Immunoassay methods used in clinical studies for the detection of anti-drug antibodies to adalimumab and infliximab.

We examined the assay formats used to detect anti-drug antibodies (ADA) in clinical studies of the anti-tumour necrosis factor (TNF) monoclonal antibodies adalimumab and infliximab in chronic inflammatory disease and their potential impact on pharmacokinetic and clinical outcomes. Using findings of a recent systematic literature review of the immunogenicity of 11 biological/biosimilar agents, we conducted an ancillary qualitative review of a subset of randomized controlled trials and observational studies of the monoclonal antibodies against anti-TNF factor adalimumab and infliximab. Among studies of adalimumab and infliximab, the immunoassay method used to detect antibodies was reported in 91 of 111 (82%) and 154 of 206 (75%) adalimumab and infliximab studies, respectively. In most adalimumab and infliximab studies, an enzyme-linked immunosorbent assay or radioimmunoassay was used [85 of 91 (93%) and 134 of 154 (87%), respectively]. ADA incidence varied widely among assays and inflammatory diseases (adalimumab, 0-87%; infliximab, 0-79%). Pharmacokinetic and clinical outcomes were only reported for ADA-positive patients in 38 of 91 (42%) and 61 of 154 (40%) adalimumab and infliximab studies, respectively. Regardless of assay format or biological used, ADA formation was associated with lower serum concentrations, reduced efficacy and elevated rates of infusion-related reactions. Consistent with previous recommendations to improve interpretation of immunogenicity data for biologicals, greater consistency in reporting of assay methods and clinical consequences of ADA formation may prove useful. Additional standardization in immunogenicity testing and reporting, application of modern, robust assays that satisfy current regulatory expectations and implementation of international standards for marketed products may help to improve our understanding of the impact of immunogenicity to biologics.

Sperm motility in the semen analysis affects the outcome of superovulation intrauterine insemination in the treatment of infertile Asian couples with male factor infertility.

OBJECTIVE: To ascertain the relationship between the initial and unprocessed sperm parameters and pregnancy rates in SOIUI, for Asian couples with male factor infertility. DESIGN: Retrospective study. SETTING: A large government tertiary-care women's hospital with 15,000 deliveries per year. POPULATION: One thousand four hundred and seventy nine couples undergoing 2846 cycles of SOIUI. METHODS: All couples enrolled in the SOIUI programme were analysed, comparing initial sperm parameters and the post-processed total motile sperm, against pregnancy rates per cycle. MAIN OUTCOME MEASURES: Pregnancy rates in relation to initial sperm parameters and post-processed total motile sperm. RESULTS: Ninety-three percent of the couples had male factor infertility. The average normal forms for these men was 14.7%. Overall pregnancy rate was 12.1% per completed SOIUI cycle. We found a significant drop in pregnancy rates if the percentage of motile sperms in the unprocessed sperm sample fell below 30%. We also found that insemination of at least 1 million motile sperm resulted in a significant increase in pregnancy rates. CONCLUSIONS: We recommend SOIUI as an effective treatment of suitable couples with male infertility, before embarking on IVF. However, if the initial percentage of motile sperm fell below 30%, or if after processing, the total motile sperm count was fewer than 1 million, these couples should consider in vitro fertilisation.

Lyme arthritis synovial gamma delta T cells respond to Borrelia burgdorferi lipoproteins and lipidated hexapeptides.

Lyme arthritis synovial fluid contains a large proportion of gamma delta T cells that proliferates upon stimulation with the causative spirochete, Borrelia burgdorferi. A panel of Borrelia-reactive gamma delta T cell clones was derived from synovial fluid of two patients with Lyme arthritis. Each of six gamma delta clones from one patient used the V delta 1 TCR segment but had otherwise unique CDR3 sequences and diverse V gamma segment usage. Stimulation of the V delta 1 clones was optimal in the presence of Borrelia, dendritic cells, and exogenous IL-2, which was reflected by proliferation, TCR down-modulation, as well as induction of CD25 and Fas ligand expression. Stimulation by B. burgdorferi-pulsed dendritic cells withstood chemical fixation and was not restricted to class I or class II MHC, CD1a, CD1b, or CD1c. In contrast, anti-gamma delta antibody potently inhibited proliferation. Extraction of B. burgdorferi lipoproteins with Triton X-114 enriched for the stimulatory component. This was confirmed using lipidated vs nonlipidated hexapeptides of Borrelia outer surface proteins. These observations suggest that synovial V delta 1 T cells may mediate an innate immune response to common lipoprotein products of spirochetes.

Apoptosis of Fashigh CD4+ synovial T cells by borrelia-reactive Fas-ligand(high) gamma delta T cells in Lyme arthritis.

The function of the minor subset of T lymphocytes bearing the gamma delta T cell antigen receptor is uncertain. Although some gamma delta T cells react to microbial products, responsiveness has only rarely been demonstrated toward a bacterial antigen from a naturally occurring human infection. Synovial fluid lymphocytes from patients with Lyme arthritis contain a large proportion of gamma delta cells that proliferate in response to the causative spirochete, Borrelia burgdorferi. Furthermore, synovial gamma delta T cell clones express elevated and sustained levels of the ligand for Fas (APO-1, CD95) compared to alpha beta T cells, and induce apoptosis of Fashigh CD4+ synovial lymphocytes. The findings suggest that gamma delta T cells contribute to defense in human infections, as well as manifest an immunoregulatory function at inflammatory sites by a Fas-dependent process.

Purification of a [3H]dihydrotetrabenazine-binding protein from bovine adrenal medulla.

A high affinity binding site for [3H]dihydrotetrabenazine is thought to be present on the monoamine transport protein from chromaffin granules. We describe a procedure for purification of this binding activity from frozen bovine adrenal tissue, and we partially characterize the purified preparation. Binding activity solubilized with sodium cholate and soybean lecithin was fractionated on wheat germ lectin-Sepharose, phenyl-Sepharose, Mono Q, and hydroxylapatite. Denaturing electrophoresis of the purified binding activity, followed by silver staining, revealed a single broad band centered at an apparent molecular weight of 85,000. This preparation bound [3H]dihydrotetrabenazine with an apparent dissociation constant of 2.7 nM and had a site density of 10 nmol/mg. Treatment of the purified protein with neuraminidase reduced the apparent molecular weight by 9000, indicating the presence of terminal sialic acids on the oligosaccharide portion of this molecule.

Interaction of dihydrotetrabenazine binding activity with immobilized lectins.

High-affinity dihydrotetrabenazine binding activity was solubilized by treatment of crude bovine striatal synaptosomes with cholate and incubated with lectins immobilized on sepharose. Both concanavalin A and wheat germ lectin sepharose removed dihydrotetrabenazine binding activity from the cholate extract, and this removal was prevented by inclusion of the appropriate hapten sugars. These results imply that the dihydrotetrabenazine binding subunit of the synaptic vesicle catecholamine/serotonin (5-HT) transporter, or a vesicle component tightly associated with it, is glycosylated.

Distribution of [3H]dihydrotetrabenazine binding in bovine striatal subsynaptic fractions: enrichment of higher affinity binding in a synaptic vesicle fraction.

[3H]Dihydrotetrabenazine bound to a single class of binding sites in bovine striatal synaptic vesicles with an apparent dissociation constant of 3-9 nM. This is comparable to the inhibitory potency of dihydrotetrabenazine in catecholamine transport assays. In contrast to these results, [3H]dihydrotetrabenazine bound to at least two classes of sites in all other subsynaptic fractions investigated. The higher affinity class of sites was comparable in affinity to that of synaptic vesicles, whereas the lower affinity sites exhibited an apparent dissociation constant of 95-400 nM. Higher affinity sites were most abundant in the synaptic vesicle fraction, and little higher affinity binding was observed in mitochondrial and myelin fractions, or in highly purified synaptic plasma membranes. Lower affinity binding was not enriched in any subsynaptic fraction and was the only class of binding sites detected in homogenates of liver and diaphragm. The distribution of the presynaptic vesicle marker synaptophysin corresponded with that of higher affinity but not lower affinity binding. These results are consistent with the expectation that the higher affinity sites are associated primarily with synaptic vesicles and other neuronal entities that are in communication with these organelles.