 antibody was also capable of binding to the surface of LA-N-1 cells and inhibiting the binding of human anti-NB IgM in a dose-dependent manner. CONCLUSIONS: Collectively, our data suggest that NB-p260, the apoptosis-mediating receptor for natural human anti-NB IgM, represents a novel surface protein of human NB cells containing polypeptide sequences related to the subunit c of the mitochondrial ATP synthase and the heat shock protein HSP90.

Animals↗
PubMed2001

N-linked oligosaccharides of cobra venom factor contain novel alpha(1-3)galactosylated Le(x) structures.

Cobra venom factor (CVF), a nontoxic, complement-activating glycoprotein in cobra venom, is a functional analog of mammalian complement component C3b. The carbohydrate moiety of CVF consists exclusively of N-linked oligosaccharides with terminal alpha1-3-linked galactosyl residues, which are antigenic in human. CVF has potential for several medical applications, including targeted cell killing and complement depletion. Here, we report a detailed structural analysis of the oligosaccharides of CVF. The structures of the oligosaccharides were determined by lectin affinity chromatography, antibody affinity blotting, compositional and methylation analyses, and high-resolution (1)H-NMR spectroscopy. Approximately 80% of the oligosaccharides are diantennary complex-type, approximately 12% are tri- and tetra-antennary complex-type, and approximately 8% are oligomannose type structures. The majority of the complex-type oligosaccharides terminate in Galalpha1-3Galbeta1-4(Fucalpha1-3)GlcNAcbeta1, a unique carbohydrate structural feature abundantly present in the glycoproteins of cobra venom.

Carbohydrate Sequence↗
PubMed2001

Structural studies on the cobra venom factor: isolation, purification, crystallization and preliminary crystallographic analysis.

Cobra venom factor (CVF) is the complement-activating protein in cobra venom. It is a three-chain glycoprotein with a molecular weight of 149,000 Da. In serum, CVF forms a bimolecular enzyme with the Bb subunit of factor B. The enzyme cleaves C3 and C5, causing complement consumption in human and mammalian serum. CVF is frequently used to decomplement serum to investigate the biological functions of complement and serves as a tool to investigate the multifunctionality of C3. Furthermore, CVF bears the potential for clinical application to deplete complement in situations where complement activation is involved in the pathogenesis of disease. CVF was isolated from Indian cobra (Naja naja naja) venom. The protein was crystallized at room temperature using the sitting-drop vapour-diffusion technique. The crystals diffract to 2.7 A resolution and belong to the tetragonal space group P4(1), with unit-cell parameters a = b = 62.7, c = 368.1 A.

Animals↗
PubMed1999

Human natural immunoglobulin M antibodies induce apoptosis of human neuroblastoma cells by binding to a Mr 260,000 antigen.

Sera of healthy humans contain natural cytotoxic IgM antibodies that specifically recognize a Mr 260,000 antigen (NB-p260) on the surface of human neuroblastoma (NB) cells. Here we demonstrate that anti-NB IgM antibodies prepared from different healthy individuals induce, in all human NB cell lines analyzed thus far, typical morphological and biochemical features of apoptosis including nuclear fragmentation, chromatin condensation, and DNA fragmentation. Both the binding of human anti-NB IgM to NB cells and the induction of apoptosis could be inhibited by preincubation of NB cells with murine IgG raised against purified NB-p260. Furthermore, preincubation of human anti-NB IgM with purified NB-p260 immobilized onto a solid support abolished its ability to induce apoptosis in NB cells. Natural human anti-NB IgM failed to bind to and induce apoptosis in control tumor cell lines that lack expression of NB-p260. The anti-NB IgM-induced apoptotic response was also observed in vivo in xenografted human NB tumors. After a single i.v. injection of anti-NB IgM into nude rats bearing solid NB xenografts, many areas of pyknotic cells with fragmented nuclei were observed that stained positive using the terminal dUTP nick end labeling method. In conclusion, the data demonstrate that natural anti-NB IgM antibodies in the sera of healthy individuals are potent mediators of apoptotic cell death of NB cells both in vitro and in vivo. The NB-p260 antigen was identified as the apoptosis-inducing receptor for anti-NB IgM. Whereas natural anti-NB IgM and NB-p260 may be useful tools for immunotherapy of NB, their biological significance remains to be determined.

Animals↗
PubMed1999

Effect of complement consumption by cobra venom factor on the course of primary infection with simian immunodeficiency virus in rhesus monkeys.

Cobra venom factor (CVF)-induced consumption of complement proteins was used to investigate the role of complement in vivo in the immunopathogenesis of simian immunodeficiency virus of macaques (SIVmac) infection in rhesus monkeys. Repeated administration of CVF was shown to deplete complement to <5% of baseline hemolytic activity of serum complement for 10 days in a normal monkey. Three groups of SIVmac-infected animals were then evaluated: monkeys treated with CVF resulting in complement depletion from days -1 to 10 postinfection, monkeys treated with CVF resulting in complement depletion from days 10 to 21 postinfection, and control monkeys that received no CVF. CD8+ SIVmac-specific cytotoxic T lymphocyte (CTL) generation and CD4+ T lymphocyte depletion during primary infection were not affected by CVF treatment. Viral load, assessed by measurements of plasma p27gag antigen and viral RNA, was transiently higher during the first 4 weeks following infection in the CVF-treated monkeys and the subsequent clinical course in these treated animals was accelerated. These results suggest that complement proteins may participate in immune defense mechanisms that decrease virus replication following the initial burst of intense viremia during primary SIVmac infection. However, we cannot rule out that the observed increased virus replication was induced by immune activation resulting from the administration of a foreign antigen to these monkeys.

Animals↗
PubMed1999

A nonhuman primate model for the selective elimination of CD8+ lymphocytes using a mouse-human chimeric monoclonal antibody.

Nonhuman primates provide valuable animal models for human diseases. However, studies assessing the role of cell-mediated immune responses have been difficult to perform in nonhuman primates. We have shown that CD8+ lymphocyte-mediated immunity in rhesus monkeys can be selectively eliminated using the mouse-human chimeric anti-CD8 monoclonal antibody cM-T807. In vitro, this antibody completely blocked antigen-specific expansion of cytotoxic T cells and decreased major histocompatibility complex class I-restricted, antigen-specific lysis of target cells but did not mediate complement-dependent cell lysis. In vivo administration of cM-T807 in rhesus monkeys resulted in near total depletion of CD8+ T cells from the blood and lymph nodes for up to 6 weeks. This depletion was not solely complement-dependent and persisted longer in adults than in juveniles. Preservation of B cell and CD4+ T cell function in monkeys depleted of CD8+ lymphocytes was demonstrated by their ability to develop humoral immune responses to the administered chimeric monoclonal antibody. Furthermore, during CD8+ lymphocyte depletion, monkeys developed delayed-type hypersensitivity reactions comprised only of CD4+ T cells but not CD8+ T cells. This CD8+ lymphocyte depletion model should prove useful in defining the role of cell-mediated immune responses in controlling infectious diseases in nonhuman primates.

Animals↗
PubMed1999

Serotype distribution and secretory acid proteinase activity of Candida albicans isolated from the oral mucosa of patients with denture stomatitis.

Denture stomatitis is the most common form of oral Candida infection in humans. In the current study, the distribution of Candida albicans serotype A and B as well as the activity of the secreted acid proteinase were determined in clinical isolates from patients with denture stomatitis. It was found that 70% of individuals with clinical signs of denture stomatitis exhibited fungal growth, with C. albicans representing the most frequently isolated species (75%). Of the C. albicans isolates, 75% were serotype A and 25% were serotype B, representing a significant increase of serotype B compared to a control group of non-denture-wearing HIV-seronegative individuals with oral candidiasis, but no significant difference compared with isolates from HIV-seropositive patients, who also exhibited a high percentage of serotype B. The mean secretory acid proteinase activity of C. albicans isolates from denture stomatitis patients (2796 +/- 819 U/l) was statistically not different from the mean secretory acid proteinase activity in non-denture-wearing HIV-seronegative individuals (2324 +/- 1487 U/l). Both values were significantly lower than the mean secretory acid proteinase activity of C. albicans from HIV-seropositive individuals (4256 +/- 2372 U/l). No correlation exists between the C. albicans serotype and the amount of secreted acid proteinase, indicating that serotype and secretory acid proteinase expression are two independent pathogenetic factors in oral candidiasis. These results indicate that there seems to be strain selection for C. albicans serotype B in denture stomatitis. These results further indicate that increased secretion of the acid proteinase seems to be of pathogenetic significance in the candidiasis of HIV-seropositive patients but not in denture stomatitis. Nevertheless, the secretory acid proteinase is likely to be an important pathogenetic factor also in denture stomatitis, where an increased secretion of the acid proteinase may not be required because of decreased salivary flow and a low pH under the denture, which will result in a high enzymatic activity.

Adult↗
PubMed1997

Additive cytotoxicity of different monoclonal antibody-cobra venom factor conjugates for human neuroblastoma cells.

Insufficient numbers of antigen molecules and heterogeneity of antigen expression on tumor cells are major factors limiting the immunotherapeutic potential of the few clinically useful monoclonal antibodies capable of mediating complement cytotoxicity and antibody-dependent cellular cytotoxicity. To overcome this limitation, we converted two non-cytotoxic monoclonal anti-neuroblastoma antibodies, designated 3E7 (IgG2b) and 8H9 (IgG1), and the non-cytotoxic F(ab')2 fragment of the cytotoxic monoclonal anti-GD2 antibody 3F8 (IgG3) into cytotoxic antibody conjugates by covalent attachment of cobra venom factor (CVF), a structural and functional homologue of the activated third component of complement. Competitive binding experiments confirmed the different specificities of the three antibodies. In the presence of human complement, all three antibody-CVF conjugates mediated selective complement-dependent lysis of human neuroblastoma cells. Consistent with the kinetics of the alternative pathway of complement, approximately seven hours incubation were required to reach maximum cytotoxicity of up to 25% for the 3E7-CVF conjugate, up to 60% for the 8H9-CVF conjugate, and up to 95% for the 3F8 F(ab')2-CVF conjugate. The different extent of maximal cytotoxic activity of the three conjugates was reflected by corresponding differences in the extent of binding of both unconjugated antibodies and the respective conjugates. Any combination of the three antibody-CVF conjugates caused an additive effect in complement-mediated lysis. Using a cocktail of all three conjugates, the extent of complement-mediated killing could be increased up to 100%. These data demonstrate that by coupling of CVF the relative large number of non-cytotoxic monoclonal anti-tumor antibodies of interesting specificity can be used to design cocktails of cytotoxic conjugates and, thereby, to overcome the problem of insufficient and heterogeneous antigen expression on tumor cells for immunotherapy.

Antibodies, Monoclonal↗
PubMed1997

Expression of a 260 kDa neuroblastoma surface antigen, the target of cytotoxic natural human IgM: correlation to MYCN amplification and effects of retinoic acid.

Human neuroblastoma cells contain a 260 kDa surface-associated antigen (NB-p260) that is recognised by natural cytotoxic IgM antibodies. In this study we demonstrate that NB-p260 is expressed in vivo in a neuroblastoma tumour specimen but not in normal human tissues of neuronal origin. Since MYCN amplification is a clinical marker of neuroblastoma disease progression, we analysed the expression of NB-p260 in human neuroblastoma cell lines with different MYCN amplification status. However, both amplified and non-amplified neuroblastoma cell lines exhibited comparable NB-p260 expression. Treatment of neuroblastoma cells with the differentiation-inducing agent retinoic acid (RA) also had no effect on the expression of NB-p260. Collectively, the data suggest that expression of NB-p260 on human neuroblastoma cells is independent of malignancy and differentiation status of neuroblastoma.

Antigens, Neoplasm↗
PubMed1997

Mechanisms of in vivo anti-neuroblastoma activity of human natural IgM.

Normal human sera of healthy adults contain natural IgM antibodies which are cytotoxic for human neuroblastoma cells. In this study, we evaluated the anti-neuroblastoma activity of these natural IgM antibodies in nude rats bearing solid human neuroblastoma tumours. A single intravenous (i.v.) injection of purified cytotoxic IgM led to uptake of IgM in the tumours with massive perivascular complement activation and accumulation of neutrophil granulocytes after 24 h. Five consecutive i.v. injections of purified cytotoxic IgM into neuroblastoma-bearing animals resulted in complete growth arrest of even large established solid tumours which lasted for several weeks after discontinuation of the injections, whereas tumours of control animals continued to grow exponentially during the observation period. These studies suggest that natural anti-neuroblastoma IgM may have a potential as a novel therapeutic modality in the treatment of human neuroblastoma.

Adult↗
PubMed1996

Normal human serum contains a natural IgM antibody cytotoxic for human neuroblastoma cells.

Neuroblastoma (NB) is characterized by the second highest spontaneous regression of any human malignant disorder, a phenomenon that remains to be elucidated. In this study, a survey of 94 normal human adult sera revealed a considerable natural humoral cytotoxicity against human NB cell lines in approximately one-third of the tested sera of both genders. Specific cell killing by these sera was in the range of 40% to 95%. Serum cytotoxicity was dependent on an intact classical pathway of complement. By several lines of evidence, IgM antibodies were identified as the cytotoxic factor in the sera. Further analyses revealed that a 260-kDa protein was recognized by natural IgM of cytotoxic sera in Western blots of NB cell extracts. The antigen was expressed on the surface of seven human NB cell lines but not on human melanoma or other control tumor cell lines derived from kidney, pancreas, colon, bone, skeletal muscle, lymphatic system, and bone marrow. Furthermore, no reactivity was observed with normal human fibroblasts, melanocytes, and epidermal keratinocytes. The antigen was expressed in vivo as detected by immunohistochemistry in both the tumor of a NB patient and NB tumors established in nude rats from human NB cell lines. Most interestingly, the IgM anti-NB antibody was absent from the sera of 11 human NB patients with active disease. The anti-NB IgM also could not be detected in tumor tissue obtained from a NB patient. Collectively, our data suggest the existence of a natural humoral immunological tumor defense mechanism, which could account for the in vivo phenomenon of spontaneous NB tumor regression.

Adolescent↗
PubMed1996

Growth arrest of solid human neuroblastoma xenografts in nude rats by natural IgM from healthy humans.

Neuroblastoma (NB) is the most common extracranial solid neoplasm of infancy and is associated with very poor prognosis in patients with advanced disease. Current therapeutic regimens of advanced NB which combine surgical resection with radiation therapy and/or chemotherapy brought some improvements, but in a significant number of patients, a cure remains elusive. Normal human serum of healthy adults contains natural IgM antibodies that are cytotoxic for human NB cells. In this study, we evaluated the anti-NB activity of these natural IgM antibodies in nude rats bearing solid human NB tumors. A single intravenous (i.v.) injection of purified cytotoxic IgM led to uptake of IgM into the tumors with massive perivascular complement activation and accumulation of neutrophil granulocytes after 24 hours. Five consecutive i.v. injections of purified cytotoxic IgM into NB-bearing animals resulted in complete growth arrest of even large and established solid tumors which lasted for several weeks after discontinuation of the injections, whereas tumors of control animals continued to grow exponentially during the observation period. These studies suggest that natural anti-NB IgM may have a potential as a novel therapeutic modality in the treatment of human NB.

Adult↗
PubMed1995

Classical complement pathway activation on nucleated cells. Role of factor H in the control of deposited C3b.

The restriction of alternative complement pathway activation in fluid phase or on nonactivator surfaces has been described as the major physiologic function of the complement regulatory protein factor H. In this study, we provide evidence that factor H is also a restriction factor of classical pathway activation on the surface of nucleated cells. We found that C3b was rapidly converted to inactivated C3b (iC3b) on human SK-MEL-93-2 melanoma cells after classical pathway activation with the murine monoclonal IgG3 Ab R24 directed against the disialoganglioside surface Ag GD3. The SK-MEL-93-2 cells are nonactivators of the alternative pathway and express neither CR1 (CD35) nor the C3b-cleaving protease p65. The cells are further characterized by the expression of only moderate amounts of DAF (CD55) and approximately 5 x 10(3) MCP (CD46) molecules/cell. FACS analysis and direct quantitation using [125I]factor H revealed high level binding of factor H to the melanoma cells (5.6 x 10(6) molecules/cell) during classical pathway activation. The binding of factor H could be inhibited under conditions that inactivate the classical complement pathway (EGTA and heat treatment), but not by factor B depletion of the serum, demonstrating that classical pathway activation was responsible for factor H binding. Treatment of factor B-depleted serum with neutralizing concentrations of polyclonal anti-factor H resulted in the prolonged presence of intact C3b on the cells and a significantly reduced generation of iC3b. The increased amount of C3b on these cells correlated with a 2.65-fold greater rate of cell death. In contrast, the increase in cell death effected by neutralizing concentrations of anti-CD46 or anti-CD55 Ab was only 0.13- or 0.35-fold, respectively. In addition, the supplementation of serum with purified factor H decreased the extent of lysis of the cells. Collectively, these data provide experimental evidence that factor H, through its cofactor activity for C3b degradation, is involved in the restriction of the classical pathway of complement on the surface of nucleated cells, a function that to date has been exclusively attributed to the membrane regulatory proteins CD35 and CD46.

Complement Activation↗
PubMed1995

Increased expression of Candida albicans secretory proteinase, a putative virulence factor, in isolates from human immunodeficiency virus-positive patients.

The increased prevalence and the severity of oropharyngeal candidiasis in human immunodeficiency virus (HIV)-positive patients are attributed exclusively to the virus-induced immune deficiency of the host. The present study was aimed at answering the question of whether Candida albicans secretory proteinase, a putative virulence factor of the opportunistic C. albicans yeast, has any potential influence on the clinical manifestation of oropharyngeal candidiasis in HIV-positive patients. We measured the secretory proteinase activities of clinical C. albicans isolates from the oropharynges of either HIV-positive individuals (n = 100) or a control group (n = 122). The mean secretory proteinase activity of C. albicans isolates from the HIV-positive group (4,255 +/- 2,372 U/liter) was significantly higher compared with that of isolates from the control group (2,324 +/- 1,487 U/liter) (P < 0.05). The higher level of secretory proteinase activity in the culture supernatants of individual C. albicans isolates correlated with the increased level of proteinase expression on the cell surface, as revealed by cytofluorometry, and with higher levels of secretion of the immunodetectable protein, as shown by Western blotting (immunoblotting). Proteinase activity within the population of C. albicans isolates from HIV-positive individuals was independent of the patient's clinical disease stage and the CD4+/CD8+ cell numbers. Furthermore, no correlation of the proteinase activities with the C. albicans serotype was found, although C. albicans serotype B was significantly more frequent in the HIV-positive group (40%) compared with that in the control group (12%). However, a positive correlation of proteinase activity to antifungal susceptibility was evident. The C. albicans isolates from the HIV-positive group that were characterized by higher levels of proteinase activity were also less susceptible to the widely used azole antifungal ketoconazole and fluconazole. Collectively, the present data are consistent with a concept of early preferential selection of a subpopulation of C. albicans in HIV-infected patients.

Adult↗
PubMed1994

Molecular cloning and derived primary structure of cobra venom factor.

Cobra venom factor (CVF) is the complement-activating protein in cobra venom. Like C3b, CVF forms with factor B and factor D in human and mammalian serum the bimolecular C3/C5 convertase. This functional similarity of CVF and C3 correlates with many structural similarities, which led to the suggestion that CVF is evolutionally related to C3. We report here the molecular cloning and derived primary structure of CVF. CVF mRNA is > 5924 nucleotides in length. It contains a single open reading frame of 4926 nucleotides, coding for a pre-pro-protein of 1642 amino acids. The deduced amino acid sequence reveals approximately 70% protein similarity to mammalian and human C3 and exceeds 91% in the case of cobra C3. The single-chain pre-pro-CVF consists of a 22-amino acid signal sequence, a 627-amino acid alpha-chain, and a 989-amino acid precursor chain for the CVF gamma- and beta-chains. The processing of pro-CVF involves the removal of 4 arginine residues between the alpha- and precursor chains as well as of the C3a-like and C3d-like domains from the precursor chain, thereby confirming the predicted chain homologies to C3. Pro-CVF contains five potential N-glycosylation sites, of which only three can be expected to be glycosylated in mature CVF. Like C3, pro-CVF contains 27 cysteine residues and a homologous thioester site in the C3d-like region.

Animals↗