Effects of administration of two growth hormone-releasing hormone plasmids to gilts on sow and litter performance for the subsequent three gestations.

OBJECTIVE: To determine whether a novel optimized plasmid carrying the porcine growth hormone-releasing hormone (GHRH) wild-type cDNA administered at a lower dose was as effective at eliciting physiologic responses as a commercial GHRH plasmid approved for use in Australia. ANIMALS: 134 gilts. PROCEDURES: Estrus was synchronized and gilts were bred. Pregnant gilts were assigned to 2 treatment groups (40 gilts/group) or 1 untreated control group (24 gilts). Gilts in one of the treatment groups received the commercial GHRH plasmid, whereas gilts in the other treatment group received a novel optimized GHRH plasmid; both plasmids were administered IM in the right hind limb, which was followed by electroporation. Sow and litter performance were monitored for the 3 gestations after treatment. RESULTS: A significant increase in insulin-like growth factor-I concentrations, decrease in perinatal mortality rate, increase in the number of pigs born alive, and increase in the weight and number of pigs weaned were detected for both groups receiving the GHRH-expressing plasmids, compared with values for the control group. Additionally, there was a significant decrease in sow attrition in GHRH-treated females, compared with attrition in the control group, during the 3 gestations after treatment. CONCLUSIONS AND CLINICAL RELEVANCE: Both of the GHRH plasmids provided significant benefits for sow performance and baby pig survivability for pregnant and lactating sows and their offspring during the 3 gestations after treatment, compared with results for untreated control gilts. Use of a novel optimized plasmid reduced the effective plasmid dose in these large mammals.

Short-lived, transitory cell-cell interactions foster migration-dependent aggregation.

During embryonic development, motile cells aggregate into cohesive groups, which give rise to tissues and organs. The role of cell migration in regulating aggregation is unclear. The current paradigm for aggregation is based on an equilibrium model of differential cell adhesivity to neighboring cells versus the underlying substratum. In many biological contexts, however, dynamics is critical. Here, we provide evidence that multicellular aggregation dynamics involves both local adhesive interactions and transport by cell migration. Using time-lapse video microscopy, we quantified the duration of cell-cell contacts among migrating cells that collided and adhered to another cell. This lifetime of cell-cell interactions exhibited a monotonic decreasing dependence on substratum adhesivity. Parallel quantitative measurements of cell migration speed revealed that across the tested range of adhesive substrata, the mean time needed for cells to migrate and encounter another cell was greater than the mean adhesion lifetime, suggesting that aggregation dynamics may depend on cell motility instead of the local differential adhesivity of cells. Consistent with this hypothesis, aggregate size exhibited a biphasic dependence on substratum adhesivity, matching the trend we observed for cell migration speed. Our findings suggest a new role for cell motility, alongside differential adhesion, in regulating developmental aggregation events and motivate new design principles for tuning aggregation dynamics in tissue engineering applications.

Gene therapy by electroporation for the treatment of chronic renal failure in companion animals.

BACKGROUND: Growth hormone-releasing hormone (GHRH) plasmid-based therapy for the treatment of chronic renal failure and its complications was examined. Companion dogs (13.1+/-0.8 years, 29.4+/-5.01 kg) and cats (13.2+/-0.9 years, 8.5+/-0.37 kg) received a single 0.4 mg or 0.1 mg species-specific plasmid injection, respectively, intramuscularly followed by electroporation, and analyzed up to 75 days post-treatment; controls underwent electroporation without plasmid administration. RESULTS: Plasmid-treated animals showed an increase in body weight (dogs 22.5% and cats 3.2%) compared to control animals, and displayed improved quality of life parameters including significant increases in appetite, activity, mentation and exercise tolerance levels. Insulin-like growth factor I (IGF-I, the downstream effector of GHRH) levels were increased in the plasmid treated animals. Hematological parameters were also significantly improved. Protein metabolism changes were observed suggesting a shift from a catabolic to an anabolic state in the treated animals. Blood urea nitrogen and creatinine did not show any significant changes suggesting maintenance of kidney function whereas the control animal's renal function deteriorated. Treated animals survived longer than control animals with 70% of dogs and 80% of cats surviving until study day 75. Only 17% and 40% of the control dogs and cats, respectively, survived to day 75. CONCLUSION: Improved quality of life, survival and general well-being indicate that further investigation is warranted, and show the potential of a plasmid-based therapy by electroporation in preventing and managing complications of renal insufficiency.

Growth hormone-releasing hormone plasmid treatment by electroporation decreases offspring mortality over three pregnancies.

LifeTideSW5 is a growth hormone-releasing hormone (GHRH)-expressing plasmid delivered by intramuscular (IM) electroporation (EP), and the first therapeutic plasmid delivered by this physical method to be approved for use in food animals. Gestating sows (n = 997) were treated once with a single 5-mg GHRH-plasmid by EP or served as controls. Data on offspring from three parities subsequent to treatment were collected. No adverse effects related to treatment were noted. First parity post-treatment offspring from treated sows displayed a 2.93 kg (P < 0.0001) increase in carcass weight (CW), 1.0 mm (P < 0.0001) less back-fat (P2), and a 27.0 g CW/day (P < 0.0001) increase in rate of gain (ROG) compared with controls. An increase of 21.6% was recorded in the number of offspring surviving. In the second and third parities post-treatment, offspring from treated females displayed higher number of born alive and total born number, and lower stillborn rates. Third parity offspring from treated sows displayed a 1.6 kg advantage in CW (P < 0.05), 1.0 mm less P2 (P < 0.05), and a 10.0 g CW/day benefit in ROG. Furthermore, offspring from treated females had a 19.04% lower post-wean loss rate. Overall, plasmid GHRH administration decreased morbidity and mortality in treated females and their offspring over three consecutive pregnancies.

Parameters for DNA vaccination using adaptive constant-current electroporation in mouse and pig models.

Enhancing the expression of DNA vaccines requires that specific conditions of delivery are optimized. We describe experiments using adaptive constant-current electroporation (EP) in mice and pigs examining parameters such as target muscle, delay between plasmid delivery and onset of EP pulses and DNA vaccine formulation; our studies show that concentrated formulations result in better expression and immunogenicity. Furthermore, various conditions of EP that limit the amount of muscle damage were measured. The results of these studies will help to advance the success of DNA vaccines in animals into success in human clinical trials.

Double-blinded, Placebo-controlled plasmid GHRH trial for cancer-associated anemia in dogs.

The use of growth hormone releasing hormone (GHRH) plasmid-based therapy to treat companion dogs with spontaneous malignancies and anemia receiving a cancer-specific treatment was examined in a double-blinded, placebo-controlled trial. The dogs (age 10.5 +/- 2.5 years, weight 24.9 +/- 12.9 kg) received a single 0.35 mg dose of plasmid or placebo intramuscularly (i.m.), followed by electroporation (EP), and were analyzed for up to 120 days. The response rate was defined as > or = 5% increase above the nadir in the red blood cell (RBC), hemoglobin (Hb), and hematocrit (Ht) levels. Plasmid-treated dogs had at least a 7% increase in all three parameters. The initial response rates for the plasmid-treated dogs were 40.6 and 35.5%, respectively on days 40 and 60, which increased to 54.2% on day 90. Although the response rate reduced to 47.1% by day 120, it was still 22.1% higher than in the control dogs. Post-hoc analysis of the GHRH-treated group showed that responder dogs survived 84% longer, 178 +/- 26 days post-treatment, while nonresponders and controls survived for 95 +/- 16 and 97 +/- 31 days post-treatment, respectively. The quality of life, defined by 10 different parameters, dramatically improved with treatment. Overall, the possibility of a GHRH plasmid-based therapy for anemia in cancer-afflicted subjects is important enough to deserve further investigation.

Automated quantitative analysis of epithelial cell scatter.

Epithelial cell scatter is a well-known in vitro model for the study of epithelial-mesenchymal transition (EMT). Scatter recapitulates many of the events that occur during EMT, including the dissociation of multicellular structures and increased cell motility.Because it has been implicated in tumor invasion and metastasis,much effort has been made to identify the molecular signals that regulate EMT. To better understand the quantitative contributions of these signals, we have developed metrics that quantitatively describe multiple aspects of cell scatter. One metric (cluster size)quantifies the disruption of intercellular adhesions while a second metric (nearest-neighbor distance) quantifies cell dispersion. We demonstrate that these metrics delineate the effects of individual cues and detect synergies between them. Specifically, we find epidermal growth factor (EGF), cholera toxin (CT) and insulin to synergistically reduce cluster sizes and increase nearest-neighbor distances. To facilitate the rapid measurement of our metrics from live-cell images, we have also developed automated techniques to identify cell nuclei and cell clusters in fluorescence images. Taken together, these studies provide broadly applicable quantitative image analysis techniques and insight into the control of epithelial cell scatter, both of which will contribute to the understanding of EMT and metastasis.

A microtiter assay for quantifying protein-protein interactions associated with cell-cell adhesion.

Cell-cell adhesions are a hallmark of epithelial tissues, and the disruption of these contacts plays a critical role in both the early and late stages of oncogenesis. The interaction between the transmembrane protein E-cadherin and the intracellular protein beta-catenin plays a crucial role in the formation and maintenance of epithelial cell-cell contacts and is known to be downregulated in many cancers. The authors have developed a protein complex enzyme-linked immunosorbent assay (ELISA) that can quantify the amount of beta-catenin bound to E-cadherin in unpurified whole-cell lysates with a Z' factor of 0.74. The quantitative nature of the E-cadherin:beta-catenin ELISA represents a dramatic improvement over the low-throughput assays currently used to characterize endogenous E-cadherin:beta-catenin complexes. In addition, the protein complex ELISA format is compatible with standard sandwich ELISAs for parallel measurements of total levels of endogenous E-cadherin and beta-catenin. In 2 case studies closely related to cancer cell biology, the authors use the protein complex ELISA and traditional sandwich ELISAs to provide a detailed, quantitative picture of the molecular changes occurring within adherens junctions in vivo. Because the E-cadherin: beta-catenin protein complex plays a crucial role in oncogenesis, this protein complex ELISA may prove to be a valuable quantitative prognostic marker of tumor progression.

Plasmid-based expression technology using growth hormone releasing hormone: a novel method for physiologically stimulating long-term growth hormone secretion.

Novel DNA-based technologies were recently introduced for various purposes, such as screening of targets identified from genomic projects, shuffled molecules for vaccination, or to direct the in vivo production of hormones and other peptides for therapeutic or preventative applications. We have used a plasmid-based technology to deliver growth hormone releasing hormone (GHRH) to various animal species for screening, toxicology and therapy. A single intramuscular injection of a low dose of plasmid followed by electroporation can ensure that the target species will produce physiological levels of GHRH for extended periods of time, which would replace costly, frequent injections of the recombinant hormone and improve the quality of life and compliance of patients. This therapeutic modality is of particular importance in circumstances requiring long-term administration of small molecules with naturally short half-life (e.g. treatment of anemia and cachexia associated with renal failure, cancer or other chronic disability). A similar technique was used to create, test and validate protease-resistant analogs of GHRH with significantly longer half-life. Analysis of the characteristics of each of the plasmid components and tissue-specific transcription factors and the choice of target tissue is imperative when designing plasmids for therapeutic applications. Using the species-specific sequences of GHRH or other molecule along with the appropriate choice of plasmid backbone and expression cassette components can result in long and steady expression of the transgene product.

Lymphocyte-dendritic cell interactions and mucosal acquisition of SIV/HIV infection.

PURPOSE OF REVIEW: Several previous models of HIV dissemination implicated dendritic cells as viral conduits to the lymphatics. However, recent macaque transmission and microbicide studies have highlighted a more complex situation. RECENT FINDINGS: Resting CD4 lymphocytes are observed to be the major infected population in mucosal tissue after vaginal challenge with SIV. Resting lymphocytes appear to bridge infection over short distances, whereas activated lymphocytes provide long-distance virus dissemination as a result of greater virus amplification. In addition, dendritic cells might be early carriers of virus, transmitting virus to T cells locally and to the lymph nodes, and thus support parallel mechanisms in transmission. Microbicide studies using agents against CCR5 corroborate a model that infection at the mucosa must occur for transmission to be successful. The fast-rate dendritic cell trafficking of virus to the lymphatics may not result in immediate and efficient viral replication in lymphatic tissue. As dendritic cells might also be infected at the mucosa before lymphatic trafficking, this would enable them to transfer virus in this region at a later timepoint. SUMMARY: There are now several models that can be attributed to the mucosal acquisition of SIV/HIV. One feature that unites these models is that infection in the mucosa must occur for dissemination to take place. Whether this is a feature of CD4 lymphocytes, dendritic cells or macrophage infection is still unclear. A model that intertwines one or more of the above cell types would be more prudent than addressing each in isolation.

Highly efficient constant-current electroporation increases in vivo plasmid expression.

Electroporation has been demonstrated as an effective technique for enhancing the delivery of plasmids coding for DNA vaccines and therapeutic proteins into skeletal muscle. Nevertheless, constant-voltage techniques do not take into account the resistance of the tissue and result in tissue damage, inflammation, and loss of plasmid expression. In the present study, we have used a software-driven constant-current electroporator to deliver plasmids to mice and small and large pigs. The voltage, amperage, and resistance of the tissue during pulses were recorded and analyzed. Optimal conditions of electroporation were identified in both species, and found to be highly dependent on the individual tissue resistance. Six- to 10-week-old pigs had higher muscle resistance compared to 1- to 2-year-old pigs, but both values were four to five times lower than the resistance of the mouse muscle. In mice, optimum amperage, pulse length, and lag time between plasmid injection and electroporation were identified to be 0.1 Amps, 20 msec and 0 sec. The electroporation pulse pattern among the electrodes also affected plasmid expression. These results indicate that age- and tissue-specific resistance, pulse pattern, and other variables associated with the electroporation need to be optimized for each separate species to achieve maximum plasmid expression.

Immunomodulatory effects of HSV-2 infection on immature macaque dendritic cells modify innate and adaptive responses.

Herpes simplex viruses (HSV) infect human and murine dendritic cells (DCs) and interfere with their immunostimulatory functions in culture. HSV-2 infection increases human immunodeficiency virus (HIV) spread in patients, and DCs also promote HIV infection. We have studied these topics in rhesus macaque monocyte-derived DCs (moDCs) to set the stage for future studies of these issues in animals. We provide the first evidence that macaque DCs become infected by HSV-2. Structural viral proteins (ICP5 [infected cell protein 5], glycoprotein D [gD], envelope) were detected in the cell periphery, and a functional protein (infected cell protein 8 [ICP8]) was predominantly found in the nucleus after infection. Infectious HSV-2 induced apoptotic death, decreased expression of HLA-DR, CD40, CD80, CD83, and CD86, and increased release of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-1alpha (MIP-1alpha) (CCL3), and RANTES (regulated on activation normal T cells expressed and secreted) (CCL5) but not IL-12 or interferon-alpha (IFN-alpha) by macaque DCs. This coincided with HSV-2-infected DCs stimulating weak T-cell responses, including impaired SIV-specific responses. Comparable HSV-2 protein expression, DC apoptosis, as well as membrane immunophenotype and functional modifications were observed in HSV-2-exposed human moDCs. Such HSV-2-induced modifications of macaque and human DCs could augment DC-driven immunodeficiency virus infection. This work affords the basis for future macaque studies to explore how HSV-2 impacts the efficacy of strategies being developed to prevent HIV transmission.

Elimination of retroviral infectivity by N-ethylmaleimide with preservation of functional envelope glycoproteins.

The zinc finger motifs in retroviral nucleocapsid (NC) proteins are essential for viral replication. Disruption of these Cys-X2-Cys-X4-His-X4-Cys zinc-binding structures eliminates infectivity. To determine if N-ethylmaleimide (NEM) can inactivate human immunodeficiency virus type 1 (HIV-1) or simian immunodeficiency virus (SIV) preparations by alkylating cysteines of NC zinc fingers, we treated infectious virus with NEM and evaluated inactivation of infectivity in cell-based assays. Inactivation was rapid and proportional to the NEM concentration. NEM treatment of HIV-1 or SIV resulted in extensive covalent modification of NC and other internal virion proteins. In contrast, viral envelope glycoproteins, in which the cysteines are disulfide bonded, remained intact and functional, as assayed by high-performance liquid chromatography, fusion-from-without analyses, and dendritic cell capture. Quantitative PCR assays for reverse transcription intermediates showed that NEM and 2,2'-dipyridyl disulfide (aldrithiol-2), a reagent which inactivates retroviruses through oxidation of cysteines in internal virion proteins such as NC, blocked HIV-1 reverse transcription prior to the formation of minus-strand strong-stop products. However, the reverse transcriptase from NEM-treated virions remained active in exogenous template assays, consistent with a role for NC in reverse transcription. Since disruption of NC zinc finger structures by NEM blocks early postentry steps in the retroviral infection cycle, virus preparations with modified NC proteins may be useful as vaccine immunogens and probes of the role of NC in viral replication.

Growth hormone releasing hormone plasmid supplementation, a potential treatment for cancer cachexia, does not increase tumor growth in nude mice.

Growth hormone releasing hormone (GHRH) is known to have multiple anabolic effects and immune-stimulatory effects. Previous studies suggest that treatment with anabolic hormones also has the potential to mitigate the deleterious effects of cancer cachexia in animals. We studied the effects of plasmid-mediated GHRH supplementation on tumor growth and the role of antitumor immune cells with two different human tumor cell lines, NCI-H358 human bronchioalveolar carcinoma and MDA-MB-468 human breast adenocarcinoma, subcutaneously implanted in nude mice. GHRH supplementation by delivery of human GHRH from a muscle-specific GHRH expression plasmid did not increase tumor progression in tumor-bearing nude mice. Male animals implanted with the NCI-H358 tumor cell line and treated with the GHRH-expressing plasmid exhibited a 40% decrease in the size of the tumors (P<.02), a 48% increase in white blood cells (P<.025) and a 300% increase in monocyte count (P<.0001), as well as an increase in the frequency of activated CD3+ and CD4+ cells in the tumors, compared to tumors of control animals. No adverse effects were observed in animals that received the GHRH-plasmid treatment. The present study shows that physiological stimulation of the GHRH-GH-IGF-I axis in mice with cancer does not promote tumor growth and may provide a viable treatment for cancer cachexia in humans.

Induction of neutralising antibodies restricts the use of human granulocyte/macrophage colony stimulating factor for vaccine studies in rhesus macaques.

Granulocyte/macrophage-colony stimulating factor (GM-CSF) is a valuable adjuvant to enhance induction of cellular immune responses in rodents. Less information is available regarding its use as an adjuvant in primates or humans. We explored recombinant human GM-CSF for potential vaccine studies in rhesus macaques and focused on its effect on peripheral monocytes as progenitors of dendritic cells and its potential immunogenicity. Application of human GM-CSF to nine animals led to an average 32-fold increase in monocyte numbers. This was not observed upon re-treatment, which coincided with GM-CSF-specific neutralising antibodies. These also neutralised the activity of rhesus macaque GM-CSF. The data underscore the need to use species-specific GM-CSF for immunomodulation in primates.

CpG-C immunostimulatory oligodeoxyribonucleotide activation of plasmacytoid dendritic cells in rhesus macaques to augment the activation of IFN-gamma-secreting simian immunodeficiency virus-specific T cells.

There are two principle subsets of dendritic cells (DCs); CD11c(+)CD123(-) myeloid DCs (MDCs) and CD11c(-)CD123(+) plasmacytoid DCs (PDCs). DC activation via TNF-TNFRs (e.g., CD40L) and TLRs (e.g., immunostimulatory oligodeoxyribonucleotides (ISS-ODNs)) is crucial for maximal stimulation of innate and adaptive immunity. Macaque DC biology is being studied to improve HIV vaccines using the SIV macaque model. Using lineage (Lin) markers to exclude non-DCs, Lin(-)HLA-DR(+)CD11c(+)CD123(-) MDCs and Lin(-)HLA-DR(+)CD11c(-)CD123(+) PDCs were identified in the blood of uninfected macaques and healthy macaques infected with SIV or simian-human immunodeficiency virus. Overnight culture of DC-enriched Lin-depleted cells increased CD80 and CD86 expression. IL-12 production and CD80/CD86 expression by MDC/PDC mixtures was further enhanced by CD40L and ISS-ODN treatment. A CpG-B ISS-ODN increased CD80/CD86 expression by PDCs, but resulted in little IFN-alpha secretion unless IL-3 was added. In contrast, a CpG-C ISS-ODN and aldrithiol-2-inactivated (AT-2) SIV induced considerable PDC activation and IFN-alpha release without needing exogenous IL-3. The CpG-C ISS-ODN also stimulated IL-12 release (unlike AT-2 SIV) and augmented DC immunostimulatory activity, increasing SIV-specific T cell IFN-gamma production induced by AT-2 SIV-presenting MDC/PDC-enriched mixtures. These data highlight the functional capacities of MDCs and PDCs in naive as well as healthy, infected macaques, revealing a promising CpG-C ISS-ODN-driven DC activation strategy that boosts immune function to augment preventative and therapeutic vaccine efficacy.

Blockade of attachment and fusion receptors inhibits HIV-1 infection of human cervical tissue.

Identification of cellular factors involved in HIV-1 entry and transmission at mucosal surfaces is critical for understanding viral pathogenesis and development of effective prevention strategies. Here we describe the evaluation of HIV-1 entry inhibitors for their ability to prevent infection of, and dissemination from, human cervical tissue ex vivo. Blockade of CD4 alone or CCR5 and CXCR4 together inhibited localized mucosal infection. However, simultaneous blockade of CD4 and mannose-binding C-type lectin receptors including dendritic cell-specific intercellular adhesion molecule-grabbing integrin was required to inhibit HIV-1 uptake and dissemination by migratory cells. In contrast, direct targeting of HIV-1 by neutralizing mAb b12 and CD4-IgG2 (PRO-542) blocked both localized infection and viral dissemination pathways. Flow cytometric analysis and immunostaining of migratory cells revealed two major populations, CD3(+)HLA-DR(-) and CD3(-)HLA-DR(+) cells, with a significant proportion of the latter also expressing dendritic cell-specific intercellular adhesion molecule-grabbing integrin. Bead depletion studies demonstrated that such HLA-DR(+) cells accounted for as much as 90% of HIV-1 dissemination. Additional studies using immature monocyte-derived dendritic cells demonstrated that although mannose-binding C-type lectin receptors and CD4 are the principal receptors for gp120, other mechanisms may account for virus capture. Our identification of the predominant receptors involved in HIV-1 infection and dissemination within human cervical tissue highlight important targets for microbicide development.

Short-term Flt3L treatment effectively mobilizes functional macaque dendritic cells.

In vivo administration of soluble Flt3L increases dendritic cell (DC) numbers to favor improved DC targeting of vaccine antigens, augmenting vaccine efficiency. In addition to confirming the effectiveness of human Flt3L in macaques, we strove to determine the optimal regimen to elevate numbers of functional DCs. Circulating DCs were identified within lineage(-)human leukocyte antigen-DR(+) cells, which comprised CD11c(-)CD123(+) plasmacytoid DCs (PDCs) and CD123(-) cells including CD11c(+)CD123(-) myeloid DCs as well as CD11c(-)CD123(-) cells. Traditionally, DCs have been monitored 1-2 days after 10- to 14-day treatments with Flt3L (100 microg/kg/day). We demonstrate that although standard treatment increased macaque DC percentages, as little as 5-7 days of treatment was sufficient, if not more effective at mobilizing DCs. Moreover, DC frequency continued to escalate over the ensuing days, peaking at approximately 4 days post 7 days of treatment and ultimately decreasing thereafter. As expected, there was a more pronounced increase in the percentages and actual numbers of CD123(-) cells (CD11c(+) and CD11c(-) subsets) compared with PDCs. Flt3L-mobilized DCs exhibited slightly increased CD80/CD86 expression but typically still that of immature DCs and were resilient to freeze-thawing. Overnight culture activated the cells, up-regulating CD80/CD86 expression as well as interleukin-12 release, typically being boosted by CD40L. This was even more apparent for enriched DC cultures. These data verify that peak mobilization of large numbers of functional macaque DCs occurs a few days, not immediately, after short-term Flt3L dosing. This has important implications for improved DC-targeting vaccine strategies to prevent infection with human immunodeficiency virus and other pathogens.

Immunodeficiency virus uptake, turnover, and 2-phase transfer in human dendritic cells.

HIV-1 subverts antigen processing in dendritic cells (DCs) resulting in viral uptake, infection, and transfer to T cells. Although DCs bound monomeric gp120 and HIV-1 similarly, virus rarely colocalized with endolysosomal markers, unlike gp120, suggesting HIV-1 alters endolysosomal trafficking. Virus within DC intracellular compartments rapidly moved to DC-CD4+ lymphocyte synapses when introduced to CD4+ lymphocyte cultures. Although viral harboring and transfer from nonlysosomal compartments was transient, given DC-associated virus protein, nucleic acids, and infectious HIV-1 transfer to CD4+, lymphocytes decayed within 24 hours. However a second long-term transfer phase was apparent in immature DCs after 48 hours as a zidovudine-sensitive rise in proviral DNA. Therefore, DCs transfer HIV-1 to CD4+ lymphocytes in 2 distinct phases. Immature and mature DCs first divert virus from the endolysosomal pathway to the DC-T-cell synapse. Secondly, the later transfer phase from immature DCs is through de novo HIV-1 production. Thus, the controversy of DCs being infected or not infected for the mechanics of viral transfer to CD4+ lymphocytes can be addressed as a function of time.

Optimization of electroporation parameters for the intramuscular delivery of plasmids in pigs.

Increased transgene expression after plasmid transfer to the skeletal muscle is obtained with electroporation in many species, but optimum conditions are not well defined. Using a plasmid with a muscle-specific secreted embryonic alkaline phosphatase (SEAP) gene, we have optimized the electroporation conditions in a large mammal (pig). Parameters tested included electric field intensity, number of pulses, lag time between plasmid injection and electroporation, and plasmid delivery volume. Electric pulses, between 0.4 and 0.6 Amp constant current, applied 80 sec after the injection of 0.5 mg SEAP-expressing plasmid in a total volume of 2 mL produced the highest levels of expression. Further testing demonstrated that electroporation of a nondelineated injection site reduces the levels of SEAP expression. These results demonstrate that electroporation parameters such as amperage, lag time, and the number of pulses are able to regulate the levels of reporter gene expression in pigs.