Francisco J. Candal

Seeding of intracoronary stents with immortalized human microvascular endothelial cells

Intracoronary stents are effective in decreasing the complications associated with acute closure during coronary angioplasty. A major complication associated with the use of coronary stents is acute thrombotic occlusion. It has been postulated that the stent loses its thrombogenic potential after it becomes covered with a layer of endothelial cells. Human dermal microvascular endothelial cells were transfected with a plasmid containing the simian virus 40 large T-antigen gene. Stents were placed in culture media with cells for 2 weeks. Seeding efficiency of the stent with the endothelial cells was assessed by scanning electron microscopy. Balloon-expandable coronary stents placed in cell culture with immortalized human microvascular endothelial cells showed near-complete coverage after 2 weeks. After balloon inflation, persistence of cells on the stent was noted only on the lateral aspect of the balloon-expanded stents. If these stents were placed in culture, complete recovery of the monolayer was noted after 3 days. Stents were then covered with endothelial cells and frozen for 4 days. After thawing, the cells adhered to the devices and divided to form a monolayer in tissue culture. Seeded balloon-expandable stents were frozen for 4 months, thawed, and then implanted in a pig coronary artery. Human endothelial cells were identified on the stent 4 hours after deployment. These studies demonstrate the feasibility of using a human microvascular endothelial cell line to seed an uncoated metal stent. The cells remain adherent to the stent, are functional after freezing, and remain on the stent at least 3 hours after intracoronary implantation.

Human Endothelial Cells as an Alternative to DH82 Cells for Isolation of Ehrlichia chaffeensis, E. canis, and Rickettsi rickettsii

Ehrlichia chaffeensis, etiologic agent of human ehrlichiosis, and Rickettsia rickettsii, etiologic agent of Rocky Mountain spotted fever (RMSF), are both tick-borne agents that cause nonspecific symptoms that may be indistinguishable from each other early in the course of infection. E. canis is a canine pathogen closely related to E. chaffeensis and was initially suspected of being the causative agent of human ehrlichiosis. If a febrile illness is reported, after tick exposure, neither ehrlichiosis nor RMSF can be immediately ruled out. When attempts are made to isolate the agent from blood, a very limited amount of blood is often available; we, therefore, sought a tissue-culture cell line that would support the growth of both R. rickettsii and E. chaffeensis. A newly established human microvascular endothelial immortal cell line (CDC/EU.HMEC-1) was evaluated for supporting the growth of both agents. Our results demonstrate that HMEC-1 supports the growth of R. rickettsii, E. chaffeensis, and E. canis and may be a useful tool for the isolation of these agents.

Development, expression, and murine testing of a multistage Plasmodium falciparum malaria vaccine candidate.

A synthetic gene encoding twelve B cell epitopes, six T-cell proliferative epitopes, and three cytotoxic T lymphocyte (CTL) epitopes from nine stage-specific antigens, representing the sporozoite, liver stage, asexual blood-stage, and sexual-stage antigens of Plasmodium falciparum, was constructed by assembling overlapping oligonucleotides followed by PCR extension and annealing. A three-step PCR protocol using twelve long oligonucleotides was employed to generate a 1053 base-pair synthetic gene, the identity of which was confirmed by sequencing. This synthetic gene, named CDC/NII MAL VAC-1, was cloned, and the recombinant protein was expressed in the Baculovirus Expression Vector System (BEVS). The selection of malarial epitopes for inclusion in this vaccine construct was based on immunoepidemiological studies in malaria endemic area, in vitro, and in vivo protection studies in model systems. The 41 kDa BEVS-expressed recombinant protein reacted with mouse antibodies specific for individual B cell epitopes in the vaccine construct and with sera from clinically immune Kenyan adults. An immunization study in three strains of mice that differ at the H-2 locus demonstrated that the BEVS-expressed recombinant protein is immunogenic; the candidate vaccine antigen induced high titer antibodies, and lymphocyte proliferative and IFN-gamma responses. These results demonstrate that individual B and T cell epitopes can be assembled to create synthetic genes that encode proteins capable of eliciting specific antibody and T cell responses.

PEC‐A: An immortalized porcine aortic endothelial cell

Abstract: Cultured porcine aortic endothelium was transfected with sequences that encode the SV40 T antigen, resulting in an immortalized cell line that retains the differentiated properties of normal aortic endothelial cells. Specifically, these cells form cobblestone monolayers, synthesize von Willebrand factor, and endocytose acetylated LDL. These cells can be readily propagated in culture and have been passaged over a year in culture. The cells form tubular structures when grown on Matrigel. The cells in culture express class I but do not express MHC class II antigens. Both class I and class II expression can be induced by treatment with recombinant swine interferon‐γ. Expression of CD44 and several other cell surface antigens have been observed. Co‐culture of these cells with purified human CD4+ T cells resulted in a significant T‐cell proliferative response similar to that observed for primary porcine EC. Finally, the cells are readily susceptible to transfection and express exogenous genes. These cells should be valuable for study of human anti‐porcine endothelial responses.

Influence of adjuvants in inducing immune responses to different epitopes included in a multiepitope, multivalent, multistage Plasmodium falciparum candidate vaccine (FALVAC-1) in outbred mice.

FALVAC-1, a vaccine against Plasmodium falciparum was developed by joining 21 epitopes from P. falciparum vaccine antigens and an universal T helper epitope from tetanus toxoid. Since adjuvants influence different aspects of immune responses, in this study we investigated the effect of four adjuvants aluminum hydroxide (alum), nonionic copolymer adjuvant P1005 (water-in-oil emulsion), CpG oligodeoxynucleotides (ODN), and QS-21 in eliciting immune responses in outbred mice. QS-21 and copolymer adjuvants were the best formulations in inducing higher and long-lasting antibody titers to the whole vaccine compared to alum and CpG. QS-21 was the only adjuvant to elicit predominantly IgG2a response and antibodies reactive with all epitopes incorporated in the vaccine construct. Vaccine elicited antibodies recognized sporozoites and asexual blood-stage parasites. FALVAC-1 immunized mice induced lymphoproliferative and IFN-gamma response to the vaccine. QS-21 and CpG adjuvants were able to elicit T proliferative responses to 20 of the 22 epitopes in the vaccine. In conclusion, this study demonstrated that with suitable adjuvant such as QS-21, it is possible to elicit immune responses to most of the epitopes included in the FALVAC-1 vaccine.

Inhibition of induced angiogenesis in a human microvascular endothelial cell line by ET-18-OCH3

Alkyl-lysophospholipids are a group of anticancer compounds that have previously been shown to have the unique feature of being selectively toxic to neoplastic tissues. One of these compounds ET-18-OCH3, has been used for purging bone marrow of cancer cells in phase I clinical trials. Tumor-induced angiogenesis has been directly correlated with tumor growth and metastasis. In this study, we examined the effect ET-18-OCH3 has on a human microvascular endothelial cell line (HMEC-1), including the following functions: angiogenesis, cell-adhesion molecule expression, and cell-junction integrity. We found that ET-18-OCH3 (in vitro) reversibly inhibited induced angiogenesis at levels that did not affect viability. At lower concentrations, ET-18-OCH3 down-regulated the expression of cell-adhesion molecules and affected the integrity of cell-to-cell junctions., This observation demonstrates this versatile family of compounds to have additional targets of action.

IDENTIFICATION AND AUTHENTICATION OF ANIMAL CELL CULTURE BY POLYMERASE CHAIN REACTION AMPLIFICATION AND DNA SEQUENCING

SummaryPolymerase chain reaction (PCR) amplification and deoxyribonucleic acid (DNA) sequence analysis were used to identify the species origin of cell lines used in a cell culture facility where various cell lines of different species are routinely propagated. The aldolase gene family was selected for PCR amplification because the DNA sequences of this gene are highly conserved over a wide range of animals and humans. A total of 36 cell lines representing 13 different species were selected for this study. The DNA from each cell line was amplified, and PCR products were analyzed by agarose gel electrophoresis. The results showed unique profiles of amplified bands on agarose gels that allowed differentiation among non-closely related species. However, DNA amplification of closely related species, including rat and mouse or human and primate, resulted in similar and indistinguishable banding patterns that could be further differentiated by DNA sequence analysis. These results suggested that aldolase gene amplification coupled with DNA sequence analysis is a useful tool for identification of cell lines and has potential application for use in identification of interspecies cross-contamination.

Viral susceptibility of an immortalized human microvascular endothelial cell line

CDC/EU.HMEC-1 is the first immortalized human microvascular endothelial cell line that retains morphologic, phenotypic, and functional characteristics of a normal human microvascular endothelial cell. This study evaluates a variety of viruses and their effects on this human endothelial cell line. The data indicate that adenoviruses, some herpesviruses, reoviruses and most picornaviruses grow well in HMEC-1, with distinctive cytopathic effects. The paramyxoviruses, however, do not appear to propagate, nor does HIV. The findings indicate that microvascular endothelial cells may act as a reservoir of these viruses; it also suggests the possibility that microvascular endothelium could be involved in the processing and presentation of antigen to immune cells.

Destruction of Human Microvascular Endothelial Cell Capillary-Like Microtubules by Brazilian Purpuric Fever-Associated Haemophilus influenzae Biogroup Aegyptius

When grown in the presence of Matrigel, monolayers of an immortalized human microvascular cell line (HMEC-1) form capillary-like microtubule networks. Previous work, using HMEC-1 monolayers, demonstrated a significant difference in in vitro cytotoxicity between Brazilian purpuric fever (BPF)-associated Haemophilus influenzae biogroup aegyptius (HAE) strains and non-BPF-associated HAE strains. The present study demonstrates that BPF-related cytotoxic differences can also be observed in HMEC-1 microtubule networks. At a multiplicity of infection (MOI) of 2 x 10(-2) bacteria/tissue culture cell, BPF-associated strain F3031 disrupted the microtubule network, producing random clumps of rounded cells at 48 h of incubation. Infection with non-BPF-associated strain F1947 at the same MOI produced no observable microtubule disruption. The ability of HMEC-1 microtubule model to differentiate virulent and avirulent HAE in vitro will further aid in the study of BPF pathogenesis. In addition, the fact that the HMEC-1 cells can be induced to form microtubules make it an excellent model system for the general study of many of the agents of vascular purpura.

Possibilities of vaccine manufacture in human diploid cell strains with a serum replacement factor

Cell lines MDCK (canine kidney), BGM (Buffalo green monkey kidney) and human embryonic lung fibroblast will support viral growth efficiently in medium without serum. Both MRC-5 and WI-38 cell strains have been approved by the Food and Drug Administration for manufacturing viral vaccines against cytomegalovirus and varicella-zoster virus. In this study we examine these two cell lines and viruses for their ability to grow in medium containing a serum replacement. The serum substitute used is LPSR-1 (low protein serum replacement). Using LPSR-1 in defined medium, we demonstrate multipassage cell growth and viral cultivation and replication equivalent to those obtained in medium containing fetal bovine serum (FBS). Viral growth after complete elimination of FBS varies and depends on cell line and virus. Serum substitutes can eliminate FBS in the viral growth phase of vaccine production and reduce costs.