Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infection caused by a novel Bunyavirus. Analysis on the dynamic changes of clinical, laboratory, and immunological abnormalities associated with SFTS in a concurrent study is lacking. Thirty-three SFTS patients were admitted to Jiangsu People's Hospital, Nanjing, China, and diagnosis was made based on the clinical symptoms and positive viral RNA detected by RT-PCR. Four patients deceased and twenty-nine survived. Blood samples were collected every other day between Day 5 and Day 15 from the onset of fever. Samples from healthy volunteers were used as normal controls. Peak viral RNA load, serum enzymes, IL-6, and IL-10 were significantly higher in deceased patients compared to survivors. Viral load, serum enzymes, and cytokines declined in survivors within 2 weeks from onset of fever. CD69+ T cells were elevated early after infection while HLA-DR+ and CTLA4+ T cells were elevated during the recovery phase of those who survived. High level SFTSV viral load was concurrently observed with reduced PLT, elevated serum enzymes, elevated pro-inflammatory and anti-inflammatory cytokines, and activation of CD69+ T cells. The degree and pattern of changes in these parameters may indicate the clinical outcome in SFTSV-infected patients.

Over the past several years, investigators in this laboratory and elsewhere have been studying tumor localization by pretargeting with streptavidin and biotin or with avidin and biotin. Despite encouraging results, difficulties related to endogenous biotin and the immunogenicities of streptavidin and avidin have made a search for alternative strategies sensible. Recently, we have considered the use of DNAs and peptide nucleic acids for this purpose because oligomers can have hybridization affinities equivalent to that of biotin for streptavidin or avidin without the associated difficulties. We now report on the use of a morpholino (MORF), another commercially available synthetic oligomer, for pretargeting applications. MORFs support the nitrogenous bases by nonionic phosphorodiamidate linkages and, besides being nuclease resistant, can display good water solubility. METHODS: An 18mer MORF and its 18mer complementary MORF (cMORF) were obtained with a primary amine through a 3-member alkyl linker on the 3' equivalent end. An anti--carcinoembryonic antigen IgG antibody (MN14) was conjugated with MORF, whereas cMORF was conjugated with N-hydroxysuccinimide-mercaptoacetyltriglycine (MAG3) to permit radiolabeling with (99m)Tc. The biodistribution of labeled cMORF was first evaluated in normal CD-1 mice. Subsequently, nude mice bearing LS174T tumors received 50 microg conjugated antibody 48 h before the administration of 1.0 microg (7.4 MBq) (99m)Tc-MAG3-cMORF. Control animals received the labeled cMORF without prior administration of the antibody. A clearing step was not used. RESULTS: Biodistributions in normal mice showed that (99m)Tc-MAG3-cMORF was excreted rapidly through the kidneys, with only 7 percentage injected dose (%ID) remaining within the whole body (excluding urine) at 3 h. In tumor-bearing mice at 24 h, only 11 %ID of the radioactivity remained in the whole body of study animals, and of this amount, 2 %ID/g was in tumor tissue. The sites with the highest %ID were the kidneys, at 4 %ID/g, and the blood, at 0.5 %ID/g; all other organs had <1 >%ID/g. At the same time, values for the control animals were 5 %ID (whole body), 0.05 %ID/g (tumor), and 3 %ID (kidneys). All images reflected high uptake in the tumors and low uptake in the normal tissues of the study mice. CONCLUSION: Pretargeting using MORFs was effective in a mouse tumor model.

One objective of this investigation was to determine whether chemical modifications of oligonucleotides to permit radiolabeling with gamma- or positron emitters interferes with hybridization and target cell accumulation. A second objective was to establish to a reasonable extent whether cellular accumulation of radiolabeled oligonucleotides can be explained by an antisense mechanism. METHODS: An 18mer uniform phosphorothioate DNA antisense to the messenger RNA (mRNA) of the type I regulatory subunit alpha of cyclic adenosine monophosphate-dependent protein kinase A (RI alpha) was conjugated with the N-hydroxysuccinimidyl derivative of S-acetylmercaptoacetyltriglycine (MAG3) through a primary amine/linker and investigated in vitro in cell culture. RESULTS: By surface plasmon resonance, the association kinetics between native (i.e., without amine/linker) DNA and MAG3-amide/linker-DNA were identical. Melting temperatures were also identical for native DNA, amine/linker-DNA, and MAG3-amide/linker-DNA, indicating that these chemical modifications had no detectable influence on hybridization. However, cellular accumulation of (99m)Tc-MAG3-DNA was lower than that of (35)S-MAG3-DNA, suggesting that chemical modifications can have an important influence on cellular accumulation. In tissue culture studies of ACHN tumor cells (a human renal adenocarcinoma cell type), an antisense effect was suggested by 3 findings: an increased accumulation of (35)S- or (99m)Tc-labeled antisense versus sense DNA, an increased accumulation of (99m)Tc-antisense DNA in another RI alpha-positive tumor cell line (LS174T) but not in a murine transfected control cell line (HC-2), and the disappearance of the increased cellular accumulation of (99m)Tc-antisense DNA with increasing dosage of antisense DNA. Higher than expected cellular accumulations of about 10(5) antisense DNAs per cell over 24 h suggest stabilization of the target mRNA or increased mRNA production by the presence of the antisense DNA. In support of this suggestion, we observed, first, an increased incorporation of uridine-5'-triphosphate into RNA in cells exposed to the antisense DNA but not to the control DNA and, second, an increase in target mRNA expression in cells exposed to the antisense DNA but not to the control DNA. CONCLUSION: This evidence suggests tumor cell accumulation by an antisense mechanism. Moreover, the high level of DNA accumulation suggests that a rapid target mRNA turnover or transcription rate may be an important determinant of tumor counting rates.