Distinction of two subtypes of human leukocyte interferon (IFN-alpha) on B cell activation. B cell proliferation by two subtypes of IFN-alpha

We examined the effect of interferon (IFN), with particular emphasis on the effects of the two subtypes of IFN-alpha (IFN-alpha A and IFN-alpha B) on the B cell proliferation induced by Staphylococcus aureus Cowan I bacterium (SpA Col). An increase of SpA Col-induced proliferation was observed in the presence of 100 to 1000 U/ml of IFN-alpha, but a decrease of SpA Col-induced proliferation was observed in the presence of 1000 to 10,000 U/ml of IFN-beta. The two subtypes of IFN-alpha had different effects on cell proliferation; a significant enhancement was shown in the presence of 1000 to 10,000 U/ml of IFN-alpha A, but inhibition was shown in the presence of 1000 to 10,000 U/ml of IFN-alpha B. In the reconstitution test of the two subtypes of IFN-alpha, the boundary between enhancement and inhibition of SpA Col-induced proliferation was revealed when the proportion of IFN-alpha A and IFN-alpha B (IFN-alpha A:IFN-alpha B) ranged between 8:2 and 9:1. Toward the SpA Col-induced responses, the above IFN were all found to act on B cells directly, independent of the presence of T cells. Proliferative responses by IFN-alpha and IFN-alpha A, however, were shown to be slightly dependent on the presence of monocytes. The lymphocyte proliferation induced by other mitogens (phytohemagglutinin, concanavalin A, pokeweed mitogen, and protein A of S. aureus) were all inhibited by the above IFN.     

Universal antibodies against the highly conserved influenza fusion peptide cross-neutralize several subtypes of influenza A virus

Dialysis related amyloidosis (DRA) is a serious complication to long-term hemodialysis treatment which causes clinical symptoms such as carpal tunnel syndrome and destructive arthropathies. The disease is characterized by the assembly and deposition of β₂-microglobulin (β₂m) predominantly in the musculoskeletal system, but the initiating events leading to β₂m amyloidogenesis and the molecular mechanisms underlying amyloid fibril formation are still unclear. Glycosaminoglycans (GAGs) and metal ions have been shown to be related to the onset of protein aggregation and to promote de novo fiber formation. In this study, we show that fibrillogenesis of a cleavage variant of β₂m, ΔK58-β₂m, which can be found in the circulation of hemodialysis patients and is able to fibrillate at near-physiological pH in vitro, is affected by the presence of copper ions and heparan sulfate. It is found that the fibrils generated when heparan sulfate is present have increased length and diameter, and possess enhanced stability and seeding properties. However, when copper ions are present the fibrils are short, thin and less stable, and form at a slower rate. We suggest that heparan sulfate stabilizes the cleaved monomers in the early aggregates, hereby promoting the assembly of these into fibrils, whereas the copper ions appear to have a destabilizing effect on the monomers. This keeps them in a structure forming amorphous aggregates for a longer period of time, leading to the formation of spherical bodies followed by the assembly of fibrils. Hence, the in vivo formation of amyloid fibrils in DRA could be initiated by the generation of ΔK58-β₂m which spontaneously aggregate and form fibrils. The fibrillogenesis is enhanced by the involvement of GAGs and/or metal ions, and results in amyloid-like fibrils able to promote the de novo formation of β₂m amyloid by a scaffold mechanism.     

Antipeptide antibodies differentiate between long and short isoforms of the D2 dopamine receptor.

We have developed specific antibodies directed against two synthetic peptides corresponding to defined sequences in the D2 dopamine receptor. One peptide is from a region that is present only in the 'long' isoform of the receptor, whereas the other is from a region that is common to both. These antibodies are able to recognize the native receptor as judged by immunocytochemical staining of cells transfected with dopamine receptor DNA. One antibody was shown to be specific for the 'long' form of the receptor and reacts only with cells transfected with the 'long' DNA subtype and not with those transfected with the 'short' DNA subtype. This recognition is specific and can be inhibited by the corresponding free peptide and not by a non-relevant peptide.     

Specificity of anti-peptide antibodies elicited against synthetic peptides mimicking conserved regions of HIV1 envelope glycoprotein

Comparison of HIV1_Bru and HIV2_Rod external envelope glycoprotein sequences enabled us to select ten highly conserved peptide sequences. The corresponding peptides were chemically synthesized, then coupled to bovine serum albumin before injection in rabbits. Although all peptides were immunogenic, only antibodies directed against peptides P1 (amino acid residues 33–55), P22 (418–462), P8 (487–508) and P21 (487–534) were able to interact with significant affinity (K_0.5 about 10⁻⁶ and 10⁻⁸ M) with the native glycoprotein by radioimmunoassay. Noteworthy was the capacity of anti-P1 antibodies to also recognize the glycoprotein of HIV2. Anti-peptide antibodies were tested for their ability to interfere with the gp120-CD4 interaction, membrane fusion and virus replication. Preincubation of gp 120 with antibodies directed to the region previously described as the putative CD4-binding site, P22 (418–462), did not abolish gp120 binding to CD4-positive cells.     

Monoclonal antibodies recognizing amino-terminal and carboxy-terminal regions of human aldolase A: probes to detect conformational changes of the enzyme.

Three monoclonal antibodies (MAbs1A2, 3C5, and 4C2) for human aldolase A [EC 4.1.2.13] were established. MAbs1A2, 3C5, and 4C2 were shown to belong to subclasses IgM, IgG1, and IgG2a, respectively. None of the MAbs inhibits aldolase A activity. Their epitopes were mapped in detail on the molecule by examining the reactivities of the MAbs to chimeric proteins between aldolases A and B [Kitajima et al. (1990) J. Biol. Chem. 265, 17493-17498] in ELISA and to the CNBr-cleaved fragments of aldolase A in immuno-blotting. MAbs1A2 and 3C5 reacted with sites located within amino acid residues 306-363 at the C-terminal region of the enzyme. MAb4C2 recognized an epitope of the enzyme present within amino acid residues 34-108 at the N-terminal region. In a competitive binding assay, MAbs1A2 and 3C5 competed with each other for binding to the antigen and also interfered with the binding of MAb4C2, whereas MAb4C2 failed to inhibit the binding of MAbs1A2 and 3C5 to the antigen. MAb3C5 showed a species-specificity in the reaction with the antigen; it reacted with human and rabbit aldolase A with similar reactivity but not at all with the rat and mouse enzymes, which differ from the human and rabbit enzymes in two amino acid residues at positions 328 and 348. Reactivities of MAbs to aldolase A were further examined with engineered enzymes containing an amino acid substitution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antibodies against a peptide representative of a conserved region of human IFN-alpha. Differential effects on the antiviral and antiproliferative effects of IFN

mAb generated from mice immunized with a synthetic peptide representative of a conserved region (amino acids 133 to 147) of human IFN-alpha are described. Antisera from mice immunized with the peptide coupled to keyhole limpet hemocyanin were able to specifically bind to the peptide and also to bind and precipitate human IFN-alpha. Binding of the antibodies to IFN-alpha was inhibited by the immunizing peptide, but not by an unrelated peptide. Immunized mice were used to obtain three hybridomas producing mAb able to recognize both the immunizing peptide and human IFN-alpha, as determined by RIA and immunoprecipitation. These antibodies also neutralized the antiviral effect of human leukocyte IFN. In contrast, none of the mAb significantly affected the inhibition of Daudi cell proliferation induced by IFN-alpha.     

Monoclonal antibodies that distinguish between subspecies of human interferon-alpha and that detect interferon oligomers

Monoclonal antibodies to human interferons (HuIFN) of the alpha-class have been prepared by screening against 125I-labeled IFN in a rapid liquid-phase radioimmunoassay. All of the six antibodies produced react with HuIFN-alpha 2 and with some components of HuIFN-alpha N (Namalwa); three of the antibodies also bind HuIFN-alpha 1, and these either do not bind or bind very weakly the 25K component of Namalwa. Reaction of the antibodies with IFN components blotted onto nitrocellulose after separation on reducing gels suggests that two of the antibodies are against conformational determinants, whereas the epitopes recognized by the other antibodies are not destroyed by reduction or SDS treatment; these antibodies can be used to detect the presence of oligomers in IFN preparations. From the reaction of the antibodies with different alpha-IFN in immunoblots, in an antiviral assay, and in an ELISA, it was concluded that at least five different epitopes are recognized by the six antibodies, only one of which is non-neutralizing.     

Biological activities of rabbit antibodies against synthetic human thyrotropin receptor peptides representing thyrotropin binding regions.

Recently, we have shown that the thyrotropin (TSH) binding regions of human thyrotropin receptor (TSHR) reside in two areas within residues 12-44 and 308-344. Serial antisera were raised against four overlapping synthetic peptides representing these two regions of TSHR (peptides 12-30, 24-44, 308-328, and 324-344) and were investigated for their ability to stimulate or block the cultured porcine thyroid cells. In addition, serum concentrations of triiodothyronine (T3) and thyroxine (T4) in serial sera obtained from each rabbit were examined. It was shown that residues of 12-30 and 324-344 of TSHR, respectively, are the site (at least a part of the site) where stimulating (TSAb) and blocking type (TSBAb) immunoglobulins are directed.     

Antipeptide antibodies reveal interrelationships of MBP 200 and MBP 235: unique apoB-specific receptors for triglyceride-rich lipoproteins on human monocyte-macrophages

Two human monocyte-macrophage (HMM) membrane binding proteins, (MBP) 200 and 235, are receptor candidates that bind to the apolipoprotein (apo)B-48 domain in triglyceride-rich lipoproteins for uptake independent of apoE. Microsequence analysis of the purified reduced MBP 200R characterized tryptic peptides of MBP 200R. A synthetic peptide mimicking a unique, unambiguous 10-residue sequence (AEGLMVTGGR) induced antipeptide antibodies that specifically recognized MBP 200, 235 and 200R, in 1- and 2-dimensional analyses, indicating 1) the ligand binding protein was sequenced and 2) MBP 200 and 235 yielded MBP 200R upon reduction. These antibodies identified the MBPs in human blood-borne, THP-1, U937 MMs, and endothelial cells (EC) but not in human fibroblasts or Chinese hamster ovary (CHO) cells. Fluorescence activated cell sorting (FACS) analysis located the MBPs on the MM surface as necessary for receptor function. The 10-residue, unambiguous MBP 200-derived sequence is unique, with no matches in extant protein databases. Antipeptide antibodies bind to the MBPs in reticuloendothelial cells that have this receptor activity, but not to proteins in cells that lack this receptor activity. These studies provide the first direct protein sequence and immunochemical data that a new, unique apoB receptor for triglyceride-rich lipoproteins exists in human monocytes, macrophages, and endothelial cells.     

Antipeptide antibodies to the carboxy terminal and the DCCD binding region of the human mitochondrial ATP synthase beta-subunit.

Antibodies to defined epitopes on the human ATP synthase would provide a powerful tool in the definition of the subunit composition of the enzyme complex and in the characterization of any defect in its assembly in diseases associated with mitochondrial disorders. Antibodies have been thus raised against synthetic peptides, corresponding to two regions on the human ATP synthase beta-subunit: the C-terminal region, and a region which includes the two dicyclohexylcarbodiimide (DCCD)-reactive glutamic acid residues suggested to be involved in the enzyme catalytic activity. The antibodies to the C-terminal peptide reacted with the ATP synthase beta-subunit in ELISA, in Western immunoblotting and in immunohistochemical experiments, and had the ability to immunoprecipitate the enzyme complex. The antibodies to the DCCD-binding region peptide did not react to the ATP synthase beta-subunit in its native configuration, although reacted well under Western immunoblotting conditions.     

Overproduction of single-chain antibodies against human IFN-alpha2B in Escherichia coli and their isolation in biologically active form

The gene encoding mouse single chain antibody (ScFv) against human interferon alpha2b (IFN-alpha2b) was cloned into the plasmid vector under the control of promoter from phage T7 and the recombinant protein was expressed in Escherichia coli as inclusion bodies. After the isolation of inclusion bodies the desired protein containing affinity tail "6His tag" was solubilized and purified under denaturing conditions by immobilized-metal affinity chromatography. The soluble and purified ScFv was obtained by "on column" refolding and the recovery of biological activity were demonstrated. The higher levels of ScFv production for intracellular expression system in comparison with ScFv obtained by secretion were shown. The advantages of described refolding method are simplicity and high efficacy, moreover, refolding using a chromatographic process represents the manufacturable approach because it is easily automated using commercially available materials and preparative chromatography systems and also can be combined with simultaneous purification.     

Poliovirus empty capsid morphogenesis: evidence for conformational differences between self- and extract-assembled empty capsids.

In this paper we describe the use of specific proteinases, surface-specific radioiodination, and antigenic reactivity in conjunction with isoelectric focusing for probing the conformations of different polioviral empty capsid species. Naturally occurring empty capsids (called procapsids) with an isoelectric point of 6.8 were resistant to proteolytic digestion by trypsin or chymotrypsin, as were empty capsids assembled in vitro in the presence of a cytoplasmic extract prepared from poliovirus-infected HeLa cells. In contrast, self-assembled empty capsids (isoelectric point, 5.0) were sensitive to both proteinases. Capsid proteins VP0 and VP1 were attacked predominantly, whereas VP3 was resistant to cleavage. Unpolymerized 14S particles possessed a trypsin sensitivity which was qualitatively similar to that of self-assembled empty shells. Surface-specific iodination of virions and procapsids labeled VP1 exclusively. In contrast, radioiodination of self-assembled empty capsids labeled predominantly VP0. After radioiodination the sedimentation coefficient corrected to water at 20 degrees C, the isoelectric point, and the trypsin resistance of the procapsids remained unchanged. Procapsids and extract-assembled empty capsids were N antigenic, whereas self-assembled empty capsids were H antigenic. Self-assembled empty capsids were not converted to pH 6.8 trypsin-resistant structures by incubation with a virus-infected cytoplasmic extract. However, 14S particles assembled in the presence of a mock-infected extract formed empty capsids, 20% of which resembled extract-assembled empty shells as determined by the above-described criteria. These and related findings are discussed in terms of empty capsid structure and morphogenesis.     

A monoclonal antibody with broad reactivity to human interferon-alpha subtypes useful for purification of leukocyte-derived interferon

A monoclonal antibody to human interferon-alpha, termed HT-1 antibody, with a broad reactivity to various subtypes of interferon-alpha was prepared. It bound and neutralized all of the four subtypes of E. coli-derived human recombinant interferon-alpha (alpha 1, alpha 2, alpha 4, and alpha 6) tested; it also neutralized human natural leukocyte interferon but only partially. Human interferon-beta and -gamma were not bound. The antibody conjugated to Sepharose beads retained over 90% of human leukocyte interferon induced by Sendai virus. The bound interferon was recovered by acid elution in good yields and in almost pure form (specific activity was about 2 X 10(8) international units/mg protein). The purified interferon showed, in SDS-polyacrylamide gel electrophoresis, an activity profile with major peaks in a mol. wt. range of 17,000-22,000, which completely agreed with the profile shown by polyclonal antibody-purified interferon. Such purified leukocyte interferon-alpha preparations containing most of the naturally occurring subtypes can be useful for clinical and other purposes.     

Thrombin-induced conformational changes of human alpha 2-macroglobulin: evidence for two functional domains

The interaction of thrombin with alpha 2-macroglobulin (alpha 2M) was characterized by monitoring conformational changes and measuring the increase of free sulfhydryl groups during the reaction. Under experimental conditions where [thrombin] greater than [alpha 2M], the conformational change, measured by increases in the fluorescence of 6-(p-toluidino)-2-naphthalenesulfonate, and thiol group appearance displayed biphasic kinetics. The initial rapid phase results in the formation of a stable complex, the appearance of two sulfhydryl groups, the cleavage of approximately half of the Mr 180 000 subunits, and a conformational change that is not as extensive as that which occurs with trypsin. The slower phase is associated with the appearance of two additional sulfhydryl groups, increased cleavage of the Mr 180 000 subunit, and additional conformational changes. The available evidence suggests that the slow phase results from hydrolysis of the Mr 180 000 subunit(s) due to proteolysis of the alpha 2M-thrombin complex by free thrombin. Experiments with 125I-thrombin document the binding of 1 mol of thrombin/mol of alpha 2M that is not dissociated upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the complex. At higher ratios of thrombin to alpha 2M, a second mole of thrombin will reversibly associate with the 1:1 alpha 2M-thrombin complex. Under conditions where [thrombin] less than [alpha 2M], biphasic kinetics were not observed, and the conformational change, sulfhydryl appearance, and hydrolysis of the Mr 180 000 subunit were found to follow second-order kinetics.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of monoclonal antibodies directed against distinct conserved epitopes of human immunodeficiency virus type 1 core proteins

Summary Two mouse hybridoma cell lines secreting antibodies to the Human Immunodeficiency Virus (HIV) p25 major core protein and its precursors p55 and p41, were developed after immunization with the highly cytopathic Zaïrian HIV-1 isolate, NDK. These monoclonal antibodies also react with the gag gene products from HIV-1-BRU prototype and present cross reaction with HIV-2-ROD, and SIV-AGM. They map into topographically distinct areas of p25 and define epitopic regions topographically separated from those recognized by four other anti-p25 mAb suggesting the existence of at least 6 spatially distinct epitopic regions on HIV-1-p25 core protein.Abbreviations HIV Human Immunodeficiency Virus - SIV Simian Immunodeficiency Virus - HTLVI Human T cell Leukaemia Virus - AIDS Acquired Immune Deficiency Syndrome - mAb Monoclonal Antibody - ELISA Enzyme Linked Immunosorbent Assay - PBS Phosphate Buffered Saline     

Monoclonal antibodies against defined regions of the muscular dystrophy protein, dystrophin.

Nineteen monoclonal antibodies which bind to native dystrophin in the plasma membrane of frozen muscle sections were obtained using a recombinant fusion protein as immunogen. On Western blots of normal mouse muscle extracts, the antibodies bind specifically to a 400,000 Mr protein which is absent from dystrophic mouse (mdx) muscle. At least four distinct epitopes have been identified by cleavage mapping methods. Although the fusion protein contained 25% of the human dystrophin sequence (Cys816-Asp1747; Mr 108,000), most of the monoclonal antibodies (15 out of 19) recognize a single fragment of Mr 27,500.     

Identification of a conserved sequence in the non-coding regions of many human genes.

We have analyzed a sequence of approximately 70 base pairs (bp) that shows a high degree of similarity to sequences present in the non-coding regions of a number of human and other mammalian genes. The sequence was discovered in a fragment of human genomic DNA adjacent to an integrated hepatitis B virus genome in cells derived from human hepatocellular carcinoma tissue. When one of the viral flanking sequences was compared to nucleotide sequences in GenBank, more than thirty human genes were identified that contained a similar sequence in their non-coding regions. The sequence element was usually found once or twice in a gene, either in an intron or in the 5' or 3' flanking regions. It did not share any similarities with known short interspersed nucleotide elements (SINEs) or presently known gene regulatory elements. This element was highly conserved at the same position within the corresponding human and mouse genes for myoglobin and N-myc, indicating evolutionary conservation and possible functional importance. Preliminary DNase I footprinting data suggested that the element or its adjacent sequences may bind nuclear factors to generate specific DNase I hypersensitive sites. The size, structure, and evolutionary conservation of this sequence indicates that it is distinct from other types of short interspersed repetitive elements. It is possible that the element may have a cis-acting functional role in the genome.     

Novel strategy for selection of monoclonal antibodies against highly conserved antigens: phage library panning against ephrin-B2 displayed on yeast

Ephrin-B2 is predominately expressed in endothelium of arterial origin, involved in developmental angiogenesis and neovasculature formation through its interaction with EphB4. Despite its importance in physiology and pathological conditions, it has been challenging to produce monoclonal antibodies against ephrin-B2 due to its high conservation in sequence throughout human and rodents. Using a novel approach for antibody selection by panning a phage library of human antibody against antigens displayed in yeast, we have isolated high affinity antibodies against ephrin-B2. The function of one high affinity binder (named as 'EC8') was manifested in its ability to inhibit ephrin-B2 interaction with EphB4, to cross-react with murine ephrin-B2, and to induce internalization into ephrin-B2 expressing cells. EC8 was also compatible with immunoprecipitation and detection of ephrin-B2 expression in the tissue after standard chemical fixation procedure. Consistent with previous reports on ephrin-B2 induction in some epithelial tumors and tumor-associated vasculatures, EC8 specifically detected ephrin-B2 in tumors as well as the vasculature within and outside of the tumors. We envision that monoclonal antibody developed in this study may be used as a reagent to probe ephrin-B2 distribution in normal as well as in pathological conditions and to antagonize ephrin-B2 interaction with EphB4 for basic science and therapeutic applications.     

Specificity of monoclonal antibodies against human thyroglobulin; comparison with autoimmune antibodies.

Ten monoclonal antibodies (mAb) directed against human thyroglobulin (hTgb) were produced, purified and characterized. The mAb avidity for hTgb ranged from 10(-10) to 10(-6) M. The species specificity of the mAb was as follows: eight mAb reacted with monkey Tgb, three with dog Tgb and one with pig Tgb; none with bovine and ovine Tgb. The binding of mAb to hTgb was not significantly inhibited in the presence of Tgb carbohydrate moieties, tyrosine, iodotyrosines and iodothyronines. The topology of the antigenic determinants recognized by the 10 mAb on hTgb was explored by inhibition of Tgb binding of radiolabeled mAb by the other antibodies. Six distinct clusters of reactivity were described. Localization of the antigenic determinants recognized by mAb on hTgb was attempted using tryptic fragments of hTgb to inhibit the binding of mAb to hTgb. The inhibitory effect of hydrolysis products was different for each mAb but exhibited partial analogies between mAb of the same cluster of reactivity. Anti-hTgb autoimmune antibodies (aAb) purified from sera of Graves patients cross-reacted essentially with mAb of one out of the six clusters. These results demonstrate that the large number of antigenic determinants presented by the hTgb are not disseminated on the molecule but are clustered in antigenic regions. Furthermore, from the six antigenic regions evidenced in this paper, only one is involved in autoimmune antibody production in Grave's disease.