We describe the development of multifunctional nanoparticle probes based on semiconductor quantum dots (QDs) for cancer targeting and imaging in living animals. The structural design involves encapsulating luminescent QDs with an ABC triblock copolymer and linking this amphiphilic polymer to tumor-targeting ligands and drug-delivery functionalities. In vivo targeting studies of human prostate cancer growing in nude mice indicate that the QD probes accumulate at tumors both by the enhanced permeability and retention of tumor sites and by antibody binding to cancer-specific cell surface biomarkers. Using both subcutaneous injection of QD-tagged cancer cells and systemic injection of multifunctional QD probes, we have achieved sensitive and multicolor fluorescence imaging of cancer cells under in vivo conditions. We have also integrated a whole-body macro-illumination system with wavelength-resolved spectral imaging for efficient background removal and precise delineation of weak spectral signatures. These results raise new possibilities for ultrasensitive and multiplexed imaging of molecular targets in vivo.

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In vivo cancer targeting and imaging with semiconductor quantum dots

In vivo transduction of nondividing cells by human immunodeficiency virus type 1 (HIV-1)-based vectors results in transgene expression that is stable over several months. However, the use of HIV-1 vectors raises concerns about their safety. Here we describe a self-inactivating HIV-1 vector with a 400-nucleotide deletion in the 3' long terminal repeat (LTR). The deletion, which includes the TATA box, abolished the LTR promoter activity but did not affect vector titers or transgene expression in vitro. The self-inactivating vector transduced neurons in vivo as efficiently as a vector with full-length LTRs. The inactivation design achieved in this work improves significantly the biosafety of HIV-derived vectors, as it reduces the likelihood that replication-competent retroviruses will originate in the vector producer and target cells, and hampers recombination with wild-type HIV in an infected host. Moreover, it improves the potential performance of the vector by removing LTR sequences previously associated with transcriptional interference and suppression in vivo and by allowing the construction of more-stringent tissue-specific or regulatable vectors.

https://jvi.asm.org/content/jvi/72/12/9873.full.pdf

Self-Inactivating Lentivirus Vector for Safe and Efficient In Vivo Gene Delivery

FLAVIVIRUSES 1103 Background and Classification 1103 Structure and Physical Properties of the Virion 1104 Binding and Entry 1105 Genome Structure 1106 Translation and Proteolytic Processing 1107 Features of the Structural Proteins 1108 Features of the Nonstructural Proteins 1109 RNA Replication 1112 Membrane Reorganization and the Compartmentalization of Flavivirus Replication 1112 Assembly and Release of Particles from Flavivirus-infected Cells 1112 Host Resistance to Flavivirus Infection 1113

Flaviviridae :T he Viruses and Their Replication

Fibroblasts can condense a hydrated collagen lattice to a tissue-like structure 1/28th the area of the starting gel in 24 hr. The rate of the process can be regulated by varying the protein content of the lattice, the cell number, or the con- centration of an inhibitor such as Colcemid. Fibroblasts of high population doubling level propagated in vitro, which have left the cell cycle, can carry out the contraction at least as efficiently as cycling cells. The potential uses of the system as an immu- nologically tolerated "tissue" for wound hea ing and as a model for studying fibroblast function are discussed.

Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative

Purpose Talimogene laherparepvec (T-VEC) is a herpes simplex virus type 1‐derived oncolytic immunotherapy designed to selectively replicate within tumors and produce granulocyte macrophage colony-stimulating factor (GM-CSF) to enhance systemic antitumor immune responses. T-VEC was compared with GM-CSF in patients with unresected stage IIIB to IV melanoma in a randomized open-label phase III trial. Patients and Methods Patients with injectable melanoma that was not surgically resectable were randomly assigned at a two-to-one ratio to intralesional T-VEC or subcutaneous GM-CSF. The primary end point was durable response rate (DRR; objective response lasting continuously 6 months) per independent assessment. Key secondary end points included overall survival (OS) and overall response rate. Results Among 436 patients randomly assigned, DRR was significantly higher with T-VEC (16.3%; 95% CI, 12.1% to 20.5%) than GM-CSF (2.1%; 95% CI, 0% to 4.5%]; odds ratio, 8.9; P .001). Overall response rate was also higher in the T-VEC arm (26.4%; 95% CI, 21.4% to 31.5% v 5.7%; 95% CI, 1.9% to 9.5%). Median OS was 23.3 months (95% CI, 19.5 to 29.6 months) with T-VEC and 18.9 months (95% CI, 16.0 to 23.7 months) with GM-CSF (hazard ratio, 0.79; 95% CI, 0.62 to 1.00; P .051). T-VEC efficacy was most pronounced in patients with stage IIIB, IIIC, or IVM1a disease and in patients with treatment-naive disease. The most common adverse events (AEs) with T-VEC were fatigue, chills, and pyrexia. The only grade 3 or 4 AE occurring in 2% of T-VEC‐treated patients was cellulitis (2.1%). No fatal treatment-related AEs occurred.

Talimogene Laherparepvec Improves Durable Response Rate in Patients With Advanced Melanoma

To assess the RNA helicase activity of hepatitis C virus (HCV) nonstructural protein 3 (NS3), a polypeptide encompassing amino acids 1175 to 1657, which cover only the putative helicase domain, was expressed in Escherichia coli by a pET expression vector. The protein was purified to near homogeneity and assayed for RNA helicase activity in vitro with double-stranded RNA substrates prepared from a multiple cloning sequence and an HCV 5' nontranslated region (5'-NTR) or 3'-NTR. The enzyme acted successfully on substrates containing both 5' and 3' single-stranded regions (standard) or on substrates containing only the 3' single-stranded regions (3'/3') but failed to act on substrates containing only the 5' single-stranded regions (5'/5') or on substrates lacking the single-stranded regions (blunt). These results thus suggest 3' to 5' directionality for HCV RNA helicase activity. However, a 5'/5' substrate derived from the HCV 5'-NTR was also partially unwound by the enzyme, possibly because of unique properties inherent in the 5' single-stranded regions. Gel mobility shift analyses demonstrated that the HCV NS3 helicase could bind to either 5'- or 3'-tailed substrates but not to substrates lacking a single-stranded region, indicating that the polarity of the RNA strand to which the helicase bound was a more important enzymatic activity determinant. In addition to double-stranded RNA substrates, HCV NS3 helicase activity could displace both RNA and DNA oligonucleotides on a DNA template, suggesting that HCV NS3 too was disposed to DNA helicase activity. This study also demonstrated that RNA helicase activity was dramatically inhibited by the single-stranded polynucleotides. Taken altogether, our results indicate that the HCV NS3 helicase is unique among the RNA helicases characterized so far.

The helicase activity associated with hepatitis C virus nonstructural protein 3 (NS3).

To study perinatal transmission of hepatitis C virus (HCV), 15 anti-HCV-positive carrier mothers without human immunodeficiency virus coinfection were recruited. At delivery, maternal blood was taken and anti-HCV titer was determined and HCV RNA measured in each serum sample by reverse transcription polymerase chain reaction (PCR). A competitive PCR was used in selected samples to quantitate HCV concentration. The 15 neonates were followed regularly for 1 year and their sera were also assayed for anti-HCV and for HCV RNA by reverse transcription PCR. All the mothers were positive for HCV RNA. Only one normal spontaneously delivered neonate of a mother with extremely high titer of anti-HCV (1:20,000) and HCV concentration (10(10) copies/mL) had both anti-HCV and HCV RNA in serum for up to 6 months of age. In contrast, none of the remaining 14 neonates born to mothers with low- to high-titer anti-HCV (1:4-1:1000) and moderate amounts of HCV RNA (10(5)-10(6) copies/mL) contracted HCV infection. The results imply that high-titer maternal viremia and normal spontaneous delivery may allow more HCV to infect the neonate intrapartum, therefore establishing perinatal transmission.

Possible Role of High-Titer Maternal Viremia in Perinatal Transmission of Hepatitis C Virus

Ribavirin enhancement of hepatitis C virus core antigen-specific type 1 T helper cell response correlates with the increased IL-12 level

An active immunotherapy strategy with cytokine-assisted tumor vaccine, although often effective for small tumor burdens, is much less so for large tumor burdens. This study examines how large tumors might suppress the T cell functions and escape from the immune responses elicited by a granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting tumor vaccine. According to our results, the T cells isolated from the tumor-bearing mice treated late with the vaccine failed to confer protective activity on naive mice against a wild-type tumor challenge, unlike those isolated from the early-treated group. Nevertheless, the antitumor activity of the inactive T cells could be restored on in vitro stimulation. Expression of transforming growth factor beta (TGF-beta) and interleukin 10 (IL-10), the potent immunosuppressive factors, was detected in the parental tumor cell line RLmale 1 (a murine T leukemia cell line), as well as in the tumor region, the levels of which correlated with tumor progression. An in vitro assay of T cell functions revealed that the TGF-beta in the conditioned medium of RLmale 1 cells mainly affected the activation, whereas the IL-1male affected the activation to a lesser extent, but significantly affected the cytolytic activity, of tumor-specific T cells. The immunosuppressive activity of IL-10 was also signified by the findings that administration of the conditioned medium of RLmale 1 cultured in a serum-free medium, in which the TGF-beta activity was then lost while the IL-10 activity still remained, or of recombinant IL-10 to the early-treated group of mice abrogated the known efficacy of tumor vaccine on the small tumors. These data suggested that the efficacy of cytokine-secreting tumor vaccine was blocked by the immunosuppressive factors secreted from the large tumors. The results have important implications for the clinical design of immunotherapeutic strategies for advanced cancer patients.

Lih Hwa Hwang

Papers

The helicase activity associated with hepatitis C virus nonstructural protein 3 (NS3).

Possible Role of High-Titer Maternal Viremia in Perinatal Transmission of Hepatitis C Virus

Ribavirin enhancement of hepatitis C virus core antigen-specific type 1 T helper cell response correlates with the increased IL-12 level

Tumor-Induced Immunosuppression: A Barrier to Immunotherapy of Large Tumors by Cytokine-Secreting Tumor Vaccine

Heat Shock Protein 72 Is Associated with the Hepatitis C Virus Replicase Complex and Enhances Viral RNA Replication