Porphyromonas gingivalis Fimbriae Use β2 Integrin (CD11/CD18) on Mouse Peritoneal Macrophages as a Cellular Receptor, and the CD18 β Chain Plays a Functional Role in Fimbrial Signaling

In this study, we demonstrate that Porphyromonas gingivalis fimbriae use molecules of β₂ integrin (CD11/CD18) on mouse peritoneal macrophages as cellular receptors and also show that the β chain (CD18) may play a functional role in signalling for the fimbria-induced expression of interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNF-α) genes in the cells. Using a binding assay with ¹²⁵I-labeled fimbriae, we observed that fimbrial binding to the macrophages was inhibited by treatment with CD11a, CD11b, CD11c, or CD18 antibody but not by that with CD29 antibody. Western blot assays showed that the fimbriae bound to molecules of β₂ integrin (CD11/CD18) on the macrophages. Furthermore, Northern blot analyses showed that the fimbria-induced expression of IL-1β and TNF-α genes in the cells was inhibited strongly by CD18 antibody treatment and slightly by CD11a, CD11b, or CD11c antibody treatment. Interestingly, intracellular adhesion molecule 1 (ICAM-1), a ligand of CD11/CD18, inhibited fimbrial binding to the cells in a dose-dependent manner. In addition, ICAM-1 clearly inhibited the fimbria-induced expression of IL-1β and TNF-α genes in the cells. However, such inhibitory action was not observed with laminin treatment. These results suggest the importance of β₂ integrin (CD11/CD18) as a cellular receptor of P. gingivalis fimbriae in the initiation stage of the pathogenic mechanism of the organism in periodontal disease.     

Modulation of Fc receptors on human peripheral blood lymphocytes by antisera against β2 microglobulin, Ia or immunoglobulin

The association between Fc receptors and other surface molecules was examined by EA-rosette (EAR) inhibition experiments. Twenty to 30% EAR were detected in suspensions of peripheral blood lymphocytes from normal individuals. Anti-β₂ microglobulin (βMi) sera fully suppressed EAR whereas anti-Ig, anti-Ia sera and heat aggregated IgG inhibited only 50–60% EAR. Thus almost half of the detected EAR were apparently not surface Ig positive B cells. Rabbit and monkey anti-βMi sera suppressed EAR effectively whereas rat and chicken antisera, despite strong βMi binding capacity, inhibited EAR poorly. The latter result was ascribed on the basis of immunofluorescent analysis to inadequate capping of βMi. Incubation of PBL with whole antisera suppressed EAR to a similar degree at either 0° or 37°, whereas F(ab′)₂ fragments were inhibitory only at 37°. Taken together the results suggest that Fc receptors can be inhibited by antibodies with specificity against any cell surface antigen. The blocking mechanism may be due to steric hindrance by the Fc part of antibody molecules and/or F(ab′)₂ fragment mediated co-capping.     

Affinity-purified cardiolipin-binding antibodies show heterogeneity in their binding to oxidized low-density lipoprotein

Antiphospholipid antibodies in autoimmune sera have been shown to react with a complex of phospholipids (cardiolipin) and a plasma phospholipid-binding protein, β₂-glycoprotein I (apolipoprotein H). The binding of these antibodies was inhibited by oxidized low-density lipoprotein (LDL) in sera from patients with systemic lupus erythematosus (SLE), suggesting cross-reactivity between antiphospholipid antibodies and antibodies binding to oxidized LDL. We purified antiphospholipid antibodies by cardiolipin-polyacrylamide column from seven SLE sera and studied the reactivity of eluted fractions with cardiolipin–β₂-glycoprotein I complex and oxidized LDL (malondialdehyde-conjugated LDL) in solid-phase enzyme immunoassay. In four sera the binding of IgG antibodies to cardiolipin–β₂-glycoprotein I complex and to oxidized LDL appeared in the same fractions, whereas in three sera reactivities against cardiolipin and oxidized LDL were observed, at least in part, in separate fractions. The binding to solid-phase cardiolipin was dependent on the presence of exogenous β₂-glycoprotein I in all fractions. Our findings show that antiphospholipid antibodies are heterogeneous in their binding to oxidized LDL, indicating that these two antibodies may have different subspecificities. Some eluted fractions reacted only with oxidized LDL, and did not show binding to cardiolipin–β₂-glycoprotein I complex, suggesting that the lipid part in the antigenic complex might be responsible for the cross-reactivity of these antibodies. Accordingly, the biological functions of antibodies against phospholipid–β₂-glycoprotein I complex and antibodies against oxidized LDL may also be different.     

Expression of human–Torpedo hybrid acetylcholine receptor (AChR) for analysing the subunit specificity of antibodies in sera from patients with myasthenia gravis (MG)

The nicotinic AChR, a pentamer composed of α₂βγ(or ε)δ subunits, is the autoantigen in the human autoimmune disease MG. Anti-AChR antibodies in MG sera bind mainly to conformational epitopes, therefore determination of their specificities requires the use of intact AChR. Indirect antibody competition studies have suggested that most MG antibodies are inhibited from binding to AChR by MoAb to the main immunogenic region (MIR) on the α-subunits. More recently, based on the knowledge that MG antibodies show little detectable cross-reaction with Torpedo AChR, we have shown, using mouse–Torpedo hybrid AChR, that most MG antibodies that detectably cross-react with the mouse AChR bind to the α-subunit. To analyse the whole anti-AChR antibody repertoire in MG sera, we expressed on stably transfected fibroblasts a novel human α+Torpedoβγδ AChR and compared the antibody titres against human, Torpedo, and the hybrid AChR. Direct information was provided for the subunit specificity of several MoAbs and sera from 50 MG patients. On average, at least 48% of the anti-AChR antibodies in the sera were directed against the α-subunit. Interestingly, the anti-α-subunit antibodies predominated in low titre (0.6–7.4 nm) but not in high titre (10–386 nm) sera, where they comprised on average 68% versus 23% of the antibodies, respectively. Finally, the directly determined anti-α-subunit antibodies and the anti-MIR antibodies defined by antibody competition were significantly correlated, thus suggesting that at least a significant fraction of the anti-MIR antibodies in MG sera bind to the α-subunit.     

Human Reagins to Grass Pollens and Moulds: Their Purification and Physico-chemical Characterization

Chromatography of reaginic sera (to grass pollens and to moulds) on DEAE-cellulose leads to the concentration of reagins in three well separated fractions. They emerge (a) together with siderophilin, (b) with the early albumin-containing fractions, and (c) in association with strongly adsorbed proteins that require a comparatively high ionic strength buffer for their elution. The proportion of reagins in each of these fractions varies with different sera and with small alterations in the experimental procedure. All three reaginic fractions contain small amounts of γ globulins (referred to as R globulins) of mobilities slightly less than that of siderophilin; (c) contains much α_2M globulin and there are traces of α_2M in (a) and (b). Yet the bulk of the γ globulins are shown to be free from reaginic activity, and the same is true of the α_2M and the β_2M globulins, of siderophilin and of albumin. The purer the reaginic fractions are the smaller is the portion of the reagins that can be precipitated with the γ globulins by half saturation with ammonium sulphate. In contrast to the bulk of the γ globulins, R globulins and reagins appear to associate readily with other serum proteins, particularly with α_2M globulins. Fractionation with sodium sulphate produces three fractions of similar potency although they have little in common; one consists of the bulk of the γ globulins (0–15 per cent w/v), the most active fraction of the remaining globulins (15–18 per cent) and the third fraction (supernatant from the 18 per cent precipitate) of albumin containing some α globulins, but only a trace of γ globulin.

Ultracentrifugation studies on three main reaginic DEAE-fractions show that the bulk of the reagins are not macroglobulins although misinterpretation can arise from complex formation with α_2M globulin.

High agglutination titres (Stavitsky's method, Stavitsky and Arquilla, 1958) for pollen proteins are associated only with the unretarded non-reaginic γ globulins of post-treatment sera (which contain the blocking antibodies) although traces of agglutinating antibodies can be demonstrated in many fractions, including the reaginic fractions derived from the sera of untreated hay fever subjects.

     

Preferential expression of IgG1 antibodies specific for the L2C leukaemia IgM idiotypic determinants in tumour-protected strain 2 guinea-pigs

Immunization of strain 2 guinea-pigs with 10⁷ syngeneic Ia⁺ L2C leukaemia cells in adjuvant leads to L2C tumour protection. After subsequent challenges with L2C tumour cells, the sera of twelve out of seventy protected guinea-pigs had detectable L2C reactivity as determined by a [¹²⁵I]-protein A binding assay. The antibodies bound equally well to Ia⁺ and Ia^- L2C tumour cells, but did not bind to L1 and L10 guinea-pig hepatocarcinoma cells or normal guinea-pig B and T lymphocytes. The binding was blocked appreciably by F(ab')₂ reagents specific for the L2C IgM idiotype but not by those specific for Ia or B.1 alloantigens or β₂ microglobulin. These results lead to provisional identification of anti-idiotype among the syngeneic antibody population. After ion-exchange chromatography, the L2C reactivity in eleven of the twelve immune sera analysed was exclusively in the IgG1 fraction. The syngeneic anti-idiotypic antibodies precipitated only IgM molecules from the NP-40 extracts of L2C tumour cells and were dissociated from the L2C leukaemia cells more readily than the xenogeneic anti-idiotypic antibodies at pH 6.5 and 6.0. These results suggest that the L2C IgM idiotype may function as a tumour-associated antigen or is near the antigenic complex recognized by the low affinity L2C antibodies. The preferential expression of IgG1 antibodies suggests that humoral immunity effects a minimum level of protection because this isotype, in the guinea-pig, has a restricted capacity to mediate tumour rejection by secondary immune mechanisms.     

Analysis of Human Serum Immunoglobulin G against O-Acetyl-Positive and O-Acetyl-Negative Serogroup W135 Meningococcal Capsular Polysaccharide

The capsular polysaccharide of Neisseria meningitidis serogroup W135 is expressed in both O-acetyl-positive (OA⁺) and O-acetyl-negative (OA⁻) forms. This study investigates the impact of OA status (OA⁺ versus OA⁻) on serological measurements of anti-W135 immunoglobulin G (IgG) antibodies in immunized adults. W135-specific serum antibody assignments were made for 28 postimmunization sera from adults by enzyme-linked immunosorbent assay using the meningococcal standard reference serum CDC1992. The established IgG concentration in micrograms per milliliter ([IgG]μg/ml) for CDC1992 against OA⁺ antigen (16.2 μg/ml) was used as a reference to assign a concentration of 10.13 μg/ml IgG against OA⁻ antigen by cross-standardization. Overall, the IgG assignments for these sera were higher against OA⁺ antigen (geometric mean concentration [GMC] = 7.16 μg/ml) than against OA⁻ antigen (GMC = 2.84 μg/ml). However, seven sera showed higher specific [IgG]μg/ml values against the OA⁺ antigen than against the OA⁻ antigen. These sera were also distinguished by the inability of fluid-phase OA⁻ antigen to compete for antibody binding to OA⁺ solid-phase antigen. Although there was no overall difference in functional activity measured by complement-mediated serum bactericidal assay (SBA) against OA⁺ and OA⁻ target bacteria (geometric mean titers of 9,642 and 9,045, respectively), three serum specimens showed a large difference in SBA antibody titers against OA⁺ versus OA⁻ W135 target bacteria, which may reflect different epitope specificities for these sera. Our data indicate that, for some sera, the agreement in anti-OA⁺ versus anti-OA⁻ W135 IgG assignments is serum specific and does not reflect the functional (killing) activity in vitro.     

Effect of Antibody on Heterologous and Homologous Cells in Tissue Culture

In order to compare antibody against heterologous and homologous tissue in the same serum, rabbits were immunized with rat kidney and complete adjuvant. The resulting sera showed antibody against both rat and rabbit kidney. Cultures of rat kidney cells were killed by exposure to these sera. A concentration of 0.3 per cent γ globulin (ammonium sulphate fraction) was adequate to kill cultures in the absence of complement, but smaller concentrations were effective when guinea-pig complement was added. The cell surfaces were shown to have taken up antibody by the fluorescent antibody technique. Cytoplasmic staining could only be shown in cells which had previously been injured by freezing and thawing or by fixation. Rabbit kidney cells in culture were unaffected when exposed to whole rabbit anti-rat serum, but were killed and their cell membranes stained on exposure to γ globulin derived by (NH₄)₂SO₄-fractionation from such serum and having the same complement-fixation titre as the parent serum. At least 0.6 per cent γ globulin had to be added to kill rabbit cell cultures. It was found that normal rabbit serum had a partial protective effect against this antibody. Fractionation of sera by gradient centrifugation or chromatography on DEAE-cellulose showed that while antibody against heterologous tissue was found both in the 7S γ globulin and macroglobulin fractions, antibody against homologous tissue was confined to the latter. It is considered that the findings do not support a concept of an in vivo pathogenic role for circulating antibody.     

Separation of mouse homocytotropic antibodies by biological screening

An attempt was made to separate mouse γ₁ antibody from mouse reaginic antibody by injecting mouse antiserum containing both antibodies into normal mice and then at 6 and 24 hours bleeding the animals and testing their sera for passive cutaneous anaphylaxis (PCA) activity. This was termed biological screening. The 2-hour homologous PCA activity was used as a measurement of mouse γ₁ and the rat PCA activity of the mouse antisera was used as a measurement of mouse reaginic antibody. These experiments showed that in vivo screening of mouse antisera containing both mouse and rat PCA activity results in removal of the rat PCA activity of these sera whereas the mouse PCA activity remains practically unchanged. It is concluded that a separation of the mouse serum PCA activity due to γ₁ antibody from that due to mouse reaginic antibody can be achieved by biological screening. Screening experiments using very early mouse antisera collected 8 days after a single antigenic stimulation resulted in the simultaneous disappearance of both homologous and heterologous PCA activity. Heating of these very early antisera resulted in complete inactivation of heterologous PCA activity and almost complete inactivation of homologous PCA activity. Absorption of these same antisera with rabbit anti-mouse γ₁ caused no change in homologous or heterologous PCA activity. It is suggested that the PCA activity of the very early antisera is due almost entirely to mouse reaginic antibody.     

Immunological Identification of the Pathological Cold Auto-Antibodies of Acquired Haemolytic Anaemia as β2M-Globulin

Cold antibodies were separated from the sera of six patients suffering from the cold-haemagglutinin syndrome and from one patient with acquired haemolytic anaemia secondary to lymphosarcoma by dissociation of the specific antigen-antibody complexes. The eluted antibodies were studied (a) by immuno-electrophoresis along with the parent sera against horse anti-human serum and (b) by double diffusion in agar gel along with electrophoretically separated `γ<sub>1</sub> globulin'† against anti-19S `γ-globulin' rabbit sera.

The protein forming the cold antibody was localized in the β₂-M position on immuno-electrophoresis in each instance. It was found by double diffusion in agar gel to be immunologically identical with the protein forming the abnormal `γ₁-globulin' electrophoretic peak in the parent serum.

The results of these experiments indicate that the cold antibodies derived from patients with the cold-antibody type of acquired haemolytic anaemia are macro-molecular globulins.

     

Effect of α2-macroglobulin on the lymphocyte response

We studied the effect of α₂-macroglobulin (α₂MG) on the proliferative response of lymphocytes to several different lectins. This response to concanavalin A (Con A) and phytohaemagglutinin (PHA) was markedly reduced by addition of α₂MG to the reaction mixture, while that to lipopolysaccharide (LPS) was reduced to a lesser extent. Preincubation of lymphocytes with α₂MG had little effect on the response of the lymphocytes to lectin, when the protein was washed out before addition of the lectin. The response of the lymphocytes to lectin was reduced when α₂MG was added within 12 hr after the addition of the lectin, while the reduction was not apparent when the protein was added 24 hr after the lectin.

¹²⁵I-α₂MG prepared by the chloramine T method which maintained a 30% trypsin-protein esterase (TPE) activity did not bind to the cells, while the labelled protein prepared using the lactoperoxidase method which had full TPE activity, did bind to the cells. The binding of α₂MG with Con A was also demonstrated. Thus, the inhibitory effect of the protein on the proliferative response of lymphocytes to lectin may be due to binding of the protein to lymphocytes and consequently blocks the binding of the lectin to the cells or due to interaction of the protein with the lectin so as to diminish the number of the lectin molecules acting on the cells.

     

Specific antisera to C1r. Quantitation of C1r in normal and pathological human sera

Monospecific neutralizing and precipitating antisera to C1r, a subunit of the first complement component, were obtained. These antisera neutralized C1r activity in purified preparations and in macromolecular C1 and did not react with C1q or C1s. They formed one line of precipitation in the β-globulin region with normal human serum, C1^hu and C1r at various stages of purification. Using anti-C1r antiserum and a radial immunodiffusion technique, the concentration of C1r was determined in normal, SLE and RA sera. It was 101 μg/ml in normal sera and lower in sera of active SLE patients (69·7 μg/ml). No significant variations from normal were found in sera of SLE patients in periods of remission or in RA patients.     

Detection of Human Toxoplasma-Specific Immunoglobulins A, M, and G with a Recombinant Toxoplasma gondii Rop2 Protein

The Toxoplasma gondii rhoptry protein Rop2 was expressed in Escherichia coli as a fusion protein containing 44 kDa of the 55-kDa mature Rop2, supplied with six histidyl residues at the N-terminal end (Rop2_196–561). Humoral response during Toxoplasma infection of humans was analyzed by immunoglobulin G (IgG), IgA, and IgM enzyme-linked immunosorbent assay with Rop2_196–561 as the antigen substrate. The analyzed sera were divided according to T. gondii-specific serological tests (IgG, IgA, or IgM indirect immunofluorescence and IgA or IgM immunosorbent agglutination assay) as group A (IgG⁺ IgA⁻ IgM⁻; n = 35), group B (IgG⁺ IgA⁺ IgM⁺; n = 21), group C (IgG⁺ IgA⁺ IgM⁻; n = 5), and group D (IgG⁺ IgA⁻ IgM⁺; n = 16). Twenty-six T. gondii-seronegative sera from individuals with other infections were also included (group E). Anti-Rop2 IgG antibodies were detected in 82.8% of group A sera and in 97.6% of the sera with acute-phase marker immunoglobulins (groups B, C, and D). The percentage of IgA antibody reactivity against Rop2_196–561 was 17.1% in group A, 50% in group D, and 80.8% in groups B and C. The percentage of IgM antibody reactivity was 0% in groups A and C and 62% in groups B and D. Sera from group E failed to show IgA, IgM, or IgG antibody reactivity. Since T. gondii Rop2 elicits a strong humoral response from an early stage of infection, it is suggested that recombinant Rop2_196–561 would be suitable for use in diagnostic systems, in combination with other T. gondii antigens, to detect specific IgG, IgA, and IgM antibodies.     

The Significance of Multiple Antibody Components in Serum of Immunized Rabbits

The electrophoretic patterns of six sera from rabbits immunized by two or more courses of intravenous injections of killed pneumococci type III showed multiple peaks in the γ-globulin region. Such sera contained large amounts of antibody (up to 85 per cent of the total γ globulin) against the capsular polysaccharide. One serum contained a cryoglobulin, which contained almost as great a proportion of specific antibody as did the remaining γ globulin.

The electrophoretic patterns and antibody contents were similar in the water-soluble and water-insoluble fractions of γ globulin.

The sedimentation constant and diffusion coefficient of a water-soluble fraction of γ globulin, containing 85 per cent specific antibody, were measured. The values, at 0.4 per cent protein concentration, were S_20.w = 6.97×10^-13 and D_20.w = 4.16×10^-7 cm.² sec.^-1, corresponding to molecular weight 159,000.

The antibody-containing globulin from one serum was separated by zone electrophoresis into three fractions with different electrophoretic mobilities. These contained 53–71 per cent of antibody precipitable by type III pneumococcus capsular polysaccharide. Only doubtfully significant differences were found in respect of amino-acid composition, hexose and hexosamine contents, or antigenic characteristics.

A method was devised for detecting small amounts of antibody against capsular polysaccharide by means of red cells sensitized with culture filtrates of capsulated pneumococci.

The antibody was also fractionated by chromatography on anion-exchange cellulose, and numerous fractions with antibody activity were obtained. It was shown by labelling the γ globulin with ¹³¹I that similar fractionation occurred both in the presence and absence of other serum components. All the chromatographic fractions of γ globulin were found to contain approximately similar proportions of antibody. By electrophoresis in starch gel the fractions were found to differ from one another and to be heterogeneous.

The implications are discussed of the finding that antibody against type III pneumococcus capsular polysaccharide can occur over the entire range of γ-globulin molecules.

     

Passive sensitization of skin and lung by guinea-pig immunoglobulins

Guinea-pig γ₁- and γ₂-globulins have been purified by preparative electrophoresis followed by chromatography. No γ₁-globulin was detectable in purified γ₂-globulin, but purified γ₁-globulin always contained fast γ₂-globulin. Normal guinea-pig serum contained much less γ₁-globulin than immune serum. Antisera prepared against normal guinea-pig serum did not contain useful amounts of antibody specific for γ₁-globulin.

Guinea-pig lung tissue was sensitized by very low concentrations of guinea-pig γ₁-globulin (of the order of 6×10^-10 molar) but γ₂-globulin antibodies were almost inactive. No evidence was found that the trace of activity in γ₂-globulin was not due to very slight contamination with γ₁-globulin antibodies.

The finding that γ₁-globulin antibodies are far more potent than γ₂-globulin antibodies in sensitizing skin has been confirmed, but several lines of evidence suggest that γ₂-globulin antibodies may also have weak activity. Thus quantitative passive cutaneous anaphylaxis (PCA) tests showed that whenever the γ₂-globulin fraction contained antibody it appeared far more potent relative to γ₁-globulin than when the same proteins were tested on lung tissue. The PCA activity of moderate amounts of purified γ₂-globulin antibodies disappeared faster than the skin sensitization produced by small amounts of γ₁-globulin antibodies, and the γ₂-globulin preparations did not contain enough γ₁-globulin impurity to account for their PCA activity. No inhibition of skin responses was observed with the largest doses of antigen tested.

The most plausible explanation of these results is that, under the conditions of our experiments, γ₂-globulin antibody had weak PCA activity. Objections to this hypothesis are discussed. The PCA activity of γ₂-globulin antibody probably involves a mechanism different from that of the sensitization produced by the highly potent γ₁-globulin antibody.

     

Regulation of Fungal Infection by a Combination of Amphotericin B and Peptide 2, a Lactoferrin Peptide That Activates Neutrophils

To establish a novel strategy for the control of fungal infection, we examined the antifungal and neutrophil-activating activities of antimicrobial peptides. The duration of survival of 50% of mice injected with a lethal dose of Candida albicans (5 × 10⁸ cells) or Aspergillus fumigatus (1 × 10⁸ cells) was prolonged 3 to 5 days by the injection of 10 μg of peptide 2 (a lactoferrin peptide) and 10 μg of α-defensin 1 for five consecutive days and was prolonged 5 to 13 days by the injection of 0.1 μg of granulocyte-monocyte colony-stimulating factor (GM-CSF) and 0.5 μg of amphotericin B. When mice received a combined injection of peptide 2 (10 μg/day) with amphotericin B (0.5 μg/day) for 5 days after the lethal fungal inoculation, their survival was greatly prolonged and some mice continued to live for more than 5 weeks, although the effective doses of peptide 2 for 50 and 100% suppression of Candida or Aspergillus colony formation were about one-third and one-half those of amphotericin B, respectively. In vitro, peptide 2 as well as GM-CSF increased the Candida and Aspergillus killing activities of neutrophils, but peptides such as α-defensin 1, β-defensin 2, and histatin 5 did not upregulate the killing activity. GM-CSF together with peptide 2 but not other peptides enhanced the production of superoxide (O₂⁻) by neutrophils. The upregulation by peptide 2 was confirmed by the activation of the O₂⁻-generating pathway, i.e., activation of large-molecule guanine binding protein, phosphatidyl-inositol 3-kinase, protein kinase C, and p47^phox as well as p67^phox. In conclusion, different from natural antimicrobial peptides, peptide 2 has a potent neutrophil-activating effect which could be advantageous for its clinical use in combination with antifungal drugs.     

Full-Length Plasmodium falciparum Circumsporozoite Protein Administered with Long-Chain Poly(I·C) or the Toll-Like Receptor 4 Agonist Glucopyranosyl Lipid Adjuvant-Stable Emulsion Elicits Potent Antibody and CD4+ T Cell Immunity and Protection in Mice

The Plasmodium falciparum circumsporozoite (CS) protein (CSP) is a major vaccine target for preventing malaria infection. Thus, developing strong and durable antibody and T cell responses against CSP with novel immunogens and potent adjuvants may improve upon the success of current approaches. Here, we compare four distinct full-length P. falciparum CS proteins expressed in Escherichia coli or Pichia pastoris for their ability to induce immunity and protection in mice when administered with long-chain poly(I·C) [poly(I·C)LC] as an adjuvant. CS proteins expressed in E. coli induced high-titer antibody responses against the NANP repeat region and potent CSP-specific CD4⁺ T cell responses. Moreover, E. coli-derived CS proteins in combination with poly(I·C)LC induced potent multifunctional (interleukin 2-positive [IL-2⁺], tumor necrosis factor alpha-positive [TNF-α⁺], gamma interferon-positive [IFN-γ⁺]) CD4⁺ effector T cell responses in blood, in spleen, and particularly in liver. Using transgenic Plasmodium berghei expressing the repeat region of P. falciparum CSP [Pb-CS(Pf)], we showed that there was a 1- to 4-log decrease in malaria rRNA in the liver following a high-dose challenge and ∼50% sterilizing protection with a low-dose challenge compared to control levels. Protection was directly correlated with high-level antibody titers but not CD4⁺ T cell responses. Finally, protective immunity was also induced using the Toll-like receptor 4 agonist glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE) as the adjuvant, which also correlated with high antibody titers yet CD4⁺ T cell immunity that was significantly less potent than that with poly(I·C)LC. Overall, these data suggest that full-length CS proteins and poly(I·C)LC or GLA-SE offer a simple vaccine formulation to be used alone or in combination with other vaccines for preventing malaria infection.     

A kinetic and quantitive analysis of antibody and protein synthesis during the primary response to keyhole limpet haemocyanin

Rabbits were injected with alum-precipitated keyhole limpet haemocyanin. On various days after immunization popliteal lymph node cells were prepared for synthesis of antibody as well as non-antibody proteins by incorporation of ¹⁴C-amino acids. Antibody and proteins were characterized by gel filtration, radio-immunoelectrophoretic and zone electrophoretic analyses of the culture media. Some `natural' antibody appeared to be synthesized by lymph node cells from uninjected animals. The first definite increase in numbers of nucleated lymph node cells and in antibody synthesis occurred on the third day after immunization. The peak of cell numbers and antibody synthesis was attained on the sixth day after antigenic stimulation. The antigen-stimulated cells also synthesized increased amounts of non-antibody proteins, including IgM and IgG, and proteins tentatively identified as α₂ macroglobulin, α₁ globulin and a microglobulin. Most of the radioactive protein which was synthesized following injection of antigen was demonstrably not antibody. Both IgM and IgG antibody were synthesized at all times, but relatively more radioactivity was incorporated into IgM antibody on day 3 and into IgG antibody on day 6 after immunization.     

Thalassemia and erythroid transcription factor KLF1 mutations associated with borderline hemoglobin A2 in the Thai population

IntroductionElevated hemoglobin (Hb) A₂ is an important diagnostic marker for β-thalassemia carriers. However, diagnosis of cases with borderline Hb A₂ may be problematic. We described the molecular characteristics found in a large cohort of Thai subjects with borderline Hb A₂.Material and methodsExamination was done on 21,657 Thai subjects investigated for thalassemia at Khon Kaen University, Thailand. A total of 202 subjects with borderline Hb A₂ (3.5–4.0%) were selectively recruited and hematological parameters were recorded. DNA variants in α-, β-, δ-globin, and Krüppel-like factor 1 (KLF1) genes were examined using PCR.ResultsAmong 202 subjects, DNA analysis identified carriers of α⁺-thalassemia (n = 48; 23.8%), β-thalassemia (n = 22; 10.9%) and KLF1 mutations (n = 48; 23.8%). No molecular defect was observed in the remaining 84 (41.5%) subjects. Interaction of KLF1 and α-thalassemia was observed in 10 cases. Of the 22 β-thalassemia carriers, five β⁺-thalassemia mutations were identified with lower MCV and higher Hb A₂. Seven KLF1 mutations were detected in 10 genotypes in subjects with higher MCV and Hb F. No β⁰-thalassemia, α-globin gene triplication or δ-globin gene mutation was detected.ConclusionsA large proportion of subjects with borderline Hb A₂ are not β-thalassemia carriers and for those with β-thalassemia, only mild β⁺-thalassemia mutations were detected. Evaluation of the patients using Hb A₂, Hb F and MCV values will help in selecting cases for further molecular analysis. The results should explain the unusual phenotype of the cases and facilitate a thalassemia screening program in the region.     

Identification of the Components of Complement Participating in the Antiglobulin Reaction

Red cells sensitized with a complement-binding antibody and then incubated with fresh serum have been shown to be coated with β_1C and β_1E globulin, two components of the complement system that have been isolated recently. Red cells presumably in the state EAC′_1,4 reacted with anti-β_1E, and cells presumably in the state EAC′_1,4,2,3a reacted with anti β_1E and with anti-β_1C. Agglutination of complement-coated cells by `broad spectrum' antiglobulin sera was effectively inhibited by purified β_1E and β_1C globulin. Red cells from certain patients with acquired haemolytic anaemia were found to be coated in vivo with β_1E and β_1C globulin. The function and significance of the two serum components have been discussed.