A surface-labeled 18 kilodalton antigen in Schistosoma mansoni

A mild surface-labeling procedure was applied to various developmental stages of Schistosoma mansoni. An 18 kDa protein was preferentially labeled in freshly transformed schistosomula. The labeled protein was equally present on skin-penetrated and mechanically prepared schistosomula and it disappeared upon digestion of intact parasites with proteolytic enzymes. The 18 kDa protein could be specifically precipitated with an antiserum raised against 3-h schistosomula. Six-day lung forms also presented a single major labeled protein component, but the apparent molecular weight of this protein in acrylamide gels was higher than 18 000. Fourteen-day-old and adult schistosomes showed only weak labeling distributed in several bands. The radioactivity pattern of adult worms (but not of schistosomula) could also be obtained by incubating fresh parasites in a medium which had previously been used to label schistosomes and to which a 100 000-fold excess of 127I over 125I had been added. Post-labeling incubation of parasites was found to be essential for the detection of stable surface proteins.     

Antibody diversity in amphibians. Noninbred axolotls used the same unique heavy chain and a limited number of light chains for their anti-2,4-dinitrophenyl antibody responses

Noninbred axolotls (Ambystoma mexicanum, amphibia, urodela) were immunized with trinitrophenylated sheep red blood cells (TNP-SRBC) and anti-2,4-dinitrophenyl (DNP)/TNP antibodies were individually purified by affinity chromatography. The isolated IgM-like antibodies were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF) under reducing conditions. The SDS-PAGE and IEF-separated heavy (H) and light (L) chains were electroblotted onto nitrocellulose, probed with mouse monoclonal antibodies specific for H or L axolotl Ig chains and stained by a rabbit anti-mouse Ig horseradish peroxidase conjugate. The specific detection of axolotl anti-DNP/TNP H chain spectrotypes shows for each of the 14 individually analyzed samples a very similar pattern of 4-5 ordered spaced bands. This suggests that all animals express the same VH chain segment representing the germinal expression of a unique VH gene. When the same analysis was performed starting from a pool of nonimmunized axolotl sera, a low background of natural anti-DNP antibodies was detected. When analyzed by IEF, the H chains of the pooled anti-DNP natural antibodies display the same pattern of restricted heterogeneity when compared to the H chain spectrotypes of the individual immune anti-DNP/TNP antibodies. The specific detection of the axolotl anti-DNP/TNP L chain spectrotypes indicates at the individual level more heterogeneous and polymorphic patterns compared with H chains, although most animals share the majority of their bands. Our experiments indicate that in axolotl, the production of antibodies to DNP results from the germinal expression of a very limited set of V genes, already expressed as naturally occurring anti-DNP antibodies before immunization. This seriously restricts the possible extension of the antibody repertoire and perhaps even the nature of antibody "specificity" in this primitive vertebrate.     

Isolation and characterization of surface antigens from Schistosoma mansoni schistosomula

Surface antigens of Schistosoma mansoni schistosomula were isolated using antibodies produced in rat and human schistosomiasis. Three immunoreactive surface proteins of 40 000, 37 000 and 32 000 daltons were identified by SDS—PAGE analysis of immune complexes formed by incubation of a detergent extract of surface labelled schistosomula with infected rat sera. Surface antigens of similar molecular weight were also isolated when using sera of patients with schistosomiasis. Binding of schistosomula surface antigens to specific antibodies was substantially inhibited by components released by adult worms. The results suggest that these schistosomula surface antigens could be involved in the immune response against challenge infection but their protective role in immunity still remains to be determined.     

Cercarial glycocalyx of Schistosoma mansoni activates human complement.

Human complement activation by cercariae and schistosomula of the human parasite Schistosoma mansoni was studied in vitro. Cercariae are composed of tails which are shed after infection of the host and bodies which transform into the larvae or schistosomula after infection. After incubation in fresh normal human serum (NHS), cercarial tails bound more anti-C3 antibodies than did cercarial bodies (CB), and the tails were rapidly lysed, while the attached CB remained intact. Complement activation by cercariae was dependent on the alternative pathway but was independent of antibody, as shown by C3 deposition by hypogammaglobulinemic human sera. By transmission microscopy, the fibrillar glycocalyx on both CB and tails was stained by NHS but not by heat-inactivated serum (HI-NHS). The glycocalyx was labeled with periodate and tritiated borohydride, and parasites were incubated in NHS and HI-NHS. After solubilization, the labeled glycocalyx on organisms incubated in NHS but not HI-NHS bound anti-C3 antibodies. Of the CB incubated with eserine sulfate to prevent transformation, 78% +/- 10% were dead after culture for 24 h in NHS. In contrast, 21% +/- 12% of the CB were dead after culture in HI-NHS. Schistosomula incubated in NHS bound 37% of the amount of anti-C3 antibodies bound by cercariae but were not killed by NHS. In conclusion, the cercarial glycocalyx activated human complement, and schistosomula were less susceptible to killing than cercariae because they had less glycocalyx and activated less complement.     

The exposed carbohydrates of schistosomula of Schistosoma mansoni and their modification during maturation in vivo

Lectins labeled with 125I or conjugated with fluorescein were employed to study the carbohydrates on the surface of different stages of schistosomula of Schistosoma mansoni. Newly transformed schistosomula were shown to bind concanavalin A; the 60 000 and 120 000 dalton agglutinins from Ricinus communis; the fucose-binding protein from Lotus tetragonolobus; wheat germ agglutinin and peanut agglutinin. Soybean agglutinin, Ulex europaeus agglutinin and Dolichos biflorus agglutinin, on the other hand, failed to bind to the schistosomulum surface. The binding of peanut and soybean agglutinin was unaffected by pretreatment of the parasites with neuraminidase. Binding of concanavalin A, the 120 000 dalton agglutinin from Ricinus communis, wheat germ agglutinin and peanut agglutinin to the surface of 5-day schistosomula, recovered from the lungs of mice, was also demonstrated. In each case, however, the level of binding was approximately 70% less than that observed with newly transformed schistosomula and the binding of the fucose-binding protein from L. tetragonolobus practically disappeared. In contrast with newly transformed schistosomula, lung stage schistosomula, pretreated with neuraminidase, displayed a significant increase in the binding of peanut and soybean agglutinin. The results indicate that a significant alteration in the surface carbohydrates of S. mansoni occurs during in vivo maturation of the parasite. This change may contribute to the organism's ability to survive in the vertebrate host.     

Identification of surface antigens of schistosomula of Schistosoma mansoni recognized by antibodies from mice immunized by chronic infection and by exposure to highly irradiated cercariae.

Surface components of mechanically transformed schistosomula of Schistosoma mansoni were labeled by lactoperoxidase-catalyzed iodination. After solubilization with Triton X-100, antigens were identified by immunoprecipitation. Serum from chronically infected Swiss mice reproducibly precipitated seven major polypeptides with approximate molecular weights (X 10(3] of 94, 68, 45, 40 to 32, 22, and 16. The antigens of molecular weights (X 10(3] of 94, 40 to 32, 22, and 16 were shown to be exposed on the parasite surface by interaction of the antibodies with intact labeled schistosomula. Sera from several strains of infected inbred mice precipitated the same polypeptides. The antibodies produced during chronic infection were found to be stimulated by adult worms since sera from 6-week-infected animals precipitated none of the surface antigens, and the pattern produced by precipitation with antibodies from a mouse infected with male worms only was indistinguishable from the pattern obtained with sera from mice with bisexual infections. Antibodies from mice immunized with highly irradiated cercariae reproducibly precipitated major polypeptides of approximately (X 10(3] 94, 68, 45, 32, 22, 19, and 15 daltons. The antigens of (X 10(3] 94, 43, 32, 22, and 15 daltons were shown to be exposed on the parasite surface by interaction of the antibodies with intact labeled schistosomula. The 15 X 10(3)-dalton surface protein was recognized by sera from vaccinated, but not chronically infected, mice, suggesting that it represents a stage-specific immunogen present on schistosomula but not on adult worms. Sera from two inbred strains of mice which develop different degrees of immunity recognized the same antigens.     

牛精子膜伴刀豆素A结合糖蛋白的分离与定位

用ＮＰ－４０处理牛精子获得膜抽提液，经伴刀豆素Ａ－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析，分离出与伴刀豆素Ａ结合的糖蛋白，经ＳＤＳ－聚丙烯酰胺凝胶电泳显示出分子量为７２、５７和３１ＫＤ的３条蛋白质带。将分离到的糖蛋白电转移在硝酸纤维膜上，用酶标的伴刀豆素Ａ检测，３条蛋白质带均呈阳性，用分离的糖蛋白免疫兔，制备抗血清。用抗血清对牛精子膜ＮＰ－４０抽提物进行免疫印迹法分析，检测出３条阳性带，与亲和层析法分离的     

The predominant heavily glycosylated glycoproteins at the surface of rat lymphoid cells are differentiation antigens.

Rat lymphoid cells have been labeled with sodium 3H-borohydride after periodate oxidation. The labeled glycoproteins were solubilized in detergent and analyzed by fluorography after polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Major bands were found at 150 000, 95 000 and 25 000 apparent mol.wt. for thymocytes; at 170 000 and 95 000 mol. wt. for T lymphocytes and at 200 000 mol.wt. for B lymphocytes. Bone marrow cells showed a diffuse band at 100 000 mol.wt. with relatively minor bands around 150 000 mol.wt. With the exception of the 95 000 mol. wt. bands, all these glycoproteins bound to lentil lectin. Using monoclonal or monospecific antibodies in immunoprecipitation and on antibody affinity columns, each of these glycoprotein bands was identified as a previously defined lymphocyte differentiation antigen. The bands at 150 000 mol.wt. on thymocytes, at 170 000 on T lymphocytes, at 200 000 on B lymphocytes, and at 130 000 to 150 000 on bone marrow cells all consist of a leukocyte-common antigen, which has previously been shown to be present on leukocytes but not on other tissues. At least a part of the 95 000 mol.wt. band on thymocytes, T lymphocytes and bone marrow cells is the W3/13 antigen previously shown to be on mature T lymphocytes, polymorphonuclear cells, and in brain. The 25 000 mol.wt. band of thymocytes is the Thy-1 antigen. Similar experiments were carried out on thymocytes labeled with ¹²⁵I by the lactoperoxidase method. An intense band at 150 000 mol. wt. was identified as the leukocyte-common antigen by immunoprecipitation. A labeled band, which did not bind to lentil lectin, was immunoprecipitated at 95 000 mol. wt. with W3/13 antibody. Rat Thy-1 antigen was not labeled with ¹²⁵I.     

Differences in expression and exposure of promastigote and amastigote membrane molecules in Leishmania tropica.

The accessibility of particular Leishmania tropica promastigote (extracellular) and amastigote (intracellular) membrane molecules might be related to the relative abilities of the two stages to induce host immune responses. To examine the exposure of membrane antigens on resident macrophage-susceptible promastigotes and resident macrophage-resistant amastigotes, both stages were analyzed by polyacrylamide gel electrophoresis and immunoblotting after specific labeling and extraction procedures. Protein compositional studies, using metabolic labeling of promastigotes and amastigotes, demonstrated that both forms possessed numerous endogenously synthesized proteins. In addition, a marked difference was revealed in the external exposure of promastigote and amastigote membrane constituents when analyzed by 125I surface labeling or Western blot analysis. Whereas nine promastigote proteins were intensely to moderately iodinated, only one amastigote membrane component was similarly labeled (9.5K band). Western blot analyses with serum from a rabbit immunized with a mixture of both L. tropica stages indicated that the majority of promastigote molecules accessible to 125I may also react with immune serum. However, Western blots of extracted amastigotes identified several bands not seen on radiographs and thus not accessible to 125I. The external exposure of these amastigote molecules was confirmed in that immune serum adsorbed with viable, intact amastigotes was no longer reactive with amastigote extracts. Further, by Western blot analyses of sodium dodecyl sulfate- but not Nonidet P-40-extracted amastigotes, three amastigote-specific membrane antigens not previously observed with nonionic detergent extraction methods were identified. The autofluorographic pattern of amastigotes intrinsically labeled with N-[3H]acetylglucosamine, an amino sugar which is incorporated into membrane carbohydrates, was in excellent agreement with the pattern of antigens reactive with antibody in Western blots. Thus, with these cell surface labeling and extraction methods, promastigote and amastigote membranes were shown to be significantly different. Amastigotes possessed several membrane-associated molecules, but few appeared to be either accessible or reactive with 125I. Moreover, the majority of molecules not reactive with 125I, but reactive with antibodies, may be glycosylated. These observations are discussed relative to the ability of amastigotes both to survive within the degradative milieu of macrophage phagolysosomes and to evade host immune reactivity.     

Estimation of the number of infectious measles viruses in live virus vaccines using quantitative real-time PCR

The two sets of connected membranes induced in Kunjin virus-infected cells are characterized by the presence of NS3 helicase/protease in both, and by RNA-dependent RNA polymerase (RdRp) activity plus the associated double-stranded RNA (dsRNA) template in vesicle packets (VP), or by the absence of both the VP-specific markers in the convoluted membranes/paracrystalline arrays (CM/PC). Attempts were made to separate flavivirus-induced membranes by sedimentation or flotation analyses in density gradients of sucrose or iodixanol, respectively, after treatment of cell lysates by sonication, osmotic shock, or tryptic digestion. Only osmotic shock treatment provided suggestive evidence of separation. This was explored by flow cytometry analysis (FCA) of RdRp active membrane fractions from a sucrose gradient, using dual fluorescent labelling via antibodies to NS3 and dsRNA. FCA revealed the presence of a dual labelled membrane population indicative of VP, and in a faster sedimenting fraction a membrane population able to be labelled only in NS3, representative of CM/PC and associated (R)ER. It was postulated that osmotic shock ruptured the bounding membrane of the VP, releasing the enclosed small vesicles associated with the Kunjin virus replication complex characterized previously. Notably, the presence of the full spectrum of nonstructural proteins in some membrane fractions was not a reliable marker for RdRp activity. These experiments may provide the opportunity for isolation of relatively pure flavivirus replication complexes in their native membrane-associated state by fluorescence-activated cell sorting.     

Phosphorylation of rat kidney proximal tubular brush border membranes

A possible correlation between cyclic-AMP dependent protein phosphorylation and altered sodium dependent transport of inorganic phosphate was analyzed in isolated rat renal proximal tubular brush border membrane vesicles. In transiently opened vesicles (opened by an osmotic shock), the addition of gamma-32P-ATP leads to 32P-incorporation into several membrane proteins. The simultaneous addition of cyclic-AMP leads to increased phosphorylation of several proteins (e.g. apparent molecular weights: 40 kD, 46 kD, 55 kD). The addition of ATP, GTP and ITP to the osmotic shock medium leads to an (non-specific) inhibition of the sodium gradient dependent phosphate uptake. No further inhibition of the sodium dependent phosphate transport was observed when membrane vesicles were phosphorylated by ATP in the presence of cyclic-AMP. These data show a lack of correlation between cyclic-AMP dependent protein phosphorylation and altered sodium gradient dependent phosphate transport. Thus, there is no experimental support for the involvement of cyclic-AMP dependent protein phosphorylation as one of the final events in the regulation of phosphate transport across the rat renal proximal tubular brush border membrane.     

Radioiodination of new protein antigens on the surface of Plasmodium knowlesi schizont-infected erythrocytes

Schizont-infected red blood cells (SI-RBCs) from Plasmodium knowlesi-infected rhesus monkeys (Macaca mulatta) have been radioiodinated and the ¹²⁵I-proteins and ¹²⁵I-antigens compared with those of uninfected RBCs. New ¹²⁵I-proteins of M_r 230 000, 180 000, 165 000, 155 000, 135 000, 107 000, 72 000 and 65 000 were shown to be parasite-dependent components of SI-RBCs, the M_r 230 000 and 72 000 bands being quantitatively the major new components. New ¹²⁵I-antigens of SI-RBCs that were absent from uninfected cells and were only immunoprecipitated by sera from infected monkeys had M_r 230 000, 200 000, 180 000, 165 000, 155 000, 135 000, 130 000, 107 000, 72 000, 65 000 and 47 000. The following approaches were used to test which new radioiodinated components are on the SI-RBC outer membrane: analysis of radioactivity in haemoglobin; electron microscopic analysis of the integrity and purity of the SI-RBCs; treatment of intact ¹²⁵I-SI-RBCs with trypsin to ascertain the sensitivity of new proteins to exogenous protease; immunoprecipitation of antigen-antibody complexes after addition of antibody to intact ¹²⁵I-SI-RBCs. Although each test has inherent disadvantages, the accumulated evidence suggests that many, if not all of the new antigens identified for the first time in this report are on the SI-RBC outer membrane. The SICA variant antigen on P. knowlesi SI-RBCs was not identified by this approach.     

Identification of Schistosoma mansoni antigens by means of biologically active monoclonal antibodies

Two monoclonal antibodies of the IgE class (54.10) and of the IgG₁ class (27.21), that were shown previously to possess biological activity against Schistosoma mansoni larvae, were used for identification of surface antigens of the cercariae and schistosomula. This was performed by immunoprecipitation, immunoaffinity chromatography and immunoblotting. The epitope reactive with 27.21 mcIgG₁ is present on four polypeptides (60, 50, 27 and 19 kDa) derived from the parasite. The 60 kDa is specific to cercariae, whereas the 50 kDa is a glycoprotein shared both by cercariae and schistosomula. The antigen reactive with the 54.10 mcIgE was isolated by affinity chromatography on 54.10 column, and contained three major peptides of 125, 94 and 30 kDa. The 125 and 94 kDa band probably originate from the same protein, since they yield almost identical peptide maps. The isolated antigen retained its biological activity as demonstrated in the basophils degranulation assay.     

Translation of Schistosoma mansoni antigens in Xenopus oocytes microinjected with mRNA from adult worms

Oocytes from Xenopus laevis microinjected with RNA isolated from Schistosoma mansoni adult worms translated antigens recognized by sera from infected rats, humans, and from immunized rabbits. The pattern of immunoprecipitated proteins analysed by SDS-polyacrylamide gel electrophoresis was species specific in rats. Serum from infected Fischer rats recognized antigens of 20, 27 and several bands in the 50-60 kDa range whereas serum from infected Brown Norway rats also immunoprecipitated major bands at 29, 43 and 100 kDa. Human infection sera gave a very variable pattern of immunoprecipitation not apparently dependent on the patients' age. At least 20 different antigenic species could be identified ranging from 14 to 150 kDa. Some S. mansoni antigenic proteins could be isolated from the membrane fraction of the oocytes whereas notably the 29 kDa band was present mainly in the soluble fraction. N-Glycosylation of S. mansoni antigens occurred as evidenced by the effects of tunicamycin treatment and concanavalin A binding. A multiple series of bands between 50 and 60 kDa, present in the membrane fraction, were glycosylated and secreted from the oocytes. Monoclonal antibodies to larval stage surface antigens failed to immunoprecipitate oocyte translation products, but sera absorbed with live schistosomula identified at least three putative surface antigens of 100, 43 and 29 kDa. However, the 29 kDa molecule was neither synthesized into membranes, nor secreted from oocytes.     

Generation of monoclonal murine anti-DNP-IgE, IgM and IgG1 antibodies: biochemical and biological characterization.

Monoclonal DNP-specific IgG (lambda 2 epsilon 2), IgM (kappa 2 mu2) and IgG [kappa 2 (gamma 1)2] were isolated fom the culture supernatant of hybridomas by affinity chromatography with 2,4-dinitrophenol bovine serum albumin (DNP-BSA) sepharose and characterized by biochemical and biological methods. The molecular weights were 84,200 for the epsilon chain, 55,400 for the gamma chain and 77,500 for the mu chain as determined by sodium dodecylsulphate polyacrylamide del electrophoresis (SDS-PAGE). The association constants for [3H]-DNP-lysine determined by equilibrium dialysis were 0 . 87 X 10(7) l/mol for IgE and 1 . 91 X 10(8) 1/mol for IgG1. The isoelectric focusing of the purified monoclonal antibodies revealed for IgG1 seven bands at a pH range of 6 . 3 - 7 . 2 and for IgE sixteen bands at a pH range of 4 . 5 to 6 . 8. the binding of 125I-anti-IgE to rat basophilic leukaemia (RBL) and rat mast cells which had been preincubated with various amounts of monoclonal IgE was studied. At saturation conditions of IgE, about 2 . 14 X 10(5) molecules of anti-IgE were bound per rat mast cell. Rat mast cells coated with monoclonal anti-DNP IgE were triggered for the release of histamine in the presence of either the antigen or guinea-pig anti-mouse IgE. A mutual inhibition of the passive cutaneous anaphylaxis (PCA) reaction in the rat by either mixing mouse reaginic serum directed against ovalbumin or rat reaginic serum directed against Nippostrongylus brasiliensis with monoclonal mouse anti-DNP IgE was demonstrated.     

Staphylococcal toxic shock toxin specifically binds to cultured human epithelial cells and is rapidly internalized.

Staphylococcal toxic shock syndrome toxin (TST) was labeled with 125I under mild conditions without apparent destruction of the molecule. [125I]TST bound specifically to human epithelial (Chang) cells in culture; the binding was inhibited by a 100-fold excess of unlabeled toxin. Scatchard analysis of the binding data indicated about 10(4) receptor sites per cell and a dissociation constant (Kd) of 4 X 10(-9) M. When cells pretreated with TST at 4 degrees C were swiftly transferred to 37 degrees C, the amount of surface-bound toxin rapidly declined, as determined by release of noninternalized label from the cell surface. Half-time (t1/2) of internalization was about 1.5 min. Ultrastructural studies showed that toxin labeled with ferritin-conjugated antibodies entered the cytoplasm via coated pits forming coated vesicles in the first 2 min of incubation at 37 degrees C. The coated vesicles coalesced with transport vesicles that are ultrastructurally unlike receptosomes. Thus, the unusual ultrastructural pattern of this internalization suggests that TST is initially internalized by receptor-mediated endocytosis and then enters an alternate pathway involving translocation in special transport vesicles, perhaps to other cells.     

Intravesicular NAD has no effect on sodium-dependent phosphate transport in isolated renal brush border membrane vesicles

The effects of intravesicular NAD on Na⁺-dependent³²P_i uptake were investigated in isolated rat kidney brush border membrane vesicles (BBMV). NAD was introduced into the vesicles by osmotic shock, and extravesicular NAD was removed by passing the vesicles through a anion exchange column. The effectiveness of the osmotic shock procedure and the hydrolysis of extra- and intravesicular NAD were controlled by enzymatic analysis and thin layer chromatography. ADP-ribosylation of the membrane proteins was analyzed in vesicles osmotically shocked in the presence of either [adenylate-³²P]-NAD or [adenine-2,8-³H]-NAD by SDS-polyacrylamide gel electrophoresis.It was found that the Na⁺-dependent P_i uptake was inhibited when the BBMV were incubated with NAD at alkaline pH, which resulted in rapid NAD hydrolysis. When NAD was present in the intravesicular space only, the Na⁺-dependent P_i uptake was not inhibited.³²P from NAD was rapidly incorporated into a number of brush border membrane proteins, but no incorporation of³H-adenine could be detected.The results provide evidence that NAD does not inhibit P_i transport by a direct interaction with the cytoplasmic side of the brush border membrane. No evidence of ADP-ribosylation of the brush border membrane protein(s) was found.     

Enhancement of IgE-dependent eosinophil cytotoxicity to dinitrophenylated schistosomula by a nematode infection

In order to obtain direct evidence for the involvement of IgE in the eosinophil-mediated cytotoxicity, dinitrophenylated (DNP) schistosomula of Schistosoma japonicum coated with monoclonal IgE antibodies were examined as to whether they were killed by rat peritoneal eosinophils. The results indicated that the cytotoxicity of the eosinophils was dependent on a mouse monoclonal anti-DNP IgE antibody, but not on another monoclonal antibody specific to ovalbumin. Moreover, an enhanced cytotoxicity was observed when eosinophils were obtained from rats 4 weeks after infection with Nippostrongylus brasiliensis.     

Immunomagnetic enrichment and detection of isolated tumor cells in bone marrow of patients with epithelial malignancies

The detection of isolated tumor cells (ITC) in the bone marrow of patients with epithelial malignancies is an independant prognostic factor for several entities as breast cancer, colorectal cancer or non-small lung cancer. However, with conventional immunocytology using Ficoll density gradient and APAAP staining, only a small proportion of the bone marrow samples can be scanned for cytokeratin-positive (CK+) cells. To improve detection rates, we evaluated the enrichment of ITC by magnetic activated cell sorting (MACS) compared to regularly stained cytospins. Recovery experiments with a CK+ breast cancer cell line (SKBR3) were performed to calculate the MACS enrichment rate. Bone marrow was obtained by aspiration from 20 patients with carcinomas of epithelial origin and from 17 controls. ITC were enriched and stained with magnetically labeled CAM 5.2 antibodies directed to cytokeratin 7 and 8. MACS of SKBR3 seeded in peripheral blood revealed average recovery rates of 62% and 48% and average enrichment factors of 104-fold and 8139-fold of the CK+ cells after one and after two separations, respectively. After immunomagnetic enrichment, CK+ cells were detected in 16 of 20 (80%) cancer patients, whereas only 7 (35%) patients showed CK+ cells without magnetic enrichment (P=0.002). Ten of twelve (83%) patients with metastatic disease (stage M₁) and six of eight (75%) patients without any overt metastases (M₀) had CK+ cells in their bone marrow. None of the negative controls showed any CK+ cells. Enrichment with magnetically labeled anti cytokeratin antibodies increases the detection rate of epithelial cells in bone marrow of cancer patients compared to immunocytology. This revised version was published online in July 2006 with corrections to the Cover Date.     

Characterization of the outer membrane proteins of Bordetella avium.

The outer membrane proteins of Bordetella avium were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sarkosyl-insoluble outer membrane protein-enriched profiles from 50 virulent B. avium isolates, containing major 21,000- and 37,000-molecular-weight proteins (21K and 37K proteins, respectively) and at least 13 less intensely stained proteins with molecular weights ranging from 13,500 to 143,000, were very similar. The 21K, 27K, 31K, and 37K outer membrane proteins were shown to be associated noncovalently with the underlying peptidoglycan layer. It was necessary to treat cell envelopes with 2% sodium dodecyl sulfate and at temperatures in excess of 60 degrees C for 15 min to release these proteins. Exposure of proteins on the cell surface of B. avium was assessed by labeling with 125I followed by electrophoresis. As many as 13 bands were present in profiles from labeled whole cells. Of the surface-labeled bands, eight corresponded to bands in a radiolabeled outer membrane preparation. The outer membrane protein profile of B. avium was compared with profiles from other Bordetella spp., including 20 B. avium-like and 16 B. bronchiseptica strains isolated from turkeys. The outer membrane protein profile of B. avium was distinctly different from those of the other bordetella. The effect of variations in the growth medium on the expression of outer membrane proteins of B. avium was examined. Expression of 22K, 26K, 56K, and 73K proteins was decreased or eliminated by addition of 50 mM MgSO4 to the medium.