Soluble HLA Antigens Present in Normal Human Serum

HLA antigens have been detected in normal human serum by inhibition of monospecific HLA antisera in the complement-dependent cytotoxicity test. The antigens present in serum were the same as those found on the donor's lymphocytes. HLA-A9 was unique in that it was present in serum at inhibitory titers of 1:8 to 1:32 whereas the majority of A-and B-locus antigens were present at titers of 1:4 or less. Serum HLA-A9 antigens were found in the high density lipoprotein fraction with a molecular weight greater than 200,000. In the presence of detergent they have an apparent molecular weight of 86,000 which is reduced to 46,000 when treated with papain. These values are similar to those obtained for detergent-solubilized cellular HLA antigens.     

Neutralizing Monoclonal Antibody to Edema Toxin and Its Effect on Murine Anthrax

Edema factor (EF) is a component of an anthrax toxin that functions as an adenylate cyclase. Numerous monoclonal antibodies (MAbs) have been reported for the other Bacillus anthracis toxin components, but relatively few to EF have been studied. We report the generation of six murine hybridoma lines producing two IgM and four IgG1 MAbs to EF. Of the six MAbs, only one IgM neutralized EF, as assayed by an increase in cyclic AMP (cAMP) production by Chinese hamster ovary (CHO) cells. Analysis of the variable gene elements revealed that the single neutralizing MAb had a different binding site than the others. There was no competition between the neutralizing IgM and the nonneutralizing IgG MAbs indicative of different specificity. MAb-based capture enzyme-linked immunosorbent assay (ELISA) detected EF in liver lysates from mice infected with B. anthracis Sterne 34F2. Administration of the neutralizing IgM MAb to A/JCr mice lethally infected with B. anthracis strain Sterne had no significant effect on median time to death, but mice treated with the MAb were more likely to survive infection. Combining the neutralizing IgM to EF with a subprotective dose of a neutralizing MAb to protective antigen (PA) prolonged mean time to death of infected mice, suggesting that neutralization of EF and PA could produce synergistic beneficial effects. In summary, the results from our study and literature observations suggest that the majority of Abs to EF are nonneutralizing, but the toxin has some epitopes that can be targeted by the humoral response to generate useful Abs that may contribute to defense against anthrax.Bacillus anthracis is a Gram-positive, spore-forming bacterium and the causative agent of anthrax (4, 24). B. anthracis spores are naturally found in the soil, and the disease primarily affects animals such as sheep and cattle that ingest or inhale spores while grazing. Human anthrax is rare, and most cases occur in individuals who contract the disease via contact with farm animals or spore-infested animal hides. However, in recent years, B. anthracis has emerged as a potent biological weapon and there has been great interest in understanding its pathogenesis and developing new therapies (12). B. anthracis produces two large plasmids, pXO1 and pXO2, which encode the genes necessary for toxin production and formation of a poly-d-glutamic acid capsule, respectively. A major virulence factor, the toxins are made up of three protein components known as protective antigen (PA), lethal factor (LF), and edema factor (EF). PA interacts in a binary fashion with EF to produce edema toxin (EdTx) and LF to produce lethal toxin (LeTx) (4). PA interacts with EF and LF via a heptameric structure that facilitates their entry into the cell. EdTx acts as a calcium- and calmodulin-dependent adenylate cyclase that leads to cellular edema, whereas LeTx is a zinc metalloprotease, which cleaves mitogen-activated protein kinase kinases activating various cellular pathways and ultimately leading to cell death (4, 16). In considering the various treatment options for anthrax, the delivery of preformed antibodies (Abs) has some advantages over other measures of postexposure prophylaxis, such as antimicrobial agents (5). Due to their low side effect profile, high specificity, lack of selection for antimicrobial drug resistance, and ability to bind to preformed toxins, Abs could reduce damage during infection. Several monoclonal antibodies (MAbs) to PA were isolated and determined to show protective effects against LeTx in mice (1, 18, 31). Other studies have shown that LF MAbs are also able to protect against LeTx activity in rats (17) and mice (38); however, relatively few attempts have been made to isolate neutralizing MAbs to EF. Little et al. described 10 MAbs to EF; however, only 2 had a moderate effect in disrupting ¹²⁵I-EF binding to cell-bound PA in vitro, with just one of the two inhibiting the rounding of CHO cells, a feature known to result from exposure to EdTx (19). Recently, a neutralizing MAb to EF was described that mediated protection by interfering with the toxin interaction with calmodulin (3).Apart from their potential usefulness as therapeutic reagents, MAbs can also be used to define protective and nonprotective epitopes and for serological assays. Since there has been relatively little work done in the area of neutralizing MAbs against EF, there is an interest in trying to isolate additional MAbs to evaluate the efficacy of humoral immunity and for the design of toxin detection assays. This study describes the isolation of six MAbs against EF, one of which has potent neutralizing activity in vitro and modest protective abilities in vivo.     

Serum Inhibitory Factor in Lepromatous Leprosy: Its Effect on the Pre-S-Phase Cell-Cycle Kinetics of Mitogen-stimulated Normal Human Lymphocytes

The sera of ten Egyptian men with long-standing lepromatous leprosy (LL) (mean duration 17.4 years) that had failed to respond in dapsone treatment were shown to inhibit mitogen stimulation responses of normal human lymphocytes. When first tested, the sera partly inhibited the response to phytohaemagglutinin (PHA) and pokeweed mitogen and virtually abolished that to concanavalin A (Con A): after repeated freezing and thawing, the Con A inhibition had disappeared, whereas the PHA response was still partly Inhibited. The inhibitory serum factor(s) had similar actions on lymphocytes from each of six normal donors. Although the sera varied in potency, they showed similar dose response curves when tested against lymphocytes from a single donor. The principal action of the sera was to reduce the number of cells responding to mitogen, without modifying the kinetics of recruitment or rate of volume growth during G₁-phase in those cells that were unaffected by the inhibitory substances(s). Study of PHA dose-response curves and of the effect of delayed addition of LL serum suggested that the serum factor(s) act by diminishing the responsiveness of the cells, rather than by reducing the concentrations of free mitogen or by blocking cell membrane mitogen receptors The serum from one apparently healthy attendant, who had nursed leprosy patients for 30 years but who did not have leprosy or other chronic infective disease, inhibited completely stimulation by all three mitogens in a manner different from that of LL sera. Serum from the other 13 control patients did not modify the response of normal lymphocytes to stimulation by any of the three mitogens studied. It was concluded that the inhibitory factor(s) in the scrum of patients, with LL were a consequence of the disease and not of the environment in which the patients lived. Microscopy confirmed that the techniques used for recovery of the cultured cells did not introduce bias into the volume spectroscopy measurements.     

Human Granulocyte-Macrophage Colony-Stimulating Factor (hGM-CSF): Identification of a Binding Site for a Neutralizing Antibody

Abstract

One approach to the localization of functionally active regions of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) is to map the epitopes recognized by neutralizing anti-hGM-CSF monoclonal antibodies. We have defined the epitope recognized by one neutralizing antibody (LMM102) using proteolytic fragments obtained by enzymic digestion of bacterially synthesized hGM-CSF. RP-HPLC fractionation of a tryptic digest resulted in the identification of an immunoreactive “tryptic core” peptide containing 66 amino acids (52% of the protein). Further digestion of this “tryptic core” with S. aureus V8 protease produced a unique immunoreactive hGM-CSF product comprising two peptides, residues 86–93 and 112–127, linked by a disulfide bond between residues 88 and 121. The individual peptides, generated by reduction with dithiothreitol, were not recognized by the antibody. An analog of this peptide has been synthesized chemically and shown to have similar immunoreactivity to the epitope obtained by enzymic digestion. A series of modified peptides has also been synthesized to identify further the region required for antibody recognition.     

Neutralizing Antibodies to Cytomegaloviruses in Normal Simian and Human Sera

Simian and human sera were examined for neutralizing antibodies to simian and human cytomegaloviruses (CMV). Neutralizing antibody to simian CMV was found in sera from 12 of 12 African green monkeys, 8 of 10 rhesus monkeys, and 7 of 7 baboons captured in the wild. The antibody did not cross-react with human CMV strain AD169 but cross-reacted with human strain C87, particularly in the presence of complement. Thirty-six baboons and 10 rhesus monkeys born and hand-reared in captivity remained free of neutralizing antibody both to simian and human CMV for as long as 4 years. Fifteen of 24 human sera (63%) revealed only species-specific neutralizing antibody.     

Anti-Interleukin-6 Antibodies in Normal Human Serum

High-avidity IgG antibodies to the cytokine interleukin-6 (IL-6) were found in sera of apparently healthy adult individuals. These antibodies specifically interfered with an ELISA (enzyme-linked immunosorbent assay) for IL-6 in which specific polyclonal rabbit antibodies to human recombinant IL-6 (rIL-6) were used. Furthermore, using precipitation of 125I-rIL-6 with rabbit antibodies to human immunoglobulins (Ig), the sera of 7 out of 68 Danish blood donors were found to contain specific antibodies in substantial amounts. Judged by ELISA interference, gel filtration of sera incubated with 125I-rIL-6 and second antibody precipitation of 125I-rIL-6, IgG seemed to be the dominant IL-6 binding protein in these normal sera. Using specific antibodies to human Ig light chains, it was found that the anti-IL-6 antibodies were of polyclonal origin. Moreover, there are at least two epitopes on the IL-6 molecule, because more than one IgG bound to some IL-6 molecules at the same time. The anti-IL-6 antibodies did not cross-react with a number of other human recombinant-derived and native cytokines. The antibodies recognized native as well as rIL-6, but preferentially monomeric IL-6.     

Toxin Levels in Serum Correlate with the Development of Staphylococcal Scalded Skin Syndrome in a Murine Model

Staphylococcal scalded skin syndrome (SSSS) is an exfoliative dermatitis that results from infection with exfoliative toxin-producing Staphylococcus aureus. SSSS is seen primarily in infants and children. Here we ask if there is a specific maturation process that protects healthy adults from this syndrome. For these studies, an active recombinant exfoliative toxin A (rETA) was used in a neonatal mouse model. A time course generated on the susceptibility to the toxin as a function of mouse age indicated that BALB/c mice developed the characteristic symptoms of SSSS until day 7 of life. Between day 7 and day 8 of life there was a dramatic decrease in susceptibility, such that mice at day 9 of life were resistant to the effects of the toxin. This time course corresponds approximately to the time needed for maturation of the adaptive immune response, and SSSS in adults is often identified with immunocompromised states. Therefore, mice deficient in this response were examined. Adult mice thymectomized at birth and adult SCID mice did not develop the symptoms of SSSS after injection with the toxin, indicating that the adaptive immune response is not responsible for the lack of susceptibility observed in the older mice. SSSS in adults is also associated with renal disorders, suggesting that levels of toxin in serum are important in the development of the disease. rETA was not cleared as efficiently from the serum of 1-day-old mice compared to clearance from 10-day-old mice. Ten-day-old mice were given repeated injections of toxin so that the maximal level of toxin was maintained for a sustained period of time, and exfoliation occurred in these mice. Thus, whereas the adaptive immune response is not needed for protection of adult mice from SSSS, efficient clearance of the toxin from the bloodstream is a critical factor.     

Identification of and Human Serum Reactogenicity to Neutralizing Epitopes within the Central Unglycosylated Region of the Respiratory Syncytial Virus Attachment Protein

We identified two overlapping neutralizing epitopes within residues 151 to 172 of the central unglycosylated region of the respiratory syncytial virus (RSV) attachment protein. In ∼40% of hospitalized and outpatient adults infected with RSV subtype A, these contiguous residues are the target of ≥4-fold increases in IgG response between acute- and convalescent-phase sera.A hallmark of the respiratory syncytial virus (RSV) attachment (G) protein is the central unglycosylated region that typically comprises amino acids (aa) 151 to 190 (9). Residues 164 to 176 are invariantly conserved among G sequences from all RSV isolates, and there is a loop structure comprising two cystine disulfide bonds (Fig. ​(Fig.1).1). Genetic selection for RSV that can replicate in the presence of subtype-independent, partially neutralizing anti-G monoclonal antibodies (MAbs) such as L9 yields strains bearing amino acid changes localized mostly within the central unglycosylated region of G (8, 15).Open in a separate windowFIG. 1.Central unglycosylated region of the RSV G protein. Residues 151 to 190 of the RGH subtype A5 RSV strain and those from the prototypical subtype A (A2) and B (B1) strains are identified. The positions of relevant residues are shown by numbers above the amino acid alignment. Note that invariant residues 164 to 176 are shown in bold, and the predicted disulfide bonds are shown as solid lines connecting the relevant cysteine residues. The dotted line underlies residues 151 to 172 (the PCC; see the text) bearing the epitopes for L9 and K6 MAbs.To better define the cognate epitopes for L9, as well as for the K6 MAb with similar neutralizing activities, we constructed a series of plasmids, each encoding a glutathione S-transferase (GST)-RSV G fusion protein bearing a portion of the central unglycosylated region of G (16). The fusion proteins were expressed in bacteria, purified to >95% homogeneity (data not shown), resolved on sodium dodecyl sulfate (SDS)-polyacrylamide gels under reducing and denaturing conditions, and tested for recognition by L9 and K6 MAbs in immunoblots.(Portions of this work were presented previously at the 49th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, September 2009 [10].)L9 and K6 did not recognize GST alone or the fusion protein consisting of GST and G residues 173 to 190 (GST-G173-190) but bound to purified, full-length G protein, GST-G151-190, and three forms of GST-G151-172 bearing corresponding residues from A2, B1, and RGH strains (Fig. ​(Fig.2).2). Neither MAb recognized GST-G151-161, but GST-G155-172, GST-G157-172, and GST-G162-172 were detected by K6 but not by L9 (Fig. ​(Fig.22 and data not shown). Consistent with these immunoblot results, all three forms of GST-G151-172 were recognized by both MAbs and GST-G162-172 was bound by K6 but not L9 in enzyme-linked immunosorbent assays (ELISAs) under nonreducing/nondenaturing conditions (data not shown). These results suggest that (i) RSV G residues 151 to 172 are required for recognition by L9; (ii) consistent with the subtype-independent neutralizing activities of both MAbs, K6 and L9 both recognized subtype A- and B-derived residues 151 to 172; and (iii) the K6 epitope involves aa 162 to 172. Based on the proximal location of residues 151 to 172 with respect to the G loop, we have termed these residues the proximal central core (PCC) region of the RSV G protein.Open in a separate windowFIG. 2.The cognate epitopes of L9 and K6 MAbs are localized within the PCC domain of the RSV G protein. (a to c) Representative immunoblots. Purified RSV G protein (subtype A), GST alone, or GST-RSV G fusion proteins (∼0.5 μg of protein/sample; G-derived residues are listed above the corresponding lanes) were resolved on denaturing SDS-12 or 6% PAGE gels, transferred onto nitrocellulose, probed with L9 or K6 MAbs at a 1:5,000 dilution followed by goat anti-mouse horseradish peroxidase-conjugated antibodies (diluted 1:20,000), and analyzed by using chemiluminescence. For each gel, molecular weight (MW) markers are resolved in the leftmost lane and the corresponding molecular masses (in kilodaltons) are indicated to the left. Note that in the analysis depicted in panel a, two independent preparations of GST-G173-190, one from bacterial strain DH5α (D) and another from strain Origami 2 (O), were used to test reactogenicity and that we occasionally observed a doublet in GST-G151-190 lanes. Note also that in the analysis depicted in panel c, fragments of aa 151 to 172 derived from the RGH, A2, and B1 RSV strains (as indicated by lane labels) were tested for recognition by L9 and K6 MAbs. (d) The L9 and K6 MAb epitopes within RSV G residues 151 to 172 are overlapping. On the right are various schematic diagrams, each representing a portion of the RSV G unglycosylated region that was expressed as a fusion protein in bacteria. Where relevant, the relative locations of the four cysteines (C) are shown. On the left is a table summarizing the immunoblot data obtained using both MAbs. + indicates reproducible MAb recognition of the RSV G-derived residues, while − indicates the absence of detectable MAb binding.The RSV G PCC contains most of the conserved amino acid sequence HFEVFNFVPCSIC (residues 164 to 176). Our data suggest that the K6 epitope likely exists within these residues and that the L9 epitope requires nonconserved residues 151 to 163 for correct epitope conformation. Also within the PCC is the motif (Y/F)XFXXFXF (residues 163 to 170), in which F163, F165, F168, and F170 are present in G proteins from subtype A strains (e.g., A2 and RGH) while Y163 is present in the G protein from the B1 strain (5, 17) (Fig. ​(Fig.1).1). This amino acid motif is also found in RSV-related viruses with different host specificities (e.g., ovine and bovine RSVs), suggesting an evolutionarily conserved structural and/or immunological function (6, 7). Alanine substitution for F163 but not for F165 abolishes K6 binding to GST-G162-172 in immunoblots, suggesting that the F163 residue is likely to be involved in K6-epitope interactions (data not shown).To determine the clinical and immunological relevance of the PCC-embedded epitopes in human RSV infections, we assayed serum reactogenicity to GST-G151-172 in ELISAs using paired acute- and convalescent-phase sera from RSV subtype A-infected hospitalized and outpatient adults (3). Among paired sera from 32 RSV-infected hospitalized adults, samples from 14 patients (44%) showed a ≥4-fold increase in the anti-RSV G PCC titers from acute- to convalescent-phase sera. Similarly, among serum samples from 19 outpatient adults, samples from 8 (42%) showed a ≥4-fold increase in the anti-G PCC antibody response. For both populations, the increase in the mean anti-G PCC titer (expressed as the log₂ reciprocal serum dilution ± the standard deviation) after RSV infection was statistically significant (for hospitalized patients, the mean titer increased from 8.3 ± 1.8 to 10.1 ± 2.8; for outpatients, it increased from 8.1 ± 1.5 to 9.9 ± 1.6; for both populations, the P value was <0.005 by the Wilcoxon signed-rank test). These data suggest that (i) an acute rise in anti-G PCC titers is found in ∼40% of RSV subtype A-infected adults and (ii) such titer increases are observed with no obvious correlation to the severity of illness as defined by the initial clinical evaluation in hospital versus outpatient settings.Our results have implications for the structure, function, and immunogenicity of the RSV G PCC region. Due to hydrophobic interactions involving F165, F168, F170, V171, P155, and P156, residues 149 to 177 of RSV G likely form a disk-like structure with two hydrophobic faces (4). Within the G central unglycosylated domain, residues 166 to 170 (EVFNF) may be involved in the multimerization of RSV G and/or interactions with a cellular RSV G protein receptor (4). Our results suggest that ≥1 neutralizing epitope is found within and flanking RSV G residues 166 to 170 and raise the possibility that L9 and other MAbs recognizing the G PCC-embedded epitopes may directly or indirectly affect RSV G structure (e.g., by destabilizing multimerization) and/or function (i.e., by blocking interactions with the host target cell). Previously, we reported the isolation of an L9-resistant virus that bore mutations outside the G PCC region; perhaps these mutations represent second-site/compensatory changes that counteract the action(s) of L9 MAb on RSV G structure/function (15).Within the RSV G protein, short “protective” B-cell epitopes have been identified, of which two (aa 152 to 163 and 165 to 172) are located within the RSV G PCC region (13). It should be noted that L9 and K6 are both neutralizing and subtype independent but that none of the protective epitope-recognizing MAbs were neutralizing and that it is unclear whether the protective effect against viral challenge was subtype independent. These differences in the functional profiles of the various MAbs may be due to the immunogen (purified, native RSV G protein from RSV-infected mammalian cells versus a bacterially derived, refolded RSV G fragment) used to generate the respective MAbs.The human serological characterization of RSV G epitopes, especially those within the G PCC domain, remains incomplete. A very limited number of adult human sera (n = 2) were used to study reactogenicity to RSV G-derived protective epitopes (13). Other RSV G-based human serological screening studies utilized bacterially synthesized, genetically hypervariable regions flanking the central unglycosylated region or G-derived peptides (overlapping or nonoverlapping) to screen adult or pediatric sera (1, 2, 11, 12, 14). In this study, we demonstrate a ≥4-fold increase in serum reactogenicity to the PCC domain of RSV G in a significant proportion of RSV subtype A-infected adults. These data suggest that the G PCC domain is immunologically significant in human RSV infections and may be a target of prophylactic and/or therapeutic agents against RSV.     

Isoforms of Murine and Human Serum Amyloid P Component

Isoelectric focusing (IEF) and immunofixation of murine serum amyloid P component (SAP), purified and in serum, showed a distinct and strain-dependent isoform pattern with up to seven bands (pI 5.1–5.7). Neuraminidase treatment caused a shift of the isoforms to more basic pI values, but did not affect their number. When the acute-phase response was analysed in three mouse strains, CBA/J and C3H/HeN initially showed seven SAP isoforms in serum and C57BL/6 J three or four. The responses in all three strains peaked at day 2 and were normalized within 14 days. On days 2 and 4, CBA/J and C3H/HeN mice showed one more acidic isoform and an increase in the concentration of the most basic isoform. C57BL/6 J mice exhibited two to three new isoforms during the acute-phase response. This appears to be the first demonstration of the physiological existence of SAP isoforms. In contrast, demonstration of isoforms of human SAP required the presence of urea and higher SAP concentrations. IEF and immunofixation of SAP monomers showed five to eight isoforms, ranging from pI 4.7–5.7. IEF of SAP in human serum resulted in a less distinct pattern and more acidic isoforms. As with murine SAP, neuraminidase treatment caused a shift of the isoforms, but no reduction in isoform number. Two-dimensional gel electrophoresis confirmed the existence of multiple isoforms of human SAP monomers.     

Serum Antibodies to Albumins in Normal Human Pregnancy

Sera from 72 pregnant women were studied for the presence of antibodies to human serum albumin (HSA), bovine serum albumin (BSA) and ovalbumin (OA) by passive haemagglutination assay. Anti-albumin antibodies were found in 96% of the cases and distributed throughout the 3 trimesters of pregnancy.     

Serum Antibodies to Albumins in Normal Human Pregnancy

Sera from 72 pregnant women were studied for the presence of antibodies to human serum albumin (HSA), bovine serum albumin (BSA) and ovalbumin (OA) by passive haemagglutination assay. Anti-albumin antibodies were found in 96% of the cases and distributed throughout the 3 trimesters of pregnancy.     

健康儿童、青少年血清生长激素的参考值调查

采用放射免疫法测定286例昆明市3～18岁健康儿童及青少年血清生长激素(GH)浓度。儿童、少年期组(3～12岁 ,n=163)和青春期组(13～18岁 ,n=123)血清GH浓度(x+s)分别为3.02±2.01 ;6.98±5.05 ,用百分位数法求出双侧P2.5和P97.5的值。参考值范围儿童、少年期组0.20～12.8ng/ml,青春期组0.70～28.46ng/ml。青春期组明显高于儿童、少年期组。其GH浓度与生长发育及年龄相关 ,分泌量的高峰出现在青春期。本文为临床提供了该年龄段GH水平的参考指标。     

Mechanisms of Immunity in Typhus Infections III. Influence of Human Immune Serum and Complement on the Fate of Rickettsia mooseri Within Human Macrophages

Preincubation of Rickettsia mooseri with human typhus convalescent serum, which is not rickettsiacidal but which confers passive protection to animals, opsonizes the rickettsiae for enhanced phagocytosis by monocyte-derived human macrophages in cell culture and renders them susceptible to destruction within the macrophages. Nonspecific opsonization by preincubation of the rickettsia with methylated bovine serum albumin enhances phagocytosis, but the rickettsiae are not prepared for intracellular destruction. Instead, they grow within the macrophages and eventually destroy these cells. Thus, immune serum and macrophages, neither of which is capable of killing these rickettsiae alone, act in concert to destroy the virulent organisms. In this system, immune serum appears to exert two distinct, possibly dissociable, actions on the rickettsiae: enhancement of phagocytosis and preparation for intracellular destruction. Complement is not required for this action but, when present with immune serum, markedly enhances phagocytosis of the rickettsiae, often leading to rapid destruction of the macrophage.