Multivariate evaluation of the effectiveness of treatment efficacy of cypermethrin against sea lice (Lepeophtheirus salmonis) in Atlantic salmon (Salmo salar).

BACKGROUND: The sea louse Lepeophtheirus salmonis is the most important ectoparasite of farmed Atlantic salmon (Salmo salar) in Norwegian aquaculture. Control of sea lice is primarily dependent on the use of delousing chemotherapeutants, which are both expensive, and toxic to other wildlife. The method most commonly used for monitoring treatment effectiveness relies on measuring the percentage reduction in the mobile stages of Lepeophtheirus salmonis only. However, this does not account for changes in the other sea lice stages and may result in misleading or incomplete interpretation regarding the effectiveness of treatment. With the aim of improving the evaluation of delousing treatments, we explored multivariate analyses of bath treatments using the topical pyrethroid, cypermethrin, in salmon pens at five Norwegian production sites. RESULTS: Conventional univariate analysis indicated reductions of over 90% in mobile stages at all sites. In contrast, multivariate analyses indicated differing treatment effectiveness between sites (p-value < 0.01) based on changes in the proportion and abundance of the chalimus and PAAM (pre-adult and adult males) stages. Low water temperatures and shortened intervals between sampling after treatment may account for the differences in the composition of chalimus and PAAM stage groups following treatment. Using multivariate analysis, such factors could be separated from those which were attributable to inadequate treatment or chemotherapeutant failure. CONCLUSIONS: Multivariate analyses for evaluation of treatment effectiveness against multiple life cycle stages of L. salmonis yield additional information beyond that derivable from univariate methods. This can aid in the identification of causes of apparent treatment failure in salmon aquaculture.

Ayahuasca and Public Health: Health Status, Psychosocial Well-Being, Lifestyle, and Coping Strategies in a Large Sample of Ritual Ayahuasca Users.

Assessing the health status of ayahuasca users has been challenging due to the limitations involved in randomized clinical trials and psychometric approaches. The main objective of this study is the implementation of an approach based on public health indicators. We developed a self-administered questionnaire that was administered to long-term ayahuasca users around Spain. The questionnaire was administrated face-to-face to participants (n = 380) in places where ayahuasca ceremonies were occurring. Public health indicators were compared with Spanish normative data, and intergroup analyses were conducted. Long-term ayahuasca use was associated with higher positive perception of health or with a healthy lifestyle, among other outcomes. Fifty-six percent of the sample reported reducing their use of prescription drugs due to ayahuasca use. Participants who used ayahuasca more than 100 times scored higher in personal values measures. The main conclusion of this study is that a respectful and controlled use of hallucinogenic/psychedelic drugs taken in communitarian settings can be incorporated into modern society with benefits for public health. This new approach, based on the use of health indicators that were not used in previous ayahuasca studies, offers relevant information about the impact of long-term exposure to ayahuasca on public health.

Age-related gene expression profiles of rhesus monkey bone marrow-derived mesenchymal stem cells.

The objective of this study was to elucidate age-related differences in gene expression profiles of rhesus monkey bone marrow-derived mesenchymal stem cells (rhMSC) obtained from fetal, infant, and adult donors relevant to their growth and other properties. Although a high degree of similarity was observed in the rhMSC gene expression profiles when comparing the three age groups, significant differences were found that strongly parallel gene expression profiles of human MSC. In general, there was a trend towards increased abundance of transcripts associated with differentiation and growth arrest with increasing donor age. Conversely, transcripts involved in RNA processing and the negative regulation of gene expression showed a downward trend with increasing donor age. Overall, the observed gene expression profiles were found to be similar to observations that MSC from older individuals show diminished proliferative capacity. These data highlight the importance of use of non-human primates to study the properties of stem and progenitor cells, and for future therapies.

Effects of busulfan dose escalation on engraftment of infant rhesus monkey hematopoietic stem cells after gene marking by a lentiviral vector.

OBJECTIVE: Non-myeloablative cytoreduction is used in clinical hematopoietic stem cell gene therapy trials to increase engraftment of gene-modified cells. We utilized an infant rhesus monkey model to identify an optimal dosage of busulfan that results in efficient long-term gene marking with minimal toxicities. METHODS: Bone marrow (BM) was harvested, followed by a single 2-hour intravenous infusion of busulfan at escalating dosages of 0 to 160 mg/m(2). CD34(+) cells were immunoselected from BM, transduced overnight with a simian immunodeficiency virus-based lentiviral vector carrying a non-expressed marker gene, and injected intravenously 48 hours post-busulfan administration. Pharmacokinetics were assessed, as well as adverse effects and peripheral blood and BM gene marking. RESULTS: Increasing dosages of busulfan resulted in increased area-under-the-curve (AUC) with some variability at each dosage level, suggesting interindividual variation in clearance. Blood chemistries were normal and no adverse effects were observed as a result of busulfan infusion. At 120 and 160 mg/m(2), transient neutropenia and thrombocytopenia were noted but not lymphopenia. Over the 6 months of study posttransplantation, a busulfan dosage-related increase in gene marking was observed ranging from undetectable (no busulfan) up to 0.1% gene-containing cells in animals achieving the highest busulfan AUC. This corresponds to a more than 100-fold increase in gene marking over the busulfan dosage range studied. CONCLUSIONS: These data indicate that increased gene marking of hematopoietic stem cells can be achieved by escalating busulfan dosages from 40 to 160 mg/m(2) without significant toxicity in infant nonhuman primates.

Fetal gene transfer using lentiviral vectors: in vivo detection of gene expression by microPET and optical imaging in fetal and infant monkeys.

Fetal intraperitoneal administration of human immunodeficiency virus (HIV)-l-derived lentiviral vectors (10(7) infectious particles/fetus) has consistently shown high levels of transduction and gene expression in the omentum, peritoneum, and diaphragm when assessed by polymerase chain reaction (PCR) and whole tissue fluorescence. In vivo imaging techniques were explored with early-gestation long-tailed macaques that were administered the vesicular stomatitis virus-glycoprotein (VSV-G)-pseudotyped HIV-1-derived lentiviral vector expressing a mutant herpes simplex virus type 1 thymidine kinase (HSV-1-sr39tk) and firefly luciferase under the control of the cytomegalovirus (CMV) promoter. Fetuses were monitored sonographically and twice during gestation 9-[4-[18F]Fluoro-3-(hydroxymethyl)butyl]guanine (18F-FHBG) was injected into the fetal circulation under ultrasound guidance in preparation for microPET imaging. All newborns were delivered at term by cesarean section and raised in the nursery for postnatal studies. At 2 months postnatal age, animals were imaged and biodistribution was assessed. Optical imaging for firefly luciferase expression was also performed every 2 months postnatal age. Under all imaging conditions gene expression was observed in the abdominal region, and closely paralleled findings from prior studies based on whole tissue fluorescence. These investigations have shown that HSV-1-sr39tk and firefly luciferase can be used to safely detect transgene expression at multiple time points in fetal and infant monkeys in vivo and without evidence of adverse effects.

Fetal gene transfer using lentiviral vectors and the potential for germ cell transduction in rhesus monkeys (Macaca mulatta).

Genetic modification of germ cells in somatic gene therapy protocols is a concern, particularly with fetal approaches. This study focused on the potential for germ cell gene transfer post-fetal gene delivery using a human immunodeficiency virus type 1 (HIV-1)-derived lentiviral vector pseudotyped with the vesicular stomatitis virus-glycoprotein (VSV-G). Rhesus monkey fetuses (n = 47) were administered vector supernatant (10(7) infectious particles per fetus) via the intraperitoneal (IP), intrapulmonary (Ipu), or intracardiac routes (Ica) under ultrasound guidance. Tissue harvests were performed near term or 3 months postnatal age, and genomic DNA obtained to analyze for vector sequences from collected sections of gonads and gonadal cells obtained by laser capture microdissection (germ cells, stroma, epithelium). Results indicated no evidence of germ cell gene transfer in fetuses or infants with Ipu or Ica routes of administration. However, evidence of the transgene (1.33 +/- 0.78 enhanced green fluorescent protein [EGFP] copies per copy epsilon-globin) was found in females, but not males, when using the IP administration approach (p < 0.05). The highest EGFP copies were detected on the surface epithelium (p < 0.05). The results of these studies suggest that the HIV-1-derived lentiviral vector pseudotyped with VSV-G may transduce a subpopulation of gonadal cells in female fetuses with IP administration, whereas no evidence of gene transfer was shown to occur in males or with organ-targeting approaches.

Fetal CD34+ cells in the maternal circulation and long-term microchimerism in rhesus monkeys (Macaca mulatta).

BACKGROUND: Studies in humans have shown that during pregnancy fetal cells can enter the maternal circulation and persist for many years. While we have previously reported the presence of cell-free fetal DNA during pregnancy in rhesus monkeys, it is unknown whether cells circulate and persist long term in maternal tissues. In this study, we asked whether fetal CD34 cells can be found in the maternal circulation and if male fetal cells persist in maternal tissues postdelivery. METHODS: The presence of the Y chromosome in maternal blood and tissues was assessed using real-time PCR assays for the sex determining region Y (SRY) and testes specific protein Y (TSPY) genes. Analysis was done on CD34 and CD34 cells isolated from maternal blood collected at select time points during gestation from gravid animals with male or female fetuses, and tissues were analyzed from nongravid animals that had previously delivered male offspring. RESULTS: All animals with male fetuses tested positive for the Y chromosome in CD34 cells (0-30 cells/50,000 genome equivalents). Y sequences were also found in one or more maternal tissues collected up to 3-years postdelivery (thyroid, heart, spleen, liver, pituitary, adrenals, skin, inguinal lymph nodes). CONCLUSION: These studies suggest transfer of fetal CD34 cells during pregnancy and persistent fetal microchimerism in the rhesus model. Thus, rhesus monkeys can be used to further our understanding of fetal:maternal microchimerism and the role of fetal cells in maternal health and disease.

Intrapulmonary and intramyocardial gene transfer in rhesus monkeys (Macaca mulatta): safety and efficiency of HIV-1-derived lentiviral vectors for fetal gene delivery.

Fetal gene transfer was studied using intrapulmonary and intramyocardial transfer of SIN HIV-1-derived lentiviral vectors expressing EGFP in rhesus monkeys. Fetuses were monitored sonographically during gestation and tissue analyses performed at term or 3 months postnatal age. Animals remained healthy during the study period as evidenced by normal growth, development, hematology, clinical chemistry, echocardiography, and pulmonary function tests. Strong pulmonary fluorescence was observed postnatally after fetal intrapulmonary delivery of lenti-CMV, but not lenti-SP-C, and compared to nontransferred controls. High EGFP copy numbers were found by quantitative PCR with both vector constructs in lung lobes (

Fetal gender determination in early first trimester pregnancies of rhesus monkeys (Macaca mulatta) by fluorescent PCR analysis of maternal serum.

Non-human primate fetal gender determination can be a powerful tool for research study design and colony management purposes. The recent discovery of the presence of fetal DNA in maternal serum has offered a new non-invasive approach for identification of fetal gender. We present a rapid and simple method for the sexing of developing rhesus monkeys in the first trimester by polymerase chain reaction (PCR) analysis of maternal serum. Serum samples were obtained from 72 gravid rhesus monkeys during 20-32 days of gestation (term 165 +/- 10 days). Fetal gender and the quantity of circulating fetal DNA were determined by real-time PCR analysis of the rhesus Y-chromosomal DNA sequences. The sensitivity for identifying a male fetus was 100% by 30 days gestation, and no false-positive results were observed. This study demonstrates that fetal gender can be reliably determined in the early first trimester from maternal serum samples, a non-invasive method for routine gender screening.

Quantitative analysis of male fetal DNA in maternal serum of gravid rhesus monkeys (Macaca mulatta).

The isolation of human fetal DNA from the maternal circulation has provided a source of fetal material for prenatal diagnosis. The objective of this study was to investigate whether a similar pattern could be observed in the maternal circulation of male-bearing gravid rhesus monkeys. A real-time PCR TaqMan system for the rhesus Y-chromosome sex determining region was used to determine fetal sex and to quantify fetal DNA concentrations. Results in 14 healthy pregnancies indicated that fetal male DNA could be routinely detected in maternal serum by 50 d of gestation (late first trimester; term 165 +/- 10 d). Fetal DNA concentrations increased with advancing gestation, reaching a mean of 341 genome equivalents/mL of serum (range 11-1570 copies/mL) in the last trimester of gestation, similar to findings in humans. The fetal DNA concentration corresponded to 2.7% of the total maternal serum DNA in the third trimester. Similar to findings in humans, male fetal DNA sequences were not detected postpartum (through 4 wk postpartum) or in animals with a previous history of delivering male offspring. These data indicate that fetal male DNA is present in the maternal circulation of gravid rhesus monkeys comparable to findings in humans and further support the use of this nonhuman primate species as a model to investigate fetomaternal cell trafficking and microchimerism.

Growth inhibition of an orthotopic glioblastoma in immunocompetent mice by cationic lipid-DNA complexes.

The effect of a cationic liposome non-coding plasmid DNA complex on the growth of an intracerebral glioblastoma in an immunocompetent syngeneic mouse strain was evaluated. Previous studies of extraneural tumors in mice have demonstrated that such complexes containing plasmid DNA are capable of stimulating a potent Th-1 cytokine immune-mediated response with a dramatic inhibition of tumor growth. A DOTIM-cholesterol cationic liposome complexed to non-coding plasmid DNA (EV-CLDC) was administered intravenously (i.v.) at weekly intervals to 6-week-old male mice of the B6D2F1 strain at either 3, 10 or 17 days post-inoculation (DPI) of 4C8 glioblastoma cells. Tumor growth was monitored by volumetric image analysis obtained from sequential weekly magnetic resonance imaging studies of the brain. Experiments were terminated between 30 to 38 DPI. Terminal tumor volumes calculated from histological sections directly correlated with tumor volumes from corresponding MR images. The EV-CLDC administered at 3 DPI resulted in a statistically significant (P<0.0001) sustained inhibition of tumor growth compared with tumors in mice administered only individual components of the EV-CLDC. The EV-CLDC similarly inhibited growth of longer established glioblastomas. Histopathologic evaluation of terminal tumors did not find any hemorrhage, edema or necrosis in either the EV-CLDC-treated or control tumors. The results indicate that an i.v.-administered EV-CLDC can significantly inhibit the growth of a brain tumor in immunocompetent syngeneic mice.