The Sequence of Delta24-RGD and TMZ Administration in Malignant Glioma Affects the Role of CD8+T Cell Anti-tumor Activity.

The conditionally replicating oncolytic adenovirus Delta24-RGD (Ad) is currently under investigation in clinical trials for glioblastoma, including in combination with temozolomide (TMZ), the standard chemotherapy for this tumor. Previously, we showed that the efficacy of Delta24-RGD in a murine model is primarily dependent on the virus-induced anti-tumor immune response. As observed with most chemotherapies, TMZ has pronounced immune-modulating effects. Here, we studied the combined effects of these treatments in a murine glioma model. In vitro, we observed a synergistic activity between Delta24-RGD and TMZ. In vivo, C57BL/6 mice bearing intracranial GL261 tumors were treated with TMZ for 5 days either prior to intratumoral Delta24-RGD injection (TMZ/Ad) or post virus injection (Ad/TMZ). Notably, the Ad/TMZ regimen led to similar tumoral CD8+ T cell influx as the virus-only treatment, but increased the ability of CD8+ T cells to specifically recognize the tumor cells. This was accompanied by improved survival. The TMZ/Ad regimen also improved survival significantly compared to controls, but not compared to virus alone. In this group, the influx of dendritic cells is impaired, followed by a significantly lower number of tumor-infiltrating CD8+ T cells and no recognition of tumor cells. Depletion of either CD4+ T cells or CD8+ T cells impaired the efficacy of Delta24-RGD, underscoring the role of these cells in therapeutic activity of the virus. Overall, we show that the addition of TMZ to Delta24-RGD treatment leads to a significant increase in survival and that the order of sequence of these treatments affects the CD8+T cell anti-tumor activity.

Multi-level analyses of spatial and temporal determinants for dengue infection.

BACKGROUND: Dengue is a mosquito-borne viral infection that is now endemic in most tropical countries. In Thailand, dengue fever/dengue hemorrhagic fever is a leading cause of hospitalization and death among children. A longitudinal study among 1750 people in two rural and one urban sites in northern Thailand from 2001 to 2003 studied spatial and temporal determinants for recent dengue infection at three levels (time, individual and household). METHOD: Determinants for dengue infection were measured by questionnaire, land-cover maps and GIS. IgM antibodies against dengue were detected by ELISA. Three-level multi-level analysis was used to study the risk determinants of recent dengue infection. RESULTS: Rates of recent dengue infection varied substantially in time from 4 to 30%, peaking in 2002. Determinants for recent dengue infection differed per site. Spatial clustering was observed, demonstrating variation in local infection patterns. Most of the variation in recent dengue infection was explained at the time-period level. Location of a person and the environment around the house (including irrigated fields and orchards) were important determinants for recent dengue infection. CONCLUSION: We showed the focal nature of asymptomatic dengue infections. The great variation of determinants for recent dengue infection in space and time should be taken into account when designing local dengue control programs.

Spatial patterns of and risk factors for seropositivity for dengue infection.

Spatial patterns of and risk factors for seropositivity of dengue infection were studied in three sites in northern Thailand. A survey was conducted in 2001 among 1,750 persons. Potential risk factors for dengue infection were measured by questionnaire and IgM antibodies against dengue were detected by an enzyme-linked immunosorbent assay. The role of landscape as a risk factor was studied using land cover maps and a geographic information system. Logistic regression identified risk factors for dengue seropositivity. Spatial patterns of seropositive cases were determined by cluster analyses. Six percent of the study population was seropositive. Risk factors for dengue seropositivity differed per site, demonstrating variation in local infection patterns. In the periurban site, seropositivity depended on human behavior and factors related to housing quality rather than environmental factors. In both rural sites, older persons had a higher risk of seropositivity and persons living in houses surrounded by natural and agricultural land covers had a lower risk of seropositivity.

Detection of immune-complex-dissociated nonstructural-1 antigen in patients with acute dengue virus infections.

Accurate and timely diagnosis of dengue virus (DEN) infections is essential for the differential diagnosis of patients with febrile illness and hemorrhagic fever. In the present study, the diagnostic value of a newly developed immune-complex dissociated nonstructural-1 (NS-1) antigen dot blot immunoassay (DBI) was compared to a commercially available DEN antigen detection kit (denKEY Blue kit; Globio Co., Beverly, Mass.) and a reverse transcription-PCR (RT-PCR) kit. Serial serum or plasma samples (n = 181) obtained from 55 acute DEN-infected patients were used. In samples obtained from 32 of these 55 DEN-infected patients, viral RNA could be detected by RT-PCR. DEN antigen was detected in only 10 of these 55 patient samples by using the denKEY kit. When these samples were treated with acid to release the immune-complex-associated NS-1 antigen for detection by DBI, 43 of these 55 patients were found to be positive for DEN NS-1 antigen. In nondissociated samples, 22 of these patients were found to be positive by the DBI. In the presence of DEN-specific immunoglobulin M antibodies, both viral RNA and DEN (NS-1) antigen could be detected. The number of positive samples identified by RT-PCR and DBI from these patients with primary DEN infections varied between 28 and 78%. In secondary DEN infections, the number of samples that tested positive by the DBI after immune-complex dissociation (DIS-DBI) was 25% higher than the number of samples that tested positive by RT-PCR and was 35% higher than that determined by nondissociated antigen (NDIS-DBI) detection. We conclude that the denKEY kit has limited diagnostic value for acute DEN infections compared to the RT-PCR and the NDIS-DBI and DIS-DBI methods. We clearly demonstrate that in secondary DEN infections the dissociation of NS-1 immune complexes is essential for early diagnosis of DEN infections.

Serological reactivity of baculovirus-expressed Ebola virus VP35 and nucleoproteins.

Ebola virus (EBOV) is a member of the family Filoviridae and is classified as a biosafety level 4 virus. This classification makes the preparation of antigen and performance of diagnostic assays time-consuming and complicated. The objective of this study was to evaluate the value of EBOV immunoassays based on recombinant nucleoprotein (r-NP) and recombinant VP35 (r-VP35) using large serum panels of African origin and from primates. Furthermore, we investigated whether the results obtained with EBOV r-VP35 enzyme-linked immunosorbent assay (ELISA) could improve on the findings obtained with the EBOV r-NP ELISA. The full-length EBOV NP and VP35 of the EBOV subtype Zaire were expressed as histidine-tagged recombinant proteins in the baculovirus expression system. The antigenic reactivity and specificity of these recombinant proteins were determined by Western blotting and ELISA using EBOV specific monoclonal antibodies. The results obtained with the r-NP and r-VP35 ELISAs were compared with the results obtained in an indirect immunofluorescence assay based on native EBOV subtype Zaire. EBOV specific monoclonal antibodies reacted specifically with the respective proteins in both Western blot and ELISA. Five hundred and twenty six samples from humans and primates were tested with r-NP and r-VP35 ELISAs. Monkey serum samples positive for EBOV subtype Reston and Zaire were both positive in the EBOV r-NP ELISA, whereas only the EBOV Zaire infected monkeys were positive in the r-VP35 ELISA. The sensitivity and specificity values of the EBOV recombinants' ELISAs compared to those of the immunofluorescence assay were 92% and 99% for r-NP and 44% and 100% for r-VP35. r-NP ELISA proved to be a sensitive and specific assay for EBOV diagnosis and for epidemiological studies for both EBOV subtypes Reston and Zaire. The use of r-VP35 in an ELISA format has no additional value for EBOV serodiagnosis.