Detection of IgG rheumatoid factor by concanavalin A treatment and complement fixation with IgG rheumatoid factor

Concanavalin A (Con A) froms precipitates with carbohydrate-rich protein such as IgM, IgD, IgE, and IgA. Since IgG contains little carbohydrate and does not react with Con A, the activity of IgG-rheumatoid factor (RF) can be measured in the supernate of the Con A-treated serum. When the latex fixation test (LFT) and the sensitized sheep cell agglutination test (SSCA) were perfromed in the supernate for the detection of IgG-RF, LFT was positive in 32-1% of sera, out of 137 sera originally positive for LFT, and SSCA was positive in 18-5% of sera, out of 119 sera originally positive for SSCA. IgG-RF exhibited lower complement fixing ability than IgM-RF and correlated with agglutination titres of IgG-RF, while the CH50 of the original serum did not correlate with haemolytic activities of either IgM-RF or IgG-RF.     

IgG3 reactive rheumatoid factor in rheumatoid arthritis: etiologic and pathogenic considerations

Rheumatoid factor (RF) is a polyclonal autoantibody directed against the Fc portion of IgG. Although the role of RF in patients with rheumatoid arthritis (RA) is unclear, immune complexes that form between RF and IgG can activate the classical complement (C) pathway, leading to pathogenic outcomes involving inflammatory events and tissue damage. The specificity of serum RF and RF produced by rheumatoid synovial cells (RSC) is different. Serum RF has specificity for rabbit IgG and human IgG subclasses IgG1, 2, and 4, but binds poorly to IgG3. The affinity of serum RF for IgG Fc is low, having an association constant of 10(4)-10(5) M-1. RSC RF, however, has specificity for human IgG and high avidity for IgG3. Because of this greater specificity and avidity for IgG3, and because RSC RF may be pathogenically more important than serum RF, an important role for IgG3-reactive RF in RA may exist. Binding of RF to IgG may be dependent on the allotype and glycosylation of IgG. Infectious agents present in RA patients may directly or indirectly induce the production of certain RF. In this communication, we review and expand on several observations examining the role of IgG3-reactive RF in RA including: 1) binding differences between RF derived from RSC and serum; 2) glycosylation characteristics of IgG and its interaction with RF; 3) apparent allotype dependent binding of IgG3-reactive RF; and 4) possible relationship between infectious agents and the production of IgG3-reactive RF. Taken together, these observations suggest an important role for IgG3-reactive RF in better understanding the etiology and pathogenesis of RA.     

Chromosome location of genes encoding human blood groups

In this study the transactivation potential and DNA binding activities of p53 protein were examined following exposure of A2780 cells, a human ovarian carcinoma cell line, to the DNA damaging agent, cis-diamminedichloroplatinum II (cisplatin). The endogenous murine double minute-2 gene (mdm-2) was used to monitor the ability of p53 to transactivate genes. Northern analysis showed an induction of mdm-2 mRNA upon cisplatin treatment. It was further demonstrated, using an RNase protection assay, that the p53-responsive, mdm-2 promoter (P2) was activated in cisplatin-treated A2780 cells. However, when p53 protein DNA binding activity was analyzed, there was no detectable increase in p53 sequence-specific DNA binding activity during the period of time following DNA damage when mdm-2 mRNA was induced. Instead the increase in p53 protein observed in nuclear, cytoplasmic, and whole cell extracts correlated with a latent conformation of p53 that lacked sequence-specific DNA binding activity. At low doses of cisplatin, these latent pools of p53 increased in parallel with mdm-2 gene activation and were detectable as early as 4 h following cisplatin treatment. In vitro attempts to convert the latent p53 into an active, sequence-specific DNA binding conformation were unsuccessful. Even though cisplatin-induced p53 lacked sequence-specific DNA binding activity, it does possess an increased affinity for cisplatin-damaged duplex DNA molecules. This represents the first identification where cisplatin treatment induces a p53 protein, lacking sequence-specific DNA binding but with an increased affinity for platinated DNA molecules.     

Lack of N regions in antigen receptor variable region genes of TdT-deficient lymphocytes

During the assembly of immunoglobulin and T cell receptor variable region genes from variable (V), diversity (D), and joining (J) segments, the germline-encoded repertoire is further diversified by processes that include the template-independent addition of nucleotides (N regions) at gene segment junctions. Terminal deoxynucleotidyl transferase (TdT)-deficient lymphocytes had no N regions in their variable region genes, which shows that TdT is responsible for N region addition. In addition, certain variable region genes appeared at increased frequency in TdT-deficient thymocytes, which indicates that N region addition also influences repertoire development by alleviating sequence-specific constraints imposed on the joining of particular V, D, and J segments.     

Sticky egyptians: a technique for assembling genes encoding constrained peptides of variable length

Naturally occurring peptides, such as those produced by the poisonous marine snails of the genus Conus , have the ability to form tight, highly specific molecular interactions. The rigidity of the peptide framework which promotes these interactions is usually maintained by disulphide bonds, and it seems that the overall main chain conformation (or fold) of the peptide is determined by its length and the sequence distribution of the pairs of cysteine residues participating in these bonds. The fold of the peptide in turn is largely responsible for its shape. Since highly effective molecular interactions occur between species complementary in shape, we reasoned that peptides with the greatest potential in therapy or diagnosis would be found in a library of shapes, those peptides with a shape complementary to a given target being identified, for example, by selection. As a first step towards constructing such a peptide shape library, we have developed a method for assembling DNA fragments which encode an even number of cysteine residues and which are of variable length. We describe this method here.     

Vectors for the expression of PCR-amplified immunoglobulin variable domains with human constant regions

Cassette vectors have been constructed for mammalian expression of complete immunoglobulin heavy and light chain genes whose variable regions are produced by the polymerase chain reaction (PCR). The light and heavy chain vectors have promoter, leader, partial intron, enhancer and constant region segments within modified pSV2-gpt and pSV2-neo plasmids, respectively. Variable (V) regions are obtained by PCR using a two step process: 1) the V gene is amplified from genomic or cDNA, cloned into an intermediate vector and sequenced; 2) the first PCR product serves as the template for a second amplification in which restriction enzyme recognition sites and limited flanking intron sequence are added. The second PCR product is inserted into the expression vector, which is then transfected into mouse myeloma cells. These vectors contain human constant regions and may be used to express chimeric, humanized or human Ig genes. This report describes the design of these vectors and their application for the expression of chimeric 60.3, an anti-CD18 antibody.     

Common polymorphisms in genes encoding the human mineralocorticoid receptor and the human amiloride-sensitive sodium channel

We have examined the human mineralocorticoid receptor gene and the genes encoding the three subunits of the human amiloride-sensitive epithelial sodium channel. Eight new common polymorphisms were identified in these genes which may be useful in genotyping and linkage analysis.     

Binding of human monoclonal IgG rheumatoid factors to Fc is influenced by carbohydrate

OBJECTIVE: The precise nature of the epitope on the Fc portion of the IgG molecule to which IgG rheumatoid factors (RF) bind has not been identified. As patients with rheumatoid arthritis (RA) have abnormal glycosylation of the Fc portion of IgG, we investigated the impact of the sugar present in the Fc on the binding of IgG RF. METHODS: Binding of monoclonal IgG RF to Fc fragments with varying oligosaccharide chains was detected using an immunoblot assay. RESULTS: We demonstrated that the binding of human hybridoma derived monoclonal IgG RF was strongly influenced by the presence of carbohydrate and was maximal when the carbohydrate molecule was intact. The RF did not bind directly to the carbohydrate moiety itself. CONCLUSION: This suggests that conformational changes in the polypeptide chain induced by the carbohydrate are responsible for the observed binding patterns.     

The mouse and human genes encoding the recognition component of the N-end rule pathway

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. The N-end rule pathway is one proteolytic pathway of the ubiquitin system. The recognition component of this pathway, called N-recognin or E3, binds to a destabilizing N-terminal residue of a substrate protein and participates in the formation of a substrate-linked multiubiquitin chain. We report the cloning of the mouse and human Ubr1 cDNAs and genes that encode a mammalian N-recognin called E3alpha. Mouse UBR1p (E3alpha) is a 1,757-residue (200-kDa) protein that contains regions of sequence similarity to the 225-kDa Ubr1p of the yeast Saccharomyces cerevisiae. Mouse and human UBR1p have apparent homologs in other eukaryotes as well, thus defining a distinct family of proteins, the UBR family. The residues essential for substrate recognition by the yeast Ubr1p are conserved in the mouse UBR1p. The regions of similarity among the UBR family members include a putative zinc finger and RING-H2 finger, another zinc-binding domain. Ubr1 is located in the middle of mouse chromosome 2 and in the syntenic 15q15-q21.1 region of human chromosome 15. Mouse Ubr1 spans approximately 120 kilobases of genomic DNA and contains approximately 50 exons. Ubr1 is ubiquitously expressed in adults, with skeletal muscle and heart being the sites of highest expression. In mouse embryos, the Ubr1 expression is highest in the branchial arches and in the tail and limb buds. The cloning of Ubr1 makes possible the construction of Ubr1-lacking mouse strains, a prerequisite for the functional understanding of the mammalian N-end rule pathway.     

Cloning and characterization of the gene encoding human osteoprotegerin/osteoclastogenesis-inhibitory factor

Cri-du-chat is a human contiguous gene deletion syndrome resulting from hemizygous deletions of chromosome 5p. Here we describe the isolation from within this interval of the human Semaphorin F (SEMAF) gene, a member of a family of proteins that has been implicated in axonal pathfinding. The human SEMAF gene covers at least 10% of the deleted region and defines a new class within this large gene family characterized by the presence of seven type 1 thrombospondin repeats. Prominent expression of murine semaphorin F (Semaf) was observed in the mouse brain, consistent with a role for semaphorin F as a signaling molecule that guides axons or migrating neuronal precursors during development. The known functions of semaphorins and the interesting pattern of expression for Semaf suggest that haploinsufficiency for SEMAF may disrupt normal brain development and might lead to some of the features of Cri-du-chat.     

Significance of IgG for the activity of factor XII measured in human plasma

The plasma level of factor XII (FXII) was measured in samples from healthy young men. The activated contact factor was assayed as prekallikrein activator (PKA), as S-2222 amidase, and in radial immunodiffusion tests. By removing the bulk of IgG on protein G columns before the activation procedure, the functional activities increased to about 135%. In such test preparations, PAGE immunoblot experiments with polyclonal antibodies against FXII showed, in addition to FXIIa (80 kD), a double band with a molecular weight of about 46 kD. This protein could also be detected with a light-chain-specific monoclonal antibody to FXII, but not with such an antibody directed against its heavy chain. The 46-kD band was also observed in plasma deficient in FXII. The amidase assays indicated that the minor part of FXIIa was present in some kind of association with another protease. To obtain a correct estimation of total FXIIa in the amidase assays a sufficiently high level of FXI was required compared to that of FXII. The PKA assays were generally carried out with a prekallikrein (PK) substrate containing IgG. By replacing this substrate by PK free from IgG additional PKA activity was observed, the activity appearing also in plasma deficient in FXII.     

Sequence analysis of genes encoding rodent homologues of the human tumor-rejection antigen SART-1

OBJECTIVE: To assess pregnant women's knowledge of, and attitudes towards, antenatal HIV testing, and its acceptability to them. SETTING: Antenatal clinic at Guy's Hospital, London, six community antenatal clinics and a midwifery group practice. POPULATION: Eight hundred and forty-three women attending the antenatal clinics. METHOD: The women received a leaflet explaining HIV testing, and completed a questionnaire before and after their booking appointment. This included an assessment of their knowledge of, and attitudes towards HIV testing, and its acceptability. RESULTS: Seven hundred and eighty-nine women (94%) completed questionnaires. Fifty-one percent (n = 405) were Caucasian, 25% (n = 195) African, 11% (n = 86) West Indian and 13% (n = 100) were from other ethnic groups. Fifty-eight percent received the HIV information leaflet, of whom 86% had read it. Knowledge relating to HIV was good, the median knowledge score being 6 out of a possible 8, but it was less in non-Caucasian women and those with lower educational qualifications. Knowledge was not related to uptake of testing. Thirty-five percent of women accepted the offer of an HIV test, rates being higher in hospital clinics (41%) than in the midwifery group practice (10%) and the community clinics (30%). Women more likely to accept the offer of an HIV test were non-Caucasian (P = 0.0443), those who had thought about the HIV test before this pregnancy (P = 0.0298) and those seeing one particular midwife (P = 0.0003). Most women (67%) thought that all pregnant women should be offered the HIV test and then make their own decision. Overall, 64% women did not change their original pre-discussion decision on testing for HIV. Thirty-six percent of women changed their decision from 'yes' to 'no' or 'don't know' after seeing the midwife. Women attending the community clinics (P = 0.003) and those who had been tested before (P = 0.0451) were more likely to change their decision. CONCLUSION: This study, in a multiethnic population, has shown that knowledge regarding HIV is good but does not increase the uptake of testing. Women prefer to be offered the HIV test and make their own choice regarding whether to accept it.     

Cloning of the genes encoding two murine and human cochlear unconventional type I myosins

The predominance of Th2 cytokine-secreting pattern in allergic asthma has been known as a cause and an accelerating factor, and Th1 suppresses these allergic phenomena, but the role of Th0 clones is obscure. Because Th1/Th2 differentiation has been determined by cytokine environment, we investigated how mite-specific helper T cells stimulated in different cytokine environments actually influenced IgE and IgG4 synthesis, which are known to be regulatory immunoglobulins for allergic response. Th0 clones, which were mainly established in the presence of IL-12, provided a great deal of help for IgG4 and IgG1 synthesis, but did not provide help for IgE synthesis, whereas Th2 clones helped IgE synthesis prominently, and IgG4 and IgG1 synthesis marginally. These characteristics of Th0 clones were also true for Th0 clones obtained from patients who were successfully treated with desensitization therapy. Furthermore, the differences in helper activity between Th0 and Th2 clones were not ascribed solely to soluble factors. These data indicate that IgE and IgG4 synthesis is differentially regulated by antigen-specific T cells, and that conversion or selection from Th2 to Th0 by the addition of IL-12 may exhibit therapeutic effects.     

Multiple regions of human Fc gamma RII (CD32) contribute to the binding of IgG

The low affinity receptor for IgG, Fc gamma RII (CD32), has a wide distribution on hematopoietic cells where it is responsible for a diverse range of cellular responses crucial for immune regulation and resistance to infection. Fc gamma RII is a member of the immunoglobulin superfamily, containing an extracellular region of two Ig-like domains. The IgG binding site of human Fc gamma RII has been localized to an 8-amino acid segment of the second extracellular domain, Asn154-Ser161. In this study, evidence is presented to suggest that domain 1 and two additional regions of domain 2 also contribute to the binding of IgG by Fc gamma RII. Chimeric receptors generated by exchanging the extracellular domains and segments of domain 2 between Fc gamma RII and the structurally related Fc epsilon RI alpha chain were used to demonstrate that substitution of domain 1 in its entirety or the domain 2 regions encompassing residues Ser109-Val116 and Ser130-Thr135 resulted in a loss of the ability of these receptors to bind hIgG1 in dimeric form. Site-directed mutagenesis performed on individual residues within and flanking the Ser109-Val116 and Ser130-Thr135 domain 2 segments indicated that substitution of Lys113, Pro114, Leu115, Val116, Phe129, and His131 profoundly decreased the binding of hIgG1, whereas substitution of Asp133 and Pro134 increased binding. These findings suggest that not only is domain 1 contributing to the affinity of IgG binding by Fc gamma RII but, importantly, that the domain 2 regions Ser109-Val116 and Phe129-Thr135 also play key roles in the binding of hIgG1. The location of these binding regions on a molecular model of the entire extracellular region of Fc gamma RII indicates that they comprise loops that are juxtaposed in domain 2 at the interface with domain 1, with the putative crucial binding residues forming a hydrophobic pocket surrounded by a wall of predominantly aromatic and basic residues.     

Expression of the genes encoding myelin basic protein and proteolipid protein in human malignant gliomas

Pathological differentiation of oligodendroglioma and mixed oligoastrocytoma from astrocytoma is difficult, relying on morphological characteristics due to the lack of reliable immunohistochemical stains. Oligodendrocytes, the presumed cell of origin of oligodendrogliomas, highly express the genes encoding myelin basic protein (MBP) and proteolipid protein (PLP). We analyzed the expression of these genes to determine whether they might be useful molecular markers of oligodendrocytic tumors. MBP and PLP were highly expressed in all oligodendrogliomas and minimally expressed in glioblastomas multiforme. MBP was highly expressed in mixed oligoastrocytomas, whereas PLP expression was minimal. The association between tumor classification and expression of the MBP and PLP genes was statistically significant. Expression of these genes may serve as a useful molecular marker for some subtypes of human gliomas.