9G4+ Antibodies Isolated from HIV-Infected Patients Neutralize HIV-1 and Have Distinct Autoreactivity Profiles

Potent HIV-1 specific broadly neutralizing antibodies (BNA) are uncommon in HIV infected individuals, and have proven hard to elicit by vaccination. Several, isolated monoclonal BNA are polyreactive and also recognize self-antigens, suggesting a breach of immune tolerance in persons living with HIV (PLWH). Persons with systemic lupus erythematosus (SLE) often have elevated levels of autoreactive antibodies encoded by the VH4-34 heavy chain immunoglobulin gene whose protein product can be detected by the 9G4 rat monoclonal antibody. We have recently found that levels of these “9G4+” antibodies are also elevated in PLWH. However, the putative autoreactive nature of these antibodies and the relationship of such reactivities with HIV neutralization have not been investigated. We therefore examined the autoreactivity and HIV neutralization potential of 9G4+ antibodies from PLWH. Results show that 9G4+ antibodies from PLWH bound to recombinant HIV-1 envelope (Env) and neutralized viral infectivity in vitro, whereas 9G4+ antibodies from persons with SLE did not bind to Env and failed to neutralize viral infectivity. In addition, while 9G4+ antibodies from PLWH retained the canonical anti-i reactivity that mediates B cell binding, they did not display other autoreactivities common to SLE 9G4+ antibodies, such as binding to cardiolipin and DNA and had much lower reactivity with apoptotic cells. Taken together, these data indicate that the autoreactivity of 9G4+ antibodies from PLWH is distinct from that of SLE patients, and therefore, their expansion is not due to a general breakdown of B cell tolerance but is instead determined in a more disease-specific manner by self-antigens that become immunogenic in the context of, and possibly due to HIV infection. Further studies of 9G4+ B cells may shed light on the regulation of B cell tolerance and interface between the generation of specific autoreactivities and the induction of antiviral immunity in persons living with HIV.     

Chemical synthesis and cloning of a poly(arginine)-coding gene fragment designed to aid polypeptide purification

A 43-bp DNA duplex coding for poly(arginine) [poly(arg)] has been synthesised by modified phosphotriester procedures. It has been inserted into the BglII and BamHI restriction sites of a cloned synthetic β-urogastrone (Uro) gene, under the control of the trp promoter. Subsequent induction with 3β-indole acrylic acid produces β-Uro with a C-terminal poly(arg) fusion. The raised isoelectric point of this polypeptide fusion facilitates rapid purification by cation exchange chromatography. The C-terminal poly(arg) tail can be readily removed by treatment with carboxypeptidase B.     

A mutation in apolipoprotein A-I in the Iowa type of familial amyloidotic polyneuropathy

Immunoblotting of isoelectric focusing gels of plasma and direct genomic DNA sequencing have been used to characterize a mutation in apolipoprotein A-I associated with the familial amyloidotic polyneuropathy originally described by Van Allen in an Iowa kindred. An arginine for glycine substitution in apolipoprotein A-I identified in the proband's amyloid fibrils was determined to be the result of a mutation of guanine to cytosine in the apolipoprotein A-I gene at the position corresponding to the first base of codon 26. Direct sequencing of genomic DNA of three affected individuals who died in the 1960s confirmed the inheritance of the disorder. Immunoblot analysis detected the variant apolipoprotein A-I in the proband's plasma and in several at-risk members of the kindred. In addition, allele-specific amplification by the polymerase chain reaction was used to detect carriers of the variant gene.     

Studies of enzyme polymorphisms in the Kamuela population of Drosophila mercatorum. II. Evaluation of glycolytic intermediates

A simple and effective cryogenic procedure for the extraction of glycolytic intermediates from whole Drosophila has been developed. This procedure gives consistent results when a measure (µM/liter/OD₂₆₀) is adopted which corrects for differences in extraction efficiency. Using this measure and a homozygous strain of D. mercatorum, there are no significant differences among extracts for the levels of any of the 15 glycolytic intermediate or energy molecules considered. The profile of means is consistent across experimental designs and instrument types. Coefficients of variation are well below 50% for most variables. The methodology presented has the statistical power to detect a mean change of 10 to 50% using an experimental design which requires as few as 32 observations. The estimated energy charge for resting Drosophila from these studies is the expected value of 0.86.