Triple Immunotherapy Overcomes Immune Evasion by Tumor in a Melanoma Mouse Model.

Background: Melanoma is a malignancy with increasing incidence that underlies most skin cancer-related deaths. Advanced melanoma patients still have poor prognosis despite recently developed immunotherapies. This study devises a triple immunotherapy to treat melanoma in a mouse model. The combination includes anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA4) antibodies, Monophosphoryl-lipid-A (MPLA), and an Indolamine-Dioxygenase-1 (IDO1) inhibitor. The aim of the study is, first, to rule out any major toxic effects related to this therapy and, second, to assess its antitumor effects. Methods: Cancer-free C57BL/6 mice were randomized into control groups and groups receiving single, dual, or triple therapies of the defined treatments. Clinical signs, weight gain, and histological sections from their main organs were assessed. Then, melanoma-bearing mice were segregated into similar groups, monitored for survival, and their tumor size was measured repeatedly. Finally, flow cytometry was used to analyze immune cell populations in the tumor masses including CD4+, CD8+, and regulatory T cells in addition to natural killer cells. Results: No adverse effects were detected in any of the treated groups. Survival analysis indicated that the groups receiving dual or triple therapies had prolonged survival compared to the controls. However, the group receiving triple therapy was the only group to show statistically significant increase in survival compared to the controls. Tumor size progression paralleled the survival outcome. The group receiving the triple therapy showed statistically significant smaller tumor sizes compared to all the other groups throughout the whole monitoring period. Flow cytometry used to analyze immune cell populations in the tumor mass indicated that the triple immune therapy was capable of significantly enhancing the natural killer cell counts as well as the CD3+CD4+/Treg and CD3+CD8+/Treg ratios possibly enhancing the anti-tumorigenic environment. Conclusions: Generated data rule out any major adverse events pertaining to the triple immunotherapy and reveal its enhanced effectiveness in thwarting melanoma progression over all other tested treatments.

Atorvastatin increases the production of proinflammatory cytokines and decreases the survival of Escherichia coli-infected mice.

To assess whether the immunosuppressive effects of atorvastatin outweigh its antibacterial ones in an infection, mice were infected with Escherichia coli and administered atorvastatin; survival rates were then monitored. Mice treated with atorvastatin post-infection showed a remarkable decrease in their survival rate. On the other hand, the higher the level of serum IFN-γ in the infected mice treated with atorvastatin, the lower was the survival rate. Levels of IL-4 were markedly depressed in all groups infected with E. coli and treated with atorvastatin. Since atorvastatin inhibits IFN-γ expression in the absence of bacterial infection, we examined whether bacterial lipopolysaccharide (LPS) was the element capable of overriding this inhibition. Mouse peripheral blood mononuclear cells were treated with atorvastatin and lipopolysaccharide ex vivo then proinflammatory (IFN-γ, TNFα, IL-6) and prohumoral/regulatory (IL-4, IL-13, IL-10) cytokine levels were analyzed in culture supernatants. While proinflammatory cytokine levels were decreased upon treatment with atorvastatin alone, their levels were markedly elevated by treatment with LPS, bacterial lysate or bacterial culture supernatant. On the other hand, atorvastatin exerted an inhibitory effect on production of the prohumoral/regulatory cytokines. Our data indicates that any consideration for statins as antimicrobial treatment should assess the possible adverse outcomes.

Epstein-Barr virus DNA modulates regulatory T-cell programming in addition to enhancing interleukin-17A production via Toll-like receptor 9.

Infection with the Epstein-Barr virus (EBV) has been associated with several autoimmune diseases including rheumatoid arthritis (RA). We have previously reported that DNA from this virus enhances production of the pro-autoimmune interleukin 17A (IL-17A) in mice. In this study we assessed the effect of EBV DNA on regulatory T cell programming and examined whether it mediated its effects via Toll-like receptor 9 (TLR9) in mice; moreover, we evaluated whether EBV DNA in humans had similar effects to those seen in mice. For this purpose, we assessed the linearity of the correlation between EBV DNA and IL-17A levels in RA subjects and matched controls. A modulatory effect for the viral DNA was observed for regulatory T cell markers with an inhibitory effect observed for CTLA4 expression in the EBV DNA-treated mice. To examine whether TLR9 mediated the detection of EBV DNA and enhancement of IL-17A production, mouse peripheral blood mononuclear cells were treated with the DNA in the presence or absence of the TLR9 inhibitor ODN 2088. Subsequently, IL-17A production from these cells was assessed. Treatment with the TLR9 inhibitor resulted in a significant decrease in IL-17A production indicating that TLR9 is involved in this pathway. In human subjects, examining the linearity of the correlation between EBV DNA and IL-17A levels in RA subjects showed a propensity for linearity that was not observed in controls. Our data thus indicates that EBV DNA itself acts as a modulator of the Th17 compartment as well as that of regulatory T cell mechanisms. The involvement of TLR9 in the EBV DNA-triggered induction of IL-17A suggests therapeutic targeting of this endosomal receptor in EBV positive subjects with an autoimmune flare-up or possibly for prophylactic purposes.

The Effect of Denatured Flagellin on Toll-Like Receptor-5 (TLR-5) in Mice.

BACKGROUND: Previous studies have demonstrated that flagellin, a component of bacterial flagella, engages Toll-Like receptor 5 (TLR-5) causing the activation of the Myeloid Differentiation Factor-88 (MYD-88) pathway that leads to the production of pro-inflammatory cytokines including Tumor Necrosis Factor-alpha (TNF-α) and Interleukin-12 (IL-12). In physiological levels, cytokines can aid in protection against infectious agents. However, excessive production of cytokines can lead to septic shock during sepsis. OBJECTIVE: In this study, we aimed at investigating the effect of denatured flagellin on hindering the effects induced by intact flagellin or flagellated Pseudomonas aeruginosa on the Toll-Like Receptor-5 (TLR-5) in mice. METHODS: Mouse mononuclear cells (MNCs) were cultured with intact flagellin, heat-denatured flagellin, TLR-5 antagonist, Pseudomonas aeruginosa, and TLR-4 antagonist each alone or in combinations. Supernatants were collected at 4 hours post incubation to assess the levels of IL-12 and TNF-α by Enzyme-Linked ImmunoAssay (ELISA). Furthermore, groups of BALB/c mice were injected intraperitoneally (IP) with Pseudomonas aeruginosa, LPS-RS, intact flagellin, and denatured flagellin, each alone or in different combinations. Serum levels of IL-12 and TNF-α were measured at 2, 4, and 6 hours post injections of Pseudomonas aeruginosa or intact flagellin. RESULTS: Pretreatment with denatured flagellin significantly reduced the amount of TNF-α and IL-12 produced both in vitro and in vivo by intact flagellin or Pseudomonas aeruginosa. CONCLUSION: Denatured flagellin suppressed the production of the pro-inflammatory cytokines induced by intact flagellin or Pseudomonas aeruginosa both in vitro and in vivo, probably by blocking TLR5. Denatured flagellin might be considered as an anti-septic shock agent.

Epstein-Barr Virus and Human herpes virus 6 Type A DNA Enhance IL-17 Production in Mice.

Several studies have shown a potential association between the Herpesviridae members, the Epstein-Barr virus (EBV) and Human herpes virus 6 (HHV-6), and an increased risk of autoimmune disease development. Because of the ability of these viruses to cause recurrent infections, various viral antigens, including viral DNA, are consistently shed. These antigens may then play a role in triggering autoimmune processes or contributing to autoimmune mechanisms. Therefore, this study examined whether the DNA of EBV or that of HHV-6A is capable of triggering IL-17, the autoimmune-associated cytokine, in mice. BALB/c mice were intraperitoneally injected with various copy numbers of either EBV or HHV-6A DNA. One group was injected with sterile water (the DNA solvent), and another was left uninjected. A mouse group that was administered DNA obtained from Staphylococcus epidermidis was included to ensure that any observed effects would pertain to the viral DNA tested. Mice were sacrificed and their sera were examined using an enzyme-linked immunosorbent assay for IL-17 and IL-23, as pro-autoimmune cytokines, IL-10, as an anti-inflammatory cytokine, and IFN-γ, as a pro-inflammatory cytokine, on days 3, 6, and 9 post-injection. All mouse groups injected with different copy numbers of EBV DNA or HHV-6A DNA displayed higher IL-17 levels than did the group injected with water on days 3, 6, and 9 post-injection. The highest IL-17 levels appeared to coincide with a marked increase in IL-23 and a decrease in IL-10 levels. Unlike the S. epidermidis DNA, which increased IFN-γ levels but not IL-17 or IL-23 levels, the viral DNA tested increased all three mediators, indicating that triggering Th17 responses is a specific property of EBV and HHV-6A DNA. In conclusion, EBV and HHV-6A viral DNA are capable of enhancing the production of the pro-inflammatory cytokine IL-17, which has been shown to play a role in autoimmune diseases.

The impact of prophylactic antiviral agents and statin administration on graft longevity in kidney allograft recipients.

CONTEXT: In an earlier study, we compared the duration of kidney graft survival between two groups of recipients; one on triple (cyclosporine, prednisone and mycophenolate mofetil) and the other on quadruple (cyclosporine, prednisone, mycophenolate mofetil, and sirolimus) immunosuppressive therapy. OBJECTIVE: The aim of this study was to examine the impact of antiviral and statin therapy on graft longevity. MATERIALS AND METHODS: One hundred five kidney allograft recipients were preoperatively assessed for serological markers of infection with various viral agents. All patients were on a prophylactic antiviral regimen of acyclovir and gancyclovir. Seventeen patients were on a statin. Patients were monitored for viral infections and graft rejection or loss for period of 3 years posttransplantation. RESULTS: We detected a high preoperative prevalence rate of IgG immunoglobulins versus the latency-establishing Herpesviridae viruses. Two patients who were preoperatively IgG positive for CMV had cytomegalovirus disease after transplantation. One patient who was preoperatively IgG positive for VZV had shingles after the surgery. No other confirmed viral infections were reported. Thirteen of 88 patients (14.77%) whose treatment regimen did not include a statin suffered a rejection episode or lost the graft whereas 1 of 17 patients (5.88%) on a statin had a rejection episode. CONCLUSIONS: The low rate of viral infections observed in our study population supports the utility of prophylactic administration of antiviral agents to transplant recipients. However, statins seem to have a protective effect on graft longevity (odds ratio [OR] = 0.361, 95% confidence interval [CI] = 0.044-2.957).

Identification of bacteria isolated from nasal polyps and their ability to produce superantigens and biofilms in Lebanese patients.

Staphylococcus aureus superantigens and bacterial biofilms have been implicated in the development of chronic rhinosinusitis and nasal polyps. We conducted a study of 32 Lebanese patients-21 males and 11 females, aged 15 to 71 years (mean: 39)-to identify bacteria isolated from nasal polyps and to determine if these bacteria produced superantigens and biofilms. Polyps were surgically removed, homogenized, and subjected to bacteriologic studies. The presence or absence of S aureus enterotoxin A, B, C, and D (superantigen) genes was determined in all isolates by polymerase chain reaction. Biofilm production by coagulase-negative staphylococci and Pseudomonas aeruginosa was assessed by tissue culture plate assay. A total of 34 bacterial species/groups were isolated from the nasal polyps. Of these, only 3 (8.8%) were S aureus, and only 1 possessed an enterotoxin-coding gene (enterotoxin B). Moreover, of the 21 coagulase-negative staphylococci isolates that were found, none possessed the investigated genes, and only 1 had a strong biofilm-formation property. Our results could not confirm that S aureus enterotoxins (superantigens) or biofilm-producing bacteria play a role in the development of nasal polyps in the Lebanese group studied.

Patented small molecules against psoriasis.

BACKGROUND: It is hypothesized that psoriasis is an autoimmune disease. The most recent therapeutic approach that proved to be more effective than earlier methods of treatment is the use of mAb/fusion proteins. Efforts nowadays are focused on investigating the antipsoriatic affect of small molecules that can be administered orally, some of which are capable of entering cells, and being selective in targeting intracellular pathways. OBJECTIVE: Preclinical patented small molecules that are recommended for the treatment of psoriasis are reviewed. Emphasis is placed on their mechanism of action. METHODS: http://ep.espacenet.com/ , Pubmed, Scopus and Google websites were the main sources used for the patented small molecule search. A number of patents were poorly described and difficulties were faced in trying to figure out the patentee(s) explanation. Moreover, most patents were recommended for the treatment of a number of autoimmune diseases and cancer, and not only for psoriasis. RESULTS/CONCLUSIONS: Small molecules that inhibit the activation of T lymphocytes, leukocyte trafficking, leukotriene activity/production and angiogenesis, and promote apoptosis have been patented. Small molecules that have been patented for the treatment of other autoimmune diseases and could be used for treating psoriasis are described. Moreover, other possible mechanistic approaches using small molecules are discussed.

The potential use of Toll-like receptor (TLR) agonists and antagonists as prophylactic and/or therapeutic agents.

Toll-like receptors (TLR) and their ligands are one of the main players in the initiation of innate immunity which precedes, and is required, for the establishment of adaptive immunity. Manipulating the immune response by using TLR agonists or antagonists might be of therapeutic and/or prophylactic value. This review covers; 1-TLR. their natural ligands and ligand - TLR signaling events, 2-TLR against and their use in clinical trials as vaccine adjuvants, and to treat allergy, cancer and infectious diseases, 3-TLR antagonists and their use in clinical trials to treat septic shock and autoimmune diseases. Potential drawbacks related to their potential use as prophylactic and/or therapeutic agents are discussed.