High levels of "complexed" interleukin-6 in human blood.

The biochemical nature of endogenous interleukin-6 (IL-6) as it exists in human serum or plasma was investigated. Serum from a patient following bone marrow (BM) transplantation and fresh plasma samples from patients with epidermolysis bullosa or psoriasis, as well as from normal volunteers, were fractionated through G-200 columns and each of the eluted fractions assayed for IL (interleukin)-6 content using enzyme-linked immunosorbent assays (ELISAs) based on the monoclonal antibody (mAb) pairs IG61/5IL6 or 4IL6/5IL6 and in the B9 hybridoma growth factor bioassay. The IG61/5IL6 ELISA and the B9 assay detected IL-6 in BM serum almost exclusively of molecular mass approximately 20 kDa. In contrast, the 4IL6/5IL6 ELISA detected strong IL-6 immunoreactivity in complexes of size 100-150 and 400-500 kDa. IL-6 present in the 100-150- and 400-500-kDa complexes was purified by immunoaffinity chromatography through a 5IL6 mAb column. The 5IL6 mAb immunoaffinity column eluate of the respective pools from BM serum contained IL-6 at concentrations approaching 1 microgram/ml as characterized by Western blotting. Sufficient IL-6 and associated proteins were purified by 5IL6 mAb immunoaffinity column chromatography of the 100-150-kDa complex from 0.8 ml of BM serum to allow (i) verification of three of the polypeptides as IL-6 by amino-terminal sequencing (estimate of IL-6 in original serum sample: 5-10 micrograms/ml), (ii) identification by amino acid sequencing of the "associated" proteins as complement factor C3b (carboxyl-terminal of the alpha-chain), complement factor C4b (gamma-chain), C-reactive protein, and albumin, and (iii) detection of an "associated" polypeptide consistent with the soluble IL-6 receptor. Taken together, these data establish that IL-6 is present at unexpectedly high concentrations in human blood in novel biochemical complexes that include other plasma proteins, which in turn, can camouflage IL-6 immunoreactivity and bioactivity as measured in conventional assays.     

A monoclonal antibody to Shigella dysenteriae serotype 13 cross-reacting with Shiga toxin

Abstract A monoclonal antibody (mAb ICT6) was produced against the newly described Shigella dysenteriae serotype type 13. The mAb was of IgM isotype and recognized purified Shiga toxin in ELISA and immunoblot. It also recognized periplasmic extract S. dysenteriae type 13 in immunoblot as did an affinity-purified polyclonal rabbit antiserum and a previously described monoclonal antibody to the B subunit of Shiga toxin. The mAb ICT6 did not neutralize the cytotoxic effects or S. dysenteriae type 13, Shiga toxin or periplasmic extracts of S. dysenteriae type 1 for HeLa cells.     

Granulocyte/macrophage colony-stimulating factor inhibits IL-12 production of mouse Langerhans cells

We investigated the capacity of mouse Langerhans cells (LC) to produce IL-12, a central cytokine in a Th1 type of immune responses. We prepared purified LC (>95%) from BALB/c mouse skin by the panning method using anti-I-Ad mAb. An ELISA showed that purified LC spontaneously produced IL-12 p40, and that its production was up-regulated following simultaneous stimulation with anti-CD40 mAb and IFN-gamma. Surprisingly, GM-CSF strikingly inhibited IL-12 p40 production by anti-CD40/IFN-gamma-stimulated LC (% inhibition = 97.0 +/- 0.9% at 1 ng/ml GM-CSF). Supernatants of 48-h cultured keratinocytes (KC) also caused the inhibition of LC IL-12 p40 secretion, and this effect was neutralized by anti-GM-CSF mAb. IL-1alpha (1 ng/ml)-stimulated KC produced much more GM-CSF than unstimulated KC (60.9 +/- 0.2 pg/ml vs 20.9 +/- 1.7 pg/ml), and IL-1alpha-stimulated KC supernatants strongly inhibited IL-12 p40 production by anti-CD40/IFN-gamma-stimulated LC (% inhibition = 89.4 +/- 1.4%). A bioassay using an IL-12-dependent T cell line demonstrated the correlation of the level of IL-12 p40 with the bioactivity of IL-12. These results provide important implications for the pathogenesis of atopic dermatitis, which involves the participation of LC and KC with the capacity to produce IL-12 and GM-CSF, respectively.     

The autocrine production of transforming growth factor-beta 1 during lymphocyte activation. A study with a monoclonal antibody-based ELISA

We describe the production and characterization of three mAb to transforming growth factor-beta (TGF-beta) and the use of two of them for the development of a TGF-beta 1-specific ELISA and for the study of the regulation of immune function in vitro. All three mAb bound recombinant human TGF-beta 1 (rHuTGF-beta 1) with high affinity and recognized the dimer form of this molecule in immunoblots. mAb 2G7 immunoprecipitated rHuTGF-beta 1, TGF-beta 2, and rHuTGF-beta 3 and neutralized the growth inhibitory activity of all three molecules in vitro on mink lung epithelial-like cells, Mv1Lu, indicating a shared neutralization epitope. mAb 4A11 neutralized and immunoprecipitated only rHuTGF-beta 1, and mAb 12H5 immunoprecipitated rHuTGF-beta 1 but had no effect on the bioactivity of either rHuTGF-beta 1, TGF-beta 2, or rHuTGF-beta 3. These results suggest that a second neutralization epitope may be unique to TGF-beta 1. The ELISA was developed with mAb 4A11 and 12H5, with a range of 0.63 to 40 ng/ml, i.e., a sensitivity of 0.63 ng/ml or 63 pg/sample. The assay is accurate, precise, and specific for the active but not the inactive or latent TGF-beta 1 complex and fails to react with TGF-beta 2, rHuTGF-beta 3, inhibin A, and activin A. Supernatants obtained from serum-free cultures of human PBMC from multiple donors contained significant quantities of TGF-beta 1 (3 to 15 ng/ml), which was detected in the ELISA only after pH 2 treatment to convert latent TGF-beta to the active form. Treatment of the PBMC with either recombinant human IL-2 (rHuIL-2) or PHA-P/PMA enhanced the production of latent TGF-beta 1. mAb 4A11 and 2G7, but not mAb 12H5 enhanced both the proliferative response of PBMC to rHuIL-2/rHuTNF-alpha and PHA-P and the development of the rHuIL-2/rHuTNF-alpha treated PBMC into LAK cells with cytotoxic activity against COLO target cells. These findings suggest that although PBMC secrete latent TGF-beta 1, mechanisms that convert the latent TGF-beta complex into an active form exist in vitro and that the endogenously produced TGF-beta can regulate immune functions in an autocrine fashion.     

The presence of autoantibodies specific for NZB serum factors in adult NZB mice and the establishment of monoclonal autoantibodies against these humoral factors

Humoral factors in serum of young NZB mice enhance maturation of B-lymphocyte precursors in vitro. A blot ELISA assay identified autoantibodies against the serum factors. NZB-SFs (designated NZB-SF alpha, pI 3.5-4.0, and NZB-SF beta, pI 7.8) were purified by sequential steps. Both had a molecular weight (MW) of approximately 23,000 in SDS-PAGE. NZB mice develop autoantibodies against NZB-SFs by 2 months of age; titers increased progressively with age. Non-autoimmune-prone mice did not produce autoantibodies against NZB-SFs. We then developed two hybridoma clones, IIC1C1 and IIC1M4, which produce monoclonal autoantibodies against NZB-SF alpha and NZB-SF beta, respectively. Both IgM autoantibodies could be affinity purified with a column of CNBr-Sepharose 4B gel conjugated with anti-mouse IgM antibody. Neither IIC1C1 nor IIC1M4 abolished bioactivity of recombinant mouse IL-1 alpha, human IL-1, mouse, rat, or human IL-2, mouse IL-3, or colony-stimulating factor. Neither antibody reacted to recombinant mouse IL-1 alpha, IL-4, TNF alpha, or IFN gamma in blot ELISA assays. Monoclonal autoantibodies IIC1C1 and IIC1M4 were used to purify NZB-SFs. SDS-PAGE of the affinity-purified NZB-SFs revealed bands of 23 and 60 kDa, and proteins extracted from the bands were reactive to our monoclonal autoantibodies.     

IL-18 contributes to host resistance against infection with Pseudomonas aeruginosa through induction of IFN-gamma production

Pseudomonas aeruginosa keratitis destroys the cornea in susceptible (B6), but not resistant (BALB/c) mice. To determine mechanisms mediating resistance, the role of IFN-gamma, IL-12, and IL-18 was tested in BALB/c mice. RT-PCR analysis detected IFN-gamma mRNA expression levels in cornea that were significantly increased at 1-7 days postinfection. IL-18 mRNA was detected constitutively in cornea and, at 1-7 days postinfection, levels were elevated significantly, while no IL-12 mRNA was similarly detected. To test whether IL-18 contributed to IFN-gamma production, mice were treated with anti-IL-18 mAb. Treatment decreased corneal IFN-gamma mRNA levels, and bacterial load and disease increased/worsened, compared with IgG-treated mice. To stringently examine the role of IFN-gamma in bacterial killing, knockout (-/-) vs wild-type (wt) mice also were tested. All corneas perforated, and bacterial load was increased significantly in -/- vs wt mice. Because disease severity was increased in IFN-gamma(-/-) vs IL-18-neutralized mice, and since IL-18 also induces production of TNF, we tested for TNF-alpha in both groups. ELISA analysis demonstrated significantly elevated corneal TNF-alpha protein levels in IFN-gamma(-/-) vs wt mice after infection. In contrast, RT-PCR analysis of IL-18-neutralized vs IgG-treated infected mice revealed decreased corneal TNF-alpha mRNA expression. Next, to resolve whether TNF was required for bacterial killing, TNF-alpha was neutralized in BALB/c mice. No difference in corneal bacterial load was detected in neutralized vs IgG-treated mice. These data provide evidence that IL-18 contributes to the resistance response by induction of IFN-gamma and that IFN-gamma is required for bacterial killing.     

Normal human sera contain antibodies directed at Fab of IgA

Serum samples from 26 normal volunteers were evaluated by isotype-specific ELISA for the presence of IgG and IgM antibodies directed at IgA. Although there were wide variations in antibody levels, anti-IgA antibodies of both isotypes were found in all individuals tested. The anti-IgA activity was detected against a variety of polymeric and monomeric IgA1 and IgA2 myeloma proteins containing both kappa and lambda light chains. By using Fab and Fc fragments generated by incubation of an IgA1 myeloma protein with IgA1 protease, it was shown that the anti-IgA activity was specific for the Fab portion of the IgA molecule. It was also demonstrated that the serum of two individuals contained both IgG and IgM activity directed at autologous affinity-purified IgA. IgM antibody levels against both whole IgA and Fab of IgA were significantly higher than IgG antibody levels. Cells producing anti-IgA antibodies of both isotypes were detected in lipopolysaccharide-stimulated human spleen.     

A monoclonal antibody recognizing 68- to 75-kilodalton protein(s) associated with the human IL-1 receptor

We produced an IgM mAb termed 4.9 against an EBV-containing lymphoblastoid cell line, termed 3B6. This mAb reacted with both various B and T cell lines such as HSB2 cells, with an NK-like cell line YT-C3 cells, and with human fibroblast MCR-5 cells. It also reacted with normal resting peripheral B lymphocytes, monocytes, and anti-CD2- or anti-CD3-activated T lymphocytes. The 4.9 mAb immunoprecipitated two bands estimated to be of Mr 68 and 75 kDa from iodinated 3B6 cells. The 4.9 mAb inhibited the proliferation of peripheral T lymphocytes induced either by anti-CD3 mAb or anti-CD2 mAb. The 4.9 mAb inhibited also the proliferation of murine thymocytes both in the presence of PHA and IL-1 and the proliferation of human fibroblasts in the presence of IL-1. Radiolabeled IL-1 binding on 3B6 cells revealed two types of IL-1 binding sites with high and low affinity for IL-1 (300 sites/cell with a Kd of 6 x 10(-11)M and 6000 sites/cell with a Kd of 3 x 10(-9)M). On both 3B6 and YT-C3 cells, mAb 4.9 inhibited specifically the binding of 125I-labeled rIL-1, alpha or beta, whereas the irrelevant IgM mAb did not. Conversely, rIL-1, alpha or beta, could inhibit specifically the binding of radioiodinated 4.9 mAb to 3B6 or YT-C3 cells, whereas rIL-2, rIFN, or the irrelevant IgM mAb were ineffective. 125I-4.9 mAb bound 3B6 cells with an association constant (Ka) of 2 x 10(8)/M and demonstrated 6000 binding sites/cell. We thus conclude that mAb 4.9 recognizes a protein complex (68 to 75 kDa) closely associated with the IL-1R.     

Detection of expressed IL-32 in human stomach cancer using ELISA and immunostaining

Interleukin (IL)-32 is a recently identified proinflammatory cytokine that is one of the IL-18 inducible genes, and plays an important role in autoimmune and inflammatory diseases. We produced antibodies against IL-32 and studied the expression of IL-32 in human stomach cancer. We detected IL-32 secreted from K-562 cells that werw stably transfected with IL-32 and in the sera of stomach cancer patients, by a sandwich ELISA using a monoclonal antibody KU32-52 and a polyclonal antibody. In order to optimize a sandwich immunoassay, recombinant IL-32alpha was added, followed by the addition of a biotinylated KU32-52 into microtiter plate wells precoated with a goat anti-IL-32 antibody. The bound biotinylated KU32-52 was probed with a streptavidin conjugated to HRP. This sandwich ELISA was highly specific and had a minimal detection limit of 80 pg/ml (mean+/-SD of zero calibrator) and measuring up to 3,000 pg/ml. This ELISA showed no cross-reaction with other cytokines such as hIL-1alpha, hIL-1beta, hIL-2, hIL-6, hIL-8, hIL-10, hIL-18, and hTNF-alpha. Intra-assay coefficients of variation were 18.5% to 4.6% (n=10), and inter-assay coefficients were 23% to 9% (n=10). The average IL-32 level in the sera of 16 stomach cancer patients (189 pg/ml) was higher than that of 12 healthy control men (109 pg/ml). Our results indicate that serum IL-32 level can be detected by using an established ELISA, and that this immunoassay and mAb KU32-09 specific for immunohistochemistry can be used in the detection of expressed and secreted IL- 32 in stomach cancer patients.     

CD40 stimulation provides an IFN-gamma-independent and IL-4-dependent differentiation signal directly to human B cells for IgE production.

IgE induction from human cells has generally been considered to be T cell dependent and to require at least two signals: IL-4 stimulation and T cell/B cell interaction. In the present study we report a human system of T cell-independent IgE production from highly purified B cells. When human cells were co-stimulated with a mAb directed against CD40 (mAb G28-5), there was induction of IgE secretion from purified blood and tonsil B cells as well as unfractionated lymphocytes. Anti-CD40 alone failed to induce IgE from blood mononuclear cells or purified B cells. The effect of the combination of anti-CD40 and IL-4 on IgE production was very IgE isotype specific as IgG, IgM, and IgA were not increased. Furthermore, anti-CD40 with IL-5 or PWM did not co-stimulate IgG, IgM, or IgA and in fact strongly inhibited PWM-stimulated IgG, IgM and IgA production from blood or tonsil cells. IgE synthesis induced by anti-CD40 plus IL-4 was IFN-gamma independent as is the in vivo production of IgE in humans; the doses of IFN-gamma that profoundly suppressed IgG synthesis induced by IL-4, or IL-4 plus IL-6, had no inhibitory effect on anti-CD40-induced IgE production. Anti-CD23 and anti-IL-6 also could not block anti-CD40 plus IL-4-induced IgE production, but anti-IL-4 totally blocked their effect. IgE production via CD40 was not due to IL-5, IL-6 or nerve growth factor as none of these synergized with IL-4 to induce IgE synthesis by purified B cells. Finally, we observed that CD40 stimulation alone could enhance IgE production from in vivo-driven IgE-producing cells from patients with very high IgE levels; cells that did not increase IgE production in response to IL-4. Taken together, our data suggest that the signals delivered for IgE production by IL-4 and CD40 stimulation may mimic the pathway for IgE production seen in vivo in human allergic disease.     

Increased serum levels of interleukin-18 in patients with diabetic nephropathy

In the present study we measured interleukin-18 (IL-18) and tumour necrosis factor-alpha (TNF-alpha) levels by enzyme linked immunosorbent assay (ELISA) in sera from 65 diabetic [30 with type 1 insulin dependent diabetes mellitus (IDDM) and 35 with type 2 non-insulin dependent diabetes mellitus (NIDDM)] patients and 15 healthy volunteers, to investigate their associations with metabolic parameters and to elucidate their roles in the pathogenesis of diabetic complications especially diabetic nephropathy. Levels of IL-18 and TNF-alpha were significantly higher in both IDDM and NIDDM individuals as compared to the control group. Similarly, their levels in patients with diabetic nephropathy increased gradually according to the clinical stage of the disease, being highest in macroalbuminuric stage. Correlation analyses showed that the serum IL-18 and TNF-alpha concentration were positively correlated with each other and positively with fasting plasma glucose (FPG), 2h postprandial glucose, glycosylated hemoglobin (HbA1c), triglyceride, and urinary albumin levels and negative correlation between TNF-alpha and high density lipoprotein cholesterol (HDL-C) were also found in diabetic subjects. High serum levels of IL-18 and TNF-alpha suggested that they might play a role in the pathogenesis of DM and in the development of nephropathy in diabetic patients whether of type 1 or 2.     

Expression of CD5 regulates responsiveness to IL-1

The role of the CD5 surface molecule in T cell responsiveness to IL-1 was examined. A CD5-mutant Jurkat cell line was generated from a CD5+ parent cell line. This CD5- mutant subclone was infected with a defective retrovirus containing the CD5 cDNA and/or the neo gene encoding G418 resistance. The CD5+ wild type Jurkat produced IL-2 in response to anti-CD3 mAb, OKT3, cross-linked to a solid surface. IL-2 production was enhanced by co-culture with IL-1 or anti-CD5 Mab. Neither the CD5- mutant nor the CD5- G418-resistant infectant responded to anti-CD5 mAb or to IL-1. Responsiveness to IL-1 was restored by cell surface expression of CD5 in the CD5+ infectant. Both the CD5+ wild type Jurkat and the CD5+ infectant responded equivalently to purified IL-1, IL-1 alpha and rIL-1 beta. Optimal concentrations of IL-1 and anti-CD5 mAb had an additive effect on the enhancement of IL-2 production stimulated with cross-linked anti-CD3 mAb suggesting that IL-1 and CD5 act through distinct, complementary pathways to augment T cell activation. The correlation of CD5 expression and specific binding of rIL-1 beta was examined in these cell lines. Both the specific binding (at 4 degrees C) and subsequent internalization (at 37 degrees C) of 125I labeled rIL-1 beta was equivalent in the CD5+ infectant and the CD5+ wild type Jurkat cell, whereas specific binding of 125I-labeled rIL-1 beta was markedly decreased in the CD5-G418-resistant infectant. These observations strongly suggest that cell surface expression of CD5 regulates binding of and responsiveness to IL-1.     

Antitumor response of regional lymph node lymphocytes in human lung cancer

 Monoclonal antibodies (mAb) are promising substances for the treatment of colorectal carcinoma, but the efficiency of this therapy still needs further improvement. We used a flow-cytometric cytotoxicity test to determine the efficacy of the cytokines interferon α (IFNα) and γ (IFNγ), interleukin-2 (IL-2), macrophage-colony-stimulating factor (M-CSF), granulocyte/macrophage-CSF (GM-CSF) and tumor necrosis factor α (TNFα) in enhancing the antibody-dependent cellular cytoxicity (ADCC) of the mAb 17-1A and the mAb BR55-2 against the colorectal carcinoma cell line HT29. In experiments performed at an effector to target ratio of 9:1, with peripheral blood mononuclear cells from five healthy volunteers as effector cells, we found that IFNα, IFNγ and IL-2 significantly augmented the ADCC of both mAb at concentrations between 3 ng/ml and 30 ng/ml. The other three cytokines were not effective. In further experiments we examined combinations of the three effective cytokines in different concentrations. The combination of IFNα and IL-2 proved to be optimal in enhancing ADCC of both mAb. Thus, the examination of ADCC by flow cytometry may reveal potentially useful combinations of cytokines and mAb for the treatment of colorectal carcinoma. Received: 11 September 1997 / Accepted: 19 February 1998     

Restoration of natural IgM production from liver B cells by exogenous IL-18 improves the survival of burn-injured mice infected with Pseudomonas aeruginosa

Pseudomonas aeruginosa is the most common bacterium of postburn infection. In the present study we investigated the immune mechanism of susceptibility to this type of postburn infection and also examined the efficacy of IL-18 treatment. C57BL/6 mice were challenged with P. aeruginosa on day 7 after burn injury. Although the burn-injured mice showed a poor survival rate after bacterial challenge, they retained their IFN-gamma production. The burned mice showed lower serum IgM levels and a poor IgM response following P. aeruginosa challenge in comparison with the sham mice, whereas IL-18 treatment after burn injury (alternate day injections for 1 wk) greatly improved the serum IgM levels, which are P. aeruginosa-independent natural IgM before bacterial challenge, thereby increasing the survival rate after the challenge. IL-18 treatment also induced specific IgM to P. aeruginosa in the sera 5 days after bacterial challenge in the burned mice. Interestingly, CD43(+)CD5(-)CD23(-)B220(dim) cells, namely B-1b cells, increased in the liver after the IL-18 treatment and were found to actively produce IgM in vitro without any additional stimulation. Furthermore, the IL-18 treatment up-regulated the neutrophil count and the C3a levels in the blood as a result of the increased IgM level, which may thus play a critical role in the opsonization and elimination of any invading bacteria. IL-18 treatment for the burned mice and their resultant natural IgM production were thus found to strengthen the host defense against P. aeruginosa infection.     

Assessment of the biological variation of plasma tissue inhibitor of metalloproteinases-1

BACKGROUND: Tissue inhibitor of metalloproteinases-1 (TIMP-1) measurements in plasma may be useful for the early detection and prognosis of colorectal cancer (CRC). Data on analytical performance and normal intra- and interindividual biological variation are required in order to interpret the utility of TIMP-1 in CRC. The aim of this study was to establish the biological and analytical variation of plasma TIMP-1 in volunteers. MATERIAL AND METHODS: Three separate studies were undertaken. 1: Plasma was collected from 23 volunteers 6 times within a 3-week period, first in September 2004 (round [R] 1), then repeated in May 2005 (R2) and May 2006 (R3) in the same group of individuals. TIMP-1 levels were determined by the MAC15 ELISA assay and with the Abbott ARCHITECT i2000 Immunoanalyzer. 2: Circadian variation was evaluated in plasma collected 7 times within a 24-hour period (n=16). 3: Effects of physical exercise were evaluated in plasma collected before and after bicycling (n=14). In studies 2 and 3 TIMP-1 levels were determined with the MAC15 ELISA assay only. RESULTS: A significant correlation between TIMP-1 MAC15 and ARCHITECT i2000 was shown (rs=0.78, p<0.002), with consistently higher levels being detected by the ARCHITECT i2000. Median levels of TIMP-1 (ARCHITECT) at 8 a.m. in each round were 74.9 ng/mL (range 65.7-89.9) (R1), 87.3 ng/mL (range 72.7-127.9) (R2), and 81.9 ng/mL (range 66.8-113.6) (R3). The within-subject variation was 10.7%, the variation between rounds was 7.4%, and the intraclass correlation was 46.2%. Comparison between the 3 rounds and time of collection showed that TIMP-1 values decreased by 11% after storage for more than 16 months (p=0.0002). A systematic circadian variation in plasma TIMP-1 levels was not observed (p=0.17). No significant variation of plasma TIMP-1 was found in relation to physical exercise (p=0.92 [global test]). CONCLUSION: Levels of plasma TIMP-1 in volunteers show limited circadian, day-to-day, week-to-week and season-to-season variation. In addition, physical exercise has no impact on plasma TIMP-1 levels. Possible storage-dependent decreases in plasma TIMP-1 levels warrant further investigation.     

Development of a sandwich ELISA assay for measuring bovine soluble type II IL-1 receptor (IL1R2) concentration in serum and milk

The IL1R is composed of two kinds of molecule, type I (IL1R I) and type II (IL1R2). IL1R1 contributes to IL-1 signaling, whereas the IL1R2 has no signaling property and acts as a decoy for IL-1. In this study, we developed a bovine IL1R2-specific sandwich ELISA to examine the sIL1R2 concentration in serum and milk from dairy cows. The concentration of colostral IL-1beta was examined to estimate the correlation to sIL1R2. The results showed that the sIL1R2 concentration in sera and milk changes with the stages of lactation. The serum sIL1R2 concentrations were 5.56+/-0.69 ng/ml (colostrum), 3.14+/-0.72 ng/ml (the early stage of lactation) and 5.76+/-1.25 ng/ml (the late stage of lactation). The milk sIL1R2 concentrations were 1.83+/-0.47 ng/ml (colostrum), 0.73+/-0.22 ng/ml (the early stage of lactation) and 2.92+/-0.56 ng/ml (the late stage of lactation). The concentrations of IL1R2 in sera and milk were significantly higher at the late stage of lactation and colostrum than that of the early stage of lactation. The reduction rates of sIL1R2 levels from the colostrum to the early stage of lactation were 43.6% (serum) and 61% (whey). IL-1beta was detected in all the colostrum (995.9+/-346.6 ng/ml). Significant correlation was observed between the levels of colostral IL-1beta and IL1R2 (r=0.75).     

Binding characteristics and cross-reactivity of three different antilipid A monoclonal antibodies

A detailed characterization of binding specificity and cross-reactivity of three antilipid A murine mAb was performed. Binding characteristics of these three mAb were investigated against Ag (ReLPS, lipid A, derivatives of lipid A) in solid phase (ELISA) and in fluid phase (C consumption, inhibition studies), and upon incorporation in membranes (E: passive hemolysis assay, and liposomes: inhibition studies). Cross-reactivity with heterologous Ag was investigated in ELISA (LPS, Gram-negative bacteria) and immunoblot experiments (LPS). The binding specificity of mAb 26-5 (IgG2b), raised against synthetic lipid A, was located in the hydrophilic region of biphospholipid A and was also exposed after membrane incorporation of lipid A or after preincubation of lipid A with polymyxin B (PMX). mAb 26-20 (IgM), also raised against synthetic lipid A, showed binding specificity for the hydrophobic region of lipid A: no binding to membrane-associated lipid A could be demonstrated, and binding in ELISA could be blocked very efficiently by PMX. The reaction pattern of mAb 8-2 (IgM), raised against the heat-killed Re mutant of Salmonella typhimurium, was in part similar to that of mAb 26-20. However, inhibition of binding with PMX was less efficient and a high specificity for ReLPS, also after membrane incorporation of this Ag, was demonstrated. In contrast to mAb 26-5 and 26-20, mAb 8-2 showed extensive cross-reactivity with heterologous LPS preparations and heat-killed as well as live Gram-negative bacteria. It is concluded that each of the three mAb binds to a different antigenic epitope in lipid A and that exposure of those epitopes for antibody binding is restricted in a differential manner, depending on mode of Ag presentation. The here defined reaction patterns provide a basis for the interpretation of potential inhibitory effects on in vitro and in vivo biologic (and toxic) activities of endotoxins and Gram-negative bacteria.     

Human macrophages produce dimeric forms of IL-18 which can be detected with monoclonal antibodies specific for inactive IL-18

We established two monoclonal antibodies (mAbs) which specifically recognize human 'functionally inactive' recombinant IL-18, and IL-18 protein polymorphism was examined using human monocytes and macrophages (M phi). In 6 day GM-CSF-treated M phi, an 'inactive' IL-18-recognizing mAb 21 detected the IL-18 proform (24 kDa) and a 48-kDa protein, which were gradually increased concomitant with maturation stage. Majority of the 24- and 48-kDa forms were barely detectable with other mAbs recognizing 'active' IL-18. No reagents including Toll stimulators up-regulated these IL-18 populations in M phi. The 21-recognizable IL-18 species were separated using an anion-exchanger column and their IFN gamma-inducing activity was assessed with human lymphocytes plus IL-12. Virtually no as yet known activity was detected with these IL-18 species. After processed with M phi proteases, an 18-kDa form was generated to express the IFN gamma-inducing activity, although the activity was far weaker than that of control 'active' IL-18. These observations suggested that large amounts of various IL-18 species are produced with monocyte-M phi differentiation and most of these IL-18 species are functionally 'inactive' in terms of the reported IL-18 function even after proteolytic 18-kDa conversion.     

Human monoclonal anti-idiotypic antibodies. I. Establishment of immortalized cell lines from a tumor patient treated with mouse monoclonal antibodies

Patients who undergo immunotherapy with a murine anti-colon carcinoma mAb (mAb17-1A) generate high titers of anti-idiotype and anti-isotype antibodies. Specifically selected anti-idiotypic antibodies that elicit in vivo a humoral and a cellular immune response against the nominal Ag can be used as surrogate Ag for immunization. We established from the B lymphocytes of a treated patient a series of EBV-transformed cell lines. Three weeks after immortalization, the cells were selected for production of antibodies (Ab2) against the Fab fragment of the murine mAb17-1A. The selected cells were cloned and screened by ELISA for specific anti-mAb17-1A idiotypic antibodies. Thirty-six out of 89 clones were anti-idiotypes. Cell culture supernatants and the purified Ig derived from 10 clones completely inhibited the specific binding of radiolabeled mAb17-1A to HT-29 colon carcinoma cells thus resembling Ab2-gamma anti-idiotypes. These cell lines which grow now in culture for 18 mo, continuously secrete IgG,K anti-Ab1-idiotype mAb. Human anti-idiotypic mAb might be candidates for vaccines when the nominal Ag itself is not available or cannot be used as such.     

Yeast Killer Toxin-Like Candidacidal Ab6 Antibodies Elicited through the Manipulation of the Idiotypic Cascade

A mouse anti-anti-anti-idiotypic (Id) IgM monoclonal antibody (mAb K20, Ab4), functionally mimicking a Wyckerhamomyces anomalus (Pichia anomala) killer toxin (KT) characterized by fungicidal activity against yeasts presenting specific cell wall receptors (KTR) mainly constituted by β-1,3-glucan, was produced from animals presenting anti-KT Abs (Ab3) following immunization with a rat IgM anti-Id KT-like mAb (mAb K10, Ab2). MAb K10 was produced by immunization with a KT-neutralizing mAb (mAb KT4, Ab1) bearing the internal image of KTR. MAb K20, likewise mAb K10, proved to be fungicidal in vitro against KT-sensitive Candida albicans cells, an activity neutralized by mAb KT4, and was capable of binding to β-1,3-glucan. MAb K20 and mAb K10 competed with each other and with KT for binding to C. albicans KTR. MAb K20 was used to identify peptide mimics of KTR by the selection of phage clones from random peptide phage display libraries. Using this strategy, four peptides (TK 1-4) were selected and used as immunogen in mice in the form of either keyhole limpet hemocyanin (KLH) conjugates or peptide-encoding minigenes. Peptide and DNA immunization could induce serum Abs characterized by candidacidal activity, which was inhibited by laminarin, a soluble β-1,3-glucan, but not by pustulan, a β-1,6-glucan. These findings show that the idiotypic cascade can not only overcome the barrier of animal species but also the nature of immunogens and the type of technology adopted.