Genomic Stability of Lyophilized Sheep Somatic Cells before and after Nuclear Transfer

The unprecedented decline of biodiversity worldwide is urging scientists to collect and store biological material from seriously threatened animals, including large mammals. Lyophilization is being explored as a low-cost system for storage in bio-banks of cells that might be used to expand or restore endangered or extinct species through the procedure of Somatic Cell Nuclear Transfer (SCNT). Here we report that the genome is intact in about 60% of lyophylized sheep lymphocytes, whereas DNA damage occurs randomly in the remaining 40%. Remarkably, lyophilized nuclei injected into enucleated oocytes are repaired by a robust DNA repairing activity of the oocytes, and show normal developmental competence. Cloned embryos derived from lyophylized cells exhibited chromosome and cellular composition comparable to those of embryos derived from fresh donor cells. These findings support the feasibility of lyophylization as a storage procedure of mammalian cells to be used for SCNT.     

Proteosynthetic activity of immobilized Staphylococcus aureus V8 protease: application in the semisynthesis of molecular variants of alpha-globin

The proteosynthetic activity of Staphylococcus aureus V8 protease (endoproteinase Glu-C) immobilized onto cross-linked agarose beads by reductive alkylation procedure has been investigated. The overall substrate specificity of the enzyme, as judged by peptide mapping of performic acid oxidized RNase A, as well as the high propensity of the protease to slice selectively the alpha-chain of hemoglobin (Hb) A at the Glu(30)-Arg(31) peptide bond at pH 4.0 and 37 degrees C was essentially unperturbed by the immobilization process. This high susceptibility of Glu(30) of the alpha-chain for proteolysis appears to be a consequence of the conformational aspects of the polypeptide in this region. The proteolysis of two mutant forms of alpha-chain, namely, those of Hb I (K16E) and Hb Sealy (D47H) by immobilized V8 protease at the Glu(30)-Arg(31) peptide bond proceeds with the same selectivity. The immobilized protease also retained the proteosynthetic activity, i.e., the ability to ligate the unprotected alpha-globin fragments at the Glu(30)-Arg(31) peptide bond in the presence of 30% 1-propanol. The use of the insoluble enzyme simplifies the procedures for the construction of new semisynthetic, molecular variants of alpha-globin. The general applicability of the immobilized enzyme for protein semisynthesis has been demonstrated by the construction of a doubly mutated alpha-globin. The complementary fragments from two natural mutant forms of alpha-globin, viz., alpha 1-30 (K16E) from Hb I and alpha 31-141 (D47H) from Hb Sealy, are readily ligated to form the double mutant alpha 1-141 (K16E;D47H).     

Interleukin-3 or erythropoietin induced nuclear localization of protein kinase C β isoforms in hematopoietic target cells

Protein kinase C (PKC) has been implicated in the signal transduction pathways for the biological effect of both interleukin-3 (IL-3) and erythropoietin (EPO) in hematopoietic target cells. The goal of this study was to identify specific classical isoforms of PKC and their localization in hematopoietic cells in response to the growth factors, IL-3 or EPO. In addition to murine fetal liver cells as a source of normal erythroid progenitor cells, we have utilized the B6SUt.EP cell line, a non-transformed hematopoietic cell line that requires IL-3 for proliferation, but for which EPO can substitute as a growth factor. With polyclonal antibodies prepared against peptide sequences specific for the α, βI, βII and γ isoforms of PKC, we have identified βI and βII as the predominant nuclear isoforms in target cells that proliferate in response to IL-3 or EPO.     

Multi-dose Romidepsin Reactivates Replication Competent SIV in Post-antiretroviral Rhesus Macaque Controllers

Viruses that persist despite seemingly effective antiretroviral treatment (ART) and can reinitiate infection if treatment is stopped preclude definitive treatment of HIV-1 infected individuals, requiring lifelong ART. Among strategies proposed for targeting these viral reservoirs, the premise of the “shock and kill” strategy is to induce expression of latent proviruses [for example with histone deacetylase inhibitors (HDACis)] resulting in elimination of the affected cells through viral cytolysis or immune clearance mechanisms. Yet, ex vivo studies reported that HDACis have variable efficacy for reactivating latent proviruses, and hinder immune functions. We developed a nonhuman primate model of post-treatment control of SIV through early and prolonged administration of ART and performed in vivo reactivation experiments in controller RMs, evaluating the ability of the HDACi romidepsin (RMD) to reactivate SIV and the impact of RMD treatment on SIV-specific T cell responses. Ten RMs were IV-infected with a SIVsmmFTq transmitted-founder infectious molecular clone. Four RMs received conventional ART for >9 months, starting from 65 days post-infection. SIVsmmFTq plasma viremia was robustly controlled to <10 SIV RNA copies/mL with ART, without viral blips. At ART cessation, initial rebound viremia to ~10⁶ copies/mL was followed by a decline to < 10 copies/mL, suggesting effective immune control. Three post-treatment controller RMs received three doses of RMD every 35–50 days, followed by in vivo experimental depletion of CD8⁺ cells using monoclonal antibody M-T807R1. RMD was well-tolerated and resulted in a rapid and massive surge in T cell activation, as well as significant virus rebounds (~10⁴ copies/ml) peaking at 5–12 days post-treatment. CD8⁺ cell depletion resulted in a more robust viral rebound (10⁷ copies/ml) that was controlled upon CD8⁺ T cell recovery. Our results show that RMD can reactivate SIV in vivo in the setting of post-ART viral control. Comparison of the patterns of virus rebound after RMD administration and CD8⁺ cell depletion suggested that RMD impact on T cells is only transient and does not irreversibly alter the ability of SIV-specific T cells to control the reactivated virus.     

Prodromo della Flora Umbra

Defense responses of plants are activated not only in the wounded tissues but also in the remote parts of the plants. Two different methyl jasmonate (MeJA) treatments were conducted, i.e., MeJA solution spraying of entire rosettes leaves and pasting leaf surface with lanolin squares containing MeJA. Glucosinolate profiles in leaves were similar using the two methods of MeJA treatment except for indole glucosinolates. The glucosinolate profiles in local and systemic leaves showed that the accumulation of glucosinolates in systemic leaves were delayed comparing with those in local treated leaves. Comparative proteomics were used to investigate the molecular processes underlying the glucosinolate changes in response to local and systemic MeJA induction. A total of 83 unique proteins were detected as differentially expressed between the local and systemic leaves. Functional analysis showed that redirection of metabolism from growth to defense was differentially regulated in local and systemic MeJA induction. The higher contents of indole glucosinolates in systemic leaves might arise from the induction of a long-distance signal produced in local leaves as well as from JA synthesized in systemic leaves.     

Replicating adenovirus-simian immunodeficiency virus (SIV) recombinant priming and envelope protein boosting elicits localized, mucosal IgA immunity in rhesus macaques correlated with delayed acquisition following a repeated low-dose rectal SIV(mac251) challenge

We have shown that sequential replicating adenovirus type 5 host range mutant human immunodeficiency virus/simian immunodeficiency virus (HIV/SIV) recombinant priming delivered first intranasally (i.n.) plus orally and then intratracheally (i.t.), followed by envelope protein boosting, elicits broad cellular immunity and functional, envelope-specific serum and mucosal antibodies that correlate with protection from high-dose SIV and simian/human immunodeficiency virus (SHIV) challenges in rhesus macaques. Here we extended these studies to compare the standard i.n./i.t. regimen with additional mucosal administration routes, including sublingual, rectal, and vaginal routes. Similar systemic cellular and humoral immunity was elicited by all immunization routes. Central and effector memory T cell responses were also elicited by the four immunization routes in bronchoalveolar lavage fluid and jejunal, rectal, and vaginal tissue samples. Cellular responses in vaginal tissue were more compartmentalized, being induced primarily by intravaginal administration. In contrast, all immunization routes elicited secretory IgA (sIgA) responses at multiple mucosal sites. Following a repeated low-dose intrarectal (i.r.) challenge with SIV(mac251) at a dose transmitting one or two variants, protection against acquisition was not achieved except in one macaque in the i.r. immunized group. All immunized macaques exhibited reduced peak viremia compared to that of controls, correlated inversely with prechallenge serum antienvelope avidity, antibody-dependent cellular cytotoxicity (ADCC) titers, and percent antibody-dependent cell-mediated viral inhibition. Both antibody avidity and ADCC titers were correlated with the number of exposures required for infection. Notably, we show for the first time a significant correlation of vaccine-induced sIgA titers in rectal secretions with delayed acquisition. Further investigation of the characteristics and properties of the sIgA should elucidate the mechanism leading to this protective effect.     

A new type of anti-ganglioside antibodies present in neurological patients

High titers of anti-GA1 antibodies have been associated with neurological syndromes. In most cases, these antibodies cross-react with the structurally related glycolipids GM1 and GD1b, although specific anti-GA1 antibodies have also been reported. The role of specific anti-GA1 antibodies is uncertain since the presence of GA1 in the human nervous system has not been clarified. A rabbit was immunized with GD1a and its sera were screened for antibody reactivity by standard immunoassay methods (HPTLC-immunostaining and ELISA). Anti-GD1a antibodies were not detected but, unexpectedly, anti-GA1 IgG-antibodies were found. Antibody binding to GA1 was inhibited by soluble GA1 but also by GD1a. These results indicate that the rabbit produced antibodies that recognize epitopes present on the glycolipids, that are absent or not exposed on solid phase adsorbed GD1a. We investigated the presence of these unusual anti-ganglioside antibodies in normal and neurological patient sera. Approximately, 10% of normal human sera contained low titer of specific anti-GA1 IgG-antibodies but none of them recognized soluble GD1a. High titers of IgG-antibodies reacting only with GA1 were detected in 12 patient sera out of 325 analyzed. Of these, 6 sera showed binding that was inhibited by soluble GD1a and four of them also by GM1. This new type of anti-ganglioside antibodies should be considered important elements for understanding of the pathogenesis of these diseases as well as their diagnosis.     

Multiple Vaccine-Elicited Nonneutralizing Antienvelope Antibody Activities Contribute to Protective Efficacy by Reducing both Acute and Chronic Viremia following Simian/Human Immunodeficiency Virus SHIV89.6P Challenge in Rhesus Macaques

We have shown that following priming with replicating adenovirus type 5 host range mutant (Ad5hr)-human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) recombinants, boosting with gp140 envelope protein enhances acute-phase protection against intravenous simian/human immunodeficiency virus (SHIV)_89.6P challenge compared to results with priming and no boosting or boosting with an HIV polypeptide representing the CD4 binding site of gp120. We retrospectively analyzed antibodies in sera and rectal secretions from these same macaques, investigating the hypothesis that vaccine-elicited nonneutralizing antibodies contributed to the better protection. Compared to other immunized groups or controls, the gp140-boosted group exhibited significantly greater antibody activities mediating antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cell-mediated viral inhibition (ADCVI) in sera and transcytosis inhibition in rectal secretions. ADCC and ADCVI activities were directly correlated with antibody avidity, suggesting the importance of antibody maturation for functionality. Both ADCVI and percent ADCC killing prechallenge were significantly correlated with reduced acute viremia. The latter, as well as postchallenge ADCVI and ADCC, was also significantly correlated with reduced chronic viremia. We have previously demonstrated induction by the prime/boost regimen of mucosal antibodies that inhibit transcytosis of SIV across an intact epithelial cell layer. Here, antibody in rectal secretions was significantly correlated with transcytosis inhibition. Importantly, the transcytosis specific activity (percent inhibition/total secretory IgA and IgG) was strongly correlated with reduced chronic viremia, suggesting that mucosal antibody may help control cell-to-cell viral spread during the course of infection. Overall, the replicating Ad5hr-HIV/SIV priming/gp140 protein boosting approach elicited strong systemic and mucosal antibodies with multiple functional activities associated with control of both acute and chronic viremia.A major goal of human immunodeficiency virus (HIV) vaccine development is the elicitation of protective antibodies capable of neutralizing the diversity of isolates in the worldwide pandemic (6, 61). Indeed, passively administered neutralizing antibodies have been shown to protect against pathogenic HIV/simian immunodeficiency virus (SIV) challenge in rhesus macaque models (4, 44, 45, 57). However, the extent to which other antibody-mediated protective mechanisms impact HIV/SIV infection is still unclear. Whether these alternate biologic activities would augment vaccine-induced protection has not been definitively established.In HIV-infected individuals, as in SIV- or simian/human immunodeficiency virus (SHIV)-infected rhesus macaques, systemic nonneutralizing antibodies appear early during acute infection, often preceding a neutralizing antibody response (21, 55). Although neutralizing antibody activity is critical for sterilizing immunity, recent studies suggest that antibodies may contribute to protection by other functional activities, such as antibody-dependent cellular cytotoxicity (ADCC) (20, 29), antibody-dependent cell-mediated viral inhibition (ADCVI) (22, 23), and transcytosis inhibition (19, 35, 59). Antibodies in secretions may directly block viral entry into intestinal and endocervical tissues by inhibiting transcytosis across epithelium, whereas local or serum-derived antibodies that mediate ADCC or ADCVI may exert protective effects by eliminating small foci of infected cells during the brief window of time that exists between transmission of virus across an epithelial cell barrier to the lamina propria and subsequent systemic spread (32). In support of this notion, mutation of the Fc portion of the broadly neutralizing monoclonal antibody, IgGb12, thereby preventing interaction with the FcγR on effector cells, rendered the antibody less able to mediate protection upon subsequent passive transfer and challenge of rhesus macaques (34). Thus, neutralizing antibodies themselves may mediate protection by additional functional activities.ADCC bridges innate and adaptive immunity. Mechanistically, it involves FcγR-bearing effector cells, such as NK cells, macrophages, neutrophils, and γδ T cells, and antibodies specific for antigens expressed on the surface of target cells. Upon interaction of these three components, the target cells are killed. Since the effector cells are not major histocompatibility complex restricted, ADCC is broadly applicable to diverse populations. Because the antibody specificity need not be restricted to neutralizing epitopes, ADCC may increase the breadth of antibody reactivity. In fact, we have shown that an HIV clade B immunization regimen elicited antibodies that mediated ADCC across several HIV clades (28). Antibodies that mediate ADCC have been shown to arise early in infection, before neutralizing antibodies (55, 60). They are present in the majority of infected individuals, and they have been associated with slow disease progression following both HIV and SIV infection (5, 8).ADCVI is closely related to ADCC, also requiring antibody that forms a bridge between an infected target cell and an FcγR-bearing effector cell (24). However, ADCVI is a broader activity not restricted solely to target cell lysis but, rather, encompassing several mechanisms by which viral replication following infection of target cells is inhibited. Thus, it may include ADCC activity but also involve noncytotoxic mechanisms of virus control, such as the secretion of inhibitory chemokines or FcγR-mediated phagocytosis of immune complexes (24, 25).Most HIV infections occur via a mucosal route, including cervicovaginal and rectal tissues (39, 52). Several nonmutually exclusive mechanisms for HIV-1 transmission across mucosal epithelia have been proposed (13, 56). Transcytosis of infectious virus across polarized columnar epithelial cells following contact of virally infected cells with apical epithelial cell surfaces is one mechanism for mucosal HIV entry (12). Rather than fusion and infection, interactions between the viral envelope proteins and epithelial surface molecules, such as glycosphingolipid galactosyl-ceramide (GalCer) (13, 47), an important component of endocytotic “raft” membrane microdomains, lead to transcytosis of the virus across the epithelial barrier and its trapping by submucosal dendritic cells which disseminate it to their target CD4⁺ T cells. Studies have shown that mucosal immunoglobulin A (IgA) antibody, a major component of the mucosal immune response, could block mucosal HIV-1 entry via transcytosis in vitro (2, 19). Therefore, mucosal antibodies blocking adherence of virus to epithelial cells and preventing HIV-1 transcytosis across the epithelial barrier and subsequent CD4⁺ T cell infection may afford additional protection against HIV/SIV infection.We have been pursuing a replicating adenovirus (Ad)-HIV/SIV prime/protein subunit boost AIDS vaccine approach (30, 51), which has elicited strong, durable protection against HIV, SIV, and SHIV challenges (11, 18, 41, 42, 50). An underlying goal of these studies has been elucidation of immune responses that correlate with protective efficacy. Recently, we studied the contribution of novel protein boosts to immunogenicity and protective efficacy in a SHIV_89.6P model (49). Immunized rhesus macaques were primed with Ad type 5 host range mutant (Ad5hr)-HIV_89.6Pgp140, -SIV₂₃₉gag, and -SIV₂₃₉nef recombinants. One group was not boosted, one was boosted with HIV_89.6P gp140ΔCFI protein (gp140 envelope with deletions in the cleavage site, fusion peptide, and part of the interspace between the two heptad repeats) (40), and one was boosted with a novel HIV-1 polypeptide “peptomer” representing the CD4 binding site of the envelope (54). The best protection was seen in the gp140-boosted group, with significant reductions in both acute and chronic viremia. Although Env-specific antibody and cellular responses were readily detected, none directly correlated with the better protection. Furthermore, neutralizing antibodies against SHIV_89.6P did not develop until 4 weeks postchallenge. Therefore, we hypothesized that vaccine-elicited nonneutralizing anti-Env antibodies might have contributed to the better control of acute and/or chronic viremia in the gp140 group. Here, we report retrospective evaluations of sera and rectal secretions from macaques in this comparative study for serum binding antibody avidity, an important characteristic of functional antibodies (38, 58), and nonneutralizing activities of systemic and mucosal antibodies, including ADCC, ADCVI, and transcytosis inhibition.     

Role of deficient type III interferon-lambda production in asthma exacerbations

Rhinoviruses are the major cause of asthma exacerbations, and asthmatics have increased susceptibility to rhinovirus and risk of invasive bacterial infections. Here we show deficient induction of interferon-lambdas by rhinovirus in asthmatic primary bronchial epithelial cells and alveolar macrophages, which was highly correlated with severity of rhinovirus-induced asthma exacerbation and virus load in experimentally infected human volunteers. Induction by lipopolysaccharide in asthmatic macrophages was also deficient and correlated with exacerbation severity. These results identify previously unknown mechanisms of susceptibility to infection in asthma and suggest new approaches to prevention and/or treatment of asthma exacerbations.