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Biomedical subjects

Paul L Hermonat

Publications and source records attributed to Paul L Hermonat.

At least 19 recordsLinked to original sources
PubMed2006

Suppression of atherogenesis by delivery of TGFbeta1ACT using adeno-associated virus type 2 in LDLR knockout mice.

TGFbeta(1) deficiency has been attributed to the development of atherosclerosis. There is, however, little direct evidence for this concept. To examine this hypothesis, low-density lipoprotein receptor knockout (LDLR(-/-)) mice were injected via tail vein with recombinant adeno-associated virus type 2 (rAAV) carrying a bioactive TGFbeta(1) mutant (AAV/TGFbeta1ACT, n=10) or granulocyte-macrophage-colony stimulating factor (AAV/GM-CSF, n=10, a negative control) or saline (n=9, control), and then put on a high cholesterol diet. At 18 weeks, blood lipids were found to be similarly elevated in all LDLR(-/-) mice. TGFbeta1ACT and GM-CSF (DNA, mRNA, and protein) were highly expressed in the tissues of mice given TGFbeta1ACT or AAV/GM-CSF, respectively, showing sustained transfection following gene delivery by the systemic route. Saline-treated and AAV/GM-CSF-treated LDLR(-/-) mice showed extensive areas of atherosclerotic lesion formation. There was evidence of intense oxidative stress (nitrotyrosine staining), inflammation (CD68 staining), and expression of adhesion molecules and the ox-LDL receptor LOX-1 (gene array analysis) in the atherosclerotic tissues. Importantly, atherosclerotic lesion formation was markedly inhibited in the LDLR(-/-) mice given AAV/TGFbeta1ACT. Expression of adhesion molecules and LOX-1, oxidative stress, and inflammatory response all were inhibited in the mice given AAV/TGFbeta1ACT (P<0.05 vs. saline-treated or GM-CSF-treated LDLR(-/-) mice). These data for the first time demonstrate that systemic delivery of TGFbeta1ACT gene via AAV can inhibit formation of atherosclerotic lesions, possibly via anti-inflammatory and anti-oxidant mechanisms. These findings suggest a novel view of TGFbeta(1) in atherogenesis and a potential new gene therapy for treatment of atherosclerosis.

Adenoviridae↗
PubMed2006

Molecular dissection of angiotensin II-activated human LOX-1 promoter.

OBJECTIVE: LOX-1, a receptor for oxidized low-density lipoprotein, plays a critical role in atherosclerosis. Its expression is upregulated by pro-atherogenic stimuli, such as angiotensin II (Ang II). In this study, we explored LOX-1 transcriptional promoter activation in response to Ang II in human coronary artery endothelial cells (HCAECs). METHODS AND RESULTS: We constructed full-length and deletion LOX-1 promoter mutants and examined their activation in response to Ang II in HCAECs. The Ang II (1 micromol/L for 24 hours) markedly induced LOX-1 promoter activity beyond the basal level, and a 116-bp fragment (between nt -2247 and -2131) was necessary for this induction. Within this 116-bp promoter fragment, there is a potential binding motif for transcription factor NF-kappaB. By EMSA, we observed the activation of NF-kappaB by Ang II. The critical role of NF-kappaB in Ang II-induced LOX-1 promoter activation was confirmed by mutagenesis assay, and further confirmed by blocking NF-kappaB activation with the NF-kappaB inhibitor caffeic acid phenethyl ester or NF-kappaB p65 siRNA. CONCLUSIONS: This study strongly suggests that Ang II, by activating NF-kappaB, induces LOX-1 promoter activation.

Angiotensin II↗
PubMed2006

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) transcriptional regulation by Oct-1 in human endothelial cells: implications for atherosclerosis.

LOX-1, a receptor for ox-LDL (oxidized low-density lipoprotein), has recently been determined to play a critical role in the progression of atherosclerosis. LOX-1 expression (mRNA and protein) has been shown to be up-regulated by pro-atherogenic stimuli, such as ox-LDL and Ang II (angiotensin II). However, the molecular mechanisms of these up-regulations are unclear. In the present study, we explored LOX-1 transcriptional promoter activation in response to ox-LDL and Ang II. Under basal states, LOX-1 core promoter (LOX-1 -35/+36) was found to be sufficient for its basal activity in HCAECs (human coronary artery endothelial cells). More importantly, we found that ox-LDL (60 microg/ml for 24 h) induced LOX-1 promoter activity significantly and that a 105 bp fragment (between nt -1599 and -1494) was required for this activation. Within this 106 bp fragment, there is a potential binding motif for the transcription factor Oct-1 (octamer-1). By electrophoretic mobility-shift assay, we observed the activation of Oct-1 by ox-LDL. The critical role of Oct-1 in ox-LDL-induced LOX-1 promoter activation was further confirmed by mutagenesis assay. For comparison, we also examined LOX-1 promoter activation in response to Ang II (1 micromol/l for 24 h). Interestingly, another promoter region, between nt -2336 and -1990, was required for Ang II-induced LOX-1 promoter activation. In conclusion, the present study strongly suggests that ox-LDL, by activating Oct-1, induces LOX-1 promoter activation. Furthermore, this study suggests that while ox-LDL and Ang II both induce LOX-1 expression in HCAECs, the underlying mechanisms of promoter activation are different from each other.

Angiotensin II↗
PubMed2005

Lectin-like, oxidized low-density lipoprotein receptor-1 (LOX-1): a critical player in the development of atherosclerosis and related disorders.

LOX-1, a lectin-like 52-kD receptor for oxidized low-density lipoproteins (ox-LDL), is present primarily on endothelial cells. This receptor is upregulated by ox-LDL itself and by angiotensin II, endothelin, cytokines, and shear stress, all participants in atherosclerosis. This receptor is upregulated in the arteries of hypertensive, dyslipidemic, and diabetic animals. Upregulation of LOX-1 has been identified in atherosclerotic arteries of several animal species and humans, not only on the endothelial lining, but also in the neovasculature of the atherosclerotic plaque, and this receptor is often co-localized with apoptotic cells. Recent studies show upregulation of LOX-1 in the ischemic-reperfused myocardium. LOX-1 inhibition is associated with attenuation of atherosclerosis and associated ischemic injury. LOX-1 may be a novel, exciting target for drug therapy.

Apoptosis↗
PubMed2005

Inhibition of atherogenesis in LDLR knockout mice by systemic delivery of adeno-associated virus type 2-hIL-10.

Atherosclerosis is an inflammatory disease of the arteries. Interleukin-10 (IL-10) is known to be an anti-inflammatory cytokine which might be useful for counteracting the development of atherosclerosis. As long-term systemic cytokine delivery is prohibitively expensive, gene therapy might be a suitable approach. To test this idea, low-density lipoprotein receptor (LDLR) knockout mice were injected with recombinant adeno-associated virus type 2 (AAV)/interleukin-10 virus or AAV/granulocyte macrophage-colony stimulating factor (GM-CSF) virus and then put on a high-cholesterol diet. Upon harvesting the animals at 18 weeks, elevated blood lipids could be documented and AAV/IL-10 and AAV/GM-CSF DNA and mRNA could be found in various mouse organs. The mice receiving the AAV/IL-10 virus had significantly lower levels of atherogenesis (Sudan IV-staining and histology) than the untreated or the AAV/GM-CSF-treated animals, dropping from 53% to 17% (p < 0.05). The aortas of the AAV/IL-10-treated animals displayed higher IL-10 expression and lower CD68 and nitrotyrosine expression. These data are similar to those of Yoshioka et al. [Yoshioka, T, Okada, T, Maeda, Y, et al. Adeno-associatedvirus vector-mediated interleukin-10 gene transfer inhibits atherosclerosis in apolipoprotein E-deficient mice. Gene Ther 2004;11:1772-9] in which AAV/IL-10 was delivered into the tibial muscle of ApoE-deficient mice, instead of tail vein injection used here. These data indicate that systemic AAV/IL-10 gene delivery, with resulting inhibition of inflammation and oxidative stress, was able to limit atherogenesis, and suggest that this approach is worthy of further study.

Animals↗
PubMed2005

Immunological treatment of liver tumors.

Although multiple options for the treatment of liver tumors have often been described in the past, including liver resection, radiofrequency ablation with or without hepatic pump insertion, laparoscopic liver resection and the use of chemotherapy, the potential of immunotherapy and gene manipulation is still largely unexplored. Immunological therapy by gene manipulation is based on the interaction between virus-based gene delivery systems and dendritic cells. Using viruses as vectors, it is possible to transduce dendritic cells with genes encoding tumor-associated antigens, thus inducing strong humoral and cellular immunity against the antigens themselves. Both chemotherapy and radiation therapy have the disadvantage of destroying healthy cells, thus causing severe side-effects. We need more precisely targeted therapies capable of killing cancer cells while sparing healthy cells. Our goal is to establish a new treatment for solid liver tumors based on the concept of cytoreduction, and propose an innovative algorithm.

Algorithms↗
PubMed2005

An autoimmune domain-reduced HCV core gene remains effective in stimulating anti-core cytotoxic T lymphocyte activity.

Chronic hepatitis C virus (HCV) infection cases resistant to conventional therapies might be treated by immunotherapy as cytotoxic T lymphocytes (CTL) are the main mechanism through which viral infections are cleared. The HCV core gene, with the highest homology between HCV types, deleted of its autoimmune-stimulating regions (pseudo-GOR and pseudo-p450), may be an appropriate antigen for targeting HCV-infected cells. Two recombinant adeno-associated virus (rAAV) vectors, carrying either the full length (aa 1-190) or truncated (aa 49-180, deleted of the pseudo-GOR and pseudo-p450 sequences) versions of core, were generated. Both AAV/core (l-190) and AAV/core (49-180) were used to transduce/load dendritic cells (DC) at high levels (88-95%). These two genetically altered DC types then stimulated anti-core CTL. The DC and CTL were analyzed by FACS and for killing efficiency (percent target killing). The rAAV-altered DC displayed higher levels of CD80, CD83, CD86, and CD 1a than control DC. The truncated core (aa 49-180) gene stimulated equivalent and strong killing of synthetic core-positive autologous peripheral blood lymphocyte (PBL) targets to that stimulated by the full length core gene. However, the smaller core (49-180) antigen gene stimulated lower levels of killing of core-negative "self" PBL targets (GOR- and p450-positive) (p = 0.002). These AAV/core: DC-stimulated CTL displayed higher IFN-gamma expression, higher CD8:CD4 ratios, and lower CD56:CD8 ratios than controls. The rAAV-loading derived CD8+ T cells had more CD69+ cells and the CD4+ T populations had fewer CD25+ cells than controls. We conclude that the core (49-180) gene is an effect antigen, but has the advantage of stimulating less self-recognition. Thus, core (49-180) may be useful for further translational immunotherapy studies against HCV.

Autoimmunity↗
PubMed2005

Multiple human papillomavirus genes affect the adeno-associated virus life cycle.

The risk of cervical cancer, one of the most prevalent cancers in the world, is determined by two viruses. Human papillomavirus (HPV) is the main risk factor for developing cervical cancer. However, although little known, it is well substantiated that the human Parvovirus adeno-associated virus type 2 (AAV), and its encoded Rep78 protein, interacts with HPV and lowers the risk of cervical cancer. HPV also contributes to AAV inhibition by serving as a helper virus for AAV and stimulating higher AAV replication levels. Here we surveyed four HPV-16 early genes, E1, E2, E6 and E7, for their ability to increase/decrease the basal level of AAV replication in stratifying squamous epithelium (the epithelial raft culture system). It was found that the HPV-16 E1, E2 and E6 genes were able to help/enhance AAV-2 replication in epithelial raft cultures. Under these conditions, with all the HPV genes being expressed from the AAV p5 promoter, E1 appeared to have the strongest enhancing effect on AAV DNA replication (Southern blot), RNA expression (RT-PCR), protein expression (Western blot) and AAV virion production (2 plate-Southern blot). Further study of E1 mutants showed that the carboxy-half of E1, the putative helicase/ATPase domain, was the main contributor of helper activity. These data are important for understanding the HPV-AAV interaction and its effect on modifying cervical cancer risk. These data also suggest the possibility that the identified HPV helper genes may be useful in the generation of recombinant (r)AAV virions for gene therapy, as rAAV is increasing in popularity for such purposes.

Capsid Proteins↗
PubMed2005

Adhesion molecule expression in fibroblasts: alteration in fibroblast biology after transfection with LOX-1 plasmids.

The endothelial lectinlike, oxidatively (ox-) modified LDL receptor LOX-1 is a critical player in the pathogenesis of atherosclerosis and myocardial ischemia. Ox-LDL binding of LOX-1 results in the expression of various adhesion molecules, which attract monocytes to endothelial cells, an initial step in atherogenesis. We wished to examine the role of the ox-LDL/LOX-1 signaling pathway in fibroblasts, which naturally express low levels of LOX-1. Rat cardiac fibroblasts were transfected with either cytomegalovirus (CMV)-LOX-1wt (amino acids [aa] 1 to 273) or CMV-LOX-1(1-261) (an ox-LDL-binding negative mutant, aa 1 to 261) plasmid. Western blots showed that LOX-1 protein expression was increased significantly in cells transfected with CMV-LOX-1wt or CMV-LOX-1(1-261) plasmid (P<0.01 vs control). Fibroblasts transfected with CMV-LOX-1wt showed ox-LDL binding, whereas fibroblasts without transfection and those transfected with CMV-LOX-1(1-261) did not bind ox-LDL. Compared with untransfected cells, ox-LDL treatment (50 microg/mL, 24 hours) markedly induced the expression of the leukocyte adhesion molecules intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM)-1 as well as matrix metalloproteinase (MMP)-1 in cells transfected with CMV-LOX-1wt (P<0.05) but not in cells transfected with CMV-LOX-1(1-261). Concurrently, ox-LDL treatment enhanced the phosphorylation of p38 mitogen-activated protein kinase (MAPK) (P<0.05 vs control) in CMV-LOX-1wt-transfected cells. These data suggest that in cardiac fibroblasts, ox-LDL binds to LOX-1 and activates p38 MAPK, followed by the expression of ICAM-1, VCAM-1, and MMP-1. Thus, fibroblasts transform into an endothelial phenotype on transfection with CMV-LOX-1wt and subsequent exposure to ox-LDL. This study provides a useful model system (plasmid-transfected fibroblasts) to study the molecular biology of LOX-1.

Animals↗
PubMed2005

Use and specificity of breast cancer antigen/milk protein BA46 for generating anti-self-cytotoxic T lymphocytes by recombinant adeno-associated virus-based gene loading of dendritic cells.

Antigen-targeted immunotherapy is an emerging treatment for breast cancer. However, useful breast cancer antigens are only found in a subset of cancer patients. BA46, also known as lactadherin, is a membrane-associated glycoprotein that is expressed in most breast cancer cells but not in general hematopoietic cell populations. Moreover, it is much more difficult to generate CTLs against self-antigens. We wished to determine if the use of recombinant adeno-associated virus (rAAV) type 2 vectors for gene-loading of dendritic cells (DCs) could generate rapid, effective cytotoxic T lymphocytes (CTLs) against BA46. We were able to demonstrate that AAV/BA46/Neo-loading of DCs resulted in: (1) BA46 expression in DCs, (2) chromosomal integration of the AAV/BA46/Neo vector within DCs, (3) strong, rapid BA46-specific, MHC class I-restricted CTLs in only 1 week, (4) T-cell populations with significant interferon-gamma (IFN-gamma) expression but low IL-4 expression, (5) high CD80 and CD86 expression in DCs, and (6) high CD8:CD4 and CD8:CD56 T cell ratios. These data suggest that rAAV-loading of DCs may be useful for immunotherapeutic protocols against self-antigens in addition to viral antigens and that the BA46 antigen is potentially appropriate for cell-mediated immunotherapeutic protocols addressing ductal breast cancer.

Antibodies↗
PubMed2005

Studying the HPV life cycle in 3A trophoblasts and resulting pathophysiology.

Human papillomavirus (HPV) productive infection has been long considered to be restricted to the squamous epithelium. However, we have demonstrated that both HPV type 16 (HPV-16) and type 31b (HPV-31b) productively replicate in a trophoblast cell line, 3A. Trophoblasts are an important cell type, as these are the cells of the placenta that directly contact maternal tissues. There are a number of studies that suggest that HPV infection may be found commonly in placental material and might be linked with spontaneous abortions. This chapter describes the preparation and transfection of HPV genomes into 3A cells, suggests ways to analyze viral replication and transcription, and describes how to assay the effect of HPV infection on the ability of the trophoblasts to bind endometrial cells.

Base Sequence↗
PubMed2005

Analysis of adeno-associated virus and HPV interaction.

It is slowly becoming accepted that adeno-associated virus (AAV) is another significant factor involved in cervical carcinogenesis. However, unlike human papillomavirus (HPV), which is positively associated with cervical cancer, AAV is negatively associated with this cancer. This negative association appears to be through a direct and complex bi-directional interaction between AAV and HPV. Essentially all assays used for studying HPV can be used for studying the AAV-HPV interaction. This is because both viruses are productive in the same tissue, the stratified squamous epithelium (skin). Their relationship can be studied on the level of the complete virus and their complete life cycle using the organotypic epithelial raft culture system, which generates a stratified squamous epithelium. Their relationship can be studied in various other tissue-culture models measuring oncogenic potential. Their interaction can also be studied on the component level, as both protein-protein and protein-DNA interactions are known. Their relationship has even been studied using transgenic animals. The AAV-HPV relationship can be broken down into two halves--AAV-encoded products, which affect HPV biology, and HPV-encoded products, which affect AAV biology. To date, the former are much better studied than the latter. The rep gene and its largest product, Rep78, are responsible for most of AAV's effects upon HPV. This chapter largely focuses on AAV's effect on the HPV life cycle.

Animals↗
PubMed2004

A morphologic and statistical comparative study of small-cell carcinoma and non-Hodgkin's lymphoma in fine-needle aspiration biopsy material from lymph nodes.

Small-cell carcinoma (SmC) and high-grade non-Hodgkin's lymphoma (NHL) are aggressive neoplasms that require prompt diagnosis and treatment. An immediate diagnosis can be obtained using fine-needle aspiration biopsy (FNAB) material from lymph nodes (LNs), which are clinically or radiologically suspicious for tumor involvement. However, in aspirates from LNs, the cytologic distinction of SmC from NHL can be challenging. The purpose of this study was to evaluate the usefulness of various cytologic features that can be used during a rapid on-site evaluation to differentiate these two entities. Twenty-seven metastatic SmC and 50 NHLs cases diagnosed by FNAB of LNs were reviewed. All NHL diagnoses (neck, 29; abdomen, 9; axilla, 6; groin, 5; and parotid, 1) were confirmed with tissue sections, flow cytometry, or immunohistochemistry. These cases were classified as follicular, 21 (42%); diffuse large B cell, 13 (26%); small lymphocytic, 7 (14%); mantle cell, 4 (8%); anaplastic large cell, 2 (4%); and 1 each (2%), Burkitt, lymphoplasmacytic, and peripheral T-cell lymphomas. Immunochemistry confirmed the cytologic diagnoses of all SmC cases (neck, 16; mediastinum, 9; abdomen, 1; and axilla, 1) with either positive chromogranin or synaptophysin. All specimens were reviewed independently by three cytopathologists who were unaware of the original diagnoses. The presence and proportion of single (noncohesive) tumor cells, lymphoglandular bodies, nuclear fragments, paranuclear blue inclusions, nuclear molding, evenly dispersed fine-granular chromatin, crush artifact, and composition of cell clusters (monomorphic vs. polymorphic) were statistically evaluated. The presence of evenly dispersed fine-granular chromatin, paranuclear blue inclusions, and nuclear fragments was each statistically significant in differentiating SmC when compared with NHL (P < 0.01). The remaining features were not significant in distinguishing SmC from NHL in LN aspirates. The identification of distinct cytologic findings such as evenly dispersed fine-granular chromatin, paranuclear blue inclusions, and nuclear fragments can be a valuable aid to accurately diagnose and differentiate metastatic SmC from NHL in FNAB preparations from LNs.

Biopsy, Fine-Needle↗
PubMed2004

Anoxia-reoxygenation stimulates collagen type-I and MMP-1 expression in cardiac fibroblasts: modulation by the PPAR-gamma ligand pioglitazone.

BACKGROUND: Cardiac remodeling after ischemic injury is a major cause of heart failure. In this process, fibroblast growth and collagen synthesis and degradation play a critical role. Recent studies indicate that ligands of the peroxisome proliferator-activated receptors-gamma (PPAR-gamma) alter cardiac remodeling during chronic ischemia. This study was designed to investigate if the PPAR-gamma ligand pioglitazone would modulate fibroblast growth and collagen type-I synthesis (and expression) in cardiac fibroblasts exposed to anoxia-reoxygenation (A-R). METHODS AND RESULTS: Cardiac fibroblasts were exposed to anoxia (95% N2/5% CO2) and then reoxygenation (95% air/5% CO2). A-R increased fibroblast growth (MTT assay) as well as collagen type-I synthesis (H-proline incorporation) and protein expression (Western analysis). Concurrently, there was a parallel increase in the expression of matrix metalloproteinase-1 (MMP-1) in fibroblasts. Pretreatment of cardiac fibroblasts with pioglitazone (10 M) reduced all these effects of A-R. Further, A-R stimulated intracellular reactive oxygen species (ROS) generation and activated the redox-sensitive transcription factor NF-kappaB (both P < 0.05). Both these phenomena were inhibited by pretreatment of cells with pioglitazone. CONCLUSION: Thus, it appears that A-R stimulates fibroblast cell growth, collagen type-I synthesis, and MMP-1 expression in cardiac fibroblasts, most likely a result of ROS generation. Inhibition of ROS generation and induction of NF-kappaB in cardiac fibroblasts during A-R may be a mechanism of action of pioglitazone.

Animals↗
PubMed2004

Potential of gene therapy for myocardial ischemia.

PURPOSE OF REVIEW: Gene therapy-based treatment for myocardial ischemia is envisioned to involve multiple approaches. RECENT FINDINGS: We discuss the various approaches and viral vectors available for this emerging field of cardioprotection by gene-based therapy. The prevention of arterial occlusion by the inhibition of clot formation or even atherosclerotic disease process is one approach. Another is the treatment of ischemia with genes that limit cardiac injury due to hypoxia. The molecular pathways that lead to cell damage and death are not yet fully understood. Thus, strides in the understanding of the disease process must be made. SUMMARY: In spite of the lack of precise knowledge, delivery of certain genes has shown promise, and the development of various gene delivery techniques to the heart has shown progress.

Acute Disease↗
PubMed2004

Sperm protein 17 is expressed in human somatic ciliated epithelia.

It was once believed that sperm protein 17 (Sp17) was expressed exclusively in the testis and that its sole function was to bind to the oocyte during fertilization. However, immunohistochemistry of the human respiratory airways and reproductive systems show that it is abundant in ciliated cells but not in human cells with stereocilia and microvilli. The high degree of sequence conservation throughout its N-terminal half, and the presence of an A-kinase anchoring protein (AKAP)-binding motif within this region, suggest that Sp17 plays a regulatory role in a PKA-independent AKAP complex in both male germinal and ciliated somatic cells.

Antigens, Surface↗