IgM Antibody-secreting Cells Research Articles

Outpatient studies of the safety and immunogenicity of an auxotrophic Escherichia coli K-12-Shigella flexneri 2a hybrid vaccine candidate, EcSf2a-2

A phase II study was conducted in 244 volunteers at Fort Ord, CA, to determine the safety and immunogenicity of EcSf2a-2, a live, oral Shigella vaccine constructed by transfer of genes from Shigella flexneri to Escherichia coli K-12. In this placebo-controlled study, four doses of vaccine ranging from 2.3 to 9.0 x 10(8) colony-forming units were given on days 0, 3, 14 and 17. Vaccine shedding occurred from 1 to 3 days after each dose. The vaccine was well tolerated at every dose tested. Significant levels of IgA, IgG or IgM antibody-secreting cells (ASC) recognizing S. flexneri 2a lipopolysaccharide (LPS) were found in 94% of a volunteer subset tested 7 days after the first dose of EcSf2a-2. Seven days after the third dose, ASC were detected less often (57%), and were mainly IgA. Significant rises in serum antibody to LPS were detected in 37% of vaccine recipients.

Localization of inhaled trimellitic anhydride to lung with a respiratory lymph node antibody secreting cell response

Male Sprague-Dawley rats were exposed to trimellitic anhydride (TMA) by inhalation (500 μg/m 3), 4 hours a day, for 1 to 10 days. TMA was localized to lung cells by immunoelectron microscopy. Enzyme-linked immunosorbent assay (ELISA) and Western blot analysis were used in an attempt to localize TMA to lung lavage proteins. The lung-associated lymph node (LALN) B-lymphocyte response was measured by quantitation of immunoglobulin (Ig)G, IgA, and IgM antibody secreting cells specific for TMA rat serum albumin (TMRSA) by use of the enzyme-linked immunospot assay (ELISPOT) method. The IgG, IgM, and IgA antibody response to TMRSA in serum and lavage fluid was quantitated by ELISA. Lung injury was assessed by the number of external lung hemorrhagic foci and lung weight. Immunoelectron microscopy localized TMA to alveolar and bronchial cells on all exposure days. ELISA detected trace amounts of TMA haptenized lavage proteins that could not be detected by Western blot analysis. A marked increase occurred in lung injury from day 7 to 10. The LALN IgG, IgA, and IgM antibody secreting cell response to TMRSA paralleled measures of lung injury. IgG, IgM, and IgA serum and lavage antibody to TMRSA were correlated with lung injury measures. Lavage and serum IgG antibody levels had the highest correlation with lung injury.

In vitro induction of a Haemophilus influenzae type b polysaccharide-specific antibody response in human peripheral blood lymphocytes of individuals recently vaccinated with an oligosaccharide-protein conjugate.

In this paper an in vitro culture system for the induction of an antibody response to the Haemophilus influenzae type b polysaccharide (PRP) is described. Anti-PRP IgM and IgG antibody-secreting cells (ASC) and anti-diphtheria toxoid (DT) IgG ASC were detected in cultures of blood B and T cells derived from donors 4 to 6 wk after immunization with Haemophilus influenzae type b oligosaccharide-mutant diphtheria toxin (CRM197) conjugate (HbOC) and required in vitro restimulation with HbOC. When lymphocytes from HbOC-vaccinated donors were stimulated with PRP, anti-PRP IgM and IgG ASC could be detected in 50% offGe cases. Lymphocytes from PRP-vaccinated donors or non-vaccinated donors consistently failed to generate anti-PRP antibodies after in vitro stimulation with HbOC. Optimal in vitro responses were observed at concentrations of 0.06 to 0.6 micrograms/ml of Ag. At higher doses of Ag (6 micrograms/ml) anti-PRP and anti-DT antibody responses were suppressed. The in vitro generation of anti-PRP and anti-DT ASC, as detected by a spot-forming cell assay was shown to be T cell dependent, Ag dependent, and Ag specific. This culture system provides a model for the study of human B cell activation and immunoregulation by polysaccharide-protein conjugates and polysaccharides.

Anti-Borrelia burgdorferi antibody response over the course of Lyme neuroborreliosis

Characteristic findings on examination of cerebrospinal fluid (CSF) in Lyme neuroborreliosis include mononuclear pleocytosis, oligoclonal immunoglobulin G (IgG) bands, and evidence for local production of specific antibodies. We utilized an immunospot assay to detect cells secreting anti-Borrelia burgdorferi antibodies of different isotypes over the course of disease. Such cells were detected in CSF from 13 consecutive patients with neuroborreliosis examined before treatment. IgG antibody-secreting cells were present in high numbers (mean, 32 cells per 10(4) CSF cells), whereas IgA and IgM antibody-secreting cells were found less frequently and at lower numbers (mean, 5 and 6 cells per 10(4) CSF cells, respectively). Clinical improvement after penicillin treatment was paralleled by a rapid decline of antibody-secreting cells in CSF, but they were still detected, although at lower numbers, in 5 of 10 patients examined more than 6 months after treatment. This specific B-cell response persisted despite clinical improvement. Whether it reflects persistence of antigen is unsettled.

Early diagnosis of tuberculous meningitis by detection of anti-BCG secreting cells in cerebrospinal fluid

A nitrocellulose immunospot assay to enumerate cells secreting anti-BCG antibodies was evaluated in the diagnosis of tuberculous meningitis. Among 25 Chinese patients with this disease diagnosed on clinical and cerebrospinal fluid (CSF) criteria, 24 had anti-BCG IgG antibody secreting cells in CSF, at a median value of 56 per 10(4) CSF cells. Among 6 patients examined within one week, 5 had antibody secreting cells in CSF, and all 19 patients examined 2-8 weeks after onset had such cells in CSF. Blood examined from 5 patients contained anti-BCG IgG or IgM antibody secreting cells, but usually at much lower numbers than in corresponding CSF, indicating that the specific antibody response is largely confined to CSF. Anti-BCG antibodies measured in parallel by ELISA were slightly raised in CSF in only 1 of the 6 patients examined within a week, whereas a good correspondence was seen between numbers of antibody secreting cells and antibody concentrations from the second week onwards. Detection of anti-BCG IgG antibody secreting cells has higher sensitivity and specificity than ELISA measurement of anti-BCG IgG antibodies, and represents a new, useful method for the early diagnosis of tuberculous meningitis.

Production of human antibodies to bee venom phospholipase A2 in vitro.

Phospholipase A2 (PLA) is the major antigen of bee venom. Whereas individuals frequently stung by bees, such as bee keepers, show high levels of IgG4 anti-PLA antibodies in serum, most patients sensitive to bee venom possess increased IgE anti-PLA. We have established a culture system by which anti-PLA antibodies can be induced in vitro. Peripheral blood mononuclear cells were stimulated in a first step with PLA and/or pokeweed mitogen (PWM). After 3 days of culture the cells were washed and further incubated with fresh medium. Anti-PLA antibodies were estimated by an enzyme-linked immunosorbent assay (ELISA) and anti-PLA antibody secreting cells were counted by means of an ELISA plaque assay. Cells from bee keepers, but not those from normal donors, produced anti-PLA IgG in vitro. The isotype pattern of anti-PLA antibodies produced in vitro was identical to that found in serum of the respective donor. Anti-PLA IgM antibody-secreting cells developed at low frequencies in both bee keepers and normal donors whereas PLA-specific IgE could not be detected. The formation of anti-PLA IgG was suppressed by high amounts of antigen, while the IgM response was not affected. We conclude that PWM-induced antibodies reflect the in vivo situation of the donors and that anti-PLA IgG and IgM are regulated by different pathways and mechanisms.

Enhanced antibody responses in mice by combined administration of interferon with rabies vaccine.

Exogenous interferon administered to mice at the time of, and 6 hours after the first dose of 3 daily vaccinations accelerated and enhanced the IgM and IgG antibody responses to rabies virus. The effect of interferon was not evident when the interferon was administered later in the vaccination schedules and was abrogated by prior administration of anti-interferon antibody to the mice. The number of IgM antibody secreting cells in the spleen was significantly greater in mice treated with interferon than in controls.

Frequency analysis of functional immunoglobulin C- and V-gene expression by mitogen-reactive B cells in germfree mice fed chemically defined ultra-filtered "antigen-free" diet.

The frequencies of lipopolysaccharide (LPS)-reactive B cells and their antibody specificity repertoire have been determined in the spleen and bone marrow (BM) of conventional (CV) and "antigen-free" C3H/HeCr mice of various ages. The antigen-free mice were germfree (GF)-raised and were fed an ultrafiltered solution of chemically defined (CD) low m.w. nutrients, and were thus devoid of exogenous antigenic stimulation. Spleen and BM cells were grown in a limiting dilution culture system that allows the growth and development of every newly formed LPS-reactive B cell into a clone of IgM-secreting cells which are capable of switching to other immunoglobulin (Ig) heavy chain isotypes (C-gene expression). The secretion of IgM and IgG1 was determined in the protein A plaque assay, whereas specific IgM antibody-secreting cells (V-gene expression) were detected in plaque assays specific for various heterologous erythrocytes and sheep red blood cells (SRBC) coupled with a number of different haptens. The absolute frequency of LPS-reactive B cells and their capacity to switch to IgG1-secretion was not significantly different in 8- to 12-wk-old and 52-wk-old GF-CD mice and their age-matched CV controls. Moreover, no differences were observed in the frequencies of antigen-specific B cells within the pool of LPS reactive B cells. These frequencies ranged from 1 in 20 to 1 in 50 for NIP4-SRBC and NNP2-SRBC, from 1 in 100 to 1 in 150 for NIP0.4-SRBC, from 1 in 50 to 1 in 100 for TNP30-SRBC, and from 1 in 1000 to 1 in 2000 for SRBC and horse red blood cells. Within the limitations of having determined the switching capacity of IgM to IgG1 only and having assessed only a minor fraction of the total B cell antibody-specificity repertoire, the data indicate that young and old GF-CD mice, although devoid of exogenous antigenic and/or mitogenic stimulation, generate B cells with a similar switching capacity and a similar IgM antibody specificity repertoire as CV mice.

Isotypes and specificities of immunoglobulins produced by germ-free mice fed chemically defined ultrafiltered "antigen-free" diet.

The regulation of the "spontaneously" occurring ("background") Ig synthesis of mice has been studied by determining the numbers of IgM-, IgG- and IgA-secreting cells and a part of the IgM antibody-specificity repertoire in spleen, bone marrow (BM) and mesenteric lymph nodes (MLN) of conventional and "antigen-free" mice. These antigen-free mice were germ-free raised and fed an ultrafiltered solution of chemically defined low molecular weight nutrients, and thus devoid of exogenous antigenic stimulation. The secretion of IgM, IgG and IgA by spleen, BM and MLN cells was assessed in the protein A plaque assay, while specific IgM antibody-secreting cells were detected by plaque assays specific for differently haptenated sheep red blood cells. In general, antigen-free and conventional mice were found to have roughly equal numbers of IgM-secreting cells in spleen and BM. The number of IgG-secreting cells in the spleen of antigen-free mice was the same as in the spleen of conventional mice, but in the BM their number was 3-5-fold decreased. About one half of the antigen-free mice did not have MLN, and in the half which did, 5 times less IgM- and more than 100-fold less IgG-secreting cells were found as compared with conventional mice. The number of IgA-secreting cells in antigen-free mice was drastically decreased in all three organs tested. The antibody-specificity repertoire of the "background" IgM-secreting cells in the spleen and BM of the antigen-free and conventional mice was much alike. This indicates that in antigen-free mice the available antibody repertoires are established independently of exogenous and/or mitogenic stimulation.

Frequency analysis of functional immunoglobulin C and V gene expression in murine B cells at various ages.

The frequencies of lipopolysaccharide- (LPS) reactive B cells and their antibody specificity repertoire have been determined in the spleen and bone marrow (BM) of mice at different ages. A limiting dilution culture system was employed that allows the growth and development of every LPS-reactive B cell into a clone an IgM-secreting cells that are capable of switching to other Ig heavy chain isotypes (C gene expression). The secretion of IgM and IgG1 was assessed in the protein A plaque assay, whereas specific IgM antibody-secreting cells (V gene expression) were detected with the use of plaque assays specific for various heterologous erythrocytes and sheep red blood cells (SRBC) coupled with a number of different haptens. The frequencies of LPS-reactive B cells in the spleen and BM of C3H/Tif, C57BL/Ka, BALB/c, and CBA/Rij mice appeared to be similar in 6- to 12- and 100-wk-old animals, as was the switch frequency to IgG1 secretion in three strains tested. Moreover, no age-related changes were observed in the frequencies of antigen-specific B cells within the pool of LPS-reactive B cells in the spleen and BM of C57BL/Ka mice. The frequencies ranged from 1 in 10 to 1 in 20 for NIP4- and NNP2-SRBC, from 1 in 50 to 1 in 100 for TNP30-SRBC, and from 1 in 1000 to 1 in 4000 for SRBC, HRBC, and GRBC. The specificity repertoire of the "spontaneously" occurring ("background") IgM-secreting cells in the spleen and BM, on the other hand, did differ between young and old C57BL/Ka mice. During aging the frequencies of the tested specificities decreased in the spleen but increased in the BM. Our data indicate that in unintentionally immunized mice the clonal selection of B lineage cells by antigen takes place at the level of the mature, antigen-reactive B cell.

Differences in the immunological activities of antibody-secreting cell precursors in mouse spleen selected on the basis of antigen-binding capacity.

Mouse spleen cells transfer a retarded and lower than normal IgG antibody response after removal of cells with a high antigen-binding capacity. The population enriched in these latter cells transfers a response equal to or, more often, higher than the response of the unfractionated controls and does not appear to have originated from previous priming of the mice. These cells are present in athymic nude mice and appear to be more susceptible to inactivation by an anti-Ig antiserum and complement than precursors of IgM antibody-secreting cells.

DNP-Lys-Ficoll: A T-Independent Antigen Which Elicits Both IgM and IgG Anti-DNP Antibody-Secreting Cells

The 2,4-dinitrophenyl-lysyl derivative of Ficoll (DNP-Lys-Ficoll) was prepared and examined for immunogenicity. This antigen elicited large numbers of DNP-specific plaque-forming cells (PFC) of the IgM and IgG2 class in the spleens of C57BL/6 mice. Similar responses were observed in congenitally athymic (nu/nu) mice and in their littermates indicating that DNP-Lys-Ficoll is a T-independent antigen. The responses of nu/nu mice included a large number of IgG2 DNP-specific PFC, indicating that IgG responses can be initiated in the absence of mature thymus-dependent (T) lymphocytes. Cell transfer studies confirmed the T independence of the response and indicated that priming with DNP-Lys-Ficoll induces only a very meager degree of memory. Because they can be obtained in large quantities and in relatively pure form, DNP-Lys-Ficoll and other hapten conjugates of Ficoll should prove most valuable in the delineation of the mode of activation of precursors of antibody-secreting cells by T-independent antigens.

Use of erythrocytes sensitized with purified enterotoxin from Vibrio cholerae for the assay of antibody and antibody-forming cells.

A method was developed for the sensitization of ovine erythrocytes with a purified enterotoxin from Vibrio cholerae. Sensitized cells were used for the titration of serum antibody by passive hemagglutination and in a hemolytic plaque assay for both IgM and IgG antibody-secreting cells. Inhibition experiments with various antigens of V. cholerae indicated that the toxin, whether unheated or heat-inactivated, significantly reduced the expected antitoxic plaque-forming cell response, whereas a lipopolysaccharide-rich extract from homologous vibiros was not inhibitory.

The use of erythrocytes sensitized with flagellar antigens from Salmonella for the assay of antibody and antibody-forming cells

A method is described for sensitizing sheep erythrocytes with chromic chloride and polymerized flagellin from two different Salmonellae. Sensitized cells have been used for the titration of serum antibody by passive hemagglutination and a hemolytic plaque assay for both IgM and IgG antibody-secreting cells. Results obtained with the methods described agree with previously used assay procedures, and offer the advantage of convinience and rapidity.

Enhanced hemolytic antibody response following thermal injury.

This paper presents evidence of a significantly enhanced humoral immune response to sheep red cells following thermal injury, as assayed by direct hemolytic antibody plaque formation. An increase in the number of 19 S IgM antibody-secreting cells from the spleen was observed in rats up to 16 days after a 30-percent total body surface area, third-degree, scald burn. Since this enhancement was present in animals immunized up to 20 days postburn, the possible role of adjuvants liberated by the burn injury is considered. The role of the interaction between thymus-processed and marrow-derived lymphocytes in this enchancement is also discussed.