Multisite analytical evaluation of the Abbott ARCHITECT cyclosporine assay.

The objective of this study was to evaluate the analytical performance of the Abbott ARCHITECT Cyclosporine (CsA) immunoassay in 7 clinical laboratories in comparison to liquid chromatography/tandem mass spectrometry (LC/MS/MS), Abbott TDx, Cobas Integra 800, and the Dade Dimension Xpand immunoassay. The ARCHITECT assay uses a whole blood specimen, a pretreatment step with organic reagents to precipitate proteins and extract the drug, followed by a 2-step automated immunoassay with magnetic microparticles coated with anti-CsA antibody and an acridinium-CsA tracer. Imprecision testing at the 7 evaluation sites gave a range of total % coefficient of variations of 7.5%-12.2% at 87.5 ng/mL, 6.6%-14.3% at 411 ng/mL, and 5.2%-10.7% at 916 ng/mL. The lower limit of quantification ranged from 12 to 20 ng/mL. Purified CsA metabolites AM1, AM1c, AM4N, AM9, and AM19 were tested in whole blood by the ARCHITECT assay and showed minimal cross-reactivity at all 7 sites. In particular, AM1 and AM9 cross-reactivity in the ARCHITECT assay, ranged from -2.5% to 0.2% and -0.8% to 2.2%, respectively, and was significantly lower than for the TDx assay, in which the values were 3.2% and 16.1%, respectively. Comparable testing of metabolites in the Dade Dimension Xpand assay at 2 evaluation sites showed cross-reactivity to AM4N (6.4% and 6.8%) and AM9 (2.6% and 3.6%) and testing on the Roche Integra 800 showed cross-reactivity to AM1c (2.4%), AM9 (10.7%), and AM19 (2.8%). Cyclosporine International Proficiency Testing Scheme samples, consisting of both pooled specimens from patients receiving CsA therapy as well as whole-blood specimens supplemented with CsA, were tested by the ARCHITECT assay at 6 sites and showed an average bias of -24 to -58 ng/mL versus LC/MSMS CsA and -2 to -37 ng/mL versus AxSYM CsA. Studies were performed with the ARCHITECT CsA assay on patient specimens with the following results: ARCHITECT CsA assay versus LC/MSMS, average bias of 31 ng/mL; ARCHITECT versus the Dade Dimension assay (4 sites), average biases of -7 to -228 ng/mL; ARCHITECT versus AxSYM and TDx, average biases of -4 and -53 ng/mL, respectively. Spearman correlation coefficients were >or=0.89. The ARCHITECT CsA assay has significantly reduced CsA metabolite interference relative to other immunoassays and is a convenient and sensitive semiautomated method to measure CsA in whole blood.

Superantigen-presentation by rat major histocompatibility complex class II molecules RT1.Bl and RT1.Dl.

Rat major histocompatibility complex (MHC) class II molecules RT1.B(l) (DQ-like) and RT1.D(l) (DR-like) were cloned from the LEW strain using reverse transcription-polymerase chain reaction and expressed in mouse L929 cells. The transduced lines bound MHC class II-specific monoclonal antibodies in an MHC-isotype-specific manner and presented peptide antigens and superantigens to T-cell hybridomas. The T-cell-hybridomas responded well to all superantigens presented by human MHC class II, whereas the response varied considerably with rat MHC class II-transduced lines as presenters. The T-cell hybridomas responded to the pyrogenic superantigens Staphylococcus enterotoxin B (SEB), SEC1, SEC2 and SEC3 only at high concentrations with RT1.B(l)-transduced and RT1.D(l)-transduced cells as presenters. The same was true for streptococcal pyrogenic exotoxin A (SPEA), but this was presented only by RT1.B(l) and not by RT1.D(l). SPEC was recognized only if presented by human MHC class II. Presentation of Yersinia pseudotuberculosis superantigen (YPM) showed no MHC isotype preference, while Mycoplasma arthritidis superantigen (MAS or MAM) was presented by RT1.D(l) but not by RT1.B(l). Interestingly, and in contrast to RT1.B(l), the RT1.D(l) completely failed to present SEA and toxic shock syndrome toxin 1 even after transduction of invariant chain (CD74) or expression in other cell types such as the surface MHC class II-negative mouse B-cell lymphoma (M12.4.1.C3). We discuss the idea that a lack of SEA presentation may not be a general feature of RT1.D molecules but could be a consequence of RT1.D(l)beta-chain allele-specific substitutions (arginine 80 to lysine, asparagine 82 to aspartic acid) in the extremely conserved region flanking the Zn(2+)-binding histidine 81, which is crucial for high-affinity SEA-binding.

Structure and expression of MHC class Ib genes of the central M region in rat and mouse: M4, M5, and M6.

The M region at the telomeric end of the murine major histocompatibility complex (MHC) contains class I genes that are highly conserved in rat and mouse. We have sequenced a cosmid clone of the LEW rat strain (RT1 haplotype) containing three class I genes, RT1.M6-1, RT1.M4, and RT1.M5. The sequences of allelic genes of the BN strain (RT1n haplotype) were obtained either from cDNAs or genomic clones. For the coding parts of the genes few differences were found between the two RT1 haplotypes. In LEW, however, only RT1.M5 and RT1.M6 have open reading frames; whereas in BN all three genes were intact. In line with the findings in BN, transcription was found for all three rat genes in several tissues from strain Sprague Dawley. Protein expression in transfectants could be demonstrated for RT1.M6-1 using the monoclonal antibody OX18. By sequencing of transcripts obtained by RT-PCR, a second, transcribed M6 gene, RT1.M6-2, was discovered, which maps next to RT1.M6-1 outside of the region covered by the cosmid. In addition, alternatively spliced forms for RT1.M5 and RT1.M6 were detected. Of the orthologous mouse genes, H2-M4, H2-M5, and H2-M6, only H2-M5 has an open reading frame. Other important differences between the corresponding parts of the M region of the two species are insertion of long LINE repeats, duplication of RT1.M6, and the inversion of RT1.M5 in the rat. This demonstrates substantial evolutionary dynamics in this region despite conservation of the class I gene sequences themselves.

The superantigen-induced polarization of T cells in rat peripheral lymph nodes is influenced by genetic polymorphisms in the IL-4 and IL-6 gene clusters.

In recent years, it has become clear that the polarization of T cells depends on the genetic background. However, due to the complexity of the genetic background of each animal, a direct comparison of the phenotype is difficult. In this study, a new rat strain LEW.BN-4-10 carrying the chromosomal regions on chromosomes 4 and 10, which harbor IL-6 and IL-4 gene clusters of BN, has been bred on the genetic background of LEW. It was asked whether these two gene clusters influence the polarization of T cell responses. As a model, the Mycoplasma arthritidis mitogen (MAM)-induced inflammation was used focusing on the microenvironment of the draining lymph node (LN). The effect of differences in these regions was tested by comparing LEW.BN-4-10 and LEW rats under steady-state conditions and upon injection of MAM into the forepaw. Under steady-state conditions, the two strains showed differences in the dendritic cell (DC) subset composition. When MAM was injected, the number of T cells in LEW.BN-4-10 rats producing T(h)2 cytokines such as IL-4 and IL-13 was significantly increased compared with LEW. The data suggest that these differences in the microenvironments in LN of LEW and LEW.BN-4-10 rats resulted in different susceptibility to the disease (increase of cells in LN and paw swelling). In addition, deviations in the distribution and function of injected effector T cells were found in the LN of LEW and LEW.BN-4-10 rats after MAM treatment. The data indicate that the IL-6 and IL-4 gene clusters are involved in polarizing T cell responses in vivo.

Health status of transgenic pigs expressing the human complement regulatory protein CD59.

BACKGROUND: Microinjection of foreign DNA into pronuclei of zygotes has been the method of choice for the production of transgenic domestic animals. Following microinjection the transgene is randomly integrated into the host genome which can be associated with insertional mutagenesis and unwanted pathological side effects. METHODS: Here, we evaluated the health status of pigs transgenic for the human regulator of complement activation (RCA) CD59 and conducted a complete pathomorphological examination on 19 RCA transgenic pigs at 1 to 32 months of age from nine transgenic lines. Nine wild-type animals served as controls. Expression levels of human complement regulator CD59 (hCD59) mRNA were measured by RT-PCR and distribution of hCD59 protein was determined by immunohistochemistry. RESULTS: Albeit variable transgene expression levels, no specific pathomorphologic phenotype associated with the presence of the transgene in all analyzed pig lines could be detected. CONCLUSIONS: Transgenic expression of this human RCA gene construct is not correlated with a specific pathological phenotype in pigs. This is crucial for the application of the technology and the use of transgenic pigs for biomedical and agricultural applications.

Epigenetic silencing and tissue independent expression of a novel tetracycline inducible system in double-transgenic pigs.

The applicability of tightly regulated transgenesis in domesticated animals is severely hampered by the present lack of knowledge of regulatory mechanisms and the long generation intervals. To capitalize on the tightly controlled expression of mammalian genes made possible by using prokaryotic control elements, we have used a single-step transduction to introduce an autoregulative tetracycline-responsive bicistronic expression cassette (NTA) into transgenic pigs. Transgenic pigs carrying one NTA cassette showed a mosaic transgene expression restricted to single muscle fibers. In contrast, crossbred animals carrying two NTA cassettes with different transgenes, revealed a broad tissue-independent and tightly regulated expression of one cassette, but not of the other one. The expression pattern correlated inversely with the methylation status of the NTA transcription start sites indicating epigenetic silencing of one NTA cassette. This first approach on tetracycline regulated transgene expression in farm animals will be valuable for developing precisely controlled expression systems for transgenes in large animals relevant for biomedical and agricultural biotechnology.

RT1.L: a family of MHC class Ib genes of the rat major histocompatibility complex with a distinct promoter structure.

RT1.L class I antigens have originally been identified in LEW rats by LEW.1LV3-anti-LEW.1LM1 antisera and have been classified as nonclassical. We report now that LEW.1LV3-anti-LEW.1LM1 antisera react with three different antigens, termed RT1.L1, RT1.L2, and RT1.L3. This was found by serological analysis of a panel of transfectants expressing different class I genes of strain LEW with a LEW.1LV3-anti-LEW.1LM1 antiserum and two monoclonal antibodies (mAbs HT20 and HT21) generated in the same strain combination. The antiserum reacted with all three antigens: the two mAbs with RT1.L1 and RT1.L2, respectively. Sequence analysis showed that the genes encoding RT1.L1, RT1.L2, and RT1.L3 cluster together in a phylogenetic analysis of rat and mouse alpha(1)-alpha(2) sequences and that they share an unusual MHC class I promoter in which Enhancer A and B, as well as the interferon response element (IRE), are missing. Exchange of the promoter in RT1.L2 against the classical RT1.A promoter resulted in high surface expression in appropriate transfectants, indicating that the deviant promoter is responsible for the weak surface expression of the RT1.L2 gene. The very similar promoter structures of RT1.L1 and RT1.L3 are likely to contribute also to the weak expression of these genes. As RT1.L3 maps closely to the deletion in the mutant haplotype lm1, the RT1.L family can be located in the class I region extending from Bat1 to Pou5f1. Different from other allogeneic mAbs detecting known class I molecules encoded by genes of the RT1.C/E region, HT20 and HT21 react with a wide panel of strains carrying different RT1 haplotypes. This suggests that nonclassical class I genes of the RT1.L family are present in most RT1 haplotypes.

In vivo depletion of hematopoietic stem cells in the rat by an anti-CD45 (RT7) antibody.

Anti-CD45 monoclonal antibodies (mAbs) are potentially powerful tools for the depletion of mature leukocytes. As their application for immunotherapy also depends on their effects on bone marrow (BM) progeny, the in vivo effects of an anti-CD45 mAb (anti-RT7(a) mAb) on BM precursor cells were analyzed in a rat model. Anti-RT7(a) mAb treatment was performed in LEW.1W (RT1(u) RT7(a)) rats with the use of different dosages. In addition, major histocompatibility complex (MHC)-congenic BM transplantation making use of a diallelic polymorphism (RT7(a)/RT7(b)) of rat CD45 was applied. Following injection of anti-RT7(a) mAb into normal LEW.1W rats, T cells were profoundly depleted in blood, lymph nodes, and spleen, whereas B cells were coated only by the antibody. Single injection of anti-RT7(a) mAb in a high dose induced a lethal aplastic syndrome with severe thrombocytopenia. Rescue of antibody-treated animals with BM from congenic LEW.1W-7B rats (RT1(u) RT7(b)) and transplantation of BM from LEW.1W rats pretreated with anti-RT7(a) mAb into sublethally irradiated LEW.1W-7B recipients revealed a profound effect of the mAb on progeny of myeloid and T-cell lineage. Following repeated antibody treatment of stable mixed chimeras (RT7(b)/RT7(a)), very few RT7(a)-positive B cells were still detectable after 6 months and their number declined during the subsequent year. These observations show that this anti-RT7(a) mAb effectively depletes mature T cells as well as BM precursor cells of myeloid, T-cell, and thrombocytic lineage after in vivo application. In contrast, mature B cells are not depleted, but precursors also appear to be eliminated. Overall, the findings suggest that the anti-RT7(a) mAb efficiently depletes early rat hematopoietic stem cells.