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PubMed2001

Variables pertinent to the efficiency of adeno-associated virus (AAV) vectors mediated gene transfer to human vascular endothelial cells.

Factors influencing adeno-associated virus (AAV) - mediated gene transfer to endothelial cells are not fully determined. We tested the variables pertinent to the efficiency of AAV-mediated gene transfer to human vascular endothelial cells (HUVEC) including: (i) kinetics of transduction efficiency of LacZ gene to HUVEC, (ii) the concentration and volume of vector-containing medium, (iii) the period of incubation time of AAV vectors with HUVEC, (iv) the target cell density/proliferation, (v) the duration of transgene expression. There is a dose-response relationship between moi of vectors and transduction efficiency in HUVEC. The higher moi of AAV vectors achieved more than 80% of transduction efficiency in cultured HUVEC. AAV vectors showed incubation time dependent increase in transduction efficiency of LacZ gene to the HUVEC up to 24 h of vector exposure. The foreign gene of AAV vectors preferably transduces the lower density of cells being proliferated. These results indicate that AAV-vector is efficient for gene transfer to HUVEC, and higher moi of vectors or a longer period exposure of vectors to proliferating HUVEC can facilitate efficient tranduction of foreign gene into human vascular endothelial cells in vitro.

Cell Count↗
PubMed2004

Development of herpesvirus-based episomally maintained gene delivery vectors.

Successful gene therapy aims to deliver and express therapeutic genes to cure or slow the progression of disease. However, a major obstacle in the application of gene therapy has been the development of the vectors used to deliver heterologous DNA to the cell or tissue of choice. A number of viral- and non-viral-based vector systems have undergone clinical trials with varying success. However, at present, no vector system possesses the full complement of properties that are generally believed necessary in an ideal gene delivery system. Therefore, alongside attempts to improve current gene delivery vectors, the identification and evaluation of new viral vectors is crucial for the long-term success of gene therapy. Herpesviruses are large DNA viruses which possess a number of advantages as gene delivery vectors. These relate to an ability to package large DNA insertions and establish lifelong latent infections in which the genomic material exists as a stable episome. This review aims to highlight the potential of herpesvirus vectors, in particular an alternative vector system based on herpesvirus saimiri (HVS). HVS is capable of infecting a range of human cell lines with high efficiencies, and the viral genome persists as high copy number, circular, non-integrated episomes which segregate to progeny following cell division. This allows the virus-based vector to stably transduce a dividing cell population and provide sustained transgene expression for an extended period of time both in vitro and in vivo. Moreover, the insertion of a bacterial artificial chromosome cassette into the HVS genome simplifies the incorporation of large amounts of heterologous DNA for gene delivery. These properties offer characteristics similar to an artificial chromosome combined with an efficient delivery system and merit its continual development as a possible gene delivery vector for the future.

Gene Transfer Techniques↗
PubMed2004

Ectoparasites and other epifaunistic arthropods of sympatric cotton mice and golden mice: comparisons and implications for vector-borne zoonotic diseases.

Ectoparasite and epifaunistic arthropod biodiversity and infestation parameters were compared between 2 sympatric small rodent species, the cotton mouse (Peromyscus gossypinus (Le Conte)) and golden mouse (Ochrotomys nuttalli (Harlan)), in southern Georgia from 1992 to 2003. Because the cotton mouse is known to be a reservoir of more vector-borne zoonotic pathogens than the golden mouse, we hypothesized that it would be parasitized by more ectoparasites that are known to be vectors of these pathogens. Cotton mice (n = 202) were parasitized by 19 species of arthropods, whereas golden mice (n = 46) were parasitized by 12 species. Eleven species of arthropods were recovered from both host species, whereas 7 were recorded only from cotton mice, and 1 species only from golden mice. Infestation prevalences (percent of mice parasitized) were significantly higher for 1 species of arthropod (the tropical rat mite Ornithonyssus bacoti (Hirst)) infesting cotton mice and for 4 species (the flea Peromyscopsylla scotti Fox and the mites Glycyphagus hypudaei Koch, Androlaelaps casalis (Berlese), and Androlaelaps fahrenholzi (Berlese)) infesting golden mice. Mean intensities (mean per infested mouse) were significantly higher for 2 species (the flea Orchopeas leucopus (Baker) and the blacklegged tick Ixodes scapularis Say) infesting cotton mice and for 2 species (G. hypudaei and A. fahrenholzi) infesting golden mice. Ectoparasites that are known to be vectors of zoonotic pathogens were significantly more common on cotton mice than on golden mice. These ectoparasites included the rhopalopsyllid flea Polygenis gwyni (Fox), a vector of the agent of murine typhus; I. scapularis, the principal vector of the agents of Lyme borreliosis, human granulocytic ehrlichiosis, and human babesiosis; and O. bacoti, a laboratory vector of several zoonotic pathogens. However, 2 species of ixodid ticks that can transmit zoonotic pathogens were recovered from both host species. These were the American dog tick Dermacentor variabilis (Say), the principal vector of the agent of Rocky Mountain spotted fever in eastern North America, and Ixodes minor Neumann, an enzootic vector of the agent of Lyme borreliosis. Overall, the cotton mouse was parasitized by significantly more ectoparasites that are known to be vectors of zoonotic pathogens than was the golden mouse. These data support the hypothesis that the cotton mouse has greater epidemiological importance for zoonotic vector-borne pathogen transmission than does the golden mouse.

Animals↗
PubMed2006

Highly efficient yeast-based in vivo DNA cloning of multiple DNA fragments and the simultaneous construction of yeast/ Escherichia coli shuttle vectors.

In vivo recombinational cloning in yeast is a very efficient method. Until now, this method has been limited to experiments with yeast vectors because most animal, insect, and bacterial vectors lack yeast replication origins. We developed a new system to apply yeast-based in vivo cloning to vectors lacking yeast replication origins. Many cloning vectors are derived from the plasmid pBR322 and have a similar backbone that contains the ampicillin resistance gene and pBR322-derived replication origin for Escherichia coli. We constructed a helper plasmid pSUO that allows the in vivo conversion of a pBR322-derived vector to a yeast/E. coli shuttle vector through the use of this backbone sequence. The DNA fragment to be cloned is PCR-amplified with the addition of 40 bp of homology to a pBR322-derived vector. Cotransformation of linearized pSU0, the pBR322-derived vector, and a PCR-amplified DNA fragment, results in the conversion of the pBR322-derived vector into a yeast/E. coli shuttle vector carrying the DNA fragment of interest. Furthermore, this method is applicable to multifragment cloning, which is useful for the creation of fusion genes. Our method provides an alternative to traditional cloning methods.

Cloning, Molecular↗
PubMed2002

Adenovirus as an integrating vector.

Recombinant adenoviral vectors have served as one of the most efficient gene delivery vehicles in vivo thus far. Multiply attenuated or completely gutless adenoviral vectors have been developed to achieve long-term gene expression in animal models by overcoming cellular immunity against de novo synthesized adenoviral proteins. However, since adenovirus lacks native integration machinery, the goal of gene therapy obtaining permanent expression cannot be realized with current adenoviral vector systems. Recent studies have shown that replication-incompetent adenoviral vectors randomly integrate into host chromosomes at frequencies of 0.001-1% of infected cells. To improve the integration frequencies of adenoviral vectors, a variety of hybrid vectors combining the highly efficient DNA delivery of adenovirus with the integrating machinery of retroviruses, adeno-associated viruses, and transposons, have been emerging. These hybrid vectors have shown promise, at least in in vitro systems. Furthermore, adenoviral vectors have shown potential as gene targeting vectors. These developments should eventually lead to more effective gene therapy vectors that can transduce a myriad of cell types stably in vivo.

Adenoviridae↗
PubMed2002

Separating fact from fiction: assessing the potential of modified adenovirus vectors for use in human gene therapy.

One of the major hurdles to successful gene therapy of genetic and/or acquired disease is the ability to efficiently introduce a foreign gene into the tissue of interest and, in the case of some genetic diseases, achieve long-term expression of the transgene. Due to their ability to transduce a wide variety of cell types in a cell-cycle independent fashion, adenovirus (Ad)-based vectors have received considerable attention in recent years as delivery vehicles for multiple gene therapy applications. Effective use of early "first-generation" versions of these vectors was hampered by not only the induction of strong immune responses in the host to the Ad vector and transduced cells, but also to direct acute and chronic toxicity caused by the vector itself. Furthermore, transgene expression was typically transient, lasting only a few weeks. Despite these limitations, these vectors have been used in a number of human clinical trials, eliciting both interesting as well as controversial results, some of which are summarized herein. Because of these limitations, a number of advances in adenovirus "vectorology", manifested primarily as the development of multiply attenuated Ads and vectors deleted of all viral protein coding sequences, has resulted in vectors which retain all of the advantages of Ad vectors and, in addition, do not exhibit the deleterious characteristics associated with [E1-]deleted Ads. This review focuses on the current state of the art regarding the potential for human use of Ad-based vectors, and how the use of this vector continues to offer the potential for successful use as a gene delivery tool for the treatment of a great number of human genetic and non-genetic diseases.

Adenoviridae↗
PubMed2003

Biosafety of onco-retroviral vectors.

Extensive gene therapy studies in preclinical models and in clinical trials underscore the relative safety of onco-retroviral vectors. Up until recently, no adverse effects have been reported in nearly 2000 patients that were enrolled in gene therapy clinical trials involving onco-retroviral vectors. However, the main safety concern of using onco-retroviral vectors is related to the risk of malignant transformation following oncogene activation due to random onco-retroviral genomic integration. Based on primate studies, there is an apparent low risk of malignancy that is predominately associated with the occurrence of chronic retroviremia resulting from replication-competent retroviruses (RCR), particularly in immunosuppressed recipient hosts. However, in the latest packaging cell lines and vectors, the risk of RCR-generation has been drastically reduced, primarily by minimizing the homologous overlap between vector and helper sequences. Nevertheless, results from a recent preclinical study in mice and a clinical trial in patients suffering from SCID-X1 strongly suggest that onco-retroviral vectors devoid of RCR can contribute to lymphomagenesis by insertional activation of cellular oncogenes. The risk of inadvertent germline transmission of onco-retroviral vectors appears to be low, especially relative to the endogenous rate of germline insertion, which is known to occur naturally in the human population via transmission of endogenous retro-transposons. The strict dependency of onco-retroviral gene transfer on cell division is an important safety advantage that significantly limits the risks of horizontal transmission. Since improved onco-retroviral vectors or transduction protocols may result in an increased number of retroviral integrations per cell, this may concomitantly increase the risk of malignant transformation. The use of suicide genes, self-inactivating vectors and/or chromosomal insulators is, therefore, warranted to further enhance the safety features of onco-retroviral vectors. Detailed analyses of insertion sites combined with long-term clinical follow-up may contribute to a more accurate risk assessment.

Animals↗
PubMed2004

The HVJ-envelope as an innovative vector system for cardiovascular disease.

Recently promising results of gene therapy clinical trials have been reported for treatment of peripheral vascular and cardiovascular diseases using various angiogenic growth factors and other therapeutic genes. Viral vector and non-viral vector systems were employed in preclinical studies and clinical trials. Adenoviral vector and naked plasmid have been used most in the clinical studies. HVJ (hemagglutinating virus of Japan or Sendai virus)-liposome vector, a hybrid non-viral vector system with fusion of inactivated HVJ virus particle and liposome, has developed and demonstrated high transfection efficiency in preclinical studies of many different disease models, including a wide range of cardiovascular disease models. However, some limitations exist in the HVJ-liposome technology, especially in the scalability of its production. Recently an innovative vector technology, HVJ envelope (HVJ-E) has been developed as a non-viral vector, consisting of HVJ envelope without its viral genome, which is eliminated by a combination of inactivation and purification steps. HVJ-E is able to enclose various molecule entities, including DNA, oligonucleotides, proteins, as single or multiple therapeutic remedies. The therapeutic molecule-included HVJ-E vector can transfect various cell types in animals and humans with high efficiency. In this review, vector technology for cardiovascular disease and the biology of HVJ-E vector technology is discussed.

Animals↗
PubMed2005

HSV-1-derived recombinant and amplicon vectors for gene transfer and gene therapy.

Herpes simplex virus type 1 (HSV-1) is a major human pathogen whose lifestyle is based on a long-term dual interaction with the infected host characterized by the existence of lytic and latent infections. Although in most cell types infection with HSV-1 will induce toxic effects ending in the death of the infected cells, the very deep knowledge we possess on the genetics and molecular biology of HSV-1 has permitted the deletion of most toxic genes and the development of non-pathogenic HSV-1-based vectors for gene transfer. Several unique features of HSV-1 make vectors derived from this virus very appealing for preventive or therapeutic gene transfer. These include (i) the very high transgenic capacity of the virus particle, authorizing to convey very large pieces of foreign DNA to the nucleus of mammalian cells, (ii) the genetic complexity of the virus genome, allowing to generate many different types of attenuated vectors possessing oncolytic activity, and (iii) the ability of HSV-1 vectors to invade and establish lifelong non-toxic latent infections in neurons from sensory ganglia and probably in other neurons as well, from where transgenes can be strongly and long-term expressed. Three different classes of vectors can be derived from HSV-1: replication-competent attenuated vectors, replication-incompetent recombinant vectors, and defective helper-dependent vectors known as amplicons. Each of these different vectors attempts to exploit one or more of the above-mentioned features of HSV-1. In this review we will update the current know-how concerning design, construction, and recent applications, as well as the potential and current limitations of the three different classes of HSV-1-based vectors.

Gene Transfer Techniques↗
PubMed2005

Host-seeking strategies of mosquito disease vectors.

Disease transmission by arthropods normally requires at least 2 host contacts. During the first, a pathogen (nematode, protozoan, or virus) is acquired along with the blood from an infected vertebrate host. The pathogen penetrates the vector's midgut and infects a variety of tissues, where replication may occur during an extrinsic incubation period lasting 3-30, days depending on vector and parasite physiology and ambient temperature. Following salivary-gland infection, the pathogen is usually transmitted to additional susceptible vertebrate hosts during future probing or blood feeding. The host-seeking strategies used by arthropod vectors can, in part, affect the efficiency of disease transmission. Vector abundance, seasonal distribution, habitat and host preference, and susceptibility to infection are all important components of disease-transmission cycles. Examples of 3 mosquito vectors of human disease are presented here to highlight the diversity of host seeking and to show how specific behaviors may influence disease-transmission cycles. In the African tropics, Anopheles gambiae s.s. is an efficient vector of human malaria due to its remarkably focused preference for human blood. Aedes aegypti is the main vector of dengue viruses in the New and Old World tropics and subtropics. This mosquito has evolved a domestic lifestyle and shares human habitations throughout much of its range. It prospers in settings where humans are its main source of blood. In south Florida, Culex nigripalpus is the major vector of St. Louis encephalitis (SLE) and West Nile (WN) viruses. This mosquito is opportunistic and blood feeds on virtually any available vertebrate host. It serves as an arboviral vector, in part, due to its ability to produce large populations in a short period of time. These 3 host-seeking and blood-feeding strategies make the specialist, as well as the opportunist, equally dangerous disease vectors.

Aedes↗
PubMed1994

Insecticide resistance issues in vector-borne disease control.

Vector-borne diseases are an increasing cause of death and suffering worldwide. Efforts to control these diseases have been focused on the use of chemical pesticides, but arthropod resistance (whether physiological, biochemical, or behavioral) to pesticides is now an immense practical problem. The pharmacokinetic interactions of pesticides with arthropods, mechanisms of resistance, and the strengths and shortcomings of different resistance test methods are briefly reviewed. Using malaria control as an example, the differences between the efficacy of insecticide-sprayed houses in reducing malaria transmission, and the actual effect of such treatments on vectors are discussed. Reduced malaria transmission as a result of spraying house walls occurs through some combination of killing vectors that land on sprayed walls (insecticidal effect) and by preventing vectors from entering or remaining inside long enough to bite (behavioral effects). Both insecticidal and behavioral effects of insecticides are important, but the relative importance of one versus the other is controversial. Field studies in Africa, India, Brazil, and Mexico provide persuasive evidence for strong behavioral avoidance of DDT by the primary vector species. This avoidance behavior, exhibited when malaria vectors avoid insecticides by not entering or by rapidly exiting sprayed houses, should raise serious questions about the overall value of current physiological and biochemical resistance tests. The continued efficacy of DDT in Africa, India, Brazil, and Mexico, where 69% of all reported cases of malaria occur and where vectors are physiologically resistant to DDT (excluding Brazil), serves as one indicator that repellency is very important in preventing indoor transmission of malaria. This experience with DDT has implications for future control efforts because pyrethroids also stimulate avoidance behaviors in arthropods. Each chemical should be studied early (before broad-scale use) to define types of action against vector species by geographic area, especially for impregnated bed net applications. The problems for vector control created by use of insecticides in agriculture and the potential for management of resistance in both agriculture and vector-borne disease control are discussed.

Agrochemicals↗
PubMed1994

Environmental management: a re-emerging vector control strategy.

Vector control may be accomplished by environmental management (EM), which consists of permanent or long-term modification of the environment, temporary or seasonal manipulation of the environment, and modifying or changing our life styles and practices to reduce human contact with infective vectors. The primary focus of this paper is EM in the control of human malaria, filariasis, arboviruses, Chagas' disease, and schistosomiasis. Modern EM developed as a discipline based primarily in ecologic principles and lessons learned from the adverse environmental impacts of rural development projects. Strategies such as the suppression of vector populations through the provision of safe water supplies, proper sanitation, solid waste management facilities, sewerage and excreta disposal systems, water manipulation in dams and irrigation systems, vector diversion by zooprophylaxis, and vector exclusion by improved housing, are discussed with appropriate examples. Vectors of malaria, filariasis, Chagas' disease, and schistosomiasis have been controlled by drainage or filling aquatic breeding sites, improved housing and sanitation, the use of expanded polystyrene beads, zooprophylaxis, or the provision of household water supplies. Community participation has been effective in the suppression of dengue vectors in Mexico and the Dominican Republic. Alone or combined with other vector control methods, EM has been proven to be a successful approach to vector control in a number of places. The future of EM in vector control looks promising.

Animals↗
PubMed1999

Efficient gene delivery to quiescent interleukin-2 (IL-2)-dependent cells by murine leukemia virus-derived vectors harboring IL-2 chimeric envelope glycoproteins.

Interleukin-2 (IL-2) is a cytokine that induces the proliferation of certain IL-2 receptor expressing quiescent cells. Human IL-2 was fused to the amino-terminus of amphotropic murine leukemia virus (MLV) envelope glycoproteins. Retroviral vectors were pseudotyped with both the IL-2 chimeric envelope and the wild-type amphotropic MLV envelope. The chimeric IL-2 glycoproteins were incorporated on retroviral vectors and the IL-2-displaying vector particles could bind specifically to cell surface IL-2 receptors. In addition, the IL-2-displaying vectors could infect proliferating cells through amphotropic receptors irrespective of whether the cells expressed the IL-2 receptor. IL-2-displaying vector particles could also transiently stimulate the cell cycle entry and proliferation of several IL-2-dependent cell lines. Finally, retroviral vectors displaying IL-2 could efficiently transduce G0/G1-arrested cells expressing the IL-2 receptor at a 34-fold higher efficiency compared with vectors with unmodified envelopes. This new strategy, whereby C-type retroviral vector particles display a ligand that activates the cell cycle of the target cells at the time of virus entry, may represent an alternative to lentivirus-derived retroviral vectors for the infection of quiescent cells. In addition, upon infection of an heterogeneous population of nonproliferating cells, MLV-retroviral vectors that display cytokines/growth factors will allow the transgene of interest to be integrated specifically in quiescent cells expressing the corresponding cytokine/growth factor receptor.

Cell Cycle↗
PubMed2001

Integrated pest management and allocation of control efforts for vector-borne diseases.

Applications of various control methods were evaluated to determine how to integrate methods so as to minimize the number of human cases of vector-borne diseases. These diseases can be controlled by lowering the number of vector-human contacts (e.g., by pesticide applications or use of repellents), or by lowering the proportion of vectors infected with pathogens (e.g., by lowering or vaccinating reservoir host populations). Control methods should be combined in such a way as to most efficiently lower the probability of human encounter with an infected vector. Simulations using a simple probabilistic model of pathogen transmission suggest that the most efficient way to integrate different control methods is to combine methods that have the same effect (e.g., combine treatments that lower the vector population; or combine treatments that lower pathogen prevalence in vectors). Combining techniques that have different effects (e.g., a technique that lowers vector populations with a technique that lowers pathogen prevalence in vectors) will be less efficient than combining two techniques that both lower vector populations or combining two techniques that both lower pathogen prevalence, costs being the same. Costs of alternative control methods generally differ, so the efficiency of various combinations at lowering human contact with infected vectors should be estimated at available funding levels. Data should be collected from initial trials to improve the effects of subsequent interventions on the number of human cases.

Animals↗
PubMed2002

Gene-viral vectors: a promising way to target tumor cells and express anticancer genes simultaneously.

OBJECTIVE: To develop a new kind of vector system called gene-viral vector, which combines the advantages of gene and virus therapies. METHODS: Using recombinant technology, an anti-tumor gene was inserted into the genome of replicative virus specific for tumor cells. The cell killing effect, reporter gene expression of the green fluorescence protein, anti-tumor gene expression of mouse interleukin-12 (mIL-12) and replication of virus were observed by the methods of cell pathology, fluorescence microscopy, ELISA and electron microscopy, respectively. RESULTS: A new kind of gene-viral vector system of adenovirus, in which the E1b-55 kD gene was deleted but the E1a gene was preserved, was constructed. The vector system, like the replicative virus ONYX-015, replicated and proliferated in tumor cells but not in normal ones. Our vector had an advantage over ONYX-015 in that it carried different kinds of anti-tumor genes to enhance its therapeutic effect. The reporter gene expression of the green fluorescence protein in tumor cells was much better than the adenovirus vector employed in conventional gene the rapy, and the expression in our vector system was as low as or even less than that in the conventional adenovirus gene therapy system. Similar results were observed in experiments with this vector system carrying the anti-tumor gene mIL-12. Replication and proliferation of the virus carrying the mIL-12 gene in tumor cells were confirmed by electron microscopy. CONCLUSIONS: Gene-viral vectors are new vectors with an anti-tumor gene inserted into the genome of replicative virus specific for tumor cells. Because of the specific replication and proliferation of the virus in tumor cells, expression of the anti-tumor gene is increased hundreds to thousands of times. This approach takes full advantages of gene therapy and virus therapy to enhance the effect on the tumor. It overcomes the disadvantages of conventional gene therapy, such as low transfer rate, low gene expression, lack of target tropism, and low anti-tumor activity. We believe that this is a promising means for future tumor treatment.

Adenoviridae↗
PubMed2002

[Construction of plant expression vectors containing the gene encoding cholera toxin B subunit].

OBJECTIVE: To construct the plant expression vector containing the nucleotide sequence encoding cholera toxin B (CTB) subunits. METHOD: Using high-fidelity PCR, we amplified CTB genes that were then subcloned into the transition vector pRTL2. Following confirmation of the CTB nucleotide sequence, the vector was subcloned into the plant vector pBI121 that was subsequently transferred into Agrobacterium tumefaciens LBA4404 by electroporation. RESULTS: CTB DNA that was ligated into the transition vectors resulted in the 2 vectors designated as pRCTB and pRCTBK. After the 2 vectors were ligated into the plant binary vector pBI121 respectively, new plant binary vectors, namely pBI-CTB and pBI-CTBK, were produced. Analysis with restriction endonucleases confirmed successful transfer of pBI-CTB and pBI-CTBK into Agrobacterium tumefaciens LBA4404. CONCLUSION: With appropriate technological strategy, the plant binary expression vectors encoding CTB have been constructed, which facilitates further investigation of CTB protein expressions in transgenic plant.

Cholera Toxin↗
PubMed2005

Inhibition of transforming growth factor beta (TGFbeta1) expression and extracellular matrix secretion in human peritoneal mesothelial cells by pcDU6 vector-mediated TGFbeta1 shRNA and by pcDNA3.1(-)-mediated antisense TGFbeta1 RNA.

We investigated the effect of transforming growthfactor beta (TGFbeta1) short hairpin RNA (shRNA) mediated by pcDU6 plasmid on TGFbeta1 expression in human peritoneal mesothelial cells (HPMCs) and compared that effect with the effect of antisense TGFbeta1 RNA. We designed two pairs of oligonucleotides for two selectedfragments of coding sequence containing a 21-nucleotide (nt) TGFbeta1 sequence starting with GGCC. After annealing, double-stranded DNA was formed and separately ligated to plasmid pcDU6 [pcDNA3.1(-) with U6 promoter). The inverted motif contained six spacers and four Ts, which made it possible to form shRNA (TGFbgeta1 shRNA1 and TGFbeta1 shRNA2). We generated recombinant human TGFbeta1 antisense mammalian expression vector, and we isolated HPMCs from human greater omentum by pancreatin disaggregation to establish a stable cell-culture model. We used Lipofectamine 2000 to transfect third-passage HPMCs with plasmid pcDU6 mediating the expression of TGFbeta1 and plasmid pcDNA3.1(-) mediating the expression of antisense TGFbeta1 messenger RNA (mRNA). The resulting transfected cells were then stimulated with 4.25% D-glucose and 10 microg/mL lipopolysaccharide (GS+LPS). We used semi-quantitative reverse-transcriptase polymerase chain reaction to detect the expression of TGFbeta1, fibronectin (FN), collagen 1, and plasminogen activator inhibitor type 1 (PAI-1) mRNA by the stimulated cells. The TGFbeta1, FN, and PAI-1 protein levels in the culture supernatant were measured with a sandwich enzyme-linked immunosorbent assay. Expression of TGFbeta1 was significantly upregulated in HPMCs stimulated with GS+LPS (p < 0.01). As compared with control HPMCs in serum-free F12 medium, HPMCs transfected with TGFbeta1 antisense RNA showed inhibited expression of FN, collagen 1, and PAI-1 mRNA (17%, 26%, and 9.6% respectively after 24 hours). Forty-eight hours after transfection, the FN and PAI-I proteins were inhibited by 54.55% and 61.13% respectively (p < 0.05). In the pcDU6 plasmid vector-mediated TGFbeta1 shRNA groups, TGFbeta1 expression was obviously downregulated as compared with the GS+LPS group and the pcDU6 void vector group (p < 0.01). No significant difference was observed between the pcDU6 plasmid vector-mediated TGFbeta1 shRNA groups (p > 0.05). No significant difference was observed between the pcDNA3.1(-) vector-mediated antisense RNA group and the pcDU6 void vector group (p > 0.05). The expression of TGFbeta1 in pcDU6 plasmid vector-mediated TGFbeta1 shRNA groups was obviously downregulated as compared with the pcDNA3.1(-) plasmid vector-mediated antisense RNA group (p < 0.01). In HPMCs stimulated with GS+LPS, pcDU6 plasmid vector-mediated shRNA can significantly inhibit the induced expression of TGFbeta1. These results suggest the possible application of pcDU6 plasmid vector-mediated shRNA in preventing peritoneal fibrosis in patients receiving peritoneal dialysis.

Cells, Cultured↗
PubMed1991

A review of investigations on leishmaniasis vectors in Kenya.

Investigations on vectors of leishmaniases in Kenya started only in the last 35 years when the visceral leishmaniasis assumed importance as a result of a major disease epidemic outbreak. Since then detailed investigations on vectors of visceral leishmaniasis as well as vectors of newly found species causing cutaneous leishmaniasis have resulted in identification of their main vectors. Synphlebotomus complex were shown to be the main vectors in Kitui focus while in the other foci Phlebotomus martini has been incriminated as the main vector and Sergentomyia garnhami as a suspected vector. The vectors of L. major are P. duboscqi and P. martini with Sergentomyia garnhami and S. ingrami as suspected vectors. Phlebotomus gusggisbergi has already been demonstrated as vector of L. tropica.

Animals↗