Murine complement C4 is not required for experimental autoimmune encephalomyelitis.

In vitro studies have demonstrated that myelin and myelin-derived proteins activate both the classical and alternative complement pathways. More recently, studies have shown that mice deficient in factor B, a protein required for activation of the alternative pathway, have attenuated experimental autoimmune encephalomyelitis (EAE), the animal model for multiple sclerosis. The relative contribution of the classical pathway to the pathogenesis of EAE has remained unexplored. To address this question, we performed EAE using mice deficient in C4 (C4-/-), a protein required for full activation of the classical pathway. We found that deletion of the C4 gene does not significantly change either the time of onset or the severity and tempo of myelin oligodendrocyte-induced EAE compared with controls with a fully intact complement system. We observed similar levels of cellular infiltration (CD11b+ macrophages and CD3+ T cells) and demyelination in the two kinds of mice. Despite this, ribonuclease protection assays demonstrated a two- to fourfold increase in several pro-inflammatory cytokines in C4-/- mice with EAE, including interleukin-beta (IL-1beta), IL-18, tumor necrosis factor-alpha (TNF-alpha), IP-10, and RANTES. These results support the conclusion that the contribution of murine complement to the pathogenesis of demyelinating disease is realized via the alternative pathway.

Identification and characterization of linear B-cell epitopes of beta-globulin, a major allergen of sesame seeds.

BACKGROUND: The increased consumption of foods containing sesame seeds is paralleled by an increase in reported sesame-induced allergic reactions. OBJECTIVE: This study aimed at identifying and characterizing the linear B-cell epitopes of the 14-kd beta-globulin, the major allergen of sesame seed. METHODS: A peptide containing 71 amino acids (peptide B) was previously identified by us as the IgE-binding region on beta-globulin. To determine the amino acid sequence of the IgE-binding sites on peptide B, we synthesized overlapping peptides 20 and 10 amino acid residues long that span the entire length of peptide B, which were offset from each other by 10 and 2 amino acid residues, respectively. Sera from 20 subjects given diagnoses of allergy to sesame beta-globulin served to identify the epitopes by using the dot-blot test. RESULTS: At least 9 different IgE-recognition sites were identified on peptide B. Three of them, numbers 2, 3, and 13 (corresponding to amino acids 46-55, 48-57, and 76-86, respectively, in the beta-globulin sequence), appeared to be immunodominant IgE-binding epitopes. Also, these peptides were best recognized in terms of intensity of response. There was no obvious sequence motif shared by the 9 different IgE-binding epitopes of beta-globulin. However, approximately 60% of the amino acids represented in the epitopes are hydrophobic residues. CONCLUSION: Identification of the IgE-binding epitopes might provide a better understanding of the functional role the allergens play in the disease and might have implications for immunodiagnosis and probably immunotherapy.

Comparison of methods for immobilization to carboxymethyl dextran sensor surfaces by analysis of the specific activity of monoclonal antibodies.

The authors have recently described the development of a carboxymethyl dextran-based sensor surface for biospecific interaction analysis by surface plasmon resonance. Ligands are immobilized via primary amine groups after activation of the carboxymethyl groups on the sensor surface with a mixture of N-hydroxysuccinimide and N-ethyl-N'-(dimethylaminopropyl) carbodiimide. Methods have now been developed for efficient immobilization via thiol/disulfide exchange, aldehyde coupling and biotin-avidin coupling. The specific activity of monoclonal antibodies immobilized by the four different methods was investigated by altering the immobilization conditions, e.g., activation time, protein concentration, ionic strength and the degree of modification, etc. Investigations have also been made concerning possible differences in the specific activity for antibodies immobilized using optimized conditions with respect to the four different chemistries. These studies show that, with the flexible carboxymethyl dextran matrix used here, the immobilization methods give rise to only minor differences in specific activity. Thus, with this solid support, a 'site directed' immobilization strategy for monoclonal antibodies has no advantage. In general the specific activity for optimized systems was approximately 75% for the binding of beta 2 mu-globulin to an immobilized monoclonal antibody directed against beta 2 mu-globulin. Reduced specific activities of immobilized antibodies induced by variation of the coupling conditions could be attributed to the deterioration of the active site of the antibody.

A glycoprotein secreted by lung cancer cells is present in human serum as an immunoglobulin-binding protein.

The 6B3-Ag recognized by a monoclonal antibody 6B3 to human large cell lung carcinoma cell line (HLC-2) is a high-molecular-weight glycoprotein of 1,000,000. Its serum level is increased in various adenocarcinoma patients. When a patient's serum with a high concentration of 6B3.Ag (54 micrograms/ml) or concentrated 6B3.Ag from normal human serum was analyzed by immunoelectrophoresis, 6B3.Ag showed a long bimodal precipitin line extending from the per-beta to beta globulin region. However, the precipitin line of 6B3.Ag in the HLC-2 culture medium was formed only in the pre-beta globulin region. The 6B3.Ag was purified from pooled patients' serum by salting out, precipitation by acidification at pH 4.5 and Sepharose 4B and immunoaffinity chromatographies. Western blotting indicated that the 6B3.Ag from human serum contained IgG and/or IgM. The 6B3.Ag from human serum showed a dose-dependent reaction in a sandwich enzyme-linked immunosorbent assay with anti-6B3.Ag antibody as a solid-phase antibody and anti-human IgG or anti-human IgM antibody labeled with alkaline phosphatase. The 6B3.Ag was concluded to be partly present as a complex with IgG and/or IgM in human serum, and this complex showed a precipitin line in the beta globulin region on immunoelectrophoresis.

[Placental-spermatic beta 2-globulin: its immunochemical identification, physicochemical characteristics and localization in the reproductive system]. / Platsentarno-spermal'nyi beta 2-globulin: immunokhimicheskaia identifikatsiia, fiziko-khimicheskaia kharakteristika i ego lokalizatsiia v reproduktivnoi sisteme.

A new antigen with beta 2-globulin mobility and M. W. of 20 kD, named placenta-sperm beta-globulin (PSBG), was identified in human early placental tissue. Using immunodiffusion method, PSBG was found in the seminal plasma with the concentration about 120 mg/l, in the amniotic fluid 1-5 mg/l, in the liquor 1-10 mg/l, and also in fetal kidney and stomach extracts. The examination of the male reproductive system revealed PSBG levels in the extracts of adults and fetus.

Identification of a brain-specific human cerebrospinal fluid glycoprotein, beta-trace protein.

A prominent human cerebrospinal fluid (CSF) protein, P5, identified at mass 19-24 kDa and charge 5.5, by two-dimensional electrophoresis (2DE) and silver staining, has been previously demonstrated to be reduced in quantity in the CSF of patients with multiple sclerosis and schizophrenia. We report the purification and partial amino acid sequences from five tryptic fragments of P5. These sequences are not those of any known sequence in the Protein Identification Resource (PIR release 31) database. Synthetic peptides from two of the sequences were used to raise rabbit polyclonal antibodies. These antibodies detected P5 on 2DE blots of normal CSF proteins and other proteins of the same mass with a charge distribution between 5.17-8.5. These proteins comprise 5-10% of the total CSF protein and their mass, charge, abundance and predominance in CSF over plasma are consistent with a protein that had been initially characterized with antibodies, beta-trace protein. Glycosidase studies confirm that most of these proteins are due to sialic acid modifications that are N-linked to an 18 kDa protein, but other charge and mass variations also exist. 2DE blots of 26 types of human tissue and body fluid were immunostained. Of these, anti-P5 serum detected proteins of the same mass and charge as beta-trace protein only in brain samples. Proteins of different mass and charge from beta-trace protein were clearly immunostained in samples of eight tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

[Cellular localization of human secretory beta globulin in normal and tumor tissues]. / Issledovanie kletochnoi lokalizatsii sekretornogo beta-globulina cheloveka v normal'nykh i opukholevykh tkaniakh.

Distribution of secretory beta-globulin (S beta G) which possesses affinity for steroids was investigated immunohistochemically. Tissue specificity of S beta G, produced in adult secretory epithelial cells of the seminal vesicles, salivary glands, prostate, bronchi and mammary gland was discovered. The protein was not detected in fetal and embryonal tissues. S beta G synthesis is abnormal in neoplasms: its expression partly preserves in breast cancer cells and increases in epithelium of mammary ducts near the focus of malignancy. In lung cancer and bronchial glands cells near the focus of neoplastic transformation S beta G positive reaction was not observed.

Isolation and amino terminal sequence of beta-trace, a novel protein from human cerebrospinal fluid.

beta-Trace, a 23.5 kDa glycoprotein of unknown biological functions, is present in all body fluids tested. It is found in higher concentration in human seminal fluid and cerebrospinal fluid (CSF) than in serum. A one-step procedure for the isolation of beta-trace from pooled CSF is described, by affinity chromatography using a specific antibody made against beta-trace. Amino terminal sequence analysis yields the sequence A P E A Q V S V Q P N F Q Q D K F L G with no homology to known proteins, indicating that beta-trace is a novel CSF protein.

[Evaluation of fetal antigenic stimulation in physiological and complicated pregnancy]. / Otsenka fetal'noi antigennoi stimuliatsii pri fiziologicheski protekaiushchei i oslozhnennoi beremennosti.

Fetal hemoglobin and trophoblastic beta 1-globulin levels have been determined in women with normal and complicated pregnancy in order to evaluate the rates of fetal antigenic stimulation. It was demonstrated that trophoblastic beta 1-globulin may not exactly represent fetal antigenic structures, but it is a marker of functional feto-placental activity. An elevated percentage of HbF in blood in threatened+ abortion and early toxemia of pregnancy confirmed feasibility of using it as an indirect marker of fetal antigenic stimulation and permeability of the fetoplacental barrier.

[The immunochemical identification of beta-globulin in human seminal plasma]. / Immunokhimicheskaia identifikatsiia beta-globulina v semennoi plazme cheloveka.

Using affinity chromatography there was isolated a protein from human seminal plasma, which was named beta-globulin of seminal plasma (beta-GSP). It was demonstrated, that beta-GSP possesses heterogeneity, consists of two subunits with the M. W. about 19 and 15 kD, it has electrophoretic mobility of b1-globulin and possesses affinity to immobilized 17-beta-estradiol. Relatively high concentration of beta-GSP was found in seminal plasma. Besides, this protein was determined in small amounts in saliva. One can suppose, that beta-GSP is a secretory protein.

A gene locus controlling a serum protein migrating electrophoretically in the beta region of mice and detected by using a strain derived from the Japanese wild mouse (Mus musculus molossinus).

Antigenic specificities of serum proteins from the MOL-ANJ strain of mice (a strain derived from Japanese wild mice, Mus musculus molossinus) were studied by gel precipitation with alloantisera produced by reciprocal alloimmunization between MOL-ANJ and BALB/c mice. An alloantigen which migrates immunoelectrophoretically in the beta region of serum proteins has been identified. Evidence indicates that this antigenic specificity is controlled by a co-dominant autosomal gene locus designated by the symbol Sas-2. The evidence also suggests that Sas-2 is genetically different from the previously described Sas-1 which controls a serum protein in the mouse. Sas-2 was located by linkage analysis between Idh-1 locus and Akp-1 locus on chromosome 1.

A precipitin for human serum proteins as released under stress by a marine invertebrate, the marsh snail, Littorina irrorata.

A substance released into the environment by stressed marsh snails, Littorina irrorata, precipitates with the beta globulin and albumin fractions of human serum proteins. Other diverse invertebrate species may produce a similar substance. Invertebrate precipitins have not been widely reported, and none like the one described. Some physicochemical and adaptive characteristics of the snail precipitin are presented. This precipitin can be produced in large quantities and has potential application as a diagnostic reagent.

Antigen-specific non-immunoglobulin "antibody'-like molecules in rhesus monkey serum.

The presence (in sera of rhesus monkeys) of four antigen-specific molecules with different electrophoretic mobilities apart from conventional immunoglobulins has been recognized. Rhesus monkeys have been primed with rabbit erythrocytes (RRBC) and the antigen-specific molecules in the serum have been fished out by absorption onto RRBC. Antisera have been raised in rabbits against such molecules and analysed by immunoelectrophoresis. The antigen-binding property of the four molecules, which do not resemble conventional immunoglobulins has been confirmed by enzyme immunoelectrophoresis. The possibility of these molecules being products of T lymphocytes in the light of observations reported in recent years is discussed.

The thymic differentiation markers T6 and M241 are two unusual MHC class I antigens.

The human beta 2-microglobulin (beta-2m)-associated human thymocyte differentiation antigens T6 and M241 were compared using biochemical techniques. T6 and M241 antigens reside on different molecules with apparent m.w. of 49,000 and 43,000, respectively. Here we show that both proteins have a protein backbone m.w. of 33,000. In addition, T6 and M241 have a large portion of their peptides in common. When we compared the protein backbone m.w. of T6 and M241 with the murine beta-2m-associated thymus leukemia (TL) antigens, we found a considerable difference in size, suggesting that T6 and M241 may not be human homologues of TL antigens and constitute a novel type of major histocompatibility (MHC) class I antigens.

Arginine 45 is a major part of the antigenic determinant of human beta 2-microglobulin recognized by mouse monoclonal antibody BBM.1.

Monoclonal antibody BBM.1 (Brodsky, F.M., Bodmer, W. F., and Parham, P. (1979) Eur. J. Immunol. 9, 536-545) identifies an antigenic determinant of human beta 2-microglobulin (beta 2-M). The antibody binds free and HLA-A,B-associated beta 2-M with similar affinity, showing that the BBM.1 antigenic determinant does not involve residues of beta 2-M that interact with HLA-A,B heavy chains. Peptides (SWH.1-5) synthesized from residues 35-50 of the beta 2-M sequence specifically inhibit the binding of BBM.1 to cell surfaces. Their inhibitory activity is destroyed by trypsin treatment. The observations (i) that BBM.1 does not bind to beta 2-M of species other than man, gorilla, and chimpanzee and (ii) that arginine 45 is the only human-specific residue between positions 35 and 50 suggested that this residue might be part of the BBM.1 antigenic determinant. This hypothesis was confirmed by reversible modification of arginine residues with cyclohexanedione. Modification of arginines in native beta 2-M and of the single arginine, corresponding to position 45, in the peptide SWH.5 resulted in up to 95% loss of BBM.1 inhibitory activity. Reversal of the modification by treatment with hydroxylamine resulted in complete recovery of activity. Rabbit antibodies elicited by immunization of SWH.5 conjugated to bovine serum albumin showed no detectable reaction with native beta 2-M but did specifically react with human beta 2-M after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoresis onto nitrocellulose. These results thus identify a region around residue 45 of the beta 2-M polypeptide which is exposed to the environment and not involved in binding HLA-A,B heavy chain. Analysis of the beta 2-M sequence by calculating local hydrophilicity indices (Hopp, T. P., and Woods, K. R. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 3824-3828) agree with this region being a major antigenic determinant. Models of beta 2-M structure as an immunoglobulin domain show this region of polypeptide to be part of a loop between the two layers of beta-pleated sheet, also consistent with it being a major antigenic determinant. The position of the loop favors a model in which beta 2-M interacts with HLA across the four-stranded beta-pleated sheet like an immunoglobulin constant region domain.

Intramolecular organization of Class I H-2 MHC antigens; localization of the alloantigenic determinants and the beta 2 m binding site to different regions of the H-2 Kb glycoprotein.

The intramolecular organization of the membrane integrated Class I major histocompatibility complex (MHC) molecule H-2Kb (Kb) was analyzed. After the removal of the two carbohydrate moieties by glycosidase enzymes, proteolytic digestion of the Kb molecule yielded: 1) several fragments with the beta 2 microglobulin (beta 2 m) subunit still bound and 2) one fragment carrying alloantigenic activity but lacking the beta 2 m. Isolation of the beta 2 m binding fragments showed them to be derived from the C-2 domain by partial N-terminal sequence analysis. One fragment extended to the C-terminus and the other fragment had lost the transmembrane region. Such studies conclusively show that the beta 2 m subunit is bound in the third domain, i.e., C-2, of the Kb 44,000 m.w. heavy chain. The alloantigenic fragment also isolated from the proteolytic digest consists of the first 180 residues of the 44,000 m.w. heavy chain, i.e., domains N and C-1, and carried alloantigenic determinants detected by several monoclonal antibodies as well as alloantisera. The present studies indicate that the external region of the Class I molecules has two functional regions. The first 180 residues bear the recognition elements for the immune system, and the next 90 residues (180-270) are involved in binding to beta 2 m.

Transformation of murine LMTK- cells with purified HLA class I genes. I. Modification of conformation of murine beta 2-microglobulin upon its association with HLA heavy chains.

Murine LMTK- cells were unexpectedly found to cross-react with a murine anti-human beta 2-microglobulin (beta 2-m)monoclonal antibody (m.Ab) after transformation with cosmid clones containing different purified HLA class I genes. The same cross-reactivity was observed with CTP 34 B4 (murine x human) somatic hybrid cells, which express class I molecules constituted of human HLA heavy chains and murine beta 2-m. Inhibition studies of the complement-dependent cytolysis mediated by the cross-reacting m.Ab indicated that isolated murine beta 2-m does not express the cross-reacting determinant, suggesting that its expression by the transformed cells reflects conformational modification of murine beta 2-m upon its association with HLA heavy chains. These results illustrate one of the possible post-translational mechanisms through which the antigenicity of a polypeptide chain can be modified. They might provide a serologic marker of the third domain of HLA class I heavy chains. Finally, because quantitative differences of reactivity with the anti-human beta 2-m m.Ab were observed, depending on the HLA class I genes used for transformation, these results individualize two families of HLA class I heavy chains responsible for different conformational modifications of murine beta 2-m.

Associated peptides isolated from human seminal fluid containing beta 2-microglobulin determinants.

Proteins in human semen that react with a monoclonal mouse antibody to human beta 2-microglobulin (h beta 2M) have been isolated using immunoaffinity chromatography. As well as material of the same size as h beta 2M, a peptide of approximately 18000 kdaltons was isolated. It is suggested that this substance either contains h beta 2-M sequences itself or, as is more likely, is non-covalently associated with an h beta 2M-like peptide chain.