Clonality of cold agglutinins in patients with hemolytic anemia: an analysis by high-resolution two-dimensional gel electrophoresis.

High-resolution two-dimensional gel electrophoresis (2-DGE) was used to analyse plasma samples and partially purified cold agglutinins (CA) obtained from two selected patients. Both presented an acute hemolytic anemia with CA of high thermal amplitude, normal immunoglobulin levels, no detectable paraproteinemia, and no clinical evidence of a malignant B-cell disorder. The electrophoretograms of their plasma showed evident alternations of the "normal" protein profile, which were directly related to hemolysis (absence of the spots of haptoglobin and in one case of those of hemopexin), but no monoclonal gammopathy. The electrophoretograms of their purified CA revealed two clearly different spot patterns respectively corresponding to a monoclonal IgM and to polyclonal IgM. These results show that the clonality of CA associated with hemolytic anemia can be easily determined by 2-DGE. This technique may be very useful to discriminate chronic cold agglutinin disease in the early phase from "parainfectious" CA.     

Histochemical study of pituitary adenomas with Ki-67 and anti-DNA polymerase α monoclonal antibodies,bromodeoxyuridine labeling,and nucleolar organizer region counts

Summary The growth potential of 65 pituitary adenomas was determined by histochemical analysis with Ki-67 and anti-DNA polymerase monoclonal antibodies, bromodeoxyuridine (BrdUdR) labeling, and counts of argyrophilic nucleolar organizer regions (Ag-NORs). The mean proliferating cell indices (PCIs) determined by Ki-67 and anti-DNA polymerase and the BrdUdR labeling index (LI) were generally very low [1.0±0.2%, 1.1±0.2%, and 0.5±0.1% (±SE), respectively]. Apart from adrenocorticotropic hormone-positive adenomas, which had significantly higher indices, there were no statistically significant differences in the indices among the other subtypes of pituitary adenomas. Recurrent tumors had higher Ki-67 and DNA polymerase PCIs and BrdUdR LIs (3.6%, 4.2%, 1.4%) than primary tumors (0.8%, 0.8%, 0.3%; P<0.005). The number of Ag-NORs did not correlated significantly with any of the three indices. The mean number of Ag-NORs was higher in nonfunctioning adenomas than in functioning adenomas (2.04 vs 1.66, P<0.005); among prolactin-positive adenomas, those treated preoperatively with bromocriptine had more Ag-NORs than untreated tumors (1.75 vs 1.57, P<0.005). These results suggest that the Ki-67 and DNA polymerase PCIs and the BrdUdR LI predict the growth potential of individual pituitary adenomas, whereas the number of Ag-NORs appears to correlate with hormone production rather than with the proliferative potential.Supported by grants CA-13525 and CA-50210 from the National Cancer Institute, by a grant from the Phi Beta Psi Sorority, and by Grant-in-Aid A 63771083 from the Ministry of Education, Science, and Culture of Japan     

GD3 expression by cultured human tumor cells of neuroectodermal origin

Summary Seven monoclonal antibodies (mAbs) reactive with ganglioside II³(NeuAc)₂-LacCer (GD3) were generated; four of these mAbs (DMAb-21, DMAb-22, DMAb-23, and DMAb-24) by immunizing mice with GD3 adsorbed to Salmonella minnesota and the remaining three (DMAb-7, DMAb-8, and DMAb-17) with melanoma line SK-MEL 28, which contains 1.4 nmol sialic acid of GD3 per mg protein. The specificities of the mAbs were defined by high-performance thin-layer chromatography (HPTLC) immunostain and solid-phase radioimmunoassay (SP-RIA) with a panel of purified gangliosides. DMAb-7 and DMAb-8 reacted with GD3, IV³(NeuAc)₂nLcOse₄Cer(3,8-LD1), and very weakly with IV³(NeuAc)₂II³NeuAc-GgOse₄Cer (GTla), but not with II³NeuAc-LacCer (GM3), II³NeuAcGgOse₃Cer(GM2), II³NeuAc-GgOse₄Cer(GM1), II³NeuAc, IV³NeuAcGgOse₄Cer (GD1a), II³(NeuAc)₂GgOse₃(GD2), II³(NeuAc)₂GgOse₄Cer (GD1b), IV³NeuAcII³(NeuAc)₂, GgOse₄Cer(GT1b), suggesting the binding epitope to be a terminal tetrasaccharide NeuAc2-8NeuAc2-3Gal1-4(Glc or GlcNAc). DMAb-7 and DMAb-8 were used to investigate the expression of GD3 on cultured human tumor cells of neuroectodermal origin. Thirteen of 19 gliomas, 3 of 5 medulloblastomas, 5 of 5 neuroblastomas, 2 of 2 melanomas, and 1 of 3 teratomas were shown to react with DMAb-8 and/or DMAb-7 by cell surface-RIA (CS-RIA) and immunofluorescence (IF) assays. HPTLC and densitometric analysis confirmed these results, as positive immunostains in the GD3 region were obtained with oligoganglioside fractions from 9 glioma, 1 medulloblastoma, 2 neuroblastoma, 1 melanoma, and 1 teratoma cell line. Glioma cell line U-105 MG and medulloblastoma cell line Daoy contain GD3 as shown by HPTLC immunostain analysis of extracts, although GD3 was undetectable on the cell surface as determined by CS-RIA and IF. There was no detectable GD3 found in gangliosides isolated from cell lines U-373 MG, D-54 MG, TE-671, and PA-1, which were negative for both DMAb-7 and DMAb-8 by CS-RIA and IF assay. Our results provide evidence that GD3 is expressed extensively with significant quantitative heterogeneity on cultured human neuroectodermal tumor cells including glioma, medulloblastoma, neuroblastoma, and melanoma.Supported by NIH grants R37 CA11898, NS 20023, and CA32672 and by grants from the Swedish Medical Research Council (project no. 03X-627), Swedish Cancer Society (project no. 2260-B88-01X) and the National Swedish Board for Technical Development (project no. 84-4667)     

Demonstration of microglial cells in and around senile (neuritic) plaques in the Alzheimer brain

Summary A monoclonal antibody, termed AD11/8, reactive to microglial cells, was produced by immunization of mice with partially purified amyloid fibrils of senile (neuritic) plaques. With immunoperoxidase staining on human tissues, AD11/8 also recognized macrophages in the red pulp of the spleen, Kupffer cells in the liver, and macrophages in the bone marrow. The results show that AD11/8 recognizes the antigens associated with mononuclear phagocytes lineage. In normal brains a few resting microglial cells were stained in gray matter, and less frequently in white matter. In senile dementia of the Alzheimer type numerous microglial cells were stained intensively and they often formed clusters in gray matter. By double immunostaining with AD11/8 and a polyclonal antibody against synthetic amyloid -protein, clustered microglial cells were observed in and around senile plaques with amyloid deposits. Some amyloid plaque cores were surrounded by microglial cell processes. These results indicate that microglial cells may play an important role in senile plaque formation.Supported in part by the Grant-in Aid for Scientific Research from the Ministry of Education, Science and Culture, the grants for Research of Dementia and for Primary Amyloidosis from the Ministry of Health and Welfare, Japan     

Development of an ELISA for detection of an organophosphorus compound using monoclonal antibodies

This paper describes a specific and highly sensitive ELISA system using monoclonal antibodies in order to assay an organophosphorus compound. The soman derivative methyl phosphonic acid, p-aminophenyl 1,2,2,-trimethyl-propyl diester (MATP) served as model substance. In order to obtain antibody-producing hybridomas BALB/c mice were immunized with MATP linked onto human serum albumin (HSA). The spleen cells of immunized mice were fused with syngenic plasmacytomas of the non-producer-line X63Ag8.653 with the aid of polyethylene glycol. To eliminate undesirable cross-reaction, common screening procedures were modified by directly coating the ELISA plates with hapten. Five out of 15 positive cell-lines were cloned by limiting dilution and further propagated. The respective immunoglobulin class and subclass of the obtained monoclonal antibodies was determined. Four of which were identified as IgG₁, the other as IgG_2a. After enrichment of antibodies in ascites and their isolation by protein A-sepharose, the affinity of various monoclonal antibodies was estimated in competitive inhibition enzyme immunoassay (CIEIA) by measuring the IC₅₀ rates of free MATP. The rates were found to lie between 2.5 × 10^–6 mol/l and 4.3 × 10^–4 mol/l MATP. The IC₁₀ rate for detectable MATP concentration was 5.4 × 10^–7 mol/l MATP. Test duration was 280 min. The reactivity of the monoclonal antibodies with structurally related substances was used to check their specificity. Cross-reaction turned out to be negative. In order to develop a direct competitive ELISA, MATP was linked to horse radish peroxidase (HRPO) by adding a spacer. This helped to reduce total duration to 40 min. The detection level was further reduced to 1.3 × 10^–7 mol/l MATP (corresponding to 975 pg/25 l test-buffer) using the monoclonal antibody F71D7. Likewise, MATP was detected in goat serum, chicken serum, rabbit serum, milk and company's water in concentrations between 2.1 × 10^–7 mol/l (IC₁₀, company's water) and 4.9 × 10^–8 mol/l (IC₁₀, milk).     

Immunoglobulin mimicry by Hepatitis C Virus envelope protein E2

Hepatitis C virus (HCV) establishes persistent infection in the majority of infected individuals. The currently accepted hypothesis of immune evasion by antigenic variation in hypervariable region 1 (HVR1) of glycoprotein E2 does not however, explain the lack of subsequent immune recognition. Here, we show that the N-terminal region of E2 is antigenically and structurally similar to human immunoglobulin (Ig) variable domains. E2 is recognized by anti-human IgG antibodies and also possesses common amino acid (aa) sequence features of the conserved v-gene framework regions of human Ig light chains in particular but also heavy chains and T cell receptors. Using a position specific scoring system, the degree of similarity of HVR1 to Ig types correlated with immune escape and persistence in humans and experimentally infected chimpanzees. We propose a unique role for threshold levels of Ig molecular mimicry in HCV biology that not only advances our concept of viral immune escape and persistent infection but also provides insight into host-dependent disease patterns.     

Epitope mapping and biological function analysis of antibodies produced by immunization of mice with an inactivated Chinese isolate of severe acute respiratory syndrome-associated coronavirus (SARS-CoV)

Inactivated severe acute respiratory syndrome-associated coronavirus (SARS-CoV) has been tested as a candidate vaccine against the re-emergence of SARS. In order to understand the efficacy and safety of this approach, it is important to know the antibody specificities generated with inactivated SARS-CoV. In the current study, a panel of twelve monoclonal antibodies (mAbs) was established by immunizing Balb/c mice with the inactivated BJ01 strain of SARS-CoV isolated from the lung tissue of a SARS-infected Chinese patient. These mAbs could recognize SARS-CoV-infected cells by immunofluorescence analysis (IFA). Seven of them were mapped to the specific segments of recombinant spike (S) protein: six on S1 subunit (aa 12-798) and one on S2 subunit (aa 797-1192). High neutralizing titers against SARS-CoV were detected with two mAbs (1A5 and 2C5) targeting at a subdomain of S protein (aa 310-535), consistent with the previous report that this segment of S protein contains the major neutralizing domain. Some of these S-specific mAbs were able to recognize cleaved products of S protein in SARS-CoV-infected Vero E6 cells. None of the remaining five mAbs could recognize either of the recombinant S, N, M, or E antigens by ELISA. This study demonstrated that the inactivated SARS-CoV was able to preserve the immunogenicity of S protein including its major neutralizing domain. The relative ease with which these mAbs were generated against SARS-CoV virions further supports that subunit vaccination with S constructs may also be able to protect animals and perhaps humans. It is somewhat unexpected that no N-specific mAbs were identified albeit anti-N IgG was easily identified in SARS-CoV-infected patients. The availability of this panel of mAbs also provided potentially useful agents with applications in therapy, diagnosis, and basic research of SARS-CoV.     

Effect of moderate exercise on salivary immunoglobulin A and infection risk in humans

The incidence of upper respiratory tract infections (URTI) and salivary immunoglobulin A concentrations [IgA_s] of nine individuals were examined during 12 weeks of moderate exercise training, and compared to ten sedentary controls. Changes in maximal oxygen uptake were assessed at initial, mid-point and final evaluations (T1–3), while changes in [IgA_s] and salivary immunoglobulin concentration-salivary albumin concentration ratio ([IgA_s]:[Alb_s]) were monitored at T1 and T3. During the 12 week period, symptoms of URTI were self-recorded daily. During the period of training the level of fitness significantly increased (P<0.05) in the exercise group. The number of days recording symptoms of influenza, but not of cold, and total light URTI symptoms was significantly reduced in the exercise group during the last weeks of training. A significant increase in [IgA_s] and in [IgA_s]:[Alb_s] was found in the exercise group after training. Both [IgA_s] and [IgA_s]:[Alb_s] were significantly related to the number of days showing symptoms of influenza (P<0.01) and the total number of days of sickness (P<0.05). These data provide quantitative support for the belief that regular, moderate exercise results in an increased [IgA_s] at rest and [IgA_s]:[Alb_s], which may contribute to a decreased risk of infection. Electronic Publication     

Historical evolution,overview, and therapeutic manipulation of co-stimulatory molecules

Co-stimulatory molecules are key mediators in the regulation of immune responses and knowledge of its different families, structure, and functions has improved in recent decades. Understanding the role of co-stimulatory molecules in pathological processes has allowed the development of strategies to modulate cellular functions. Currently, modulation of co-stimulatory and co-inhibitory molecules has been applied in clinical applications as therapeutic targets in diseases and promising results have been achieved.     

Stereospecific monoclonal antibodies to nicotine and cotinine and their use in enzyme-linked immunosorbent assays

Stereospecific monoclonal antibodies (McAb) have been prepared against the tobacco alkaloid (S)-(-)-nicotine and its major metabolite (S)-(-)-cotinine. Nine anti-nicotine and 4 anti-cotinine hybridomas, selected by a screening procedure that utilized immunoprecipitation of the ³H-labeled natural isomers of nicotine or cotinine, were grown in the ascites fluid of pristane-primed syngeneic BALB/c mice. Antibodies in concentrations up to 7.5 mg/ml ascites and with binding affinities that generally exceeded 10⁸ M⁻¹ were obtained. Enzyme-linked immunosorbent assays (ELISAs) were developed in which nicotine or cotinine derivatives bound covalently to poly- -lysine werecoated onto wells of polyvinyl chloride microtiter plates. Coated wells were incubated sequentially with McAb in the presence or absence of inhibitor, rabbit anti-mouse immunoglobulin, then horseradish peroxidase-labeled protein A (HRP-SpA) before addition of substrate. The antibodies are highly specific and show minimal cross-reactivity with several nicotine metabolites and other structurally related compounds. In the respective assays, only 0.25 ng (S)-(-)-nicotine and 0.12 ng (S)-(-)-cotinine are required to give 50% inhibition of antibody binding, and as little as 0.05 ng nicotine and 0.02 ng cotinine give 15% inhibition. These assays are 5–10 times more sensitive than analogous ELISAs developed with rabbit antisera and HRP-SpA or conventional radioimmunoassays (RIAs) that utilize the rabbit antisera and ³H-labeled ligands. There was good correlation between the levels of nicotine (r = 0.967) found in saliva samples from smokers and non-smokers assayed by McAb-based ELISAs and conventional RIAs.