Sperm mediated gene transfer in pig: Selection of donor boars and optimization of DNA uptake

Transgenic animals are produced primarily by microinjecting exogenous DNA into the male pronuclei of a zygote. Microinjection is successful in mice but not efficient in farm animals, limiting its general utility. We have pursued an alternative technology for producing transgenic animals: Sperm Mediated Gene Transfer (SMGT). Based on our finding that sperm cells bind and internalize exogenous DNA, we used sperm as a vector for transmitting, not only their own DNA, but also, the exogenously-introduced gene of interest to the zygote. SMGT is highly efficient (up to greater than 80%) and relatively inexpensive; it can be used in species refractory to microinjection, whenever reproduction is mediated by gametes. In this report, we describe the procedure for selection of sperm donors and optimization of DNA uptake that are the key steps for the successful outcome of SMGT. We found that the nominal parameters that boar sperm should possess to serve as a good vector for exogenous DNA are the quality of semen based on standard parameters used in conventional animal breeding programs (volume, concentration, presence of abnormal sperm cells, motility at time of collection, and high progressive motility after 2 hr) and the ability of the sperm cells to take up and internalize exogenous DNA. The results described provide significant advances in SMGT technology applied to pigs, so that transgenic pigs can be efficiently obtained. Mol.     

TRAIL-R2 (DR5) mediates apoptosis of synovial fibroblasts in rheumatoid arthritis

TRAIL has been proposed as an anti-inflammatory cytokine in animal models of rheumatoid arthritis (RA). Using two agonistic mAbs specific for TRAIL-R1 (DR4) and TRAIL-R2 (DR5), we examined the expression and function of these death receptors in RA synovial fibroblast cells. The synovial tissues and primary synovial fibroblast cells isolated from patients with RA, but not those isolated from patients with osteoarthritis, selectively expressed high levels of cell surface DR5 and were highly susceptible to anti-DR5 Ab (TRA-8)-mediated apoptosis. In contrast, RA synoviocytes did not show increased expression of TRAIL-R1 (DR4), nor was there any difference in expression of Fas between RA and osteoarthritis synovial cells. In vitro TRA-8 induced apoptosis of RA synovial cells and inhibited production of matrix metalloproteinases induced by pro-inflammatory cytokines. In vivo TRA-8 effectively inhibited hypercellularity of a SV40-transformed RA synovial cell line and completely prevented bone erosion and cartilage destruction induced by these cells. These results indicate that increased DR5 expression and susceptibility to DR5-mediated apoptosis are characteristic of the proliferating synovial cells in RA. As highly proliferative transformed-appearing RA synovial cells play a crucial role in bone erosion and cartilage destruction in RA, the specific targeting of DR5 on RA synovial cells with an agonistic anti-DR5 Ab may be a potential therapy for RA.     

Abnormal splicing of ABCA1 pre-mRNA in Tangier disease due to a IVS2 +5G>C mutation in ABCA1 gene

Two point mutations of ABCA1 gene were found in a patient with Tangier disease (TD): i) G>C in intron 2 (IVS2 +5G>C) and ii) c.844 C>T in exon 9 (R282X). The IVS2 +5G>C mutation was also found in the brother of another deceased TD patient, but not in 78 controls and 33 subjects with low HDL. The IVS2 +5G>C mutation disrupts ABCA1 pre-mRNA splicing in fibroblasts, leading to three abnormal mRNAs: devoid of exon 2 (Ex2-/mRNA), exon 4 (Ex4-/mRNA), or both these exons (Ex2-/Ex4-/mRNA), each containing a translation initiation site. These mRNAs are expected either not to be translated or generate short peptides. To investigate the in vitro effect of IVS2 +5G>C mutation, we constructed two ABCA1 minigenes encompassing Ex1-Ex3 region, one with wild-type (WTgene) and the other with mutant (MTgene) intron 2. These minigenes were transfected into COS1 and NIH3T3, two cell lines with a different ABCA1 gene expression. In COS1 cells, WTgene pre-mRNA was spliced correctly, while the splicing of MTgene pre-mRNA resulted in Ex2-/mRNA. In NIH3T3, no splicing of MTgene pre-mRNA was observed, whereas WTgene pre-mRNA was spliced correctly. These results stress the complexity of ABCA1 pre-mRNA splicing in the presence of splice site mutations.     

Cholesteryl nitrolinoleate,a nitrated lipid present in human blood plasma and lipoproteins

Nitric oxide (*NO) and *NO-derived reactive species (e.g., peroxynitrite anion, nitrogen dioxide radical) react with lipids containing unsaturated fatty acids to generate nitrated species. In the present work, we synthesized, characterized, and detected a nitrated derivative of cholesteryl linoleate (Ch18:2) in human blood plasma and lipoproteins using a high-pressure liquid chromatography coupled to electrospray ionization tandem mass spectrometry method. It was synthesized by a reaction of Ch18:2 with nitronium tetrafluoroborate, yielding a species with m/z 711, which is characteristic of the cholesteryl nitrolinoleate (Ch18:2NO2) ammonium adduct. The presence of the nitro group was confirmed by using [15N]nitrite, which gave a product with m/z 712, with the same chromatographic and spectrometric characteristics of those of m/z 711. Furthermore, a C-NO2 structure was also demonstrated in Ch18:2NO2 by infrared analysis (Vmax 1549, 1374 cm-1). A stable product with m/z of 711, showing the same chromatographic characteristics and fragmentation pattern as those of synthesized standard, was found in human blood plasma and lipoproteins of normolipidemic subjects. The presence of this novel nitrogen-containing lipid product in human plasma and lipoproteins could represent a potential indicator of the oxidative/nitrative roles that *NO or its metabolites play during in vivo lipid oxidation, generating a compensatory mechanism of protection in vascular disease.     

The desynaptic (dy) and desynaptic1 (dsy1) mutations in maize (Zea mays L) cause distinct telomere-misplacement phenotypes during meiotic prophase

During meiotic prophase, telomeres actively attach themselves to the nuclear envelope and cluster in an arrangement called the bouquet. The bouquet is unique to meiosis, highly conserved, and thought to facilitate homologous chromosome synapsis. Analy sis of three-dimensional fluorescence in situ hybridization (3-D FISH) image data has been employed to characterize the bouquet in fixed pollen mother cells of maize (Zea mays L.). In order to examine the function of the bouquet further, several meiotic mutants were screened for telomeric defects using 3-D FISH as an assay. Two mutants, desynaptic (dy) and desynaptic1 (dsy1), were found to exhibit novel telomere-misplacement phenotypes. In both cases, the telomere-associated mutant phenotypes occurred prior to what was previously reported as the earliest affected stage. Three alleles of the desynaptic1 mutation (dsy1-1, dsy1-9101, and dsy1-9307) resulted in a partial bouquet phenotype at the zygotene stage of meiotic prophase. By contrast, dy nuclei contained apparently normal bouquets, but then resulted in a premature intranuclear localization of telomeres at the pachytene stage, when telomeres normally disperse but remain attached to the nuclear envelope. The dsy1 mutation is known to impair the fidelity and progression of homologous synapsis, whereas the dy mutation is known to reduce recombination rates. If the telomere misplacements are primary defects of these mutants, then these data would be consistent with the hypothesis that meiotic telomeres have at least two separable functions, one involving proper homologous chromosome synapsis at the bouquet stage and another involving post-bouquet cross-over control.     

Cutting edge: mast cell antimicrobial activity is mediated by expression of cathelicidin antimicrobial peptide

Cathelicidins (caths) are peptides that are expressed at high levels in neutrophils and some epithelia and can act as natural antibiotics by directly killing a wide range of microorganisms. We hypothesized that caths are expressed in mast cells (MCs), because these cells have been previously associated with inherent antimicrobial activity. Cultured murine MCs contained abundant amounts of cathelin-related antimicrobial peptide (AMP), the murine cath, and this expression was inducible by LPS or lipoteichoic acid. Human skin MCs also expressed cath as detected by immunohistochemical analysis for the human cath LL-37. The functional significance of this expression was shown by comparing MCs cultured from normal mice to MCs from littermates deficient in the cathelin-related AMP gene (Cnlp(-)). MCs derived from Cnlp(-/-) animals had a 50% reduction in their ability to kill group A STREPTOCOCCUS: These MCs expressed equivalent amounts of mRNA for murine beta-defensin-4, a beta-defensin AMP. Thus, different antimicrobials can be identified in MCs, and the presence of cath is necessary for efficient bacterial killing. These observations suggest that the presence of cath is vital to the ability of mammalian MCs to participate in antimicrobial defense.     

Cutting edge: bacterial lipoprotein induces endotoxin-independent tolerance to septic shock

Tolerance to bacterial cell wall components is an adaptive host response. Endotoxin/LPS tolerance is characterized by a survival advantage against subsequent lethal LPS challenge. However, it is uncertain whether LPS tolerance can afford protection against other septic challenges. In this study, we show that tolerance induced by bacterial lipoprotein (BLP) protects mice against not only BLP-induced lethality, but also LPS-, live bacteria-, and polymicrobial sepsis-induced lethality. In contrast, LPS tolerance offers no survival benefit against the latter two challenges. Furthermore, induction of BLP tolerance results in overexpression of complement receptor type 3 and FcgammaIII/IIR on neutrophils (polymorphonuclear neutrophils) and peritoneal macrophages, with increased bacterial recognition and bactericidal activity, whereas LPS-tolerized mice exhibit an impaired ability to ingest and to kill bacteria. These results indicate that BLP tolerance is a novel adaptive host response associated with a unique protective effect during septic shock.     

OX40 ligand-transduced tumor cell vaccine synergizes with GM-CSF and requires CD40-Apc signaling to boost the host T cell antitumor response

Efficient T cell priming by GM-CSF and CD40 ligand double-transduced C26 murine colon carcinoma is not sufficient to cure metastases in a therapeutic setting. To determine whether a cellular vaccine that interacts directly with both APC and T cells in vivo might be superior, we generated C26 carcinoma cells transduced with the T cell costimulatory molecule OX40 ligand (OX40L) either alone (C26/OX40L) or together with GM-CSF (C26/GM/OX40L), which is known to activate APC. Mice injected with C26/OX40L cells displayed only a delay in tumor growth, while the C26/GM/OX40L tumor regressed in 85% of mice. Tumor rejection required granulocytes, CD4+, CD8+ T cells, and APC-mediated CD40-CD40 ligand cosignaling, but not IFN-gamma or IL-12 as shown using subset-depleted and knockout (KO) mice. CD40KO mice primed with C26/GM/OX40L cells failed to mount a CTL response, and T cells infiltrating the C26/GM/OX40L tumor were OX40 negative, suggesting an impairment in APC-T cell cross-talk in CD40KO mice. Indeed, CD4+ T cell-depleted mice failed to mount any CTL activity against the C26 tumor, while treatment with agonistic mAb to CD40, which acts on APC, bypassed the requirement for CD4+ T cells and restored CTL activation. C26/GM/OX40L cells cured 83% of mice bearing lung metastases, whereas C26/OX40L or C26/GM vaccination cured only 28 and 16% of mice, respectively. These results indicate the synergistic activity of OX40L and GM-CSF in a therapeutic setting.     

Cutting edge: cross-presentation as a mechanism for efficient recruitment of tumor-specific CTL to the brain

The number and localization of effector cells to the tumor site are crucial elements for immune rejection of solid tumors. However, for cerebral malignancies, antitumor responses need to be finely tuned to avoid neuropathologic consequences. In this study, we determine factors that regulate CTL localization and tumoricidal function after intracerebral implantation of tumors expressing model Ag. H-2(bxd) mice implanted with a CW3(+) murine glioma lacking H-2K(d) molecules necessary to present the CW3(170-179) epitope demonstrate cross-priming of H-2K(d)-restricted CTL, and moreover, Ag-dependent accumulation of functional H-2K(d)/CW3(170-179)-specific CTL within the tumor bed. This implicates a role for cross-presentation not only in priming, but also in retention of fully differentiated CTL in the tumor stroma at the effector stage of the response. Modulating cross-presentation of Ag may be the key in regulating specific immune responses in the brain: either by augmenting protective responses or by down-modulating destructive autoimmune reactions.