Statolon-induced resistance of mice to mengovirus

Mice receiving statolon intraperitoneally were 1,000 times more resistant to intraperitoneal challenge with mengovirus than were untreated controls. Protection was afforded when statolon was administered 1 day before or 1 day after intraperitoneal inoculation with the virus. No therapeutic effect was observed when treatment with statolon was delayed for 2 days or more after virus infection. Exposure of mice to a simulated space cabin environment did not increase their susceptibility to the lethal effects of mengovirus infection or eliminate the protective effect of statolon.     

Influence of Statolon on Resistance of Mice to Influenza

Various interferon inducers are known to elicit protection against lethal or infecting doses of certain viral agents. Because of the relatively high morbidity rate of influenza and its seasonal occurrence, we wished to determine whether statolon-induced interferon might be effective in controlling this disease. Mice were treated intraperitoneally with statolon and challenged with influenza A(2) virus by the intranasal route. Although interferon was present in the serum at the time of virus administration, no change in mortality rate was observed. There was, however, a significant increase in the mean survival time of treated animals. Similar results were obtained when Newcastle disease virus was used as the interferon inducer. To determine the effect of the route of challenge, other mice were treated with statolon or Newcastle disease virus and inoculated with mengovirus by the intranasal or intraperitoneal route. The results demonstrated that the treated mice were protected to similar degree against challenge by either route. It is suggested that the relative ineffectiveness of interferon in protecting mice against influenza is due to an intrinsic characteristic of the virus itself rather than the type of interferon induced or the route of virus challenge.     

Duration of Prophylactic Effect of Statolon Against Mengovirus in Mice

Intraperitoneal injection of statolon into mice is known to exert a protective effect against a subsequent infection with a variety of viruses owing to stimulation of production of interferon. To study the duration of such protection, groups of mice were treated with 350 mug of statolon at 69, 55, 41, 27, 13, 6, or 1 day prior to a challenge with mengovirus. Other groups were treated with 760 mug at 1 to 6 days before challenge. Ten days after inoculation with the virus, LD(50) determinations were made for each group. Mice which received statolon 1 to 5 days before infection were significantly more resistant to the virus than nontreated controls; no protection could be demonstrated in the other groups.     

Simian Virus 40 Transformation and the Period of Cellular Deoxyribonucleic Acid Synthesis

The antiviral agents interferon and statolon protected cells of the mouse line 3T3 against the transforming effect of simian virus 40. Loss of ability of these agents to protect when added some time after infection indicated that the transformation was already fixed. The cells of exponentially growing cultures became resistant to the protective effect of interferon at a linear rate after infection; after one cell generation, the whole population was resistant. By use of synchronous cultures, it was shown that, in cells passing though the G-1 period of the growth cycle, the transformation did not pass the interferon-sensitive stage, whereas cells in S [the period of cellular deoxyribonucleic acid (DNA) synthesis] readily passed this stage (i.e., became interferon-resistant). An irreversible step in transformation appeared to occur in cells synthesizing DNA, and it seems likely that replicating cellular DNA was the target of the viral action.     

Effect of Statolon-induced Interferon on Antibody Formation

The effect of statolon-induced interferon on immune response was investigated. Young adult Swiss albino mice were immunized with chicken red blood cells or infectious mengovirus. The red blood cells were injected three times and the virus twice at 2-week intervals, with or without treatment with statolon 24 hr before the inoculations. The effect of interferon on antibody formation was measured by determinations of hemagglutinating, hemolytic, and virus-neutralizing antibodies in the sera of treated and nontreated groups. The hemagglutinin and hemolysin titers after each successive inoculation were equal in both groups, indicating that interferon had no effect on their production. A decrease in the primary response to the viral antigen was associated with interferon induction; the booster response was unaffected. The possible use of interferon inducers as attenuants for immunization with live virulent viruses is discussed.     

Effects of intravenous silica on immune and non-immune functions of the murine host.

Silica, an agent toxic for macrophages, administered i.v. to DBA/2 mice rapidly depresses the clearance of colloidal carbon by the reticuloendothelial system and reduces the in vitro phagocytic activity of peritoneal macrophages harvested 3 days after silica injection. Silica blocks the humoral immune response to sheep erythrocytes and the cell-mediated immune response to allogeneic fibroblasts when given before antigen. Silica also induces complex alterations in spleen cell responsiveness to concanavalin A involving both local and serum factors. Silica had no significant effect on the induction of interferon by statolon or Newcastle disease virus. No unequivocal evidence was obtained that silica has a direct depressive effect on cells other that macrophages, but indirect effects on lymphocytes were produced most likely by factors released from silica-lysed macrophages. Intravenous silica may prove useful for the separation of interferon induction and immune response stimulation in studies of host resistance to infection and oncogenesis. Considerable variation exists in the immunodepressive effects of different preparations of silica.     

Interferon and Interferon Inducers in Protozoal Infections

Several interferon inducers (Newcastle disease virus, statolon, and poly rI:poly rC) as well as exogenous mouse interferon protect mice from sporozoite-induced Plasmodium berghei malaria, as long as they are administered before the end of the preerythrocytic phase of development of the parasite. The protective effect of the interferon inducers was related to their interferon-inducing effect; the protective effect of the interferon preparations was related to the interferon titer of the preparations, and it exhibited other attributes of interferon such as species specificity. In contrast to sporozoite-induced infection, blood forms-induced P. berghei malaria was only weakly susceptible to the protective effect of interferon inducers. This difference may provide an approach to study the mechanism of protection. The growth in cell cultures of another intracellular protozoon, Toxoplasma gondii, is also inhibited by interferon (22). The fact that P. berghei and T. gondii (as well as another group of intracellular parasites susceptible to interferon, the Chlamydia) have their own ribosomes raises questions, concerning the role of host cell ribosomes in the host cell-parasite relationship of these intracellular parasites and in the mechanism of interferon action against them, that can be approached experimentally. The possibility of therapeutic or prophylactic application of interferon or of its inducers to certain protozoal diseases of man and of other animals is still remote, but it has to be considered for long range planning.     

Characterization of ionic strength for X-MuLV inactivation by low pH treatment for monoclonal antibody purification

In the production of monoclonal antibodies (mAbs) intended for use in humans, it is a global regulatory requirement that the manufacturing process includes unit operations that are proven to inactivate or remove adventitious agents to ensure viral safety. Viral inactivation by low pH hold (LPH) is typically used to ensure this viral safety in the purification process of mAbs and other biotherapeutics derived from mammalian cell lines. To ascertain the effectiveness of the LPH step, viral clearance studies have evaluated LPH under worst-case conditions of pH above the manufacturing set point and hold duration at or below the manufacturing minimum. Highly acidic conditions (i.e., pH < 3.60) provide robust and effective enveloped virus inactivation but may lead to reduced product quality of the therapeutic protein. However, when viral inactivation is operated above pH 3.60 to ensure product stability, effective (>4 log₁₀ reduction factor) viral inactivation may not be observed under these worst-case pH conditions in viral clearance studies. A multivariate design of experiments was conducted to further characterize the operating space for low pH viral inactivation of a model retrovirus, xenotropic murine leukemia virus (X-MuLV). The statistically designed experiment evaluated the effect of mAb isotype, pH, temperature, acid titrant, sodium chloride (NaCl) concentration, virus spike timing, and post-spike filtration on X-MuLV inactivation. Data from the characterization study were used to generate predictive models to identify conditions that reliably achieve effective viral inactivation at pH ≥ 3.60. Results of the study demonstrated that NaCl concentration has the greatest effect on virus inactivation in the range studied, and pH has a large effect when the load material has no additional NaCl. Overall, robust and effective inactivation of X-MuLV at pH 3.65–3.80 can be achieved by manipulating either the pH or the NaCl concentration of the load material. This study contributes to the understanding of ionic strength as an influential parameter in low pH viral inactivation studies.     

Suppression of Established Friend Virus Leukemia by Statolon: I. Demonstration of a Latent Infection in Clinically Normal Mice

A single inoculation of statolon into mice with established Friend virus (FV) leukemia can suppress the viral infection and produce a clinical remission lasting many months. Eventually, however, most of the mice develop characteristic FV leukemia. Persistence of FV activity in the spleens of mice during clinical remission can be demonstrated by cell transfer and histopathologic studies. Transfer to normal mice of a large number of spleen cells (10⁷) from mice in remission produces FV leukemia, and transfer of a small number of cells (10²) produces immunity to FV challenge. Histopathologic examination reveals clusters of abnormal FV leukemia-like cells directly beneath the capsules of the spleens of mice in clinical remission.     

Effect of fatty acids on growth of Japanese encephalitis virus cultivated in BHK-21 cells and phospholipid metabolism of the infected cells.

Growth of Japanese encephalitis virus (JEV) in BHK-21 cells was stimulated in the presence of 20 to 40 mug of the sodium salt of oleic acid (cis-9-octadecenoic acid, 9-18:1) per ml supplemented in Waymouth medium. The stimulatory effect of the salt was highest when 9-18:1 was added after adsorption of the virus. Study of the effect of other fatty acids on growth of JEV showed the following results: the longer the chain length of the saturated fatty acid salt, the higher the stimulatory effect on viral growth. In contrast, polyunsaturated fatty acids had an inhibitory effect on viral growth. The effect of isomeric cis-octadecenoic acids on viral growth was variable, depending upon the position of the double bond. The cis-6-octadecenoic acid had the highest inhibitory effect on growth of JEV compared to other isomeric octadecenoic acids. The sodium salt of (1-14C) cis-9-octadecenoic acid (9-18:1, 20 mug/ml) was rapidly incorporated into control and JEV-infected cells. Specific radioactivity in phosphatidylcholine dropped 12 to 24 h after virus inoculation, whereas synthesis of phosphatidylethanolamine increased 12 to 24 h after virus inoculation in infected cells compared to uninfected cells. Results from these studies suggest that phospholipid metabolism of infected cells is markedly changed, which can be associated with altered fatty acid metabolism when using labeled 9-18:1 fatty acid as a marker.     

Resistance to Bacterial Infection Induced by Statolon and Pyran

The antibacterial properties of statolon and pyran were compared with those of bacterial endotoxin and found to be similar. Although high resistance was transient, it was followed by immunity of the same order as in mice infected without pre-treatment with the interferon inducers.     

Direct gene transfer with IP-10 mutant ameliorates mouse CVB3-induced myocarditis by blunting Th1 immune responses

Background

Myocarditis is an inflammation of the myocardium that often follows the enterovirus infections, with coxsackievirus B3 (CVB3) being the most dominant etiologic agent. We and other groups previously reported that chemokine IP-10 was significantly induced in the heart tissue of CVB3-infected mice and contributed to the migration of massive inflammatory cells into the myocardium, which represents one of the most important mechanisms of viral myocarditis. To evaluate the direct effect of IP-10 on the inflammatory responses in CVB3 myocarditis, herein an IP-10 mutant deprived of chemo-attractant function was introduced into mice to antagonize the endogenous IP-10 activity, and its therapeutic effect on CVB3-induced myocarditis was evaluated.

Methodology/Principal Findings

The depletion mutant pIP-10-AT, with an additional methionine after removal of the 5 N-terminal amino acids, was genetically constructed and intramuscularly injected into BALB/c mice after CVB3 infection. Compared with vector or no treatment, pIP-10-AT treatment had significantly reduced heart/body weight ratio and serum CK-MB level, increased survival rate and improved heart histopathology, suggesting an ameliorated myocarditis. This therapeutic effect was not attributable to an enhanced viral clearance, but to a blunted Th1 immune response, as evidenced by significantly decreased splenic CD4⁺/CD8⁺IFN-γ⁺ T cell percentages and reduced myocardial Th1 cytokine levels.

Conclusion/Significance

Our findings constitute the first preclinical data indicating that interfering in vivo IP-10 activity could ameliorate CVB3 induced myocarditis. This strategy may represent as a new therapeutic approach in treating viral myocarditis.     

Administration of fludarabine-loaded autologous red blood cells in simian immunodeficiency virus-infected sooty mangabeys depletes pSTAT-1-expressing macrophages and delays the rebound of viremia after suspension of antiretroviral therapy

A major limitation of highly active antiretroviral therapy is that it fails to eradicate human immunodeficiency virus (HIV) infection due to its limited effects on viral reservoirs carrying replication-competent HIV, including monocytes/macrophages (M/M). Therefore, therapeutic approaches aimed at targeting HIV-infected M/M may prove useful in the clinical management of HIV-infected patients. In previous studies, we have shown that administration of fludarabine-loaded red blood cells (RBC) in vitro selectively induces cell death in HIV-infected M/M via a pSTAT1-dependent pathway. To determine the in vivo efficacy of this novel therapeutic strategy, we treated six naturally simian immunodeficiency virus (SIV)-infected sooty mangabeys (SMs) with either 9-[2-(R)-(phosphonomethoxy)propyl]adenine (PMPA) only, fludarabine-loaded RBC only, or PMPA in association with fludarabine-loaded RBC. The rationale of this treatment was to target infected M/M with fludarabine-loaded RBC at a time when PMPA is suppressing viral replication taking place in activated CD4+ T cells. In vivo administration of fludarabine-loaded RBC was well tolerated and did not induce any discernible side effect. Importantly, addition of fludarabine-loaded RBC to PMPA delayed the rebound of viral replication after suspension of therapy, thus suggesting a reduction in the size of SIV reservoirs. While administrations of fludarabine-loaded RBC did not induce any change in the CD4+ or CD8+ T-cell compartments, we observed, in chronically SIV-infected SMs, a selective depletion of M/M expressing pSTAT1. This study suggests that therapeutic strategies based on the administration of fludarabine-loaded RBC may be further explored as interventions aimed at reducing the size of the M/M reservoirs during chronic HIV infection.     

“Armed” oncolytic herpes simplex viruses for brain tumor therapy

Genetically engineered, conditionally replicating herpes simplex viruses type 1 (HSV-1) are promising therapeutic agents for brain tumors and other solid cancers. They can replicate in situ, spread, and exhibit oncolytic activity via a direct cytocidal effect. One of the advantages of HSV-1 is the capacity to incorporate large and/or multiple transgenes within the viral genome. Oncolytic HSV-1 can therefore be “armed” to add certain functions. Recently, the field of armed oncolytic HSV-1 has drastically advanced, due to development of recombinant HSV-1 generation systems that utilize bacterial artificial chromosome and multiple DNA recombinases. Because antitumor immunity is induced in the course of oncolytic activities of HSV-1, transgenes encoding immunomodulatory molecules have been most frequently used for arming. Other armed oncolytic HSV-1 include those that express antiangiogenic factors, fusogenic membrane glycoproteins, suicide gene products, and proapoptotic proteins. Provided that the transgene product does not interfere with viral replication, such arming of oncolytic HSV-1 results in augmentation of antitumor efficacy. Immediate-early viral promoters are often used to control the arming transgenes, but strict-late viral promoters have been shown useful to restrict the expression in the late stage of viral replication when desirable. Some armed oncolytic HSV-1 have been created for the purpose of noninvasive in vivo imaging of viral infection and replication. Development of a wide variety of armed oncolytic HSV-1 will lead to an establishment of a new genre of therapy for brain tumors as well as other cancers.     

Effectiveness of topically administered neutralizing antibodies in experimental immunotherapy of respiratory syncytial virus infection in cotton rats.

Initial studies of the prophylactic effect of parenterally administered respiratory syncytial virus (RSV)-neutralizing antibodies in cotton rats indicated that virus replication in lung tissues was restricted when animals with preexisting antibody titers in serum of 1:100 or more (as measured by plaque reduction) were challenged intranasally with 10(4) PFU of virus. Subsequently, a therapeutic effect of parenterally administered RSV antibodies (present in human gamma globulin) was demonstrated in both cotton rats and owl monkeys. Parenteral inoculation of RSV-infected cotton rats or owl monkeys with purified human immunoglobulin licensed for intravenous administration in humans (IVIG) effected a 10(-1.7) to 10(-2.7) reduction in the level of pulmonary virus at the height of infection. Because of these encouraging results, we examined topical administration of IVIG to determine whether it was also effective and whether it offered an advantage over the parenteral route with regard to simplicity and the dose required for full therapeutic effect. IVIG (0.025 g/kg) administered topically by the intranasal route to anesthetized cotton rats at the height of RSV infection effected a 10(2.2)-fold reduction in viral titers of pulmonary tissues and a complete clearance of detectable virus in 92% of the animals within 24 h. In contrast, 4 g of IVIG per kg was required to produce a comparable therapeutic effect when the material was administered parenterally. Thus, the therapeutic effect of IVIG was 160 times greater by the topical route than by parenteral inoculation.     

Inhibitory effect of essential oils against herpes simplex virus type 2.

Essential oils from anise, hyssop, thyme, ginger, camomile and sandalwood were screened for their inhibitory effect against herpes simplex virus type 2 (HSV-2) in vitro on RC-37 cells using a plaque reduction assay. Genital herpes is a chronic, persistent infection spreading efficiently and silently as sexually transmitted disease through the population. Antiviral agents currently applied for the treatment of herpesvirus infections include acyclovir and its derivatives. The inhibitory concentrations (IC50) were determined at 0.016%, 0.0075%, 0.007%, 0.004%, 0.003% and 0.0015% for anise oil, hyssop oil, thyme oil, ginger oil, camomile oil and sandalwood oil, respectively. A clearly dose-dependent virucidal activity against HSV-2 could be demonstrated for all essential oils tested. In order to determine the mode of the inhibitory effect, essential oils were added at different stages during the viral infection cycle. At maximum noncytotoxic concentrations of the essential oils, plaque formation was significantly reduced by more than 90% when HSV-2 was preincubated with hyssop oil, thyme oil or ginger oil. However, no inhibitory effect could be observed when the essential oils were added to the cells prior to infection with HSV-2 or after the adsorption period. These results indicate that essential oils affected HSV-2 mainly before adsorption probably by interacting with the viral envelope. Camomile oil exhibited a high selectivity index and seems to be a promising candidate for topical therapeutic application as virucidal agents for treatment of herpes genitalis.     

Expression characteristics of pokeweed antiviral proteins (PAPs): Two distinct types of proteins

Pokeweed antiviral proteins (PAPs) become novel therapeutic agents in relation to application in human viral diseases and cancer, as well as potent tools in plant system for defending viral infection. We have studied the expression characteristics of PAPs in pokeweed plants by western blot analysis. PAP-I was constitutively expressed in leaves, stems and roots of the pokeweed plant, while PAP-II was not expressed in roots. The expression of PAP-II began in May and then gradually increased with development of the plants. The PAP-II expression was induced and/or stimulated not only by biotic stresses, such as insect pests and viral infection, but also by abiotic stresses, like drought. Interestingly, low-light intensity was found to be more effective than high-light in the expression of both PAP-I and PAP-II. Our results suggest that PAP-II appears to have an additive effect in terms of protection of the plant against pathogens during summer-time when the plant actively grows and is attacked by various pathogens.     

肿瘤基因治疗的靶向策略

对肿瘤组织的靶向性可以提高基因治疗的效果 ,避免对正常组织的损伤 ,并且能降低作为载体的微生物对机体的危害。对于瘤内注射的给药方法 ,靶向性似乎显得不是特别重要 ,但是如果要系统给药 ,靶向性是很关键的一个问题。靶向基因治疗肿瘤可以通过靶向基因导入和靶向基因表达来实现。近年来 ,在靶向基因导入方面的研究有很多进展 ,例如 ,用双亲性的桥连分子协助腺病毒和逆转录病毒靶向转导 ;在各种病毒载体的衣壳蛋白中插入靶向性的小肽或较大的多肽靶向结构域 ;增殖病毒作为一种很有前途的抗肿瘤制剂可有效地靶向杀伤肿瘤细胞。受体介导的DNA或DNA 脂质体复合物的靶向系统和其他一些靶向性的有疗效的载体 ,如细菌 ,也处于研究中。其中的一些载体已经进入临床实验。为了实现基因的靶向可调控表达 ,组织或肿瘤特异性的启动子和人工合成的可调控表达系统被用来调控治疗基因的表达。反义核酸、核酶以及脱氧核酶 (DNAzyme)被用来靶向抑制与肿瘤发生密切相关基因的表达。