Double Transgenic Pigs with Combined Expression of Human α1,2-Fucosyltransferase and α-Galactosidase Designed to Avoid Hyperacute Xenograft Rejection

Hyperacute rejection (HAR) depends on the response of xenoreactive antibodies principally against porcine α-Gal epitope. Methods eliminating HAR include GGTA1 inactivation, regulation of the complement system and modification of the oligosaccharide structure of surface proteins in donor’s cells. Transgenic animals designed for the purpose of xenotransplantation with single modification do not display full reduction of the α-Gal epitope level, which means that a accumulation of several modifications in one transgenic individual is needed. The aim of the study was to create a molecular and cytogenetic profile of a double transgenic animal with α1,2-fucosyltransferase and α-galactosidase expression. As a result of interbreeding of an individual with α1,2-fucosyltransferase expression with an individual with α-galactosidase expression 12 living piglets were obtained. PCR revealed the pCMVFUT gene construct was present in four individuals and pGAL-GFPBsd in three, including one with a confirmed integration of both the gene constructs. Fluorescence in situ hybridization confirmed the site of transgene integration, which corresponded to the mapping site of the transgenes which occurred in the parental generations. Karyotype analysis did not show any changes in the structure or the number of chromosomes (2n = 38, XX). As for the results pertaining to the single transgenic individuals, expression analysis demonstrated a high extent of α-Gal epitope level reduction on the surface of cells, whereas human serum cytotoxicity tests revealed the smallest decrease in longevity of cells in the obtained double transgenic individual (4.35 %). The tests suggest that the co-expression of both the transgenes leads to a considerable reduction of the α-Gal antigen level on the surface of cells and a decrease of xenotransplant immunogenicity.     

Immuno‐Quantitative Polymerase Chain Reaction for Detection and Quantitation of Prion Protein

Abstract

Immuno‐polymerase chain reaction (PCR) is an extremely sensitive detection method, combining the specificity of antibody detection and the sensitivity of PCR. We have developed an immuno‐quantitative PCR (iqPCR), exploiting real‐time PCR technology, in order to improve this immuno‐detection method and make it quantitative. To illustrate the advantages of iqPCR, we have compared it with a conventional enzyme linked immuno sorbent assay (ELISA) technique in experiments aimed at detecting the cellular and the resistant form of prion protein in bovine brain extract. The iqPCR technique proved to be more sensitive than ELISA, so it could be a technique of choice for the diagnosis of infected animals both at an ante mortem and post‐mortem stage.     

Immunocytochemical Studies of Aquaporin 4, Kir4.1, and α1-syntrophin in the Astrocyte Endfeet of Mouse Brain Capillaries

One of the most important physiological roles of brain astrocytes is the maintenance of extracellular K⁺ concentration by adjusting the K⁺ influx and K⁺ efflux. The inwardly rectifying K⁺ channel Kir4.1 has been identified as an important member of K⁺ channels and is highly concentrated in glial endfeet membranes. Aquaporin (AQP) 4 is another abundantly expressed molecule in astrocyte endfeet membranes. We examined the ultrastructural localization of Kir4.1, AQP4, α1-syntrophin, and β-spectrin molecules to understand the functional role(s) of Kir4.1 and AQP4. Immunogold electron microscopy of these molecules showed that the signals of these molecules were present along the plasma membranes of astrocyte endfeet. Double immunogold electron microscopy showed frequent co-localization in the combination of molecules of Kir4.1 and AQP4, Kir4.1 and α1-syntrophin, and AQP4 and α1-syntrophin, but not those of AQP4 and β-spectrin. Our results support biochemical evidence that both Kir4.1 and AQP4 are associated with α1-syntrophin by way of postsynaptic density-95, Drosophila disc large protein, and the Zona occludens protein I protein-interaction domain. Co-localization of AQP4 and Kir4.1 may indicate that water flux mediated by AQP4 is associated with K⁺ siphoning.     

Immunocytochemical Evidence for the Glutamate-Containing and GABA-Containing Callosal Neurons in Rats

Chemical characterization of the callosal neurons in the frontal and occipital cortex of Wistar rats was studied by means of combined HRP retrograde tracing and ABC immunocytochemistry for Glutamate (Glu) and α-aminobutyric acid (GABA). Glu-containing callosal neurons were found as large or medium sized pyramidal cells and mainly localized in layers Ⅱ/Ⅲ, Ⅴ and Ⅵ. They tended to appear as clusters. GABA-containing callosal neurons were also observed. They were medium sized nonpyramidal cells with round, elliptic, or fusiform soma and mostly distributed in     

Double Reactivity Against Actin and α-Actinin Defines a Severe Form of Autoimmune Hepatitis Type 1

Anti-filamentous actin antibodies characterize autoimmune hepatitis type 1 (AIH-1). Recently, the binding domain of alpha-actinin on actin was shown to be a predominant epitope. To test this reactivity, an anti-alpha-actinin enzyme-linked immunosorbent assay was developed, and positivity confirmed by Western blot. Anti-alpha-actinin antibody was found in 21/50 (42%) of AIH-1 patients, compared with 52/401 (12.9%) of liver disease control patients, and with 6/200 (6%) of blood donors. Anti-filamentous and anti-alpha-actinin activities were found specifically together in 66% of anti-filamentous-positive AIH-1 patients. This combination of specificities reflected clinical and histological disease activity, short duration and absence of treatment. Finally, using an actin-alpha-actinin complex assay, the binding of anti-filamentous actin to alpha-actinin-binding domain on actin was demonstrated, as well as that of anti-alpha-actinin on the actin-binding domain of alpha-actinin. Thus, the frequent combination of anti-filamentous and anti-alpha-actinin antibodies seems to be the hallmark of activity in AIH-1.     

Characteristics of Radioimmunoassays for the α- and β-Subunits of Human Luteinizing Hormone

Abstract

The binding characteristics and specificities of the National Hormone and Pituitary Program (NHPP) kits for the radioimmunoassay of the alpha- and beta-subunits of human luteinizing hormone (hLH-α and hLH-β) were studied, as well as the specificities of the anti-hLH and anti-human follicle stimulating hormone (anti-hFSH) antisera distributed by the same organization. The affinity constants of the anti-hLH-α and anti-hLH-β antisera were calculated at 157 ± 8.4 nM^?1 and 109 ± 7.4 nM^?1, respectively. Both antisera were highly specific with regard to the other subunit. However, in the homologous hLH-α RIA, native hLH cross-reacted at 21.9%, hFSH at 17.5% and hTSH at 7.9%. The alpha-subunit of the human chorionic gonadotropin, hCG-α, was equipotent with the hLH-α standard in this assay. In the homologous hLH-β RIA, hLH showed a cross-reactivity of 14.7% while the cross-reactivities of hCG-β, hFSH and hTSH were 3.5%, 1.2% and 0.6%, respectively. The anti-hFSH antiserum was highly specific, while the anti-hLH antiserum showed non parallel competition curves. With this knowledge of the specificity of each antiserum, corrections can be properly made for the assays of hLH, hLH-α and hLH-β while the hFSH RIA can be used without correction for the presence of the three other components.     

Interaction of simian virus 40 large T-antigen with cellular DNA polymerase α: studies with various T-antigen mutants of SV40

Summary Simian virus 40 (SV40) large T antigen binds to host cell DNA polymerase and the T-antigen-DNA polymerase complex is implicated in the initiation of viral DNA replication. We have examined various SV40 T-antigen mutants to test the correspondence between viral DNA replication and T-antigen-DNA polymerase complex formation. The various SV40 T-antigen mutants were used to either infect or transfect African green monkey kidney cell line CV-1, and at different time intervals we measured the production of T-antigen and host cell DNA polymerase by radioimmunoassay, complex formation by a sandwich radioimmunoassay and the amount of viral DNA synthesis by dot-blot hybridization analysis. There was a good correlation between complex formation and viral DNA synthesis in lytic mutants of SV40. Poor complex formation and correspondingly lower DNA synthesis were observed in the non-viable mutants of SV40, even though significant amounts of T-antigen and DNA polymerase were present. Our results substantiate the earlier findings of T-antigen-DNA polymerase complex formation and establish the need for formation of this complex in promoting viral DNA synthesis.     

Determination of attachment sites for N-linked carbohydrate groups of class II histocompatibility α-chain and analysis of possible O -linked glycosylation of α- and γ -chains

Two glycopeptide fractions were isolated from a tryptic digest of the human class II antigen α-subunit, by chromatography on Lens culinaris and Ricinus communis lectin columns, respectively. Partial NH₂-terminal sequence analysis of radiochemically labelled glycopeptide fractions allowed alignment with two stretches of the deduced DRα sequence, each encompassing a signal for N-linked glycosylation, i.e. Asn-X-Thr(Ser). The fraction displaying affinity for L. culinaris lectin was susceptible to the action of endo-β-N-acetylglucosaminidase H whereas the fraction adsorbed to R. communis was not. Since DRα is known to carry only one N-linked carbohydrate chain in the high-mannose form, this glycan could thereby be mapped to Asn 78. Accordingly, the complex N-linked carbohydrate is attached to Asn 118. Moreover, analysis of material released from class II antigens and γ-chains upon mild alkaline hydrolysis, indicates the presence of O-linked sugars on the α- and γ- but not the β-subunits.     

Concentrations of α- and β-defensins in plasma of patients with inflammatory bowel disease

Background:  Impaired production/release of defensins, representative endogenous antimicrobial peptides, is associated with the pathogenesis of inflammatory bowel disease (IBD). Material and methods:  Employing in house radioimmunoassay, we examined concentrations of the major forms α-defensins, human neutrophil peptides (HNP) 1–3 and human β-defensin (HBD)-2 in plasma of 55 IBD patients consisting of 29 patients with ulcerative colitis (UC) and 26 with Crohn’s disease (CD) and 57 controls. Results:  The circulating HNP 1–3, but not HBD-2, levels in IBD patients were significantly higher than those in controls. Plasma HNP 1–3 concentrations in CD patients significantly correlated with Crohn’s disease activity index, peripheral white blood cell counts, serum CRP values and TNF-α levels. Conclusions:  Elevation of circulating α-defensins levels is suggestive of their physiopathological roles in IBD. Plasma HNP 1–3 concentrations may be an indicator for CD activity and their association with CRP and TNF-α supports a possible association with the inflammatory process. Received 2 June 2008; returned for revision 20 June 2008; received from final revision 25 June 2008; accepted by C. Kasserra 19 August 2008     

Age-related changes in expression and activity of DNA polymerase α: some effects of dietary restriction

DNA polymerase α (pol α) purified from human diploid fibroblasts (HDF) and from livers of C57BL/6N mice showed age-related decreases in: (1) mRNA levels; (2) the amount of enzyme isolated per cell; and (3) enzyme activity (HDF); as well as: a) the amount of enzyme isolated; b) the specific activity; and c) the enzyme fidelity (liver). Hepatic pol α from dietary restricted (DR) mice exhibited less of a decline in specific activity and copied synthetic DNA templates with relatively higher fidelity than did enzymes from animals fed ad libitum (AL). Pol α from fetal-derived HDF exhibited increased expression compared with aged donor-derived HDF, with both fetal and old cell pol α in normal cells being expressed at lower levels than in their transformed cell corollaries. Treatment of human pol α from aged donor-derived HDF with a pol α accessory protein isolated from log phase murine cells resulted in increased pol α binding of DNA and increased pol α activity. However, highly active pol α isolated from fetal-derived or transformed HDF, or from transformed murine cells, showed little or no activity enhancement in the presence of accessory protein. These data indicate that, as a function of increased age, there is a decrease in pol α expression and specific activity in HDF, as well as decreases in specific activity and fidelity of pol α in essentially amitotic murine hepatic tissues. Dietary restriction impedes the age-related declines in both activity and fidelity of hepatic pol α in mice. The data further indicate that transformation of slowly dividing HDF is associated with increased expression of pol α, but suggest that increased expression alone is not sufficient to explain the difference in polymerase activity levels between parental and transformed HDF. Lastly, the data suggest that interaction of pol α with an essential accessory protein may be altered as a function of age, an alteration that appears to be correlated with the decline in pol α DNA binding and specific activity.     

Anti-mycobacterial activities of synthetic cationic α-helical peptides and their synergism with rifampicin

The rapid emergence of multi-drug resistant tuberculosis (TB) and the lack of effective therapies have prompted the development of compounds with novel mechanisms of action to tackle this growing public health concern. In this study, a series of synthetic cationic α-helical antimicrobial peptides (AMPs) modified with different hydrophobic amino acids was investigated for their anti-mycobacterial activity, both alone and in synergistic combinations with the frontline anti-tuberculosis drug rifampicin. The addition of thiol groups by incorporating cysteine residues in the AMPs did not improve anti-mycobacterial activity against drug-susceptible and drug-resistant Mycobacterium tuberculosis, while the enhancement of peptide hydrophobicity by adding methionine residues increased the efficacy of the primary peptide against all strains tested, including clinically isolated multidrug-resistant mycobacteria. The peptide with the optimal composition M(LLKK)₂M was bactericidal, and eradicated mycobacteria via a membrane-lytic mechanism as demonstrated by confocal microscopic studies. Mycobacteria did not develop resistance after multiple exposures to sub-lethal doses of the peptide. In addition, the peptide displayed synergism with rifampicin against both Mycobacterium smegmatis and Mycobacterium bovis BCG and additivity against M. tuberculosis. Moreover, such combination therapy is effective in delaying the emergence of rifampicin resistance. The ability to potentiate anti-TB drug activity, kill drug-resistant bacteria and prevent drug resistance highlights the potential utility of the peptide in combating multidrug-resistant TB.     

Production and characterization of monoclonal antibodies against α-globin chain-containing human hemoglobins for detecting α-thalassemia disease

Monoclonal antibodies against α-globin containing human Hbs, named AMS-Alpha1 and AMS-Alpha 2, were produced by the hybridoma technique using spleen cells enriched by the newly developed B lymphocyte enrichment protocol. These two monoclonal antibodies were of IgM class, reacting to only intact form of human Hbs A, A₂, E, and F, which contain α-globin chain. By the indirect ELISA, the AMS-Alpha1 and AMS-Alpha 2 quantified less amount of α-globin chain containing hemoglobins in HbH disease than the SEA-α thalassemia 1 carriers and normal individuals. It was thus anticipated that these monoclonal antibodies can be used for detecting Hb Bart’s hydrops fetalis in which no α-globin chain is produced.     

Genetic and physical interactions between Schizosaccharomyces pombe Mcl1 and Rad2, Dna2 and DNA polymerase α: evidence for a multifunctional role of Mcl1 in DNA replication and repair

Schizosaccharomyces pombe rad2 is involved in Okazaki fragments processing during lagging-strand DNA replication. Previous studies identified several slr mutants that are co-lethal with rad2 and sensitive to methyl methanesulfonate as single mutants. One of these mutants, slr3-1, is characterized here. Complementation and sequence analyses show that slr3-1 (mcl1-101) is allelic to mcl1⁺, which is required for chromosome replication, cohesion and segregation. mcl1-101 is temperature-sensitive for growth and is highly sensitive to DNA damage. mcl1 cells arrest with 2C DNA content and chromosomal DNA double-strand breaks accumulate at the restrictive temperature. Mcl1p, which belongs to the Ctf4p/SepBp family, interacts both genetically and physically with DNA polymerase . Mutations in rhp51 and dna2 enhance the growth defect of the mcl1-101 mutant. These results strongly suggest that Mcl1p is a functional homologue of Saccharomyces cerevisiae Ctf4p and plays a role in lagging-strand synthesis and Okazaki fragment processing, in addition to DNA repair.     

Efficacy of combination therapy with amantadine,thymosin α1 and α/β interferon in mice infected with influenza a virus

The aim of this study was to examine the effects of the antiviral drug amantadine (AMN) administered in combination with thymosin α1 (Tα1) and murine α/β interferon (IFN) on mice infected with influenza A PR8 virus. Combined treatment with AMN and Tα1, for 4 days, followed by a single injection of IFN, was initiated 1 h after intranasal viral inoculation. The effectiveness of this new chemoimmunotherapy protocol was seen in the longterm survival of a high percentage of animals and was statistically significant when compared to treatment with single agents in conjunction with chemotherapy or to chemotherapy alone. In addition, chemoimmunotherapy treatment reduces the viral titre in the lungs as well as restoring the immunological parameters tested (natural killer cell activity; cytotoxic T-lymphocyte responses; CD4⁺/CD8⁺ lymphocyte subsets) with respect to all other groups. These results suggest the potential use of these immunomodulating agents in combination with an antiviral drug in controlling PR8 influenza virus infection.     

Polymerase chain reaction and DNA probe hybridization to assess the efficacy of diminazene treatment in Trypanosoma brucei -infected cattle

Four of eight Ankole longhorn cattle experimentally infected with Trypanosoma brucei were treated with 7 mg/kg diminazene aceturate (Berenil, Hoechst AG, Germany) at day 71 postinfection. The trypanocidal activity was monitored using polymerase chain reaction (PCR) and DNA probe hybridization. When extracted parasite DNA (without host DNA) was used, as little as 1 fg per reaction, which is equivalent to about 1–10% of the DNA in a single trypanosome, produced a specific product that was visible as a 177-bp band in an agarose gel. In infected cattle, specific PCR products could be amplified at as early as 1 day postinfection. PCR signals remained positive during infection, except in one sample, although aparasitemic phases occurred. In cases where treatment resulted in a significant clinical improvement, PCR signals disappeared at 3–4 days after the administration of the drug. By contrast, in cattle that showed clinical signs of CNS involvement after treatment, although aparasitemic, and died before the termination of the experiment, specific products could be amplified on several occasions following treatment. The PCR signals generated after treatment could be further enhanced by subsequent slot-blot hybridization with a T. brucei-specific DNA probe. We conclude that PCR coupled with DNA probe hybridization provides a highly sensitive tool for the assessment of therapeutic efficiency and disease progression in trypanosome infections, especially in chronic infections when the level of parasitemia is low or when trypanosomes are sequestered at cryptic sites. Received: 29 May 1998 / Accepted: 18 August 1998