Comparative Study of Immunomodulatory Agents to Induce Human T Regulatory (Treg) Cells: Preferential Treg-Stimulatory Effect of Prednisolone and Rapamycin.

T regulatory (Treg) cells play a critical role in the maintenance of self-tolerance, as well as in inhibition of inflammation and exaggerated immune response against exogenous antigens. They develop in the thymus (tTreg cells) but also may be generated at the peripheral tissues, including tumor microenvironment (pTreg cells), or induced in vitro in the presence of transforming growth factor (TGF)-ß (iTreg cells). Since tTreg cells constitute a minor fraction of peripheral blood lymphocytes in physiological conditions, an alternative way to obtain high number of functional Treg cells for therapeutic purposes is their generation in vitro from conventional T cells. In our studies, we compared effectiveness of several pharmacological agents with suggested immunomodulatory effects on Treg development (rapamycin, prednisolone, inosine pranobex, glatiramer acetate, sodium butyrate, and atorvastatin) to optimize Treg-inducing protocols. All but one (atorvastatin) immunomodulators augmented induction of polyclonal Treg cells in cultures. They were effective both in increasing the number of CD4+CD25highFoxp3high cells and Foxp3 expression. Rapamycin and prednisolone were found the most effective. Both drugs prolonged also phenotypic stability of Treg cells and induced fully active Treg cells in a functional assay. In the assay, prednisolone appeared superior versus rapamycin. The results, on the one hand, may be helpful in planning optimal protocols for generation of Treg cells for clinical application and, on the other hand, shed some light on mechanisms of the immunomodulatory activity of some tested agents observed in vivo.

Synergistic antitumor effects of histone deacetylase inhibitor scriptaid and bortezomib against ovarian cancer cells.

Despite debulking surgery and good initial response to chemotherapy, the majority of patients with advanced ovarian cancer relapse and succumb to their disease. Thus, there is a pressing need to improve treatment outcome. In the present study, the antitumor activity of histone deacetylase (HDAC) inhibitor scriptaid in combination with bortezomib or conventional chemotherapeutics was tested in vitro against representative ovarian cancer cell lines: SKOV­3, MDAH 2774, and OVP­10. Incubation of ovarian cancer cells with scriptaid and bortezomib (or doxorubicin) led to synergistic antitumor effects. As shown in the Annexin V-FITC/PI assay and western blot analysis of caspase­3/-9 and p21 protein expression, these synergistic antitumor effects were due to both induction of apoptosis and inhibition of proliferation. Since synergistic antitumor activity of scriptaid and bortezomib appeared in suboptimal concentrations, one can assume that the administration of the combination of these agents to ovarian cancer patients can exert the therapeutic effect in parallel with limited general toxicity of the treatment.

IN VITRO INfIBITION OF HHV-1 REPLICATION BY INOSINE PRANOBEX AND INTERFERON-α.

Key issues in the development of novel antivirals are the emergence of resistant strains. The development of new drugs effective against herpes diseases has proven to be both difficult and time-consuming. Some alternative may be to optimize the efficacy and selectivity of existing antiviral drugs or combining them with other well known agents. Inosine pranobex exerts a direct antiviral effect as well as secondary effect to its immunomodulatory activity. We found that increasing concentrations of inosine pranobex (50-400 µg/mL) produced progressively growing inhibitory effect on HHV-1 replication, following infection of different cell lines. The combination of 1000 IU/mL IFN-α and inosine pranobex also resulted in enhanced anti-HHV activity. Immunotherapy may be beneficial for patients from whom strains resistant to currently known antiviral drugs have been isolated.

Immunomodulatory effects of inosine pranobex on cytokine production by human lymphocytes.

Inosine pranobex (inosine dimepranol acedoben, isoprinosine) (Inos) is an immunomodulatory and antiviral drug used in some viral infections, especially in patients with weakened immunity. In the present study, effects of Inos on the production of cytokines attributable to Th1 (IL-2, IFN-g, and TNF-a) or Th2 cells (IL-4, IL-5, and IL-10) were tested in human peripheral blood lymphocyte cultures stimulated with phytohemagglutinin (PHA). Inos enhanced TNF-a secretion significantly (in short-term--24-hour, and prolonged term--72-hour cultures) and IFN-g (in 72-hour cultures). Surprisingly, production of IL-10 by PHA-stimulated lymphocytes was suppressed by Inos in a dose-dependent manner in both 24-hour and 72-hour cultures. These results shed some light on immunomodulatory properties of Inos and suggest applicability of this agent in patients with a depressed function of the immune system.

Inhibition of adenovirus multiplication by inosine pranobex and interferon α in vitro.

There are no specific antivirals designed for adenoviral infections. Due to many cases of adenovirus infections worldwide, epidemic nature of some types of adenoviruses, and growing number of patients with severe adenoviral infections resulting from dysfunction the immune system, the need for searching an effective and safe therapy is increasing. Inosine pranobex exerts antiviral effects which are both direct and secondary to immunomodulatory activity. In the present study we evaluated in vitro effect of inosine pranobex and interferon α (IFN-α) on replication of HAdV-2 and HAdV-5. The effectiveness of inosine pranobex under these conditions has not been previously reported. In conducted study we reported that inosine pranobex reduced the titer of infectious HAdV-2 and HAdV-5 in vitro. Higher concentrations of IP strongly inhibited multiplication of viruses. Combination of inosine pranobex and IFN-α display higher efficacy than either treatment alone and suggest that both agents may increase therapeutic effectiveness without augmenting toxic effects. Combination index calculations showed that inosine pranobex and INF-α synergistically inhibit HAdV-2 and HAdV-5 titers in A549 cells.