Laparoscopic preperitoneal repair for primary falciform ligament herniation.

Epigastric hernia involving the falciform ligament is exceptionally rare. Most reported cases are incisional hernia secondary to prior abdominal surgery. We report a case of primary falciform ligament herniation into the epigastric region repaired by the laparoscopic preperitoneal approach. In this case, an accompanying vessel along the herniated falciform ligament was identified. This finding provides a basis for the hypothesis of a perforating vessel piercing the linea alba and thereby creating a weak point for hernia protrusion (Moschowitz theory). The patient had an uneventful recovery and was discharged home on the postoperative day two. A laparoscopic preperitoneal approach is feasible for the repair of primary falciform ligament herniation. The magnified endoscopic view enables surgeons to achieve definite repair without missing occult defects.

Effect of NPC15199 on [Ca²âº]i and viability in SCM1 human gastric cancer cells.

NPC15199 is a synthesized compound that inhibits inflammation in some models. However, whether NPC15199 affects Ca²âº homeostasis in human gastric cancer is unclear. This study examined the effect of NPC15199 on cytosolic free Ca²âº concentrations ([Ca²âº]i) and viability in SCM1 human gastric cancer cells. The Ca²âº-sensitive fluorescent dye fura-2 was used to measure [Ca²âº]i. NPC15199 evoked [Ca²âº]i rises concentration-dependently. The response was reduced by removing extracellular Ca²âº. NPC15199-evoked Ca²âº entry was not inhibited by store-operated channel inhibitors (nifedipine, econazole and SKF96365) and protein kinase C (PKC) activator (phorbol 12-myristate 13 acetate, PMA), or PKC inhibitor (GF109203X). In Ca²âº-free medium, treatment with the endoplasmic reticulum Ca²âº pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) nearly abolished NPC15199-evoked [Ca²âº]i rises. Conversely, treatment with NPC15199 also nearly abolished thapsigargin or BHQ-evoked [Ca²âº]i rises. Inhibition of phospholipase C (PLC) with U73122 did not affect NPC15199-evoked [Ca²âº]i rises. NPC15199 at concentrations of 100-900 µM induced concentration-dependent, Ca²âº-independent decrease in viability. Together, in SCM1 cells, NPC15199 induced [Ca²âº]i rises that involved Ca²âº entry through PKC-insensitive non-store-operated Ca²âº channels and PLC-independent Ca²âº release from the endoplasmic reticulum. NPC15199 also induced Ca²âº-independent cell death.

Lentiviruses with trastuzumab bound to their envelopes can target and kill prostate cancer cells.

In this study, we took advantage of the overexpression of human epidermal growth factor receptor 2 (HER-2) in prostate cancers to design lentiviruses with modified envelope proteins that bind antibodies to specific cell-surface antigens. When bound to trastuzumab (Herceptin, Genentech, CA), lentiviruses were able to selectively infect androgen-sensitive LNCaP and castration-resistant C4-2 human prostate cancer cell lines, both of which express high levels of HER-2. To test for a therapeutic effect, we engineered our antibody-binding lentiviruses to express thymidine kinase, which can convert the non-toxic pro-drug ganciclovir (GCV) into a cytotoxic form. LNCaP and C4-2 cells infected by these viruses were sensitive to GCV killing. In vivo, C4-2 xenograft tumors treated either intratumorally or i.v. with trastuzumab-bound lentivirus expressed luciferase, although the latter route was less tumor specific. When a prostate-specific promoter for governing luciferase expression was combined with trastuzumab-mediated delivery, there was a further enrichment in targeting viral gene expression in prostate tumors. In conclusion, we found that although prostate cancers that express high levels of HER-2 are resistant to the killing effects of trastuzumab, they can be targeted for selective gene expression and destruction by viruses with envelope proteins engineered to bind this antibody.

Lentiviral-mediated BMP-2 gene transfer enhances healing of segmental femoral defects in rats.

The objective of the present study was to assess the ability of bone marrow cells expressing BMP-2 created via lentiviral gene transfer to heal a critical sized femoral defect in a rat model. Femoral defects in Lewis rats were implanted with 5x10(6) rat bone marrow stromal cells (RBMSC) transduced with a lentiviral vector containing either the BMP-2 gene (Group I), the enhanced green fluorescent protein (LV-GFP) gene (Group IV), or RBMSC alone (Group V). We also included femoral defects that were treated with BMP-2-producing RBMSC transduced with lentivirus, 8 weeks after infection (Group III), and a group with 1x10(6) RBMSC transduced with a lentiviral vector with the BMP-2 gene (Group II). All defects (10/10) treated in Group I healed at 8 weeks compared with none of the femora in the control groups (Groups IV and V). In Group II, only one out of 10 femora healed. In Group III, 5 out of 10 femora healed. Significantly higher amounts of in vitro BMP-2 protein production were detected in Groups I, II, and III when compared to that of the control groups (p<0.05). Histomorphometric analysis revealed significantly greater total bone volume in defects in Group I and III when compared to control specimens (p<0.003). Biomechanical testing revealed no significant differences in the healed defects in Groups I and III when compared to intact, nonoperated femora with respect to peak torque and torque to failure. Our results indicate that BMP-2-producing RBMSC created through lentiviral gene transfer have the capability of inducing long-term protein production in vitro and producing substantial new bone formation in vivo.

Safrole-induced Ca2+ mobilization and cytotoxicity in human PC3 prostate cancer cells.

The effect of the carcinogen safrole on intracellular Ca2+ mobilization and on viability of human PC3 prostate cancer cells was examined. Cytosolic free Ca2+ levels ([Ca2+]i) were measured by using fura-2 as a probe. Safrole at concentrations above 10 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 350 microM. The Ca2+ signal was reduced by more than half after removing extracellular Ca2+ but was unaffected by nifedipine, nicardipine, nimodipine, diltiazem, or verapamil. In Ca2+-free medium, after treatment with 650 microM safrole, 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) failed to release Ca2+. Neither inhibition of phospholipase C with U73122 nor modulation of protein kinase C activity affected safrole-induced Ca2+ release. Overnight incubation with 0.65-65 microM safrole did not affect cell viability, but incubation with 325-625 microM safrole decreased viability. Collectively, the data suggest that in PC3 cells, safrole induced a [Ca2+]i increase by causing Ca2+ release from the endoplasmic reticulum in a phospholipase C- and protein kinase C-independent fashion, and by inducing Ca2+ influx. Safrole can decrease cell viability in a concentration-dependent manner.

Formaldehyde-treated, heat-inactivated virions with increased human immunodeficiency virus type 1 env can be used to induce high-titer neutralizing antibody responses.

The lack of success of subunit human immunodeficiency virus (HIV) type 1 vaccines to date suggests that multiple components or a complex virion structure may be required. We hypothesized that the failure of current vaccine strategies to induce protective antibodies is linked to the inability of native envelope structures to readily elicit these types of antibodies. We have previously reported on the ability of a formaldehyde-treated, heat-inactivated vaccine to induce modest antibody responses in animal vaccine models. We investigated here whether immunization for HIV with an envelope-modified, formaldehyde-stabilized, heat-inactivated virion vaccine could produce higher-titer and/or broader neutralizing antibody responses. Thus, a clade B vaccine which contains a single point mutation in gp41 (Y706C) that results in increased incorporation of oligomeric Env into virions was constructed. This vaccine was capable of inducing high-titer antibodies that could neutralize heterologous viruses, including those of clades A and C. These results further support the development of HIV vaccines with modifications in native Env structures for the induction of neutralizing antibody responses.

Induction of humoral immune responses following vaccination with envelope-containing, formaldehyde-treated, thermally inactivated human immunodeficiency virus type 1.

The lack of success of subunit human immunodeficiency virus type 1 (HIV-1) vaccines to date suggests that multiple components or a complex virion structure may be required. We previously demonstrated retention of the major conformational epitopes of HIV-1 envelope following thermal treatment of virions. Moreover, antibody binding to some of these epitopes was significantly enhanced following thermal treatment. These included the neutralizing epitopes identified by monoclonal antibodies 1b12, 2G12, and 17b, some of which have been postulated to be partially occluded or cryptic in native virions. Based upon this finding, we hypothesized that a killed HIV vaccine could be derived to elicit protective humoral immune responses. Shedding of HIV-1 envelope has been described for some strains of HIV-1 and has been cited as one of the major impediments to developing an inactivated HIV-1 vaccine. In the present study, we demonstrate that treatment of virions with low-dose formaldehyde prior to thermal inactivation retains the association of viral envelope with virions. Moreover, mice and nonhuman primates vaccinated with formaldehyde-treated, thermally inactivated virions produce antibodies capable of neutralizing heterologous strains of HIV in peripheral blood mononuclear cell-, MAGI cell-, and U87-based infectivity assays. These data indicate that it is possible to create an immunogen by using formaldehyde-treated, thermally inactivated HIV-1 virions to induce neutralizing antibodies. These findings have broad implications for vaccine development.

Cytotoxic butanolides from the stem bark of Formosan Lindera communis.

Two new butanolides, lincomolide A (1), lincomolide B (2), along with seven known compounds, isolinderanolide E, sepesteonol, beta-sitosterol, beta-sitosterol-beta-glucoside, tetradecane, nonanoic acid and decanol, were isolated from the chloroform-soluble portion of the stem bark of Lindera communis. Compound 1 showed cytotoxicity against P-388, KB16 and A549, and 2 exhibited cytotoxicity against P-388 cancer cell lines. Moreover, 1 showed marginal activity against HT-29, and 2 against KB16, A549 and HT-29 cancer cell lines. The structures of these compounds were determined by spectral analyses.

Anticancer effect of a lentiviral vector capable of expressing HIV-1 Vpr.

A lentiviral vector capable of expressing the HIV-1 vpr gene (Vpr lentiviral vector) was constructed, and its in vivo anticancer effect was determined against cutaneous tumors derived from the AT-84 oral cancer cells in immunocompetent mice. A single intratumoral injection of the Vpr lentiviral vector not only significantly reduced the primary tumor volume but also completely regressed tumors in >40% of animals. More interestingly, the mice of which the primary tumors were completely regressed by the Vpr lentiviral vector were additionally protected from a secondary challenge of AT-84 cells. These data suggest that the Vpr lentiviral vector elicits its anticancer activity in part by the activation of the immune system. The above suggestion is additionally supported by the failure of the lentiviral vector to demonstrate anticancer activity in immunocompromised nude or SCID mice. The Vpr lentiviral vector offers a powerful new strategy for cancer gene therapy and may be useful for the control of solid tumors, such as human oral squamous cell carcinomas.

Cytotoxic neolignans and butanolides from Machilus obovatifolia.

From the chloroform-soluble portion of the stem wood of Machilus obovatifolia, one new neolignan, perseal F (1), four known neolignans, perseal G (2), licarin A, licarin B, acuminatin, two butanolides, linderanolide E and isolinderanolide E, two steroids, beta-sitosterol, beta-sitosterol-beta-D-glucoside, and syringaldehyde were isolated. Perseal F (1) and G (2) are neolignans that have a C-1' formyl side chain instead of a propenyl group. Compound 2 was isolated in a mixture with acuminatin. The structure of 2 was identified by comparison with the product formed by the Lemieux-von Rudloff oxidation of licarin B. Two minor oxidative by-products, 2a and 2b, were also obtained. Linderanolide E showed cytotoxicities against P-388, KB16, A549 and HT-29, 1 against P-388, KB16 and HT-29, and isolinderanolide E against P-388, cancer cell lines, respectively. All structures were identified by means of spectroscopic analyses.

Antibody-directed targeting of retroviral vectors via cell surface antigens.

Targeted stable transduction of specific cells is a highly desirable goal for gene therapy applications. We report an efficient and broadly applicable approach for targeting retroviral vectors to specific cells. We find that the envelope of the alphavirus Sindbis virus can pseudotype human immunodeficiency virus type 1- and murine leukemia virus-based retroviral vectors. When modified to contain the Fc-binding domain of protein A, this envelope gives a significant enhancement in specificity in combination with antibodies specific for HLA and CD4 relative to that without antibody. Unlike previous targeting strategies for retroviral transduction, the virus titers are relatively high and stable and can be further increased by ultracentrifugation. This study provides proof of principle for a targeting strategy that would be generally useful for many gene therapy applications.

Sensitive and reproducible quantitation of mucosal HIV-1 RNA and DNA viral burden in patients with detectable and undetectable plasma viral HIV-1 RNA using endoscopic biopsies.

Mucosal tissue is the main portal of entry for HIV-1 infection and, in macaques, has been demonstrated to be a significant compartment for viral replication and CD4+ T lymphocyte depletion. Quantitating tissue viral burden in addition to plasma viral load provides insights into HIV-1 pathogenesis and an additional means to gauge antiretroviral response. The aim of this study was to develop reliable, reproducible, and sensitive assays to quantitate tissue viral burden of HIV-1 RNA and DNA using 1-3 endoscopically acquired, rectosigmoid biopsies. Total DNA and RNA were simultaneously extracted following homogenization from the same tissue samples. Quantitative polymerase chain reaction (PCR) assay in the HIV-1 LTR region was used to detect viral DNA and RT-PCR for viral RNA. It was determined that HIV-1 RNA and DNA can be reproducibly quantified from a single rectosigmoid biopsy with minimal intra-assay or intra-patient variability. These results reflect high recovery of extracted nucleic acids with calculated results accurately reflecting in vivo levels. The techniques outlined differ from currently available approaches by incorporating control standards to identify loss or degradation of RNA and DNA from acquisition through the in vitro assay and permit extraction with high yields of RNA and DNA from the same tissue sample.

Envelope gene of the human endogenous retrovirus HERV-W encodes a functional retrovirus envelope.

A member of the human endogenous retrovirus (HERV) family termed HERV-W encodes a highly fusogenic membrane glycoprotein that appears to be expressed specifically in the placenta. It is unclear whether the glycoproteins of the HERVs can serve as functional retrovirus envelope proteins to confer infectivity on retrovirus particles. We found that the HERV-W envelope glycoprotein can form pseudotypes with human immunodeficiency virus type 1 virions and confers tropism for CD4-negative cells. Thus, the HERV-W env gene represents the first HERV env gene demonstrated to encode the functional properties of a retrovirus envelope glycoprotein.

Lentivirus vector-mediated hematopoietic stem cell gene transfer of common gamma-chain cytokine receptor in rhesus macaques.

Nonhuman primate model systems of autologous CD34+ cell transplant are the most effective means to assess the safety and capabilities of lentivirus vectors. Toward this end, we tested the efficiency of marking, gene expression, and transplant of bone marrow and peripheral blood CD34+ cells using a self-inactivating lentivirus vector (CS-Rh-MLV-E) bearing an internal murine leukemia virus long terminal repeat derived from a murine retrovirus adapted to replicate in rhesus macaques. In vitro cytokine stimulation was not required to achieve efficient transduction of CD34+ cells resulting in marking and gene expression of the reporter gene encoding enhanced green fluorescent protein (EGFP) following transplant of the CD34+ cells. Monkeys transplanted with mobilized peripheral blood CD34+ cells resulted in EGFP expression in 1 to 10% of multilineage peripheral blood cells, including red blood cells and platelets, stable for 15 months to date. The relative level of gene expression utilizing this vector is 2- to 10-fold greater than that utilizing a non-self-inactivating lentivirus vector bearing the cytomegalovirus immediate-early promoter. In contrast, in animals transplanted with autologous bone marrow CD34+ cells, multilineage EGFP expression was evident initially but diminished over time. We further tested our lentivirus vector system by demonstrating gene transfer of the human common gamma-chain cytokine receptor gene (gamma(c)), deficient in X-linked SCID patients and recently successfully used to treat disease. Marking was 0.42 and.001 HIV-1 vector DNA copy per 100 cells in two animals. To date, all EGFP- and gamma(c)-transplanted animals are healthy. This system may prove useful for expression of therapeutic genes in human hematopoietic cells.

Fibronectin fragment CH-296 inhibits apoptosis and enhances ex vivo gene transfer by murine retrovirus and human lentivirus vectors independent of viral tropism in nonhuman primate CD34+ cells.

The fibronectin fragment CH-296 improved gene transfer to cytokine-mobilized nonhuman primate CD34+ cells irrespective of tropism to the MoMLV, GaLV, and VSV-G envelope proteins using murine stem cell virus (MSCV) and human immunodeficiency virus-1 (HIV-1)-based retrovirus vectors. For the HIV-1 lentivirus vector, CH-296 enhanced gene transfer in the absence of added hematopoietic growth factors necessary for ex vivo stem cell expansion. In the presence of CH-296, apoptosis of CD34+ cells was inhibited, and in mobilized peripheral blood CD34+ cells, cell division was stimulated as measured by cell history/tracking experiments.

New cytotoxic butanolides from Litsea acutivena.

Six new compounds, including one nor-neolignan, dehydroxymethylailanthoidol (1), and five butanolides, litseakolide D (2), litseakolide E (3), litseakolide F (4), litseakolide G (5), and isolincomolide D (6), were isolated from the leaves of Litsea acutivena. Their structures were elucidated from spectral analyses. The butanolides (2-6) showed significant cytotoxic activity against P-388, A549, and HT-29 cell lines in vitro.

Cytotoxic prenyleudesmane diterpenes from the fruits of Dysoxylum kuskusense.

Two new prenyleudesmane diterpenes, dysokusone D (1) and dysokusone E (2), were isolated from the fruits of Dysoxylum kuskusense and exhibited cytotoxicity against P-388, HT-29, and A549 cell lines. Their structures were determined by 1D and 2D NMR spectral analysis.