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Systemic lupus erythematosus (SLE) is a severe chronic inflammatory autoimmune connective tissue disease. Despite major efforts, SLE remains a poorly understood disease with unpredictable course, unknown etiology and complex pathogenesis. Apoptosis combined with deficiency in clearing apoptotic cells is an important etiopathogenic event in SLE, which could contribute to the increased load of potential autoantigen(s); however, the lack of disease-specific protein signatures deciphering SLE and the underlying biological processes is striking and represents a key limitation. In this retrospective pilot study, we explored the immune system as a specific sensor for disease, in order to advance our understanding of SLE. To this end, we determined multiplexed serum protein expression profiles of crude SLE serum samples, using antibody microarrays. The aim was to identify differential immunoprofiles, or snapshots of the immune response modulated by the disease, reflecting apoptosis, a key process in the etiology of SLE and disease activity. The results showed that multiplexed panels of SLE-associated serum biomarkers could be decoded, in particular reflecting disease activity, but potentially the apoptosis process as well. While the former biomarkers could display a potential future use for prognosis, the latter biomarkers might help shed further light on the apoptosis process taking place in SLE. [ABSTRACT FROM AUTHOR]

Priming of human mature B cells in vitro with staphylococcal enterotoxin A (SEA) prior to transplantation of the B cells into severe combined immunodeficiency (SCID) mice, together with human T-helper cells, resulted in higher and more uniform concentrations of serum IgG in the mice. This indicated that a large number of B cells had become activated, which was supported by the finding that SEA priming resulted in production of immunoglobulin displaying a more normal κ/λ ratio than was obtained in the absence of SEA priming. However, IgM concentrations were not affected by SEA priming. Immunization of mice, transplanted with SEA-primed B cells, with both primary and secondary antigens resulted in a high specific IgG response to both types of antigen. The elevated levels of specific antibodies were not merely the consequence of an unspecific stimulation of B cells caused by SEA, as the ratio of specific antibody to total IgG was much higher in animals receiving SEA-primed B cells. Thus, a co-operative effect on immunoglobulin production of stimulating B cells via surface immunoglobulin and help delivered by SEA-activated T-helper cells was indicated. A specific antigen-dependent IgM response to a secondary antigen was observed as well, but was, in contrast to the IgG response, not influenced by SEA priming of B cells. No IgM antibodies with reactivities to the primary antigens were detected in the SCID sera at any time-point after immunization. The results thus indicate that SEA might replace T-cell epitopes in antigens and efficiently recruit an abundance of T-cell help to B cells, resulting in enhanced production of specific IgG antibodies. [ABSTRACT FROM AUTHOR]

Adoptive transfer of human lymphoid cells into immunodeficient (SCID) mice lacking the ability to functionally rearrange T- and B-cell receptor genes constitutes a unique model to study and manipulate human immunocytes. We have investigated this model for the purpose of generating an antigen-specific primary humoral immune response. Peripheral blood lymphocytes (PBL) derived from blood donors were used to repopulate SCID mice, which subsequently were immunized with different B-cell epitopes coupled to either tetanus toxoid (TT), or to a promiscuous helper epitope of TT, or by incorporating the antigens into a liposome construct. By recruiting the necessary T-cell help found in the T-cell memory compartment against TT, primary immune responses were obtained against the hapten dinitrophenyl (DNP), the V3 loop peptide derived from glycoprotein (gp120) (HIV-1), the melanoma-associated GD2 ganglioside and ovine submaxillary mucin. The primary immune response against the GD2 ganglioside was induced by incapsulating TT into GD2-containing liposomes. These liposome constructs also allowed us to induce a high human IgG serotitre (3000-4000) against this normally not very immunogenic ganglioside. [ABSTRACT FROM AUTHOR]

Severe combined immunodeficient (SCID) mice, lacking mature T and B cells and virtually devoid of endogenous serum immunoglobulins, spontaneously produce large amounts of human immunoglobulin after transplantation with human peripheral blood lymphocytes (PBL). Moreover, after immunization with antigen an active immune response resulting in a production of specific antibodies can be induced. Here we report that human T cells must be co-transplanted with B cells into the SCID mice for immunoglobulin production to occur. Resting human B cells could be activated to immunoglobulin production in the absence of human monocytes and a specific antibody response to tetanus toxoid (TT) could be induced, suggesting that the human B cells could present antigen to T cells in the SCID environment. Production of human immunoglobulins, as well as specific antibodies, was obtained only if CD4+ T cells of the memory phenotype, i.e. expressing CD45RO, were present. No human immunoglobulin, either of IgM or of IgG isotype, was found in SCID sera if mice were co-transplanted with human B cells and CD45RA expressing CD4+ T cells. However, FACS analysis revealed that the transplanted CD45RA+ cells became activated and differentiated towards CD45RO+ cells within 1-2 weeks. These cells also gained the lymphokine gene expression pattern associated with CD45RO+ cells, as demonstrated by polymerase chain reaction (PCR) analysis, and could support immunoglobulin production in SCID mice transplanted with fresh B cells. In fact, after differentiation of CD4+ CD45RA+ T cells towards expression of CD45RO, either in vivo in the SCID mouse or in vitro, these cells could interact with and activate human B cells to immunoglobulin production. [ABSTRACT FROM AUTHOR]

Human-mouse hybridoma cell lines producing human monoclonal antibodies against the cardiac glycoside digoxin were established after in vitro immunization or direct immortalization of human peripheral blood lymphocytes with digoxin. Three antibodies, designated MO6, LH92 and LH114, displayed different patterns of fine specificity against digoxin and several digoxin analogues, as elucidated by inhibition ELISA. All three monoclonal antibodies had μ heavy chains, two of them (MO6 and LH114) had κ light chains and one (LH92) λ light chains. DNA encoding the variable regions of both heavy and light chains of the three antibodies were amplified from cDNA using the polymerase chain reaction (PCR). The nucleotide sequences of the amplified DNA were determined after subcloning of PCR fragments in MI3 vectors. The deduced amino acid sequences revealed considerable sequence differences in the complementarity determining regions between the three antibodies. [ABSTRACT FROM AUTHOR]

Peripheral blood lymphocytes from healthy, HIV sero-negative blood donors have been in vitro immunized using penv9, a recombinant fragment of the envelope of HIV-1. This primary in vitro immunization followed by Epstein-Barr virus (EBV) transformation and somatic cell fusion subsequently gave rise to several specific anti-penv9 monoclonal antibodies (MO28, MO30 and MO43) of μ isotype. The hybridomas have been kept in culture for over 6 months and the antibody productivity for MO30 was measured to 18 μg × (24 hr × 106 cells)-1. The fine specificity of the antibodies was mapped by a peptide inhibition enzyme immunoassay, using overlapping synthetic pentadeca peptides covering the whole penv9. These human monoclonal antibodies exhibited a similar epitope specificity directed against a non-sequential determinant, including the amino acids 632–646, 677–681 and 687–691. This specificity is very rarely found in immune sera from sero-positive patients and presently not reported in human monoclonal antibodies derived from in vivo immunized individuals, indicating that different antibody specificities can be obtained by the in vitro immunization technology. These human monoclonal antibodies did not neutralize HIV. The results presented here demonstrate the feasability of generating human monoclonal antibodies against HIV by primary in vitro immunizations, thereby avoiding the use of lymphocytes derived from infected patients when human monoclonal antibodies for therapeutic purposes are to be produced. [ABSTRACT FROM AUTHOR]

The selective cytotoxicity of the lysosomotropic methyl esters of leucine or its lysosomal condensation product leucyl-leucine has been used to investigate the effect of cytolytic cells on the clonal outgrowth, cellular proliferation and antibody secretion of Epstein-Barr virus (EBV)- transformed hdnan B cells. Large granular lymphocytes (LGL), monocytes, and a subset of T cells (CD8/CD11+) were permanently eliminated by the ester treatment. These lysosome-rich cells severely inhibit the clonal outgrowth of EBV-infected B cells, as determined by Poisson distribution calculations. Furthermore, leucyl-leucine methyl ester-treated and EBV-infected lymphocytes showed a significant increase in proliferative capability as well as immunoglobulin (Ig) production (three to I I times) compared to nonstreated but similarly infected lymphocytes. Since the effect of leucyl-leucine methyl ester treatment was also detectable in low-density (100 B cells/well) cultures, the suppression was unlikely to be exerted by EBV-specific T-cell clones, but pointed rather to the natural killer (NK) cells as effectors. [ABSTRACT FROM AUTHOR]

The requirements for a primary, antigen-specific in vitro immunization of human peripheral lymphocytes using haemocyanin, a T-cell dependent antigen, have been studied. In order to obtain a specific response in vitro the peripheral lymphocytes had to be separated into B, T, accessory (A) and dendritic (D) cells. These cells were activated and reconstituted to give a population with a B:T ratio of 1:2. If the induction was supported by MHC-restricted, radioresistant T cells, this cell population could then be antigen-specifically activated using haemocyanin. The immunization had also to be supported by cytokines, such as B-cell growth and differentiation factors, interleukin-2 and γ-interferon. A 5-day in vitro immunization using 2 μg haemocyanin/ml resulted in 200–300 cells secreting anti-haemocyanin-specific antibodies per 106 B cells. [ABSTRACT FROM AUTHOR]

Highly purified, small, resting murine Ig-positive spleen cells could be induced to proliferate by concanavalin A (Con A) if the activation was supported by allogeneic helper factors (AHF). Con A or AHF alone was unable to induce cell proliferation as determined by incorporation of (methyl-3H] thymidine. However, if AHF produced by a mixed lymphocyte culture was present together with Con A, the B lymphocytes were strongly activated exhibiting a stimulation index of approximately 2000 as compared to Con A alone. The dose-response maximum was obtained after a 4-day culture using 25% of a supernatant from a mixed lymphocyte culture as a source of AHF. The kinetics of the AHF- supported B-cell activation was also investigated. The activation could be completely inhibited by the addition of 100 mM methyl α-v-mannopyranoside at the initiation of the culture, but, if added 14-18 hr later, the cells have had the necessary induction period and a proliferation could be recorded. Aft F had to be present at the initiation of the cultures, otherwise the response immediately diminished, whereas Con A could be added up to 6 hr after initiation without affecting the proliferative response. [ABSTRACT FROM AUTHOR]

Human MoAbs of IgM class were developed against three regions of the HIV-1 envelope. Uninfected donor lymphocytes were immunized in vitro with recombinant protein pB1. Four out of five antibodies were directed to different parts of the V3 region, which contains a major neutralizing site. Two out of these antibodies were directed to more than one amino acid sequence, indicating reactivity to discontinuous sites. Two of the human MoAbs inhibited viral spread between cells in tissue culture, interpreted as reactivities to conserved amino acid sequences exposed during viral maturation. No MoAb neutralized virus, which may be explained by the relatively low avidity of the antibodies. One MoAb was directed to a region containing amino acids participating in CD4 binding. This technique appears to allow formation of antibodies with fine specificities other than those obtained in infected hosts. [ABSTRACT FROM AUTHOR]

Human monoclonal antibodies specific for human cytomegalovirus (CMV) antigens have been established using peripheral blood lymphocytes from a seropositive donor. Immortalization of antigen-specific B cells was achieved by Epstein-Barr virus transformation followed by somatic cell fusion of antigen-specific lymphoblastoid cells. Four clones producing high-affinity antibodies (0.2-7 × 109 M-1) specific for the viral matrix protein pp65 have been further characterized with respect to epitope specificity of secreted antibodies. The studied antigen represents a major protein produced by in vitro-cultivated virus, and is important in the scrodiagnosis of CMV infection. The human monoclonal antibodies recognized different epitopes. some of which proved to be overlapping. The fine specificity of these antibodies was evaluated using synthetic peptides covering the sequence of pp65. The antibody MO58 recognized a linear epitope (residues 283-288) whereas antibody MO53 recognized a discontinuous epitope involving residues 208-216 and 280-285. Despite the close proximity of these epitopes, the antibodies did not compete with each other for the same binding site on intact antigen. [ABSTRACT FROM AUTHOR]

Antigen-specific lymphocyte hybridomas have been produced by a simple in vitro immunization procedure. Non-immune spleen cells have been activated in vitro, and the B-cell blasts have subsequently been used for somatic cell hybridization. Hybrids secreting monoclonal antibodies to human myoglobin, pig insulin, and benzo(a)pyrene were obtained with splenocytes that had been immunized 5 days in culture before the hybridization. The frequency of specific hybridomas was the same as or higher than that of hybridomas produced from in vivo-immunized cells, showing the versatility of the procedure when making monoclonal antibodies to haptens and proteins with a conserved structure or to small amounts of antigen. Monoclonal antibodies of both IgG and IgM isotypes could be isolated. [ABSTRACT FROM AUTHOR]