PubMed Health⌕ Search

Biomedical subjects

Charles J Link

Publications and source records attributed to Charles J Link.

12 recordsLinked to original sources
PubMed2006

High-throughput fluorescent screening of transgenic animals: phenotyping and haplotyping.

BACKGROUND: Methods for genotyping transgenic animals currently consist of extracting genomic DNA from blood or tissue followed by PCR or Southern blot analysis. These methods when used to screen large numbers of animals can be time consuming and expensive. Therefore, we developed a novel method that allows high-throughput screening of phenotypic changes on leukocytes, resulting from the transgenic genotype. This technique allows investigators to quickly screen a large number of animals without the need to extract DNA from each one. Moreover, since blood is collected for the initial screening, putative homozygotes can be confirmed by conventional methods using the same blood samples. METHODS: We collected blood from wild-type alphagal positive and alphagal knockout mice and probed for the presence of Galalpha(1-->3)Gal (alphagal) epitopes. Also, alloantigen specific antibodies were used to determine the haplotype of our outbred mouse colony in order to develop an inbred line. RESULTS: alphagal epitopes were detected in wild-type but not alphagal knock-out samples. To validate these results, PCR was used to demonstrate the native alphagal gene in wild-type and the pGKneo construct in alphagal knock-out mice. Furthermore, haplotypes were determined and mice divided for backcrosses. CONCLUSIONS: This screening method is useful for both preliminary screening of transgenic mice and the development of an inbred mouse colony by rapid determination of MHC I haplotype. Here, we demonstrate the use of this technique and show how it can be a valuable tool, saving time and resources in both investigator effort and animal husbandry.

Animals↗
PubMed2006

Co-expression of alpha(1,3)galactosyltransferase and Bacillus thuringiensis PIPLC enhances hyperacute rejection of tumor cells.

The use of alpha(1,3)galactosyltransferase (alphaGT) as a method of inducing hyperacute rejection of tumors has been gaining interest recently. However, the approach is based in part on the sensitivity of each tumor line to the effects of complement lysis. Tumors expressing complement resistance factors such as membrane cofactor (CD46), decay accelerating factor (CD55) and protectin (CD59) have been shown to be more resistant to complement mediated lysis. Anchored to the membrane by a glycosylphosphoinositol moiety (GPI-anchored), CD55 and CD59 can be cleaved by Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PIPLC). Complement resistant A549 human lung carcinoma cells were engineered to express both the murine alphaGT gene and the B. thuringiensis PIPLC gene to alleviate complement resistance and enhance alphagal-mediated cancer killing. The PIPLC native signal sequence was replaced with the human epidermal growth factor signal sequence, EGFssPIPLC, to induce secretion from A549. Expression of EGFssPIPLC resulted in complete removal of CD55 and CD59 while sparing the non-GPI-anchored CD46. Results demonstrated that A549 cells transduced with two recombinant retroviral vectors carrying the alphaGT and EGFssPIPLC genes expressed high levels of alphagal epitope and exhibited a 5-fold increase in sensitivity to anti-alphagal mediated complement lysis.

Bacillus thuringiensis↗
PubMed2005

Effective treatment of preexisting melanoma with whole cell vaccines expressing alpha(1,3)-galactosyl epitopes.

The hyperacute immune response in humans is a potent mechanism of xenograft rejection mediated by complement-fixing natural antibodies recognizing alpha(1,3)-galactosyl epitopes (alphaGal) not present on human cells. We exploited this immune mechanism to create a whole cell cancer vaccine to treat melanoma tumors. B16 melanoma vaccines genetically engineered to express alphaGal epitopes (B16alphaGal) effectively treated preexisting s.c. and pulmonary alphaGal-negative melanoma (B16Null) tumors in the alpha(1,3)-galactosyltransferase knockout mouse model. T cells from mice vaccinated with B16alphaGal recognized B16Null melanoma cells measured by detection of intracellular tumor necrosis factor-alpha. We showed successful adoptive transfer of immunity to recipient mice bearing lung melanoma metastasis. Mice receiving lymphocytes from donors previously immunized with B16alphaGal had reduced pulmonary metastases. The transfer of lymphocytes from mice vaccinated with control vaccine had no effect in the pulmonary metastasis burden. This study unequivocally establishes for the first time efficacy in the treatment of preexisting melanoma tumors using whole cell vaccines expressing alphaGal epitopes. Vaccination with B16alphagal induced strong long-lasting cell-mediated antitumor immunity extended to B16Null. These data formed the basis for the testing of this therapeutic strategy in human clinical trials currently under way.

Animals↗
PubMed2005

Complete protection against melanoma in absence of autoimmune depigmentation after rejection of melanoma cells expressing alpha(1,3)galactosyl epitopes.

The major barrier for xenotransplantation in humans is the presence of alpha(1-3) Galactosyl epitopes (alphaGal) in xenogeneic tissue and the vast quantities of natural antibodies (Ab) produced by humans against this epitope. The binding of anti-alphaGal Ab to cells expressing alphaGal triggers a complement-mediated hyperacute rejection of target cells. The hyperacute rejection of whole cancer cells, modified to express alphaGal epitopes, could be exploited as a new cancer vaccine to treat human cancers. We tested this hypothesis in alphaGalactosyltransferase knockout (alphaGT KO) mice which, like humans, do not express alphaGal on their cell surfaces and can produce anti-alphaGal Ab. Forty-five percent of mice with preexisting anti-alphaGal Ab rejected alphaGal positive melanoma cells (B16alphaGal). These mice remained tumor-free for more than 90 days. The majority of control mice injected with B16Null, alphaGal negative cells succumbed to melanoma. The rejection of B16alphaGal induced strong long-lasting antitumor immunity against B16Null measured by the expansion of cytotoxic T lymphocytes. In addition, mice rejecting B16alphaGal were protected against melanoma since they survived a second rechallenge with B16Null. Protected mice developed antitumor immunity in the absence of autoimmune depigmentation (vitiligo). These results show that rejection of alphaGal positive melanoma cells can efficiently boost the immune response to other tumor associated antigens present in alphaGal negative melanoma cells. This study supports the concept of a novel anticancer vaccine to treat human malignancies.

Animals↗
PubMed2005

Pharmacological manipulation of ataxia-telangiectasia kinase activity as a treatment for Parkinson's disease.

Parkinson's disease (PD) is a major cause of morbidity and mortality among older individuals. Although the causes of Parkinson's disease are multifactorial, considerable evidence indicates that elevated labile iron in the substantia nigra pars compacta plays an important role in producing oxyradicals which subsequently damage nigro-striatal neurons. Based on this several researchers have suggested that blood-brain barrier crossing iron chelators might have clinical efficacy in treating PD. Work demonstrating that iron chelators protect nigro-striatal neurons in the N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and 6-hydroxydopamine-induced rodent PD models supports this hypothesis. Recently, we found that the ATM gene product (mutated in ataxia-telangiectasia, A-T), is required for cell survival and genomic stability maintenance following exposure to low labile iron concentrations. Iron chelators (desferal, quercetin, and apoferritin) also increase A-T cell genomic stability and viability, and activate ATM-dependent cellular events in normal cells. Additionally Atm-deficient mice exhibit a selective loss of dopaminergic nigro-striatal neurons. Based on this, we propose that iron chelators protect the substantia nigra pars compacta not only by chelating labile iron and reducing oxyradical formation, but also by inducing ATM activity, leading to increased oxidative stress resistance and DNA repair. Support for this hypothesis comes from the recent observation that the iron chelating flavonoid quercetin both directly activates ATM and protects neuronal cells from the toxic effects of the N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Therefore since; (1) ATM is required for iron toxicity resistance, (2) iron chelators such as quercetin, desferal, and apoferritin induce ATM activity and/or ATM-dependent events, and (3), Atm-deficient mice preferentially lose dopaminergic nigro-striatal neurons, we propose that ATM activity has an important function in PD. Furthermore, pharmacological manipulation of ATM activity via iron chelation might have clinical efficacy in PD treatment.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2004

Iron chelators increase the resistance of Ataxia telangeictasia cells to oxidative stress.

Ataxia telangeictasia (A-T) is an autosomal recessive disorder characterized by immune dysfunction, genomic instability, chronic oxidative damage, and increased cancer incidence. Previously, desferal was found to increase the resistance of A-T, but not normal cells to exogenous oxidative stress in the colony forming-efficiency assay, suggesting that iron metabolism is dysregulated in A-T. Since desferal both chelates iron and modulates gene expression, we tested the effects of apoferritin and the iron chelating flavonoid quercetin on A-T cell colony-forming ability. We demonstrate that apoferritin and quercetin increase the ability of A-T cells to form colonies. We also show that labile iron levels are significantly elevated in Atm-deficient mouse sera compared to syngeniec wild type mice. Our findings support a role for labile iron acting as a Fenton catalyst in A-T, contributing to the chronic oxidative stress seen in this disease. Our findings further suggest that iron chelators might promote the survival of A-T cells and hence, individuals with A-T.

Animals↗
PubMed2003

Effective suppression of class I major histocompatibility complex expression by the US11 or ICP47 genes can be limited by cell type or interferon-gamma exposure.

An impediment encountered in many viral-based gene therapy clinical trials has been the rapid destruction of the transgene by the host's immune response. The processing and presentation of antigens through the class I major histocompatibility complex (MHC) pathway is the initial specific response to viral infection. Disruption of the class I MHC pathway by herpes simplex virus (HSV) or the human cytomegalovirus (HCMV) results in a decrease of the CD8(+) cytotoxic T lymphocyte (CTL) response and prolongs survival of infected cells in the host. Two viral immune suppression genes that interfere with the class I MHC presentation pathway, the HSV type I ICP47 gene and HCMV US11 gene, were cloned and each incorporated into a retroviral vector. HSV ICP47 and HCMV US11 transgenes were expressed in multiple cells lines and compared for their abilities to reduce antigen presentation on the cell surface by class I MHC. Retroviral supernatants were used to transduce human, canine, and rat cell lines. Fluorescence-activated cell sorter (FACS) analysis of US11- and ICP47-transduced cell lines demonstrated substantial reductions in class I MHC cell surface expression in most cell lines except in rodent cells where ICP47 is nonfunctional. The decrease in the level of class I MHC expression for ICP47 transduced cell lines ranged from 31-98% relative to negative controls. US11 decreased class I cell surface MHC by 67-96%. When both ICP47 and US11 are expressed in human cells, a further reduction of class I MHC was observed. Next, human A375 melanoma cells were tested to determine if the resulting reduction in cell surface class I MHC would reduce in vitro cytotoxicity by CTL. A375 cells expressing either ICP47 or US11 demonstrated a twofold to threefold reduction of specific lysis by primed CD8(+) CTL. These data clearly establish an ability to convey immune protection to human cells by viral genes. However, further analysis demonstrated that interferon (IFN)-gamma could reverse part or all of the downregulation of class I MHC induced by the ICP47 or US11 genes. The ICP47 and US11 genes, when expressed in target cells, decrease class I MHC presentation and as such might be used in strategies to create local immunosuppression against transgenes or allografts.

Animals↗
PubMed2003

Desferrioxamine treatment increases the genomic stability of Ataxia-telangiectasia cells.

Ataxia-telangiectasia (AT) is an autosomal recessive disorder characterized by genomic instability, chronic oxidative damage, and increased cancer incidence. Compared to normal cells, AT cells exhibit unusual sensitivity to exogenous oxidants, including t-butyl hydroperoxide (t-BOOH). Since ferritin releases labile iron under oxidative stress (which is chronic in AT) and labile iron mediates the toxic effects of t-butyl hydroperoxide, we hypothesized that chelation of intracellular labile iron would increase the genomic stability of AT cells, with and without exogenous oxidative stress. Here we report that desferrioxamine treatment increases the plating efficiency of AT, but not normal cells, in the colony forming-efficiency assay (a method often used to measure genomic stability). Additionally, desferrioxamine increases AT, but not normal cell resistance, to t-butyl hydroperoxide in this assay. Last, AT cells exhibit increased sensitivity to the toxic effects of FeCl(2) in the colony forming-efficiency assay and fail to demonstrate a FeCl(2)-induced G(2) checkpoint response when compared to normal cells. Our data indicates that: (1) chelation of labile iron increases genomic stability in AT cells, but not normal cells; and (2) AT cells exhibit deficits in their responses to iron toxicity. While preliminary, our findings suggest that AT might be, in part, a disorder of iron metabolism and treatment of individuals with AT with desferrioxamine might have clinical efficacy.

Aspirin↗
PubMed2003

Immunity to the alpha(1,3)galactosyl epitope provides protection in mice challenged with colon cancer cells expressing alpha(1,3)galactosyl-transferase: a novel suicide gene for cancer gene therapy.

Human immunity to alpha(1,3)Galactosyl epitopes (alpha Gal) may provide the means for a successful cancer gene therapy that uses the immune system to identify and to destroy tumor cells expressing the suicide gene alpha(1,3)Galactosyltransferase (alpha GT). Innate antibody specific for cell surface alpha Gal constitutes a high percentage of circulating IgG and IgM immunoglobulins in humans and is the basis for complement-mediated hyperacute xenograft rejection and antibody-dependent cell-mediated cytotoxicity. In humans, the gene for alpha GT is mutated, and cells do not express the alpha Gal moiety. We hypothesized that human tumor cells induced to express the alpha Gal epitope would be killed by the hosts' innate immunity. Previous in vitro work by our group has demonstrated complement-mediated lysis of alpha Gal-transduced human tumor cells in culture by human serum. To induce antibodies to alpha Gal in this in vivo study, alpha GT knockout mice were used to determine whether immunization with alpha Gal could provide protection from challenge with alpha Gal-expressing murine MC38 colon cancer cells. Knockout mice were immunized either a single time, or twice, with rabbit RBC. Antibody titers to alpha Gal measured by indirect ELISA were significantly higher in mice immunized twice and approached the titers observed in human serum. Anti-alpha Gal antibodies were predominantly of the IgG1 and IgG3 subtype. Immunized knockout mice were challenged i.p. with varying doses of alpha Gal(+) MC38 colon carcinoma cells. Nonimmunized control groups consisting of alpha GT knockout mice, and wild-type C57BL/6 mice were challenged as well with MC38 cells. Immunized mice survived and exhibited slower tumor development in comparison to nonimmunized knockout and control mice. This study demonstrates, in vivo, the protective benefit of an immune response to the alpha Gal epitope. Our results provide a basis to pursue additional development of this cancer gene therapy strategy.

Animals↗
PubMed2003

The rat sodium iodide symporter gene permits more effective radioisotope concentration than the human sodium iodide symporter gene in human and rodent cancer cells.

Expression of the sodium iodide symporter (NIS) gene in tumor cells may provide a novel mechanism for treating cancer. The NIS mediates the normal physiological transport of iodide across the thyroid cell membrane. This mechanism of iodide uptake has been used to both diagnose and treat thyroid cancer. Tissue expression of the NIS is largely limited to the thyroid; therefore, expression of the NIS gene in cancer cells would allow for specific iodine uptake, radioisotope accumulation, and treatment. In this study, we directly compared the human and rat NIS (rNIS) for their ability to concentrate radioisotope into human and rodent cancer cells. Perchlorate-sensitive (125)I uptake in multiple cell lines was demonstrated following transduction with retroviral vectors expressing either the human or rNIS gene. Surprisingly, iodine uptake was consistently higher with the rNIS gene, up to 5-fold greater, when compared to the human gene, even within a variety of human tumor cell lines. This iodine uptake allowed for cell killing following (131)I treatment in NIS-transduced cells when assayed by in vitro clonogenic assays. These results demonstrate that the rNIS gene provides superior iodine uptake ability, and may be preferable for use in designing anticancer gene therapy approaches.

Animals↗
PubMed2002

Antitumor effects on human melanoma xenografts of an amplicon vector transducing the herpes thymidine kinase gene followed by ganciclovir.

Herpes simplex virus type-1 (HSV-1) has been demonstrated as a potentially useful gene delivery vector for gene therapy due to its high efficiency of in vivo transduction. The helper virus-dependent, HSV- 1 amplicon vectors were developed for easier operation and their larger capacity. In this study, the herpes simplex virus type-1 thymidine kinase (HSVtk) gene was cloned into the pHE700 amplicon vector to make an HE7tk vector and used for in vivo gene delivery. Human melanoma xenografts were established in athymic nude mice. Tumors were injected directly with HE7tk vector alone, HE7tk vector followed by ganciclovir (GCV), or a pHE700 amplicon vector carrying a green fluorescent protein (HE7GFP) gene followed by GCV. Efficient HSVtk transgene expression was found in the tumor 3 days after injection. Animals transduced with HE7tk followed by GCV had minimal tumor growth (P < .01 ). Animals that received either HE7tk vector without GCV or HE7GFP vector with GCV had some reduction in tumor growth compared to animals that were injected with buffer only. These data indicate that replication-defective HSV-1 amplicon vectors can be used effectively to deliver transgenes into solid tumors in vivo.

Animals↗