Human IgA and IgG F(ab')2 that bind to staphylococcal protein A belong to the VHIII subgroup

Staphylococcal protein A (SPA) is a bacterial membrane protein that possesses, in addition to its Fc gamma-binding activity, a distinct specificity for the Fab region of some IgM, IgA, IgG, and IgE. The Fab site that binds to SPA has been localized to the V region of the Ig H chain. In a previous study of human monoclonal and polyclonal IgM, we demonstrated that binding to SPA was highly restricted to molecules of the VHIII subgroup, and that nearly all VHIII IgM were able to bind SPA. The present study examines the VH composition of SPA-binding and SPA-nonbinding fractions of purified human polyclonal IgA, and IgG F(ab')2 fragments. We found that 22% of the IgA and 15% of the IgG F(ab')2 bound to SPA-agarose. Analysis with VH subgroup-specific antisera indicated that the SPA-binding fraction of IgA was dominated by the VHIII subgroup, and the SPA-binding fraction of IgG F(ab')2 contained only VHIII molecules. Furthermore, substantial portions of the total VHIII protein in IgA and in IgG F(ab')2 bound to SPA. We conclude that Fab binding to SPA is both restricted to and highly prevalent among human VHIII molecules, regardless of Ig class. These results suggest that protein A is an Ig superantigen.     

On the fragmentation of monoclonal IgG1, IgG2a, and IgG2b from BALB/c mice

Methods for the production and purification of F(ab')2 fragments from BALB/c monoclonal IgG1, IgG2a, and IgG2b with pepsin and other proteases were examined. The overall susceptibility to degradation is IgG2b greater than IgG2a greater than IgG1. Stable F(ab')2 can be produced in good yield from IgG1 with pepsin at pH 3.5 to 4.0 and can be made directly by pepsin treatment of ascites fluids or cell culture supernatants containing IgG1. IgG2a is cleaved in two steps by pepsin, first to F(ab')2 and then to Fab'. With carefully chosen conditions, F(ab')2 can be obtained in acceptable yield. The primary cleavage for the IgG2a heavy chain appears to be on the COOH terminal side of the interheavy chain disulfides, and secondary cleavage is on the NH2-terminal side. For IgG2b the reverse is true, and F(ab')2 has not been obtained in useful amounts; however, the primary cleavage of IgG2b appears to be assymetric with respect to the two heavy chains, and Fab/c fragments that have one Fab plus Fc can be made. Digestion with elastase resulted in the best yield of Fab/c. This finding may provide a method for retaining cytotoxicity in monoclonal antibodies against cell surface antigens while eliminating their capacity to modulate. The cleavage patterns of the three classes of IgG are rationalized in terms of the structure of their hinge regions.     

Primary antibody-Fab fragment complexes: a flexible alternative to traditional direct and indirect immunolabeling techniques.

Immunolabeling with immune complexes of primary and secondary antibodies offers an attractive method for detecting and quantifying specific antigen. Primary antibodies maintain their affinity for specific antigen after labeling with Fab fragments in vitro. Incubation of these immune complexes with excess normal serum from the same species as the primary antibody prevents free Fab fragments from recognizing immunoglobulin. Effectively a hybrid between traditional direct and indirect immunolabeling techniques, this simple technique allows primary antibodies to be non-covalently labeled with a variety of reporter molecules as and when required. Using complexes containing Fab fragments that recognize both the Fc and F(ab')2 regions of IgG, we show that this approach prevents nonspecific labeling of endogenous immunoglobulin, can be used to simultaneously detect multiple antigens with primary antibodies derived from the same species, and allows the same polyclonal antibody to be used for both antigen capture and detection in ELISA.     

Purification of recombinant chimeric B72.3 Fab' and F(ab')2 using streptococcal protein G.

Streptococcal protein G has been used extensively for the purification of antibodies using the interaction of the Fc region with protein G. Many antibodies also interact with protein G through a low-affinity binding site for the Fab region. The exploitation of this low-affinity interaction for the purification of Fab' fragments is described here. Chimeric mouse-human B72.3 Fab' and F(ab')2 fragments were expressed by CHO cells and purified from CHO cell supernatant using protein G-Sepharose. Since chimeric B72.3 Fab' bound weakly to the protein G-Sepharose it could be separated from F(ab')2 and eluted with a pH 7 wash whereas B72.3 F(ab')2 required elution at pH 2. Both Fab' and F(ab')2 were recovered with full immunoreactivity and could be further purified using gel-filtration chromatography to greater than 99% purity. This method allows the simple purification of directly expressed Fab' or F(ab')2 fragments from CHO cell supernatant.     

Influence of subunit-specific antibodies on the activity of the F0 complex of the ATP synthase of Escherichia coli. II. Effects of subunit c-specific polyclonal antibodies.

After incubation of F1-stripped everted membrane vesicles with antibodies against subunit c of the ATP synthase of Escherichia coli the proton translocation through the open F0 channel was blocked. Rebinding of F1 to those vesicles is affected by the antibody concentration used. In general, the use of F(ab')2 or Fab fragments prepared from anti-c antibodies gave similar results. However, using Fab fragments a higher amount of antigenic binding sites was necessary to block the F0 complex completely, whereas extremely low amounts of Fab fragments were necessary to inhibit the binding of F1. This can be explained by an antigenic determinant of subunit c, which is only accessible to the smaller Fab fragments with a molecular mass of approximately 50,000. Incubation of F1-containing everted membranes with anti-c antibodies showed that the binding of the antibodies resulted in a displacement of F1, while simultaneously the proton translocation through F0 has been blocked. Such a displacement can only be observed after incubation with IgG molecules or F(ab')2 fragments. Fab fragments were not able to displace the F1 part, indicating that the ability of antibodies and F(ab')2 fragments to produce cross-links is responsible for the loss of F1 from the membranes.     

Immunoglobulin G3 from polyclonal human immunodeficiency virus (HIV) immune globulin is more potent than other subclasses in neutralizing HIV type 1.

Passive antibody prophylaxis against human immunodeficiency virus type 1 (HIV-1) has been accomplished in primates, suggesting that this strategy may prove useful in humans. While antibody specificity is crucial for neutralization, other antibody characteristics, such as subclass, have not been explored. Our objective was to compare the efficiencies of immunoglobulin G (IgG) subclasses from polyclonal human HIV immune globulin (HIVIG) in the neutralization of HIV-1 strains differing in coreceptor tropism. IgG1, IgG2, and IgG3 were enriched from HIVIG by using protein A-Sepharose. All three subclasses bound major HIV-1 proteins, as shown by Western blot assay and enzyme-linked immunosorbent assay. In HIV-1 fusion assays using X4, R5, or X4R5 envelope-expressing effector cells, IgG3 more efficiently blocked fusion. In neutralization assays with cell-free viruses using X4 (LAI, IIIB), R5 (BaL), and X4R5 (DH123), a similar hierarchy of neutralization was found: IgG3 > IgG1 > IgG2. IgG3 has a longer, more flexible hinge region than the other subclasses. To test whether this is important, IgG1 and IgG3 were digested with pepsin to generate F(ab')(2) fragments or with papain to generate Fab fragments. IgG3 F(ab')(2) fragments were still more efficient in neutralization than F(ab')(2) of IgG1. However, Fab fragments of IgG3 and IgG1 demonstrated equivalent neutralization capacities and the IgG3 advantage was lost. These results suggest that the IgG3 hinge region confers enhanced HIV-neutralizing ability. Enrichment and stabilization of IgG3 may therefore lead to improved HIVIG preparations. The results of this study have implications for the improvement of passive immunization with polyclonal or monoclonal antibodies and suggest that HIV-1 vaccines which induce high-titer IgG3 responses could be advantageous.     

Single-step purification of F(ab')2 fragments of mouse monoclonal antibodies (immunoglobulins G1) by hydrophobic interaction high performance liquid chromatography using TSKgel Phenyl-5PW.

Hydrophobic interaction high performance liquid chromatography (HPLC) using TSKgel Phenyl-5PW was applicable to single-step purification of F(ab')2 fragments from pepsin digests of mouse monoclonal antibodies of IgG1 class. The digests were applied to the gel equilibrated with phosphate-buffered saline containing 1 M ammonium sulfate. F(ab')2 fragments were adsorbed onto the gel using the same buffer, and eluted by reducing the ammonium sulfate concentration to 0 M. The fraction containing F(ab')2 fragments was homogeneous (purity: higher than 98%) by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration HPLC. The recovery of the antigen binding site was 42-58%. The cycle time of the Phenyl-5PW HPLC was 45 min, and F(ab')2 of up to 2200 mg was purified in a cycle. This method could be useful especially for large scale purification of F(ab')2 fragments.     

Activation of resting T lymphocytes by anti-CD3 (T3) antibodies in the absence of monocytes

The antigen receptor molecules on human T lymphocytes are noncovalently associated on the cell surface with the CD3 (T3) molecular complex. Perturbation of this complex with anti-CD3 monoclonal antibodies induces T cell activation. Previous studies have demonstrated that this process requires the participation of monocytes. In the present report, we demonstrate that purified, resting (G0 phase) T cells incubated with monoclonal anti-CD3 antibodies proliferate in response to purified interleukin 2 (IL 2), in a lymphokine dose-dependent fashion. Anti-CD3 antibody or IL 2 alone did not trigger cell division. The effect was specific for anti-CD3 antibodies because monoclonal antibodies reactive with other surface molecules (OKT4, OKT8, L368) were inactive. Furthermore, the same phenomenon was observed when anti-CD3 antibody Leu-4 (IgG1) was incubated with cells of individuals whose monocytes cannot process antibodies of the IgG1 subclass (Leu-4 nonresponders). In addition, both F(ab')2 and Fab fragments of anti-CD3 antibody OKT3 were also capable of rendering T cells receptive to the IL 2 growth signal. These data indicate that neither monocytes nor CD3 receptor cross-linking are required absolutely for resting T cell activation, provided that IL 2 is supplied exogenously. T lymphocytes treated with anti-CD3 antibodies proliferated in response to both purified mitogen-induced and recombinant IL 2. Antibodies to the IL 2 receptor (anti-Tac) inhibited the proliferation. Thus, the most likely mechanism for anti-CD3 antibody-mediated triggering is induction of IL 2 receptors.     

抗人IgG Fc片段及Fab段单抗的鉴定及应用

本实验采用木瓜酶水解,SPA柱亲合层析等手段得到人IgGFc段及Fab段,以Sigma抗人IgGfFc段和抗人IgG Fab段单抗为标准品,鉴定了细胞库中抗人IgG系列的部分细胞株,得到特异性分泌抗人IgG Fc段和抗人IgG Fab段单抗的细胞各一株。 在上述实验基础上,用抗人IgG Fc及抗人IgG Fab单抗分别制备了Sepharose4B亲合层析柱,提纯了酶解人IgG Fc、Fab片段,经ELISA法鉴定,相互之间无交叉反应。同时用此方法制备了人抗HBe Fab片段,并将该片段进行了过氧化物酶标记,用来配制HBe ELISA诊断盒,证明其生物活性未受影响,而且消除了类风湿因子引起的HBe Ag假阳性现象。因抗HBe单抗来源困难,如采用HBe多抗制备ELISA试剂,本法将是提高质量的一个好方法。     

Requirements for the opsonic activity of human IgG directed to type 6 group A streptococci: net basic charge and intact Fc region

By two independent techniques for separating human opsonic IgG for group A type 6 streptococci into fast- and slow-migrating fractions, it was found that the opsonic activity was localized within the basic charge population. This charge dependence was found to be a characteristic of the IgG isolated from three individuals. When the fast- and slow-migrating IgG fractions were tested for their ability to bind to purified M6 protein, antibodies in both opsonic and nonopsonic populations exhibited binding activity, with the majority being located within the opsonic IgG in two of the three individuals; the third displayed greater binding in the nonopsonic population. The functional difference observed in the antibody populations to this M antigen may be a reflection of the net charge within the area of the antibody binding site, which suggests that the opsonic antibodies need to bind to acidic residues along the outer surface of the fibrillar M protein molecule. F(ab')2 fragments prepared from both human and rabbit type 6 opsonic IgG were still able to bind to the M6 molecule but were unable to mediate opsonization of type 6 streptococci. However, the F(ab')2 fragments had the capacity to enhance or amplify the opsonic activity of low concentrations of opsonic IgG molecules. The results suggest that the M protein molecule may function as an active inhibitor of phagocytosis and that F(ab')2 fragments from opsonic IgG have the capacity to neutralize the "active" determinants on the molecule, thus allowing lower concentrations of IgG with functional Fc receptors to mediate phagocytosis.     

Radiolocalisation of an anti-CEA monoclonal antibody (FO23C5) and its fragments in a colon carcinoma xenograft model

A new monoclonal antibody designated FO23C5 against a protein component of carcinoembryonic antigen (CEA) has been developed. A xenograft system of human colon cancer was used to compare the intact monoclonal IgG with its fragments (Fab')2 and Fab) and with an established anti-CEA antibody (MAb35) and the antibody AUA1 raised against the colon carcinoma cell line. We demonstrate that FO23C5 compares well with the existing anti-CEA antibody and with AUA1, and that F(ab')2 fragments perform best in achieving optimal tumour to normal tissue ratios compared with intact IgG and Fab fragment.     

Involvement of Fc gamma RI (CD64) in the mechanism of HIV-1 inhibition by polyclonal IgG purified from infected patients in cultured monocyte-derived macrophages

The aim of this study was to investigate the mechanism of HIV-1 neutralization using monocyte-derived macrophages (MDM) in comparison to PBMC as target cells. For this purpose, we analyzed neutralizing activities of different human polyclonal IgG samples purified from sera of HIV-1-infected individuals using a single cycle infection assay. We found an increase of the neutralizing titer when macrophages vs PBMC were used as target cells. Moreover, polyclonal IgG from HIV-1-infected patients that are not able to neutralize virus when PBMC are used as target cells strongly inhibit MDM infection. Similar results were obtained with neutralizing mAbs. To explore the participation of FcgammaRs in HIV-1 inhibition, F(ab')(2) and Fab of these Igs were produced. Results indicated that both F(ab')(2) and Fab are less effective to inhibit virus replication in MDM. Moreover, competition experiments with Fc fragments of IgG from healthy donors or with purified monoclonal anti-human FcgammaRs Ab strengthen the participation of the FcgammaRs, and in particular of FcgammaRI (CD64) in HIV-1 inhibition on MDM. Mechanisms by which HIV-specific IgG inhibit virus replication in cultured macrophages are proposed and the benefit of inducing such Abs by vaccination is discussed.     

Fluorine-18 labeling of monoclonal antibodies and fragments with preservation of immunoreactivity

A new method is reported for labeling proteins with the positron-emitting nuclide 18F. Initially, 4-[18F]-fluorobenzylamine was prepared in two steps from aqueous [18F]fluoride in high yield. The 18F acylation agent was formed by reaction of this product with disuccinimidyl suberate. Overall yields for the 4-[18F]fluorobenzylamine succinimidyl ester ([18F]SFBS), decay corrected to the end of cyclotron bombardment, were about 30% in a synthesis time of 60 min. After a 15-min reaction, 30-45% (decay corrected) of the [18F]SFBS could be coupled to intact antibodies and their F(ab')2 and Fab fragments. Coupling yields were dependent on protein concentration but not reaction time. HPLC purification of [18F]SFBS was necessary to obtain optimal coupling efficiency and immunoreactivity. The immunoreactivities of 18F-labeled F(ab')2 and Fab fragments of an antimyosin antibody were 89 +/- 5% and 75 +/- 9%, respectively. Biodistribution studies in normal mice demonstrated similar in vivo behavior of 18F-labeled antibody fragments and those labeled with 125I by using N-succinimidyl 3-[125I]iodobenzoate. These results indicate that this method may be useful for labeling monoclonal antibodies and other proteins and peptides with 18F.     

Single-step purification of pepsin-derived monoclonal antibody fragments from crude murine ascitic fluids by ceramic hydroxyapatite high-performance liquid chromatography

Ceramic hydroxyapatite (CHT) high-performance liquid chromatography (HPLC) is used to purify a variety of classes of monoclonal antibodies (mAbs) from crude murine ascites fluids. We report here that this method is also applicable for simple and efficient purification of many mAb fragments that are generated by pepsin treatment of crude ascites. F(ab')(2) fragments were quantitatively generated from IgG(1) mAbs in ascitic fluids by incubation with pepsin for 6 h at pH 3.9-4.1. Under the same conditions, pepsin also cleaved unwanted ascites components, such as albumin and transferrin to very low molecular weight polypeptides. The F(ab')(2) fragments, but not the low molecular weight products, selectively bound to and were eluted from the CHT column using a linear gradient of phosphate ion concentration over 15 min. The recovery of the F(ab')(2) fragments by CHT-HPLC was >90%. This method also allowed single-step purification of mAb fragments from distinct IgG subclasses (IgG(2a) and IgG(2b)) and IgM directly from crude digested ascitic samples. This CHT-HPLC method combined with direct pepsinolysis of murine ascites is a useful strategy for rapid purification and characterization of many types of mAb fragments.     

The interaction of monoclonal antibodies with MHC class I antigens on mouse spleen cells. I. Analysis of the mechanism of binding

We studied the mechanism of binding of radiolabeled, monoclonal anti-H-2 antibodies to mouse spleen cells to determine the number of H-2 antigen molecules per cell. Equilibrium and kinetic data were analyzed in detail according to theoretical models developed for different modes of antibody binding. The results of binding experiments from three monoclonal IgG antibodies (36-7-5, anti-Kk; 27-11-13, anti-DbDd; and 11-4-1, anti-Kk) and their F(ab')2 and F(ab') fragments show that for the IgG and F(ab')2 from all three antibodies, the monovalently and bivalently bound states of the antibody co-exist in rapid equilibrium with one another on the cell surface, with the bivalent state predominating. We show that the relative proportions of the monovalently and bivalently bound species can be estimated from dissociation kinetics experiments, and that once the mode of antibody binding has been established, the density of H-2 determinants on the cell surface can be estimated from equilibrium-binding data. We conclude that the average numbers of H-2K and H-2D molecules on B10.A spleen cells are 5 X 10(4) and 1.1 X 10(5) molecules/cell, respectively.     

Glycosylation in the Fc domain of IgG increases resistance to proteolytic cleavage by papain

IgG antibodies (Abs) and fragments of IgG Abs are becoming major biotherapeutics to treat an assortment of human diseases. Commonly prepared fragments of IgGs include Fc, Fab, and F(ab')2 fragments, all of which can be made using the sulfhydryl protease papain, although prolonged digestion times and/or excessive amounts of papain typically result in further cleavage of the Fc domain into smaller fragments. During our attempts to use papain to isolate Fc fragments from different IgG monoclonal Abs, it was observed that prior removal of Fc glycans resulted in a faster rate of papain-mediated degradation of the Fc domain. Subsequent time-course experiments comparing glycosylated and deglycosylated versions of IgG antibodies showed that the majority of molecules in a deglycosylated IgG sample were converted into Fab, Fc, and smaller Fc fragments in less than one hour, whereas the original glycosylated IgG required more than two hours to convert into a comparable amount of Fab and Fc fragments. Furthermore, whereas papain digestion converted almost all of a deglycosylated Fc fragment into smaller fragments of approximately 10 and approximately 12 kDa within 4 h, more than 40% of a glycosylated Fc fragment remained intact even after 24 h of digestion. These results indicate that the presence of CH(2) domain glycans in either IgGs or purified Fc fragments increases resistance to papain digestion. Increased sensitivity of non-glycosylated Fc domains to papain is consistent with the Fc domains lacking a defined structure, as exemplified by their inability to bind Fcgamma receptors, since misfolded proteins are often degraded by proteases because of increased accessibility of their proteolytic cleavage sites. Based on these observations it is possible to use papain sensitivity as a means of assessing proper Fc structure of IgG molecules.     

Monoclonal antibodies to glucose-6-phosphate dehydrogenase (G6PDH) form cyclic 1:1 complexes with G6PDH and act as regulatory subunits

A number of IgG monoclonal antibodies against L. mesenteroides glucose-6-phosphate dehydrogenase (G6PDH) have been prepared. Four of the antibodies form 1:1 enzyme-antibody complexes which are stabilized in the presence of glucose-6-phosphate (G6P) and have greatly reduced enzyme activity. In the absence of G6P, the 1:1 complexes convert gradually to a more active multimeric form. Reduction of the IgG inter-heavy chain disulfides partially relieves inhibition and removes the G6P requirement for stability. F(ab')2 fragments of one of the antibodies behave similarly to the intact IgG. Reduction of the disulfides in the G6PDH-F(ab')2 complex leads to complete recovery of activity. The activity of complexes of G6PDH with reduced antibodies or Fab with digoxin bound to the antibody or Fab sulfhydryl groups can be modulated with antibodies to digoxin. The anti-G6PDH antibodies bridge two identical epitopes of this two subunit enzyme and simulate the function of regulatory subunits in which anti-digoxin acts as an activator. The system can be used to provide a sensitive homogeneous immunoassay for digoxin.     

Further studies on the biologic properties of guinea pig IgG1 antibodies. II. In vivo activation of C3 by anti-glomerular basement membrane antibodies.

Normal rats were injected with guinea pig anti-rat glomerular basement membrane antibodies of the IgG1 or IgG2 class or with their F (ab') 2 fragments, in order to study which antibody site triggers the alternate complement pathway in vivo. Both IgG classes were able to induce a heavy proteinuria and led to C3 deposition in the glomeruli in a pattern similar to their own distribution along the glomerular basement membrane, as shown by the immunofluorescence technique. The Fab(ab')2 fragment of IgG2 did not produce C3 binding or proteinuria. The F(ab')2 fragment of IgG1 was difficult to obtain devoid of Fc determinants. A F(ab')2 fragment of IgG1 still bearing Fc determinants led to C3 binding and proteinuria, whereas the true F(ab')2 fragment of IgG1 had none of these effects in two out of three animals.     

Receptor aggregation is necessary for activation of the soluble insulin receptor kinase

Purified polyclonal human antibodies (B-8) against the receptor for insulin (anti-R IgG), and their F(ab')2 and Fab' fragments, were used to study a possible role of receptor aggregation in the process that couples insulin binding with the activation of the insulin receptor kinase. Anti-R IgG, F(ab')2, and Fab' fragments were shown to inhibit insulin binding to solubilized partially purified receptor preparations from rat liver. This suggests that the antibodies and fragments bind near or at the insulin-binding site. Only anti-R IgG and its bivalent F(ab')2 fragments were capable of stimulating the receptor kinase activity. Monovalent Fab' fragments were completely devoid of such activity. Cross-linking of anti-R Fab' with goat anti-human Fab' restored the capability of the Fab' fragments to activate the receptor kinase. These data strongly suggest that receptor cross-linking or aggregation constitutes a sufficient trigger to activate the insulin-receptor kinase and could, therefore, be an important step in the transmembrane signaling process. This step presumably precedes the activation of the receptor kinase and the resulting phosphorylation of its protein substrates.     

Anti-idiotypic antibodies to a polyomavirus monoclonal antibody recognize cell surface components of mouse kidney cells and prevent polyomavirus infection.

Anti-idiotypic antibodies have been successfully used to identify and isolate the receptor for several cell ligands. To prepare an immunologic probe for identification of the polyomavirus receptor on mouse kidney cells, polyclonal antisera against antipolyomavirus antibodies were prepared in rabbits. Fab fragments of the previously characterized monoclonal antibody E7, which neutralizes polyomavirus infection, were used for immunization (S. J. Marriott and R. A. Consigli, J. Virol. 56:365-372, 1985). Sera containing the greatest anti-idiotype activity were identified by enzyme-linked immunosorbent assay (ELISA) and purified by a series of affinity columns. The anti-idiotypic antibodies recognized the E7 idiotope in an ELISA, and anti-idiotype binding could be inhibited by preincubation of E7 monoclonal antibody with polyomavirus virions. When mixed with anti-idiotype immunoglobulin G (IgG), E7 was no longer capable of binding or immunoprecipitating polyomavirus virions or neutralizing polyomavirus infection. Direct immunofluorescence showed anti-idiotype IgG reactivity with a cell surface component of mouse kidney cells. Anti-idiotype F(ab')2 effectively competed with polyomavirus for binding to mouse kidney cells and displayed binding kinetics similar to those of polyomavirus. Virus infection of mouse kidney cells was blocked in a dose-dependent manner following treatment of the cells with anti-idiotype IgG. The anti-idiotype identified several proteins (95, 50, and 24 to 31 kilodaltons) in an immunoblot of mouse kidney cell membrane proteins but reacted predominantly with a single 50-kilodalton protein in a radioimmunoassay. The anti-idiotype failed to react with virus proteins in three assays, including ELISA, immunoprecipitation, and immunoblotting. The implications of this work for future identification and characterization of the polyomavirus receptor on mouse kidney cells are discussed.