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Biomedical subjects

M H Nahm

Publications and source records attributed to M H Nahm.

At least 19 recordsLinked to original sources
PubMed1995

Germinal center T cells exhibit properties of memory helper T cells.

Potential roles of germinal center (GC) T cells in immune memory were investigated by examining T cells in GCs lymph nodes (LNs) following immunization with myelin basic protein (MBP). MBP preferentially stimulates T cells expressing the V beta 8 TCR family in B10.PL mice. By staining lymph nodes with peanut agglutinin, GCs were first identified 8 days after immunization and could still be detected 50 days later. When T cells within GCs were examined at Day 8, the majority (> 90%) were CD45RB-negative and a large percentage (20%) had already proliferated several days before as determined by bromodeoxyuridine (BrdU) incorporation. When BrdU+ V beta 8+ cells were examined in various areas of LNs 3 and 8 weeks after immunization, BrdU+ V beta 8+ cells were approximately fourfold more frequent in GCs than other areas of LNs. When GCs were examined for IL-4 mRNA+ cells using an in situ hybridization method, only 0.01% of GC cells expressed IL-4 mRNA, corresponding to about 1% of GC T cells. Shortly following restimulation with MBP, the frequency of cells expressing IL-4 mRNA increased fivefold. Taken together, these findings indicate that GC T cells possess characteristics associated with memory T cells.

Animals↗
PubMed1995

The V-region repertoire of Haemophilus influenzae type b polysaccharide antibodies induced by immunization of infants.

Haemophilus influenzae type b (Hib) is a significant pathogen for young children, and three Hib vaccines (named PRP-OMPC, HbOC, and PRP-T) are currently available for young children. Extensive studies of anti-Hib polysaccharide (PS) antibodies (Abs) have shown that the V regions of Abs against the Hib PS comprise a VH gene in the VH3 gene family and a VL gene from various K kappa and V lambda subgroups. To study immunogenic properties of the three vaccines in young children, we determined the VL subgroups and avidities of anti-Hib-PS Abs induced by the three clinically available conjugate vaccines. Ab avidity was measured by determining the concentration of a Hib-PS oligomer that abrogates half of the binding of immunoglobulin G anti-Hib-PS Abs to microwells. The PRP-OMPC vaccine induced lower-avidity Abs than the prelicensure HbOC vaccine (P = 0.05). When we compared anti-Hib-PS Abs expressing V kappa Ia, V kappa II, and V lambda subgroups, a greater Ab response was induced by the prelicensure HbOC vaccine than other vaccines (P < 0.05). When anti-Hib-PS Abs with the V kappa III subgroup were compared, however, both PRP-T and prelicensure HbOC vaccines induced a comparable response, which in turn was greater than those induced by the PRP-OMPC or the postlicensure HbOC vaccine (P < 0.001). The VL repertoire of Abs induced with the prelicensure HbOC or PRP-T vaccine in young children is dominated (about 80%) by anti-Hib-PS Abs using subgroup V kappa II. However, anti-Hib-PS using V kappa II VL accounts for only about 40% of the total anti-Hib-PS Abs induced with the PRP-OMPC vaccine or the postlicensure HbOC. Our data suggest that immunogenic properties of Hib vaccines in young children vary depending on the vaccine preparations as well as the vaccine types.

Antibodies, Bacterial↗
PubMed1995

Functional capacities of clonal antibodies to Haemophilus influenzae type b polysaccharide.

Haemophilus influenzae type b (Hib) is an important pathogen for young children, and children can be protected with antibodies (Abs) to Hib polysaccharide (PS) capsule, a linear polymer of ribosyl ribitol phosphate. The structure of anti-Hib-PS Abs has been well characterized at the molecular level; about two-thirds of anti-Hib-PS Abs use a V kappa gene named A2, and the remaining anti-Hib-PS Abs use one of many other VL genes. In order to understand the structural basis for the variability in the function of these Abs, we prepared 18 clonally pure Abs from adults and studied their affinity, avidity, bactericidal potency in vitro, and ability to reduce bacteremia in newborn rats. Affinities and avidities were determined as the inverse of the concentrations of short (3 repeating units) and long (20 repeating units) ligands which could bind 50% of anti-Hib-PS Ab in solution, respectively. No significant correlations between the protection of newborn rats and affinity (r = 0.02) or avidity (r = 0.16) were observed. The amount of Ab required to kill 50% of bacteria in vitro decreased with avidity (r = -0.32), as expected. However, Abs with high affinity were unexpectedly found to have less bactericidal activity (r = 0.38). This suggests that avidity may be a better predictor of Ab function than affinity. Affinity and avidity results were negatively correlated (r = 0.76, P = 0.0022), and Abs that had A2 V kappa gene products had higher avidity (P < 0.05) and lower affinity (P = 0.06) than Abs that had other VL genes. A possible explanation of these observations is that the epitope for Abs with the A2 gene is within the Hib-PS chain itself, whereas the epitope for Abs with a non-A2 gene is the terminus of Hib-PS.

Animals↗
PubMed1995

Absence of lymph nodes in lymphotoxin-alpha(LT alpha)-deficient mice is due to abnormal organ development, not defective lymphocyte migration.

Mice homozygous for a targeted null mutation of lymphotoxin-alpha (LT alpha) are born without lymph nodes (LN) or Peyer's patches (PP) and with altered splenic architecture. To investigate the mechanism of failed LN organogenesis, we transferred bone marrow (BM) from Thy 1.2 LT alpha-deficient or Thy 1.2 wild type mice to lethally irradiated 8-12-week-old Thy 1.1 wild type recipients. Six to 10 weeks later, reconstitution of LN and spleen with Thy 1.2 cells was similar whether the BM was derived from LT alpha-deficient or wild type donors. In contrast, reconstitution of irradiated LT alpha-deficient mice with wild type BM did not induce the development of detectable LN, although reconstitution of the spleen occurred appropriately. The expression and regulation of the lymphocyte adhesion molecule L-selectin from the LT alpha-deficient mice appeared normal. These data indicate that LT alpha-dependent interactions must occur during development in order for LN genesis to take place; however, lymphocyte expression of LT alpha is not required for these cells to home to existing LN structures.

Animals↗
PubMed1994

Properties of human follicular dendritic cells purified with HJ2, a new monoclonal antibody.

Follicular dendritic cells (FDC) are found in germinal centers and may play an important role in germinal center (GC) B cell development. However, very little is known about FDC because it is very difficult to obtain a pure preparation of FDC. To aid in the purification of human FDC, we produced a mouse monoclonal antibody named HJ2 that immunohistochemically stains FDC in various lymphoid tissues. Isolated HJ2+ cells obtained by flow cytometric cell sorting exhibit morphological features associated with FDC and display numerous FDC-associated surface markers, but do not express T cell and B cell markers. Taken together, these findings indicate that HJ2 binds FDC and can be used to purify human FDC. Phenotypic studies of purified FDC revealed that FDC express large amounts of the complement (C)-regulatory proteins CD46, CD55, and CD59. By deactivating C, these proteins may safeguard FDC from membranolysis by surface-bound C containing immune complexes. To begin studies of FDC function, we cultured purified FDC and found they survive for at least 2 weeks in vitro. Furthermore, FDC were potent stimulators of allogeneic peripheral blood mononuclear cells and a T cell line in mixed lymphocyte cultures, and on a cell basis, FDC were more active than B cells. The purification of functionally active human FDC should facilitate studying the role of FDC in B cell development within GC.

Animals↗
PubMed1994

Comparison of nonhuman primate antibodies against Haemophilus influenzae type b polysaccharide with human antibodies in oligoclonality and in vivo protective potency.

Nonhuman primates are often used as a model for studying vaccines for humans. However, it is not always clear how closely the antibody responses in these species mimic human responses. Recent studies have characterized the human antibody response to Haemophilus influenzae type b (Hib) in great detail. In this study, we have compared the antibody response to Hib of humans with those of other primates. Studies of isoelectric points and V kappa subgroup usage show that, like humans, nonhuman primates produce oligoclonal antibodies. Also, monkey antibodies to the Hib polysaccharide are as protective as human antibodies in an in vivo model of Hib infection. Thus, we conclude that nonhuman primates produce antibodies to Hib polysaccharide that are structurally and functionally similar to human antibodies and are a good model for testing human vaccines.

Animals↗
PubMed1994

Endogenous serum antibodies that interfere with a common thyroid hormone uptake assay: characterization and prevalence.

We identified individuals whose serum contained a substance that produced falsely decreased thyroid hormone (T)-uptake values determined by the Emit (Syva) procedure. Investigation of this interference was prompted by identification of a patient with T-uptake values inconsistent with clinical assessment. IgG depletion and supplementation studies with this patient's serum suggested that the interference was due to endogenous antibodies with specificity for the thyroxine-glucose-6-phosphate dehydrogenase conjugate in the Emit T-uptake assay. The prevalence of the interference was examined by prospectively comparing routine Emit T-uptake values of 1710 patients' samples to T-uptake values obtained by another method. Discrepant samples were also assayed by a radioactive binding triiodothyronine-uptake assay. We identified eight samples that had falsely decreased T-uptake values by Emit, for an overall prevalence of 0.46%. Among 45 consecutive patients with a T-uptake value < 20%, five patients, or 11%, were falsely decreased by Emit and three of these were clearly due to an interfering IgG. We suggest that samples with abnormally low T-uptake values determined by the Emit method be confirmed by an alternative method.

Adult↗
PubMed1993

T cells within germinal centers are specific for the immunizing antigen.

A normal antibody response to T cell-dependent Ag requires physical contact between Ag-specific B and T cells. Because such Ag-specific cells are rare in vivo, we sought to identify an in vivo site where they physically contact each other. We examined the Ag specificity of T cells in germinal centers (GC) in lymph nodes, where it is known that Ag-specific B cells proliferate and mature. We investigated the Ag specificity of GC T cells in situ by examining two characteristics: 1) expression of certain V alpha and V beta TCR families; and 2) incorporation of bromodeoxyuridine into T cell DNA after exposure to Ag as an index of Ag-induced proliferation. When GC were induced in mice with cytochrome c and myelin basic protein, the GC T cells were found to preferentially express V alpha 11 and V beta 8 TCR families, which are, respectively, the dominant TCR families in these two responses. Furthermore, GC T cells have proliferated upon exposure to the Ag that induced GC formation. Taken together, these results demonstrate that GC must recruit and retain Ag-specific T cells, thus implicating the GC as an in vivo site where Ag-specific T and B cells interact.

Animals↗
PubMed1993

Clonal characterization of the human IgG antibody repertoire to Haemophilus influenzae type b polysaccharide. V. In vivo expression of individual antibody clones is dependent on Ig CH haplotypes and the categories of antigen.

Antibodies (Ab) to the polysaccharide capsule of Haemophilus influenzae type b (Hib-PS) provide protection against Haemophilus influenzae type b disease in children, and Hib-PS vaccines with different immunologic properties are widely used clinically. The repertoire of human anti-Hib-PS Ab induced by these vaccines is relatively restricted and can be divided into two types by the structure of the light chain V region. Ab using A2-V kappa II gene product, which account for the majority of anti-Hib-PS Ab response in most patients, show little somatic mutations. In contrast, non-Ab using A2-V kappa II gene product use VL genes from the V kappa I, V kappa II, V kappa III, V kappa IV, and V lambda subgroups, are variably expressed among patients, and contain somatic mutations. To further study the expression of these two types of anti-Hib-PS Ab, we have produced KB13, a mAb specific for V kappa II subgroup, and used mAb specific for various other VL subgroups to develop immunoassays specific for anti-Hib-PS Ab of each VL subgroup. When Ig allotypes were studied for the effect on the Ab repertoire, A2-V kappa II (A2) Ab were found to be expressed less in patients expressing fb or zag CH haplotypes (p < 0.05). When the T cell-independent Hib-PS carbohydrate vaccine was compared to two T cell-dependent Hib-PS protein conjugate vaccines for their effect on Ab repertoire, Ab using V kappa III VL were found to be more often elicited with the conjugate vaccines than with the Hib-PS carbohydrate vaccine (p < 0.01). Thus, individual members of the anti-Hib-PS Ab repertoire differ not only in their V region structure but also in the control of their expression.

Adult↗
PubMed1993

Cytokine expression by germinal center cells.

Germinal centers (GC) primarily consist of B cells along with a small number of T cells (5 to 10%) and follicular dendritic cells (FDC) (< or = 1%). Although extensive Ag-driven B cell proliferation and maturation occurs in GC, very little is known about the role of cytokines in the development of GC B cells. Therefore, to identify cytokines present in the GC microenvironment that may influence B cell development, we systematically examined cytokine gene expression by GC cells. GC T cells (CD57+/CD4+), GC B cells (CD77+), and FDC (HJ2+) were isolated from human tonsils by cell sorting using a flow cytometer. Freshly isolated GC cells were examined for mRNA expression for IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, TNF-alpha, and IFN-gamma using reverse transcription polymerase chain reaction. Freshly isolated GC T cells consistently expressed IL-4 mRNA (11 of 12 tonsils), whereas CD57- Th cells (mostly non-GC Th cells) were often negative for IL-4 mRNA. When the other nine cytokine mRNA were studied, freshly isolated CD57+ Th cells occasionally expressed mRNA for IL-10, TNF-alpha, and IFN-gamma. CD57- Th cells were occasionally positive for IL-1 beta, IL-10, IFN-gamma, and TNF-alpha, and negative for IL-2 and IL-6. Freshly isolated GC B cells as well as FDC failed to express detectable quantities of mRNA for all 10 cytokines that were studied. Thus, IL-4 is the only cytokine out of 10 that is consistently expressed in GC and may be important for the development of B cells in GC. After stimulation of CD57+ Th cells with PWM, production of IL-4 mRNA was dramatically reduced, whereas CD57- Th cell production of IL-4 was greatly augmented. This finding indicates that GC T cells may differ from other Th cells in cytokine gene expression and that results of cytokine production obtained after in vitro stimulation do not always reflect in vivo results.

Antigens, CD↗
PubMed1993

Comparison of naturally acquired and vaccine-induced antibodies to Haemophilus influenzae type b capsular polysaccharide.

The objective of this study was to assess qualitative differences in the types of Haemophilus influenzae type B (Hib) capsular polysaccharide (polyribosylribitol phosphate [PRP]) antibodies induced in children 15 to 27 months of age by (i) natural exposure, (ii) PRP vaccine, and by (iii) PRP-diphtheria toxoid conjugate vaccine, (iv) PRP-group B Neisseria meningitidis outer membrane vesicle conjugate vaccine, and (v) Haemophilus type B oligosaccharide conjugate vaccine (HbOC). The highest levels of total Hib-PRP antibody measured by radioimmunoassay and immunoglobulin G (IgG) measured by enzyme-linked immunosorbent assay were seen after HbOC immunization. IgG1 Hib-PRP antibodies predominated in all groups, and there were no differences between the groups in the proportion of IgG and IgA Hib-PRP antibodies. However, the proportions of IgM differed significantly by group. The highest proportions of IgM occurred in naturally acquired antibody and after PRP vaccine, and the lowest proportion occurred after HbOC vaccine. IgG light-chain V kappa type alpha PRP antibody was present in all groups, and the level correlated with the total IgG Hib-PRP antibody level. Therefore, HbOC induced the highest concentrations of V kappa II type alpha PRP antibody, and the naturally acquired antibody group had the lowest levels. IgG light-chain V kappa III antibody levels were also highest in the HbOC group, but there was no correlation between V kappa III antibody levels and total amount of IgG Hib-PRP antibody. These data demonstrate qualitative differences in the antibody repertoires induced by natural exposure, the Hib-PRP vaccine, and each of the different Hib conjugate vaccines. We doubt that there are major differences in the protection afforded by these different antibody repertoires, because these differences do not appear to correlate with differences in protective efficacy in older children.

Antibodies, Bacterial↗
PubMed1993

Endogenous antibodies that interfere with thyroxine fluorescence polarization assay but not with radioimmunoassay or EMIT.

We have identified an individual whose thyroxine (T4) concentration was undetectable with Abbott's fluorescence polarization immunoassay (FPIA) but within the reference range by radioimmunoassay or EMIT (Syva). The patient's thyrotropin, triiodothyronine, and T-uptake values were within the normal range. The T4 concentration measured by FPIA increased to normal when the immunoglobulin fraction was selectively removed from the serum. When the patient's immunoglobulin fraction was added to normal serum, the T4 content of the normal serum measured by FPIA became falsely low. The patient's antibody interfered with the T4 FPIA by binding to the fluorescein-T4 conjugate. The T-uptake was less affected by the patient's serum because of the low affinity of the patient's antibody to fluorescein-T4 (K = 3.5 x 10(8) L/mol). The patient's immunoglobulin bound preferentially to fluorescein-T4, in comparison with binding to fluorescein or T4 alone. We conclude that the patient's immunoglobulin bound to an epitope unique to the fluorescein-conjugated T4.

Adult↗
PubMed1992

Functional properties of human germinal center B cells.

Germinal centers (GCs) are histologically defined areas where B cells undergo extensive proliferation and maturation, or die of apoptosis. GC B cells isolated from human tonsils can be phenotypically identified by expression of peanut agglutinin (PNA)-binding sites and can be further divided into subpopulations based on their expression of CD77. To assess the functional potential of GC B cells, we studied CD77+ PNA+ B cells isolated from tonsils by examining their differentiation status and their ability to proliferate in vitro to various cytokines and costimulants. We found that CD77+ GC B cells are less differentiated than CD77- GC B cells; GC B cells less frequently express cytoplasmic IgG and IgM, and spontaneously secrete less Ig compared to CD77- GC B cells. To identify conditions capable of inducing GC B cell proliferation, we examined IL-4, IL-2, IFN-gamma, low molecular weight BCGF (LMW-BCGF), and an MLR supernatant along with costimulants such as anti-IgM antibody, Staphylococcus aureus Cowan I (SAC), PMA, and pokeweed mitogen (PWM). While non-GC B cells proliferate strongly in response to these stimuli, GC B cells did not proliferate. However, CD77+ as well as CD77- GC B cells mounted a rapid and strong proliferative response upon stimulation with IL-4, but only in the presence of anti-CD40 antibody. Moreover, although nine additional cytokines were examined, only IL-4 was capable of supporting CD77+ GC B cell proliferation in the presence of anti-CD40 antibody. When cells were stimulated with IL-4 and anti-CD40 antibody, we also found that IFN-gamma consistently decreased the proliferative response of CD77+ GC B cells without affecting the response of non-GC B cells. Taken together, these data indicate that GC B cells have characteristic growth requirements and that IL-4 may be important for GC B cell growth in vivo.

Antigens, CD↗
PubMed1992

The evolution of immune memory and germinal centers.

Antibody responses in homoiothermic and poikilothermic vertebrates are significantly different in their heterogeneity and affinity range, and in the speed of the secondary response following repeated antigenic stimulation. This article presents the hypothesis that the evolutionary development of unique lymphoid structures, the germinal centers, in combination with the development of a distinct B-cell lineage, is a determining feature of these differences.

Animals↗
PubMed1992

Characterization of the human IgG antibody VL repertoire to Haemophilus influenzae type b polysaccharide.

The human antibody response to the capsular polysaccharide of Haemophilus influenzae type b (Hib) is a good model for examining human V region repertoires. While the VL repertoire of human antibodies to Hib polysaccharide is relatively simple and dominated by the product of a germline V kappa II gene named A2, at least five other VL genes can be expressed by some individuals. These include at least two V kappa I products and at least one V kappa III, one V kappa IV, and one V lambda product. The epitope recognized by a monoclonal anti-idiotype antibody is on the A2 V kappa II product.

Adult↗
PubMed1992

The human antibody V region repertoire to the type B capsular polysaccharide of Haemophilus influenzae.

The V region repertoire of the human antibody response to the type b capsular polysaccharide of Haemophilus influenzae (Hib-PS) is being defined at the molecular level using antibodies purified from serum of immunized adults. The VH of this response is restricted to the VHIII subgroup while the VL can be divided into two categories. The most common VL, expressed in > 90% of adults and usually constituting the majority of a subjects anti-Hib-PS antibody response, is restricted to the product of a single V kappa II gene known as A2 that probably lacks somatic mutations. The product of the A2 gene is invariably joined to one of several J kappa products by an inserted arginine at the V kappa-J kappa junction. In contrast to the restricted nature of the dominant VL clonotype, the second category of VL constitutes a heterogeneous group of at least seven different VL gene products that often contain somatic mutations and generally exhibit crossreactivity with a related polysaccharide from E. coli. Elucidation of anti-Hib-PS V regions at the molecular level will permit examination of structure-function relationships among these clinically important antibodies and should make the V region repertoire to Hib-PS a useful model for studying human V gene responses.

Amino Acid Sequence↗