Inclusion of a non-immunoglobulin binding protein in two-site ELISA for quantification of human serum proteins without interference by heterophilic serum antibodies

Measurement of human serum molecules with two-site ELISA can be biased by the presence of human heterophilic anti-animal immunoglobulin antibodies (HAIA) that cause false-positive signals by cross-linking the monoclonal (mAb) and/or polyclonal antibodies (pAb) used for the pre- (capture) and post-analyte steps (detection). To evaluate a novel ELISA format designed to avoid interference by HAIA, a target-specific non-immunoglobulin (Ig) affinity protein (affibody) was used to replace one of the antibodies. First, a human IgA-binding affibody (Z(IgA)) selected by phage display technology from a combinatorial library of a single Staphylococcus aureus protein A domain was used. The detection range of IgA standard using an ELISA based on Z(IgA) for capture and goat pAb against IgA (pAb(IgA)) for detection was comparable with that of using pAb(IgA) for both capture and detection. Secondly, another affibody (Z(Apo)) was combined with mAb and used to detect recombinant human apolipoprotein A-1. The affibody/antibody ELISAs were also used to quantify human serum levels of IgA and apolipoprotein A1. To verify that human serum did not cause false-positive signals in the affibody/antibody ELISA format, the ability of human serum to cross-link affibodies, mAb (mouse or rat) and/or pAb (goat) displaying non-matched specificities was assessed; affibodies and antibodies were not cross-linked whereas all combinations of mAb and/or pAb were cross-linked. The combination of affibodies and antibodies for analysis of human serum molecules represents a novel two-site ELISA format which precludes false-positive signals caused by HAIA.     

N-cadherin expression in adrenal tumors: upregulation in malignant pheochromocytoma and downregulation in adrenocortical carcinoma

Cell adhesion molecules (CAMs) are important regulators of tumor growth. The aim of the present study was to evaluate the expression pattern of CAMs in adrenal tumors regarding origin (cortex vs medulla) and biologic behavior (benign vs malignant). Eighty-seven adrenal tumors were investigated by immunocytochemistry (ICC) using monoclonal antibodies against N-cadherin (NCAD), E-cadherin (ECAD), neural cell adhesion molecule (NCAM), and CD44. Western blotting was performed on 30 tumors using the same antibodies. Markers for proliferation (Ki-67) and catecholamine synthesis (tyrosine hydroxylase) were also analyzed in tumors by ICC. NCAD was expressed in 12/27 benign pheochromocytomas (BPCs) (12 familial cases), 8/8 malignant pheochromocytomas (MPCs), 28/30 adrenocortical adenomas, and 9/22 adrenocortical carcinomas. ECAD was expressed in 0/27 BPCs, 0/8 MPCs, 0/30 adrenocortical adenomas, and 2/22 adrenocortical carcinomas. NCAM was expressed in 26/27 BPCs, 7/8 MPCs, 21/30 adrenocrotical adenomas, and 17/22 adrenocortical carcinomas. CD44 was expressed in 23/27 BPCs, 6/8 MPCs, 7/30 adrenocortical adenomas, and 4/22 adrenocortical carcinomas. Both cortical and medullary adrenal tumors expressed NCAD, NCAM, and CD44 but were devoid of ECAD. The expression of CD44 and NCAM did not correlate with the malignant potential of tumors. NCAD was upregulated in MPCs, but downregulated in adrenocortical carcinoma. Thus, NCAD appears to be involved in the development of both cortical and medullary adrenal tumors.     

Characterization of a novel breast carcinoma xenograft and cell line derived from a BRCA1 germ-line mutation carrier

A human tumor xenograft (L56Br-X1) was established from a breast cancer axillary lymph node metastasis of a 53-year-old woman with a BRCA1 germ-line nonsense mutation (1806C>T; Q563X), and a cell line (L56Br-C1) was subsequently derived from the xenograft. The xenograft carries only the mutant BRCA1 allele and expresses mutant BRCA1 mRNA but no BRCA1 protein as determined by immunoprecipitation or Western blotting. The primary tumor, lymph node metastasis, and xenograft were hypodiploid by DNA flow cytometry, whereas the cell line displayed an aneuploidy apparently developed via polyploidization. Cytogenetic analysis, spectral karyotyping, and comparative genomic hybridization of the cell line revealed a highly complex karyotype with numerous unbalanced translocations. The xenograft and cell line had retained a somatic TP53 missense mutation (S215I) originating from the primary tumors, as well as a lack of immunohistochemically detectable expression of steroid hormone receptors, epidermal growth factor receptor, human epidermal growth factor receptor 2 (HER-2), and keratin 8. Global gene expression analysis by cDNA microarrays supported a correlation between the expression profiles of the primary tumor, lymph node metastasis, xenograft, and cell line. We conclude that L56Br-X1 and L56Br-C1 are useful model systems for studies of the pathogenesis and new therapeutic modalities of BRCA1-induced human breast cancer.     

An azurocidin-like protein is induced in Trichoplusia ni larval gut cells after bacterial challenge

Trichoplusia ni immune genes up-regulated in response to bacterial infection have been isolated using differential display polymerase chain reaction. Here we report the cloning and characterisation of a gut-specific immune gene encoding an azurocidin-like protein. The deduced protein is 317 amino acid residues long with a hydrophobic C-terminus and a predicted 17-residue signal peptide. The mature T. ni protein shows 30% identity to human azurocidin, an antibacterial protein. Like azurocidin, the T. ni protein contains two amino acid substitutions in the active site triad normally present in serine proteases. The T. ni protein was synthesised with a six-histidine C-terminal extension using the baculovirus expression system. Sequencing of the recombinant azurocidin-like protein confirmed the predicted cleavage of the signal peptide. Northern blots show that T. ni azurocidin-like protein is expressed solely in the larval gut and that expression is up-regulated by injecting or feeding bacteria. Expression reaches its highest level at 10 h after bacteria injection.     

Genome profiles of bilateral dysgerminomas, a unilateral gonadoblastoma, and a metastasis from a 46, XY phenotypic female

We present a case report of a 16-year-old, phenotypic female with bilateral dysgerminomas, a unilateral gonadoblastoma, and a peritoneal metastasis. The patient's constitutional karyotype was 46,XY. The chromosomal copy number, examined by the comparative genomic hybridization technique, showed 3 gains in the dysgerminoma of the right ovary, 6 gains in the dysgerminoma of the left ovary, and 2 gains and 1 loss in the gonadoblastoma of the left ovary. The metastasis showed 5 gains of which 4 were also observed in the dysgerminoma of the left ovary. The DNA ploidy classifications of the gonadoblastoma and the dysgerminoma in the right ovary were tetraploid, whereas the dysgerminoma in the left ovary and the metastasis were aneuploid. We therefore propose that the metastasis most probably developed from the dysgerminoma of the left ovary.     

Comparison of serum hepatitis C virus RNA and core antigen concentrations and determination of whether levels are associated with liver histology or affected by specimen storage time

An enzyme immunoassay has recently been developed for the hepatitis C virus (HCV) core antigen. To evaluate the possible association between core antigen and HCV RNA levels with regards to the change in liver histology over time as well as study the effect of duration of storage on viral load results, sequential sera were analyzed from 45 patients with chronic HCV infection who had undergone two or more liver biopsies. A relatively strong association was found between the core antigen and HCV RNA concentrations (r(s) = 0.8), with a core antigen level of 1 pg/ml corresponding to approximately 1,000 IU/ml. All 42 sera with detectable HCV RNA at the time of the second biopsy had core antigen concentrations above 1 pg/ml, and the three sera without detectable HCV RNA had concentrations below 1 pg/ml. No association was found between HCV RNA or core antigen levels and the stage of fibrosis in biopsy samples, progression of fibrosis, necro-inflammatory grade, steatosis, genotype, alanine aminotransferase level, or alcohol consumption. A significant association was demonstrated between the storage time of the samples and both the HCV RNA and core antigen concentrations. The median log HCV RNA concentrations (international units/milliliter) were 3.92 for the sera obtained at the time of the first biopsy (median storage time, 13.0 years) and 4.41 for the sera obtained at the time of the second biopsy (median storage time, 6.6 years) compared to 5.96, the median for 102 different routine clinical patient samples.     

Oestrogen-induced suppression of collagen arthritis; 17 beta-oestradiol is therapeutically active in normal and castrated F1 hybrid mice of both sexes.

The F1 hybrid mouse strain, from B10Q and DBA/1 parentals (the QD strain), is highly susceptible to induction of type II collagen-induced arthritis, an experimental model for rheumatoid arthritis. Males are more susceptible than females. Oophorectomy enhances susceptibility to arthritis and treatment with physiological doses of 17 beta-oestradiol (E2) suppresses disease. E2 treatment lowers the incidence of arthritis also in non-castrated and castrated males, showing that the anti-arthritic effect by oestrogen is not dependent on either sex hormone imprinting effects or interference with male sex hormones. Testosterone treatment of normal females, but not of castrated females, exaggerated development of the disease. In the testosterone-treated normal females, the oestrogen effect on vaginal smear was abolished and ovarian weight decreased, suggesting that the testosterone-mediated enhancing effect is caused by inhibition of ovarian oestrogen production. The crucial importance of oestrogens for the development of arthritis is focused on the effectiveness of treatment with gestation-related doses of E2 of normal, non-castrated females.     

Serum antibody response to filamentous hemagglutinin in patients with clinical pertussis measured by an enzyme-linked immunosorbent assay

Titers of antibodies to filamentous hemagglutinin (FHA) were determined by enzymelinked immunosorbent assay in acute and convalescent phase serum samples from 158 patients with clinical symptoms typical of whooping-cough. In 96 of the patients the diagnosis was verified by culture. Significant changes in serum levels of IgG, IgM and/or IgA antibodies against FHA were demonstrated in 126 patients (80%). Thus, demonstration of significant changes in FHA antibody titers in serum can be used for serological diagnosis of pertussis. The results also show that high levels of IgG, IgM and/or IgA antibodies in a single serum sample suggest current pertussis infection, but if the diagnosis is based on determinations of FHA antibody titers in a single serum sample the sensitivity is low. The levels of antibody to FHA were compared with previously determined levels of antibodies to pertussis toxin. A significant antibody response against both FHA and pertussis toxin was seen in 111 patients (70 %) while 147 patients (93 %) developed a significant increase in antibodies against one or both antigens.