The trimeric artesunate derivative TF27 exerts strong anti-cytomegaloviral efficacy: Focus on prophylactic efficacy and oral treatment of immunocompetent mice.

Human cytomegalovirus (HCMV) causes serious and even life-threatening diseases, particularly upon congenital or post-transplant infection. Treatment of HCMV infections with currently available drugs targeting viral enzymes is often limited by severe side effects and the emergence of drug-resistant viruses. To avoid this problem, novel therapeutic options directed to host proteins involved in virus replication are being investigated. Recently, we described the pronounced antiherpesviral activity of the trimeric artesunate derivative TF27 at low nanomolar concentrations in vitro and in vivo. In the present study, we report first data on the prophylactic efficacy of TF27 against human and murine CMV and the oncogenic avian alphaherpesvirus Marek's disease virus (MDV). The main findings of this study are (i) a pronounced activity of the experimental drug TF27 against alpha- and betaherpesviruses in vitro upon prophylactic treatment and (ii) a therapeutic and prophylactic efficacy upon oral treatment in an immunocompetent mouse model. Moreover, our data highlight (iii) the tolerability of orally administered TF27 free of compound-associated adverse events and further confirm (iv) the suitability of cellular factors as primary antiviral targets. Thus, we provide evidence for therapeutic and prophylactic antiherpesviral efficacy of TF27 upon oral treatment in immunocompetent hosts and thereby underline its potential for future antiviral drug development.

[A case of neurolymphomatosis presented as cauda equine syndrome accompanied with M-proteinemia].

A 63-year-old man developed a syndrome of cauda equine, with the numbness which is a left lower extremity from the left buttocks, weakness of left leg, and a dysfunction of bladder and bowel. Enhanced MRI revealed the enhancement of lower cauda equine, and a nerve conduction test revealed decreased F-wave persistency in the tibial nerve and increased F-wave latency in the peroneal nerve on the both sides. M-proteinemia was admitted and myeloma was suspected. By a biopsy of a vertebral arch, we diagnosed with diffuse large B-cell lymphoma. We treated with dexamethasone and R-CHOP (rituximab, cyclophosphamide, hydroxydaunorubicin, oncovin, prednisone (prednisolone)), then the symptom was improved. In case of caude equine syndrome with M-proteinemia, a possibility of the malignant lymphoma should also be considered.

Growth inhibitive effect of betulinic acid combined with tripterine on MSB-1 cells and its mechanism.

Marek's disease (MD), a highly infectious lymphoproliferative disease in chickens, is caused by a cell-associated oncogenic herpesvirus, Marek's disease virus (MDV). MSB-1 is a MD-derived lymphoblastoid cell line and can induce tumors when inoculated into susceptible chickens. Betulinic acid, which is present as one of the major effective components in many traditional Chinese medicines, has recently been reported to inhibit growth of cancer cells and employed as a potential anticancer agent. Tripterine, a major active compound extracted from the Chinese herb Tripterygium wilfordii Hook F, has now also shown anti-tumor activities in various cancers. The aim of this study was to investigate the synergistic growth-inhibitive effect of betulinic acid combined with tripterine on MSB-1 cells and its mechanism. Viability of MSB-1 cells was assessed by 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT) method. Cell apoptotic analysis was performed by fluorescence detection. NF-κB transcription activity was detected by measuring luciferase activity. Western blotting was used to analyze the expression of p65, IκB and Meq. Our results showed that the proliferation in the combination group was significantly decreased as compared with that of monotherapy using betulinic acid or tripterine, accompanied by an induction of apoptosis, inhibition of NF-κB transcriptional activity and its targeting oncogenic gene Meq. The results suggest that the combination of betulinic acid and tripterine at lower concentration may produce a synergistic inhibitive effect on MSB-1 cells that warrants further investigation for its potential clinical applications.

Sodium tanshinone IIA sulfonate inhibits the meq, ul49 and VP22 expression of Marek's disease virus.

BACKGROUND: Our previous studies have demonstrated that sodium tanshinone IIA sulfonate (STS), a natural compound derived from Salviae Miltiorrhizae Radix et Rhizoma (Danshen), could effectively inhibit Marek's disease virus (MDV) infection both in vitro and in vivo, but the underlying mechanisms remain unclear. The main objective of the study was to explore the effect of STS on the meq, ul49 and VP22 expression of MDV in vitro. METHODS: Quantitative real-time PCR (qRT-PCR) was used to analyse the effect of STS on meq and ul49 expression at both the DNA and messenger RNA (mRNA) level, and the effect of STS on VP22 was assessed by immunofluorescence assay and western blotting. RESULTS: The DNA and mRNA copy numbers of meq and ul49 significantly decreased in the groups treated with STS compared with MDV control (P<0.05), which indicated that STS could inhibit the expression of meq and ul49 at both the DNA and mRNA level. Moreover, the expression of VP22 encoded by ul49 was also significantly inhibited (P<0.05). CONCLUSIONS: STS possessed anti-MDV activity in chicken embryo fibroblasts. Its antiviral mechanisms may be ascribed to inactivating MDV directly, disturbing meq and ul49 replication and inhibiting the expression of VP22 encoded by ul49. These results suggested that STS is a promising natural compound to be further developed as an antiviral agent against MDV infection.

Effects of sodium tanshinone IIA sulfonate against Marek's disease virus in experimentally infected chickens.

Chickens experimentally infected with Marek's disease virus J-1 strain were used to evaluate the anti-Marek's disease virus (MDV) activity of sodium tanshinone IIA sulfonate (STS) in vivo. Chickens in same group were kept in one pen and control group chickens were housed in negative pressure isolator. Chickens were treated with different dose of STS or ABOB for 21 consecutive days. Peripheral T lymphocyte proliferation, expression level of IFN-γ and IL-10 in serum, and MDV load in spleen were determined. The results showed that the treatments with STS and ABOB could significantly increase stimulating index (SI) of peripheral T lymphocytes while the SI is dropping due to the MDV infection, SI of chickens in STS prevention groups were significantly higher than that in STS treatment group and ABOB group (P<0.05 or P<0.01); IFN-γ and IL-10 level of chickens in STS groups were higher than that in other groups (P<0.05 or P<0.01). The results of qPCR demonstrated that STS could inhibit the virus replication in spleen of chickens infected with MDV. These findings indicated that STS can be potentially applied as an anti-MDV drug and set a solid basis for further investigating the antiviral mechanisms of STS.

High frequency of neurolymphomatosis as a relapse disease of intravascular large B-cell lymphoma.

BACKGROUND: Intravascular large B-cell lymphoma (IVL) is characterized by lymphoma cell proliferation in the lumina of small vessels in various organs. A high incidence of neurologic symptoms associated with the central nervous system has been reported, but peripheral nerve involvement (neurolymphomatosis [NL]) rarely has been described. METHODS: The medical records from patients who were diagnosed with IVL over the past 4 years were reviewed. A diagnosis of NL was made based on the combination of neurologic symptoms and their correspondence with imaging studies, such as magnetic resonance imaging (MRI), (18) F-fluoro-2-deoxy-D-glucose (FDG)-positron emission tomography/computed tomography (PET/CT), and/or the histologic confirmation of lymphoma cells within the peripheral nerves, nerve root/plexuses, or cranial nerves. RESULTS: Four patients with NL were identified among 11 patients who had IVL. All cases of NL occurred as relapsed disease during or shortly after the completion of chemotherapy. Although MRI studies of the brains and whole spines revealed nerve infiltration by gadolinium enhancement in 2 patients, the technology was not sensitive enough to detect such infiltration in the remaining 2 patients. In contrast, FDG-PET/CT studies successfully revealed cranial or peripheral nerve lesions in all 4 patients and was useful for evaluating therapeutic response. Patients received treatment with high-dose methotrexate with or without other systemic chemotherapy, which achieved varied success. Further studies will be needed to determine the optimal treatment. CONCLUSIONS: Considering the rarity of IVL and NL, the current observations suggested that IVL may have a predilection not only for the vessels but also for both the central and peripheral nervous systems.

Intravenous treatment with liposome-encapsulated dichloromethylene bisphosphonate (Cl2MBP) suppresses nitric oxide production and reduces genetic resistance to Marek's disease.

In this study the functional effectiveness of in vivo macrophage depletion using liposome-encapsulated dichloromethylene bisphosphonate (Cl(2)MBP) was examined in the chicken. The main target organs for systemic liposome-encapsulated Cl(2)MBP treatment are the spleen and the liver. Intravenous treatment with Cl(2)MBP of B(21)/B(21) chickens, genetically resistant to Marek's disease (MD), before challenge with the very virulent strain RB-1B, increased viral load in the blood and spleen after the first week and up to 6 weeks post-infection. In addition, Cl(2)MBP treatment dramatically increased tumour incidence and tumour load, especially in the spleens and livers of sick animals, but without affecting MD-specific mortality of B(21)/B(21) chickens infected with RB-1B at 12 days of age. Nitric oxide (NO) is an important effector of the macrophage and has antiviral and antitumoural properties. NO has been shown to be one of the mechanisms triggered in resistance to Marek's disease. Intravenous treatment with Cl(2)MBP before infection with RB-1B induced a long-lasting decrease in numbers of macrophages and reduction in splenic inducible NO production associated with an absence of nitrate induction in the serum (up to 6 weeks p.i.). These results do not identify macrophage and NO production as major effector components in genetic resistance to Marek's disease, but underline their roles in limiting viraemia and tumour development in organs such as the spleen and the liver.

Antiviral and antitumoral effects of recombinant chicken myelomonocytic growth factor in virally induced lymphoma.

Chicken myelomonocytic growth factor (cMGF) is a 27-kDa glycoprotein that stimulates the growth and activation of cells from the monocyte/macrophage lineage. Recombinant cMGF was produced in a prokaryotic (Escherichia coli) expression system and purified via a C-terminal His-tag. Treatment of 2-week-old histocompatible B(13)/B(13) chickens highly susceptible to Marek's disease (MD) with rcMGF (two daily injections of 50 microg rcMGF per chicken) enhanced background and LPS-inducible systemic NO (NO3- + NO2-) responses 3 days later. NO has antiviral activity on Marek's disease virus (MDV), a herpesvirus specifically inducing T cell-lymphomas in chickens. When the very virulent strain of MDV RBI-B was inoculated 3 days after treatment with rcMGF, MDV viremia was significantly inhibited and development of visceral tumors was drastically reduced. Combination of rcMGF with partially protective vaccination using a herpesvirus of turkey (HVT) further reducedtumor burden and significantly delayed mortality, but only in very young birds. In conclusion, rcMGF might be worth considering as a stimulator of innate immune response in chickens, such as activation of macrophages and NO production, and thus be beneficial for its antiviral and antitumoral effects in vaccination against MD lymphoma.

Effect of acyclovir on the replication of turkey herpesvirus and Marek's disease virus.

The toxicity of acyclovir for chick embryo fibroblasts and its effect on the replication of turkey herpesvirus (strain FC 126) and Marek's disease virus (strain HPRS 16) multiplied on fibroblast culture was studied. The influence of using acyclovir on the development of the tumour process in birds infected with a virulent Marek's disease virus was also determined. Acyclovir used in doses below 12.5 micrograms ml-1 proved to be nontoxic for chick embryo fibroblast culture. It inhibited in vitro replication of turkey herpesvirus and Marek's disease virus. It was also shown to diminish the development of tumours in birds infected with Marek's disease virus.

Cell-specific antiviral activity of 1-(2-fluoro-2-deoxy-beta-D-arabinofuranosyl)-5-iodocytosine (FIAC) against Marek's disease herpesvirus and turkey herpesvirus.

Three new fluoroarabinosylpyrimidine nucleosides (FIAC, FIAU and FMAU) were tested for in vitro activity against oncogenic and nononcogenic strains of Marek's disease virus (MDV) and herpesvirus of turkeys (HVT). Marek's disease is a herpesvirus-induced lymphoma in chickens. Nononcogenic strains of MDV and HVT can protect against this disease. All viruses were inhibited by 1 microM of these drugs in chick kidney cell (CKC) cultures, but only FMAU and FIAU were active in chicken embryo fibroblast (CEF) and spleen cell cultures. It was determined that whereas CKC produced the enzyme 2'-deoxycytidine-deaminase which is needed to deaminate FIAC to FIAU, CEF were devoid of this enzyme activity. In addition, the deaminase inhibitor 3,4,5,6-tetrahydrouridine prevented the antiviral activity of FIAC in CKC. FMAU was not active against two Marek's disease-derived lymphoblastoid tumor cell lines.

Inhibitory activity of cytosine arabinoside on Marek's disease virus.

The presence of ara-C in growth medium at concentrations of 10(-4) to 10(-7) M completely or partially suppressed the formation of plaques specific for MDV or HVT and decreased proportionally the growth of HPRS line 1 lymphoblastoid cells. Administration of ara-C to chickens immediately after infection with MDV (1 mg/chicken/day i.p. for 5 days) reduced the incidence of Marek's disease by 50%. Thus ara-C appears to be an inhibitor of Marek's disease.

Some effects of silical treatment on Marek's disease.

Treatment of newly hatched chicks with silica by the intraperitoneal route delayed the onset of mortalities due to the JM strain of Marek's disease (MD' virus inoculated at 6 days of age. During the 88-day observation period fewer silica-treated chicks died of MD, but this difference was not usually statistically significant. Silica treatment had no effect on the susceptibility of 4-week-old birds. Silica treatment reduced the antibody response to MD but, in general, not significantly. The antibody response to bovine serum albumin was significantly enhanced if measured by the indirect hemagglutination test but not if measured by the agar gel diffusion test, whereas the response to Brucella abortus was enhanced significantly in N-line (MD-resistant) chicks but not significantly in P-line (MD-susceptible) chicks. Five days after infection, silica-treated chicks had significantly less fluorescing antigen in thymus and bursa than did untreated chicks; no difference was observed in the spleen. After silica treatment the glass-adherent cell population in the buffy coat was increased by up to 10-fold compared with untreated chicks. It is suggested that silica treatment induced macrophage proliferation, with subsequent restriction of MD virus spread, yet allowed an adjuvant-type effect with other antigens.