The Rate of Infectious Complications After Intrathecal Drug Delivery System Implant for Cancer-Related Pain Is Low Despite Frequent Concurrent Anticancer Treatment or Leukopenia.

BACKGROUND: The efficacy of intrathecal drug delivery (IDD) for cancer-related pain is well established. Cancer therapies are often associated with immunosuppression and increased risk of infection, and the rate of infection after intrathecal drug delivery system (IDDS) implant in cancer patients has been reported as 2.4%-6.3%. Our objective is to report on the rate of surgical site infections (SSI) in patients implanted with IDDS for cancer-related pain and to provide a data-driven discussion on the relationship between antineoplastic treatment, leukopenia, and other clinical or demographic characteristics and SSI. METHODS: Following local institutional review board approval, we conducted a retrospective chart review of IDDS implants from May 2014 through December 2018. Data collected included demographic data, health status, prophylactic antibiotic administration, surgery duration, presence of leukopenia (white blood cell [WBC] count of <4.0 K/µL) or moderate neutropenia (absolute neutrophil count [ANC] of <1000/µL) within the 30 days before IDDS implant, and details of antineoplastic treatment or systemic corticosteroid use in the perioperative period. This information was assessed in relation to SSI incidence up to 6 months following implant. RESULTS: Two hundred seventeen IDDS implants were identified. A majority of patients (79.3%) received ≥1 form of antineoplastic therapy within 30 days before or after implant, and 42.4% received multiple forms of antineoplastic therapy. Therapies included chemotherapy in 46.5%, immunotherapy in 28.6%, systemic steroids in 32.3%, and radiation therapy in 28.1%. One-quarter of patients (25.8%) were leukopenic within 30 days before implant, with 3.2% having moderate neutropenia. There were 2 infectious complications representing an infection rate of 0.9% (95% CI, 0.1%-3.3%), with limited shared characteristics between those experiencing SSI. CONCLUSIONS: SSI risk after IDDS placement for cancer pain is low, despite frequent concurrent antineoplastic therapy and leukopenia in the perioperative period. Concomitant cancer therapies should not be a barrier to the implementation of IDD for cancer pain.

The hematopoietic regulator, ELF-1, enhances the transcriptional response to Interferon-ß of the OAS1 anti-viral gene.

Interferon (IFN) therapy is effective in treating cancers, haematological and virus induced diseases. The classical Jak/Stat pathway of IFN signal transduction leading to changes in transcriptional activity is well established but alone does not explain the whole spectrum of cellular responses to IFN. Gene promoters contain cis-acting sequences that allow precise and contextual binding of transcription factors, which control gene expression. Using the transcriptional response to IFN as a starting point we report a high frequency of tandem GGAA motifs in the proximal promoters of Interferon stimulated genes, suggesting a key regulatory action. Utilizing the well-characterized anti-viral gene, OAS1, as an example Interferon stimulated gene promoter containing such a duplicated GGAA motif, we have demonstrated a regulatory role of this promoter in response to IFN by mutation analysis. Furthermore, we identified ELF-1 as a direct binding factor at this motif. Additionally, recruitment of RB1 and SP1 factors to the promoter following IFN stimulation is shown. ELF-1 overexpression enhanced and knockdown of ELF-1 inhibited full activation of OAS1 by IFN stimulation. Collectively, ELF-1 binds an important duplicated GGAA cis-acting element at the OAS1 promoter and in cooperation with RB1 and SP1 recruitment contributes to regulation in response to IFN stimulation.

A novel single virus infection system reveals that influenza virus preferentially infects cells in g1 phase.

BACKGROUND: Influenza virus attaches to sialic acid residues on the surface of host cells via the hemagglutinin (HA), a glycoprotein expressed on the viral envelope, and enters into the cytoplasm by receptor-mediated endocytosis. The viral genome is released and transported in to the nucleus, where transcription and replication take place. However, cellular factors affecting the influenza virus infection such as the cell cycle remain uncharacterized. METHODS/RESULTS: To resolve the influence of cell cycle on influenza virus infection, we performed a single-virus infection analysis using optical tweezers. Using this newly developed single-virus infection system, the fluorescence-labeled influenza virus was trapped on a microchip using a laser (1064 nm) at 0.6 W, transported, and released onto individual H292 human lung epithelial cells. Interestingly, the influenza virus attached selectively to cells in the G1-phase. To clarify the molecular differences between cells in G1- and S/G2/M-phase, we performed several physical and chemical assays. Results indicated that: 1) the membranes of cells in G1-phase contained greater amounts of sialic acids (glycoproteins) than the membranes of cells in S/G2/M-phase; 2) the membrane stiffness of cells in S/G2/M-phase is more rigid than those in G1-phase by measurement using optical tweezers; and 3) S/G2/M-phase cells contained higher content of Gb3, Gb4 and GlcCer than G1-phase cells by an assay for lipid composition. CONCLUSIONS: A novel single-virus infection system was developed to characterize the difference in influenza virus susceptibility between G1- and S/G2/M-phase cells. Differences in virus binding specificity were associated with alterations in the lipid composition, sialic acid content, and membrane stiffness. This single-virus infection system will be useful for studying the infection mechanisms of other viruses.

LMO3 interacts with p53 and inhibits its transcriptional activity.

High expression of LMO3 contributes to the development and aggressiveness of neuroblastoma. LMO3 belongs to the LIM-only protein family, in which de-regulation of its members is implicated in human carcinogenesis. However, the molecular mechanism of LMO3 activity in oncogenesis remained poorly characterized. We found that LMO3 is a direct interacting partner of p53 both in vitro and in vivo. The DNA-binding domain of p53 is required for this interaction. Furthermore, expression of LMO3 repressed p53-dependent mRNA expression of its target genes by suppressing promoter activation. Interestingly, chromatin immunoprecipitation assay showed that LMO3 facilitated p53 binding to its response elements. This suggests that LMO3 acts as a co-repressor of p53, suppressing p53-dependent transcriptional regulation without inhibition of its DNA-binding activity.

Role of cellular FKBP52 protein in intracellular trafficking of recombinant adeno-associated virus 2 vectors.

We have reported that tyrosine-phosphorylated forms of a cellular protein, FKBP52, inhibit the second-strand DNA synthesis of adeno-associated virus 2 (AAV), leading to inefficient transgene expression from recombinant AAV vectors. To further explore the role of FKBP52 in AAV-mediated transduction, we established murine embryo fibroblasts (MEFs) cultures from FKBP52 wild-type (WT), heterozygous (HE), and knockout (KO) mice. Conventional AAV vectors failed to transduce WT MEFs efficiently, and the transduction efficiency was not significantly increased in HE or KO MEFs. AAV vectors failed to traffic efficiently to the nucleus in these cells. Treatment with hydroxyurea (HU) increased the transduction efficiency of conventional AAV vectors by approximately 25-fold in WT MEFs, but only by approximately 4-fold in KO MEFs. The use of self-complementary AAV (scAAV) vectors, which bypass the requirement of viral second-strand DNA synthesis, revealed that HU treatment increased the transduction efficiency approximately 23-fold in WT MEFs, but only approximately 4-fold in KO MEFs, indicating that the lack of HU treatment-mediated increase in KO MEFs was not due to failure of AAV to undergo viral second-strand DNA synthesis. Following HU treatment, approximately 59% of AAV genomes were present in the nuclear fraction from WT MEFs, but only approximately 28% in KO MEFs, indicating that the pathway by which HU treatment mediates nuclear transport of AAV was impaired in KO MEFs. When KO MEFs were stably transfected with an FKBP52 expression plasmid, HU treatment-mediated increase in the transduction efficiency was restored in these cells, which correlated directly with improved intracellular trafficking. Intact AAV particles were also shown to interact with FKBP52 as well as with dynein, a known cellular protein involved in AAV trafficking. These studies suggest that FKBP52, being a cellular chaperone protein, facilitates intracellular trafficking of AAV, which has implications in the optimal use of recombinant AAV vectors in human gene therapy.

SU5416 is a potent inhibitor of hepatocyte growth factor receptor (c-Met) and blocks HGF-induced invasiveness of human HepG2 hepatoma cells.

BACKGROUND/AIMS: SU5416 is a potent inhibitor of receptor tyrosine kinases, including those of the vascular endothelial growth factor receptor, stem cell factor receptor, and platelet-derived growth factor receptor. Because of the overwhelming evidence favoring the role of aberrant hepatocyte growth factor (HGF)/Met signaling in the pathogenesis of various human cancers, various inhibitor strategies have been employed to therapeutically target this receptor. METHODS: Cell proliferation was determined by incorporation of [(3)H] thymidine. Invasiveness was assayed in Boyden Chambers with 8 microm Matrigel coated filters. Phosphorylation of ERK1/2, Akt by HGF stimulation was detected by Western blotting. RESULTS: We found that SU5416 inhibited motility scattering and the invasive activity of a hepatocellular carcinoma cell line HepG2 in vitro and growth in primary cultured hepatocytes induced by HGF. Consequently, tyrosine autophosphorylation of the c-met induced by HGF was inhibited in these cells by SU5416 in a dose-dependent manner. Furthermore, ERK1/2 and Akt phosphorylation, the signaling events down-stream of c-met activation were reduced. Moreover, SU5416 caused reversion in NIH3T3 fibroblasts transformed by the oncogenic form of the receptor, Tpr-Met. CONCLUSIONS: Inhibition of various solid tumors growth and metastasis by SU5416 may be partially attributed to blocking activation of the hepatocyte growth factor receptor.

Impaired nuclear transport and uncoating limit recombinant adeno-associated virus 2 vector-mediated transduction of primary murine hematopoietic cells.

Controversies abound concerning hematopoietic stem cell transduction by recombinant adeno-associated virus 2 (AAV) vectors. For human hematopoietic cells, we have shown that this problem is related to the extent of expression of the cellular receptor for AAV. At least a small subset of murine hematopoietic cells, on the other hand, does express both the AAV receptor and the coreceptor, yet is transduced poorly. In the present study, we have found that approximately 85% of AAV genomes were present in the cytoplasmic fraction of primary murine c-Kit(+)Lin- hematopoietic cells. However, when mice were injected intraperitoneally with hydroxyurea before isolation of these cells, the extent to which AAV genomes were detected in the cytoplasmic fraction was reduced to approximately 40%, with a corresponding increase to approximately 60% in the nuclear fraction, indicating that hydroxyurea facilitated nuclear transport of AAV. It was apparent, nonetheless, that a significant fraction of the AAV genomes present in the nuclear fraction from cells obtained from hydroxyurea-treated mice was single stranded. We next tested whether the single-stranded AAV genomes were derived from virions that failed to undergo uncoating in the nucleus. A substantial fraction of the signal in the nuclear fraction of hematopoietic cells obtained from hydroxyurea-treated mice was also resistant to DNase I. That AAV particles were intact and biologically active was determined by successful transduction of 293 cells by virions recovered from murine hematopoietic cells 48 hr postinfection. Although hydroxyurea facilitated nuclear transport of AAV, most of the virions failed to undergo uncoating, thereby leading to only a partial improvement in viral second- strand DNA synthesis and transgene expression. A better understanding of the underlying mechanism of viral uncoating has implications in the optimal use of recombinant AAV vectors in hematopoietic stem cell gene therapy.