Histochemical evidence for sulfomucins at the surface of Hela cells.

The nature of the negatively charged groups present at the surface of HeLa cells was further investigated. Therefore we applied a series of light microscopic staining techniques, widely used for the demonstration of epithelial mucosubstances on tissue sections, to HeLa cells from suspension cultures. Our histochemical findings confirmed the presence of carboxylated substances at the surface of these cells. Furthermore we obtained conclusive evidence for the presence of sulfated molecules. Both substances seem to be closely related to epithelial sialomucins and sulfomucins.     

PS exposure increases the susceptibility of cells to fusion with DOTAP liposomes

Cationic liposomes are used as efficient carriers for gene delivery into mammalian cells due to their ability to bind nucleic acids, adsorb onto the cell surface and fuse with negatively charged membranes. This last property enables the release and escape of their cargo from endosomal compartments. The efficiency of this fusion mainly depends on the surface charge of the target membranes. Here, we report that cells of two different lines, epithelial adenocarcinoma HeLa and lymphocytic leukemia Jurkat T, which externalize PS, are more susceptible to fusion with DOTAP liposomes than control cells. We compared the ability to undergo fusion of untreated and apoptotic cells. Apoptosis was induced by various pro-apoptotic agents and treatments, namely: incubation in the presence of MnCl(2), cytostatic drugs fludarabine and mitoxantrone, staurosporine and serum depletion in the case of HeLa cells. Jurkat T cells were treated similarly except apoptosis was additionally induced by incubation in the presence of 4% EtOH. Epithelial cells fused with the highest efficiencies of lipid mixing, when pretreated with staurosporine. Jurkat T cells were less susceptible to fusion, but they also displayed an increase in fusion efficiency after the induction of apoptosis. Alternatively, we treated the cells with metabolic inhibitors causing ATP-depletion in order to inactivate aminophospholipid translocase. After ATP-depletion, HeLa and Jurkat T cells fused with DOTAP liposomes with higher efficiencies than control cells. Our conclusion is that the lipid asymmetry of natural membranes may limit fusion with cationic liposomes.     

Characterization of SP41, a surface protein of Brucella associated with adherence and invasion of host epithelial cells

Brucella is an invasive organism that multiplies and survives within eukaryotic cells. The brucellae are able to adhere to the surface of cultured epithelial cells, a mechanism that may facilitate penetration and dissemination to other host tissues. However, no adhesins that allow the bacteria to interact with the surface of epithelial cells before migration within polymorphonuclear leukocytes, monocytes and macrophages have been described. Here, we show that Brucella surface proteins (SPs) with apparent molecular masses of 14, 18 and 41 kDa bound selectively to HeLa cells. However, only antibodies directed against the 41 kDa surface protein (SP41) inhibited in dose-response manner, bacterial adherence and invasion of HeLa cells. HeLa cells treated with neuraminidase did not bind SP41, suggesting the involvement of cellular sialic acid residues in this interaction. Biochemical analysis of SP41 revealed that this protein is the predicted product of the ugpB locus, which showed significant homology to the glycerol-3-phosphate-binding ATP-binding cassette (ABC) transporter protein found in several bacterial species. SP41 appears to be exposed on the bacterial surface as determined by immunofluorescence and immunogold labelling with anti-SP41 antibody. An isogenic DeltaugpB mutant showed a significant inhibitory effect on Brucella adherence and invasion of human cultured epithelial cells and this effect could be reversed by restoration of the ugpB on a plasmid. Lastly, we also show that most of the sera from individuals with acute brucellosis, but not sera obtained from healthy donors or patients with chronic brucellosis, mount antibody reactivity against SP41, suggesting that this protein is produced in vivo and that it elicits an antibody immune response. These data are novel findings that offer new insights into understanding the interplay between this bacterium and host target cells, and identify a new target for vaccine development and prevention of brucellosis.     

Lysozyme facilitates adherence of Enterococcus faecium to host cells and induction of necrotic cell death

The prevalence of infections with enterococci is increasing worldwide. However, little is known about the mechanisms which enable these opportunistic pathogens to cause infections of their host. Here we demonstrate that Enterococcus faecium in the presence of lysozyme induces necrosis in human and mouse cells after 4 h indicated by disrupted cellular membranes of epithelial (HeLa), myeloid (U937, J774A.1) and lymphoid (Jurkat J16, thymocytes), but not intestinal epithelial cells (CaCo-2, CMT-93). Using an appropriate mutant strain it was shown that the enterococcal surface-protein SgrA is involved in cell death induction in mouse cells (J774A.1, thymocytes). Microscopic analyses of epithelial cells 30 min post infection revealed that lysozyme increases adhesion of E. faecium to HeLa, but not CaCo-2 cells. At that time the phalloidin-FITC-stained cytoskeleton of infected cells was still intact, whereas 2 h post infection the F-actin network of HeLa, but not CaCo-2 cells was disrupted. Hence, the early, lysozyme-mediated increase of bacterial adherence plays an important role for cell death induction by E. faecium in HeLa cells. Moreover, bacterial extracellular hydrogen peroxide might contribute to necrosis induction, since the rate of propidium iodide-positive HeLa and J774A.1 cells was lowered after infection with a ROS-deficient E. faecium mutant.     

Human papillomavirus type 16 minor capsid protein l2 N-terminal region containing a common neutralization epitope binds to the cell surface and enters the cytoplasm

The first step of papillomavirus infection is believed to be binding of major capsid protein L1 to the cell surface without involvement of minor capsid protein L2, but the viral infectivity can be neutralized either by anti-L1 or anti-L2 antibody. To understand the role of L2 in human papillomavirus (HPV) infection, we examined a segment of HPV type 16 (HPV16) L2, which contains a neutralization epitope common to HPV6, for its involvement in adsorption and penetration of the capsids. Preincubation of monkey COS-1 cells with a synthetic peptide having amino acids (aa) 108 to 120 of HPV16 L2 reduced the susceptibility of COS-1 cells to infection with HPV16 pseudovirions. Confocal microscopy showed that the green fluorescence protein (GFP) fused with the L2 peptide was found to bind to the surface of a HeLa cell, an HPV18-positive human cancer cell line, at 4 degrees C and to enter the cytoplasm after subsequent incubation at 37 degrees C. Flow cytometry showed that fused GFP did not bind to HeLa cells that had been treated with trypsin. Besides COS-1 and HeLa cells, some human and rodent cell lines were detected by flow cytometry to be susceptible to binding with fused GFP, showing a tendency of epithelial cells toward higher susceptibility. Substitutions at aa 108 to 111 inhibited fused GFP from binding to HeLa cells and reduced the infectivity in COS-1 cells of the in vitro-constructed pseudovirions. The results suggest that L2 plays an important role in enhancing HPV infection through interaction between the N-terminal region and a cellular surface protein, facilitating penetration of the virions and determining part of the tropism of HPVs.     

A defective viral superantigen-presenting phenotype in HLA-DR transfectants is corrected by CIITA

Activation of T lymphocytes by mouse mammary tumor virus superantigen (vSAg) requires binding to MHC class II molecules. The subcellular location where functional interactions occur between MHC class II molecules and vSAgs is still a matter of debate. To gain further insight into this issue, we have used human epithelial HeLa cells expressing HLA-DR1. Surprisingly, the human cells were unable to present transfected vSAg7 or vSAg9 to a series of murine T cell hybridomas. The defect is not related to a lack of vSAg processing, because these cells can indirectly activate T cells after coculture in the presence of B lymphocytes. However, after IFN-gamma treatment, the HeLa DR1(+) cells became apt at directly presenting the vSAg. Furthermore, transfection of CIITA was sufficient to restore presentation. Reconstitution experiments demonstrated the necessity of coexpressing HLA-DM and invariant chain (Ii) for efficient vSAg presentation. Interestingly, inclusion of a dileucine motif in the DRbeta cytoplasmic tail bypassed the need for HLA-DM expression and allowed the efficient presentation of vSAg7 in the presence of Ii. A similar trafficking signal was included in vSAg7 by replacing its cytoplasmic tail with the one of Ii. However, sorting of this chimeric Ii/vSAg molecule to the endocytic pathway completely abolished both its indirect and direct presentation. Together, our results suggest that functional vSAgs-DR complexes form after the very late stages of class II maturation, most probably at the cell surface.     

Thermodynamic aspects of cell spreading on solid substrata

To verify the validity of thermodynamic approaches to the prediction of cellular behavior, cell spreading of three different cell types on solid substrata was determined in vitro. Solid substrata as well as cell types were selected on the basis of their surface free energies, calculated from contact angle measurements. The surface free energies of the solid substrata ranged from 18-116 erg cm-2. To measure contact angles on cells, a technique was developed in which a multilayer of cells was deposited on a filter and air dried. Cell surface free energies ranged from 60 erg cm-2 for fibroblasts, and 57 for smooth muscle cells, to 91 for HeLa epithelial cells. After adsorption of serum proteins, cell surface free energies of all three cell types converged to approx 74 erg cm-2. The spreading of these cell types from RPMI 1640 medium on the various solid substrata showed that both in the presence and in the absence of serum proteins in the medium, cells spread poorly on low energy substrata (Ys less than 50 erg cm-2), whereas good cell spreading was observed on the higher energy substrata. Calculations of the interfacial free energy of adhesion (delta Fadh) show that delta Fadh decreases with increasing Ys, and equals zero around 45 erg cm-2 for all three cell types in the presence of serum proteins and for HeLa epithelium cells in the absence of serum proteins. This explains the spreading of these cells on the various substrata upon a thermodynamic basis. The results clearly show that substratum surface free energy has a predictive value with respect to cell spreading in vitro, both in the presence and absence of serum proteins. It is noted, however, that interfacial thermodynamics fail to explain the behavior of fibroblasts and smooth muscle cells in the absence of serum proteins, most likely because of the relatively high surface charges of these two cell types.     

Phorbol ester-induced podosomes in normal human bronchial epithelial cells

Spreading and migration of the basal cells neighboring a wound is essential for airway epithelial repair. To gain insight into the molecular mechanisms that govern these cellular processes, we asked whether normal human airway epithelial cells can form podosomes, a cellular structure discovered from cancer and mesenchymal cells that controls migration and invasion. Herein, we report that phorbol-12, 13-dibutyrate (PDBu), a protein kinase C activator, induced reorganization of cytoskeletal structure in primary normal human bronchial epithelial cells, and in normal human airway epithelial BEAS2B cells. Z-stack scanning confocal microscopy showed that PDBu-induced podosome-like structures contain actin-rich columns that arise from the ventral surface of the cell, and also revealed the presence of circular ruffles/waves at the dorsal cell surface. The molecular components of these cytoskeletal structures were determined with immunofluorescent staining. Using in situ zymography, we demonstrated that PDBu-induced podosomes were capable of degrading fibronectin-gelatin-sucrose matrix. PDBu also increased epithelial cell invasion across Transwell chamber. Podosomes and circular dorsal ruffles may be important for epithelial cell migration and invasion, thus contributing to respiratory epithelial repair and regeneration.     

Human epithelial cell intermediate filaments: isolation, purification, and characterization

Intermediate filaments (IF) isolated from human epithelial cells (HeLa) can be disassembled in 8 M urea and reassembled in phosphate-buffered solutions containing greater than 0.1 mg/ml IF protein. Eight proteins were associated with HeLa IF after several disassembly-reassembly cycles as determined by sodium dodecyl sulfate gel electrophoresis (SDS PAGE). A rabbit antiserum directed against HeLa IF contained antibodies to most of these proteins. The immunofluorescence pattern that was seen in HeLa cells with this antiserum is complex. It consisted of a juxtanuclear accumulation of IF protein and a weblike array of cytoplasmic fibers extending to the cell border. Following preadsorption with individual HeLa IF proteins, the immunofluorescence pattern in HeLa cells was altered to suggest the presence of at least two distinct IF networks. The amino acid composition and alpha-helix content (approximately 38%) of HeLa IF proteins was similar to the values obtained for other IF proteins. One-dimensional peptide maps show extensive homology between the major HeLa IF protein of 55,000-mol- wt and a similar 55,000-mol-wt protein obtained from hamster fibroblasts (BHK-21). HeLa 55,000-mol-wt homopolymer IF assembled under conditions similar to those required for BHK-21 55,000-mol-wt homopolymers. Several other proteins present in HeLa IF preparations may be keratin-like structural proteins. The results obtained in these studies indicate that the major HeLa IF protein is the same major IF structural protein found in fibroblasts. Ultrastructural studies of HeLa cells revealed two distinct IF organizational stages including bundles and loose arrays. In addition, in vitro reconstituted HeLa IF also exhibited these two organizational states.     

Adherence of Brucella to human epithelial cells and macrophages is mediated by sialic acid residues

The basis for the interaction of Brucella species with the surface of epithelial cells before migration in the host within polymorphonuclear leucocytes is largely unknown. Here, we studied the ability of Brucella abortus and Brucella melitensis to adhere to cultured epithelial (HeLa and HEp-2) cells and THP-1-derived macrophages, and to bind extracellular matrix proteins (ECM). The brucellae adhered to epithelial cells forming localized bacterial microcolonies on the cell surface, and this process was inhibited significantly by pretreatment of epithelial cells with neuraminidase and sodium periodate and by preincubation of the bacteria with heparan sulphate and N-acetylneuraminic acid. Trypsinization of epithelial cells yielded increased adherence, suggesting unmasking of target sites on host cells. Notably, the brucellae also adhered to cultured THP-1 cells, and this event was greatly reduced upon removal of sialic acid residues from these cells with neuraminidase. B. abortus bound in a dose-dependent manner to immobilized fibronectin and vitronectin and, to a lesser extent, to chondroitin sulphate, collagen and laminin. In sum, our data strongly suggest that the adherence mechanism of brucellae to epithelial cells and macrophages is mediated by cellular receptors containing sialic acid and sulphated residues. The recognition of ECM (fibronectin and vitronectin) by the brucellae may represent a mechanism for spread within the host tissues. These are novel findings that offer new insights into understanding the interplay between Brucella and host cells.     

Role of phosphatidylserine exposure and sugar chain desialylation at the surface of influenza virus-infected cells in efficient phagocytosis by macrophages

HeLa cells infected with influenza A virus undergo typical caspase-dependent apoptosis and are efficiently phagocytosed by mouse peritoneal macrophages in a manner mediated by the membrane phospholipid phosphatidylserine, which is translocated to the surface of virus-infected cells during apoptosis. However, the extent of phagocytosis is not always parallel with the level of phosphatidylserine externalization. Here we examined the involvement of influenza virus neuraminidase (NA) in efficient phagocytosis of virus-infected cells. HeLa cells infected with an influenza virus strain expressing temperature-sensitive NA underwent apoptosis and produced viral proteins, including the defective NA, at a non-permissive temperature to almost the same extent as cells infected with the wild-type virus. The cells were, however, phagocytosed by macrophages with reduced efficiency. In addition, phagocytosis of cells infected with the wild-type virus was severely inhibited when the cells had been maintained in the presence of the NA inhibitor zanamivir. On the other hand, the binding of sialic acid-recognizing lectins to the cell surface declined after infection with the wild-type virus. The decrease in the extent of lectin binding was greatly attenuated when cells were infected with the mutant virus or when wild-type virus-infected cells were maintained in the presence of zanamivir. These results indicate that sugar chains are desialylated by NA at the surface of virus-infected cells. We conclude that the presence of both phosphatidylserine and asialoglycomoieties on the cell surface is required for efficient phagocytosis of influenza virus-infected cells by macrophages.     

Lipopolysaccharide-binding protein is vectorially secreted and transported by cultured intestinal epithelial cells and is present in the intestinal mucus of mice

Lipopolysaccharide-binding protein (LBP) is an important modulator of the host's response to endotoxin. In a previous study, we found evidence for the synthesis of LBP by intestinal epithelial cells. In this study, we explored the polarity of LBP secretion by these cells. Polarized monolayers of Caco-2 cells were used as intestinal mucosa model. Cells were stimulated apically or basally with cytokines, and LBP secretion was analyzed. Furthermore, the presence of LBP in intestinal mucus of healthy and endotoxemic mice was studied using a mucus-sampling technique. The constitutive unipolar LBP secretion from the apical cell surface was markedly enhanced when cells were exposed to cytokines at their apical surface. However, bioactive LBP was secreted from both cell surfaces after basolateral stimulation of cells. Cytokines also influenced the secretion of the acute phase proteins serum amyloid A, apoA-I, and apoB from both surfaces of Caco-2 cells. Furthermore, transport of exogenous LBP from the basolateral to the apical cell surface was demonstrated. In line with these in vitro data, the presence of LBP in intestinal mucus was strongly enhanced in mice after a challenge with endotoxin. The results indicate that LBP is present at the mucosal surface of the intestine, a phenomenon for which secretion and transport of LBP by intestinal epithelial cells may be responsible.     

CEACAM is not necessary for Neisseria gonorrhoeae to adhere to and invade female genital epithelial cells

Neisseria gonorrhoeae has a repertoire of up to 11 opacity-associated (Opa) proteins that are adhesins. Most Opa proteins adhere to CEACAM antigens and when CEACAM molecules are present on the surface of transfected epithelial cells their binding by Opa is thought to induce invasion of these cells by gonococci. In this study, we investigated whether several malignant epithelial cell lines, normal cervical and fallopian tube epithelial cell cultures, as well as normal fallopian tube tissue express several of the CEACAM molecules, and whether gonococci use these molecules for adherence and invasion of these female genital epithelial cells. A primary cervical cell culture and metastatic cervical cell line ME180 both expressed CEACAM as shown by whole cell ELISA and flow cytometry, and increased the surface expression of total CEACAM during incubation with Opa⁺ gonococci. Opa⁺ gonococci both adhered to and invaded these cells; CEACAM-specific monoclonal antibody (MAb) partially abolished this interaction. Two primary fallopian epithelial tube cell cultures, a primary cervical cell culture and two malignant cell lines, HEC-1-B and HeLa, did not express CEACAM nor was CEACAM mRNA present. No evidence of either intracellular or secreted extracellular CEACAM was found with HEC-1-B and HeLa cells. Opa⁺ gonococci both adhered to and invaded CEACAM non-expressing cells; however, Opa⁺ gonococcal association with these non-expressing cell lines could not be inhibited with CEACAM-specific MAb. These data show that CEACAM is not always expressed on female genital epithelial cells and is not essential for gonococcal adherence and invasion. However, when CEACAM is expressed, Opa⁺ gonococci exploit it for the adherence to and invasion of these cells.     

Molecular basis of host epithelial cell recognition by Trichomonas vaginalis

Parasitism of host epithelial cells by Trichomonas vaginalis is a highly specific event. Four trichomonad surface proteins (adhesins) with molecular masses of 65,000 daltons (65 kDa; AP65), 51 kDa (AP51), 33 kDa (AP33), and 23 kDa (AP23) mediate the interaction of T. vaginalis with epithelial cells. Fresh isolates, when compared with long-term-grown isolates, had greater amounts of adhesins, which corresponded with increased levels of cytoadherence. Anti-adhesin antibodies reacted by immunoblot only with the respective protein and detected, by indirect immunofluorescence, each adhesion on the parasite surface. These antibodies inhibited the binding of live parasites to epithelial cells and protected epithelial cells from contact-dependent cytotoxicity. The pretreatment of epithelial cells with a preparation of purified adhesions also blocked trichomonal cytoadherence. Moreover, HeLa cells possessed molecules which recognized and bound to adhesins on nitrocellulose blots.     

Proteomic identification of mammalian cell surface derived glycosylphosphatidylinositol‐anchored proteins through selective glycan enrichment

Glycosylphosphatidylinositol‐anchored proteins (GPI‐APs) are an important class of glycoproteins that are tethered to the surface of mammalian cells via the lipid GPI. GPI‐APs have been implicated in many important cellular functions including cell adhesion, cell signaling, and immune regulation. Proteomic identification of mammalian GPI‐APs en masse has been limited technically by poor sensitivity for these low abundance proteins and the use of methods that destroy cell integrity. Here, we present methodology that permits identification of GPI‐APs liberated directly from the surface of intact mammalian cells through exploitation of their appended glycans to enrich for these proteins ahead of LC‐MS/MS analyses. We validate our approach in HeLa cells, identifying a greater number of GPI‐APs from intact cells than has been previously identified from isolated HeLa membranes and a lipid raft preparation. We further apply our approach to define the cohort of endogenous GPI‐APs that populate the distinct apical and basolateral membrane surfaces of polarized epithelial cell monolayers. Our approach provides a new method to achieve greater sensitivity in the identification of low abundance GPI‐APs from the surface of live cells and the nondestructive nature of the method provides new opportunities for the temporal or spatial analysis of cellular GPI‐AP expression and dynamics.     

Nutritional and Metabolic Requirements for the Infection of HeLa Cells by Salmonella enterica Serovar Typhimurium

Salmonella is the causative agent of a spectrum of human and animal diseases ranging from gastroenteritis to typhoid fever. It is a food - and water - borne pathogen and infects via ingestion followed by invasion of intestinal epithelial cells and phagocytic cells. In this study we employed a mutational approach to define the nutrients and metabolic pathways required by Salmonella enterica serovar Typhimurium during infection of a human epithelial cell line (HeLa). We deleted the key glycolytic genes, pfkA and pfkB to show that S. Typhimurium utilizes glycolysis for replication within HeLa cells; however, glycolysis was not absolutely essential for intracellular replication. Using S. Typhimurium strains deleted for genes encoding components of the phosphotransferase system and glucose transport, we show that glucose is a major substrate required for the intracellular replication of S. Typhimurium in HeLa cells. We also deleted genes encoding enzymes involved in the utilization of gluconeogenic substrates and the glyoxylate shunt and show that neither of these pathways were required for intracellular replication of S. Typhimurium within HeLa cells.     

Human Immunodeficiency Virus Type 1 (HIV-1) Infection and Transcytosis Activity of a HIV-1 Susceptible Clone from HeLa Cell

In order to clarify the transmission process of human immunodeficiency virus type 1 (HIV-1) through the epithelial cell barrier, HeLa cells susceptible and non-susceptible to HIV-1 were cloned and designated as P6 HeLa and N7 HeLa cells, respectively. P6 HeLa cells could be infected with the LAI strain of HIV-1 and mediated HIV-1 transcytosis. In contrast, N7 HeLa cells exhibited neither HIV-1 infection nor transcytosis. CD4 and galactosylceramide as the receptors for HIV-1 were not detected on P6 HeLa cells, although an anti-CD4 monoclonal antibody (mAb) blocked HIV-1 infection. Since HIV-1-infected P6 HeLa cells exhibited no fusion and survived, we speculated that the P6 HeLa cells expressed molecules other than CD4 which facilitated HIV-1 infection. Two mAbs (A-14 ITK and C57 a9-9) which inhibited the HIV-1 infection of P6 HeLa cells were generated. Each mAb recognized distinct molecule(s) as shown by Western blotting. Transcytosis by the P6 HeLa cells was inhibited by C57 a9-9 but not by A-14 ITK or anti-CD4 mAb. Both infection and transcytosis may be responsible for HIV-1 transmission through epithelial cells in a complex manner. Although infection and transcytosis occurred via different mechanisms, the molecule(s) recognized by C57 a9-9 mAb may be associated with both processes.     

Heterocellular gap junctional communication between alveolar epithelial cells

We analyzed the pattern of gap junction protein (connexin) expression in vivo by indirect immunofluorescence. In normal rat lung sections, connexin (Cx)32 was expressed by type II cells, whereas Cx43 was more ubiquitously expressed and Cx46 was expressed by occasional alveolar epithelial cells. In response to bleomycin-induced lung injury, Cx46 was upregulated by alveolar epithelial cells, whereas Cx32 and Cx43 expression were largely unchanged. Given that Cx46 may form gap junction channels with either Cx43 or Cx32, we examined the ability of primary alveolar epithelial cells cultured for 6 days, which express Cx43 and Cx46, to form heterocellular gap junctions with cells expressing other connexins. Day 6 alveolar epithelial cells formed functional gap junctions with other day 6 cells or with HeLa cells transfected with Cx43 (HeLa/Cx43), but they did not communicate with HeLa/Cx32 cells. Furthermore, day 6 alveolar epithelial cells formed functional gap junction channels with freshly isolated type II cells. Taken together, these data are consistent with the notion that type I and type II alveolar epithelial cells communicate through gap junctions compatible with Cx43.     

Binding of HBs antigen to HeLa cells by use of reconstitution into liposomes and fusion by Sendai virus

Hepatitis B surface antigen (HBsAg) was reconstituted into liposomes composed of phosphatidylserin. An isolated membrane fraction of HeLa cells was mixed with the liposomes, then the liposomes were incubated with HeLa cells in the presence of Sendai virus. In this system the attachment of HBsAg to the cell surface was enhanced and became resistant to treatment with trypsin-EDTA. The presence of the HBsAg on the cell surface was revealed by immunoelectronmicroscopy. This technique may provide a model system for studying immunological reactions to HBsAg-bearing target cells in vitro.