Therapeutic expression of human clotting factors IX and X following adeno-associated viral vector-mediated intrauterine gene transfer in early-gestation fetal macaques.

Adeno-associated viral vectors (AAVs) achieve stable therapeutic expression without long-term toxicity in adults with hemophilia. To avert irreversible complications in congenital disorders producing early pathogenesis, safety and efficacy of AAV-intrauterine gene transfer (IUGT) requires assessment. We therefore performed IUGT of AAV5 or -8 with liver-specific promoter-1 encoding either human coagulation factors IX (hFIX) or X (hFX) into Macaca fascicularis fetuses at ∼0.4 gestation. The initial cohort received 1 × 1012 vector genomes (vgs) of AAV5-hFIX ( n = 5; 0.45 × 1013 vg/kg birth weight), resulting in ∼3.0% hFIX at birth and 0.6-6.8% over 19-51 mo. The next cohort received 0.2-1 × 1013 vg boluses. AAV5-hFX animals ( n = 3; 3.57 × 1013 vg/kg) expressed <1% at birth and 9.4-27.9% up to 42 mo. AAV8-hFIX recipients ( n = 3; 2.56 × 1013 vg/kg) established 4.2-41.3% expression perinatally and 9.8-25.3% over 46 mo. Expression with AAV8-hFX ( n = 6, 3.12 × 1013 vg/kg) increased from <1% perinatally to 9.8-13.4% >35 mo. Low expressers (<1%, n = 3) were postnatally challenged with 2 × 1011 vg/kg AAV5 resulting in 2.4-13.2% expression and demonstrating acquired tolerance. Linear amplification-mediated-PCR analysis demonstrated random integration of 57-88% of AAV sequences retrieved from hepatocytes with no events occurring in or near oncogenesis-associated genes. Thus, early-IUGT in macaques produces sustained curative expression related significantly to integrated AAV in the absence of clinical toxicity, supporting its therapeutic potential for early-onset monogenic disorders.-Chan, J. K. Y., Gil-Farina I., Johana, N., Rosales, C., Tan, Y. W., Ceiler, J., Mcintosh, J., Ogden, B., Waddington, S. N., Schmidt, M., Biswas, A., Choolani, M., Nathwani, A. C., Mattar, C. N. Z. Therapeutic expression of human clotting factors IX and X following adeno-associated viral vector-mediated intrauterine gene transfer in early-gestation fetal macaques.

Durable engraftment of genetically modified FVIII-secreting autologous bone marrow stromal cells in the intramedullary microenvironment.

Genetically modified FVIII-expressing autologous bone marrow-derived mesenchymal stromal cells (BMSCs) could cure haemophilia A. However, culture-expanded BMSCs engraft poorly in extramedullary sites. Here, we compared the intramedullary cavity, skeletal muscle, subcutaneous tissue and systemic circulation as tissue microenvironments that could support durable engraftment of FVIII-secreting BMSC in vivo. A zinc finger nuclease integrated human FVIII transgene into PPP1R12C (intron 1) of culture-expanded primary canine BMSCs. FVIII-secretory capacity of implanted BMSCs in each dog was expressed as an individualized therapy index (number of viable BMSCs implanted × FVIII activity secreted/million BMSCs/24 hours). Plasma samples before and after implantation were assayed for transgenic FVIII protein using an anti-human FVIII antibody having negligible cross-reactivity with canine FVIII. Plasma transgenic FVIII persisted for at least 48 weeks after implantation in the intramedullary cavity. Transgenic FVIII protein levels were low after intramuscular implantation and undetectable after both intravenous infusion and subcutaneous implantation. All plasma samples were negative for anti-human FVIII antibodies. Plasma concentrations and durability of transgenic FVIII secretion showed no correlation with the therapy index. Thus, the implantation site microenvironment is crucial. The intramedullary microenvironment, but not extramedullary tissues, supported durable engraftment of genetically modified autologous FVIII-secreting BMSCs.

Stable human FIX expression after 0.9G intrauterine gene transfer of self-complementary adeno-associated viral vector 5 and 8 in macaques.

Intrauterine gene transfer (IUGT) offers ontological advantages including immune naiveté mediating tolerance to the vector and transgenic products, and effecting a cure before development of irreversible pathology. Despite proof-of-principle in rodent models, expression efficacy with a therapeutic transgene has yet to be demonstrated in a preclinical nonhuman primate (NHP) model. We aimed to determine the efficacy of human Factor IX (hFIX) expression after adeno-associated-viral (AAV)-mediated IUGT in NHP. We injected 1.0-1.95 × 10(13) vector genomes (vg)/kg of self-complementary (sc) AAV5 and 8 with a LP1-driven hFIX transgene intravenously in 0.9G late gestation NHP fetuses, leading to widespread transduction with liver tropism. Liver-specific hFIX expression was stably maintained between 8 and 112% of normal activity in injected offspring followed up for 2-22 months. AAV8 induced higher hFIX expression (P = 0.005) and milder immune response than AAV5. Random hepatocellular integration was found with no hotspots. Transplacental spread led to low-level maternal tissue transduction, without evidence of immunotoxicity or germline transduction in maternal oocytes. A single intravenous injection of scAAV-LP1-hFIXco to NHP fetuses in late-gestation produced sustained clinically-relevant levels of hFIX with liver-specific expression and a non-neutralizing immune response. These data are encouraging for conditions where gene transfer has the potential to avert perinatal death and long-term irreversible sequelae.

Using Sterile Flocked Swabs as an Alternative Method for Rodent Health Monitoring.

Routine health monitoring is an integral part of managing SPF rodent colonies. In recent years, rack-level environmental sampling has been introduced as an adjunct method or replacement for exposure of sentinel rodents to soiled bedding. However, rack-level environmental monitoring is not compatible with rodent housing systems that have cage-level filtration. The current study investigated whether exposure of sterile flocked swabs to soiled bedding can be an alternative sampling method for routine health monitoring in mice, thus replacing the use of sentinels in soiled-bedding cages. Flocked swabs were placed in cages containing pooled samples of soiled bedding but no mice; swabs remained there for 90 d, with weekly agitation and biweekly swabbing of the cage floor to mimic the agitation of soiled bedding by sentinel mice and facilitate the collection of dust particles. Fecal samples were collected from both colony and sentinel mice. For environmental samples, exhaust debris was collected from the rack plenum, and dust samples were collected from the exhaust hose. All samples were collected on days 88 through 91 and were tested for multiple pathogens by using real-time PCR assays. To determine the diagnostic agreement of flocked swab sampling with the other methods, we used κ statistics to compare the test results from flocked swabs with those from sentinel feces, exhaust debris, and colony animal feces; we found excellent agreement between the colony feces and the flocked swab methods. The sterile flocked swab method detected all enzootic pathogens in the colonies tested. Results from flocked swab samples had the least agreement with sentinel feces, which also failed to detect the presence of fur mites. This study supports the use of sterile flocked swabs as alternative to using sentinel mice, thus conforming to the guiding principles of replacement and reduction in the use of animals for routine colony health monitoring.

Investigation of meticillin-resistant Staphylococcus aureus in pigs used for research.

Meticillin-resistant Staphylococcus aureus (MRSA) was unexpectedly isolated from a pig used for streptozotocin-induced diabetes research. To investigate the possible source of the MRSA isolate, nasal swabs were obtained from the animal herd, and from animal holding rooms, and veterinary and research staff involved in the handling of the animals. Overall, four MRSA isolates were cultured from three pigs and from a clinician/scientist. Two were ST22-MRSA-IV, a human strain type associated with epidemic spread. The other two were ST398-MRSA-V, a strain type associated with pigs. Thus, care should be taken to prevent cross-transmission of MRSA.

Laboratory Animal Laws, Regulations, Guidelines and Standards in China Mainland, Japan, and Korea.

China, Japan, and Korea have spent decades developing and amending laws, regulations, and guidelines to address the humane care and use of laboratory animals. This process began in 1983 in China, 1973 in Japan, and 1991 in Korea and has continued to the present. The governmental oversight of research varies between these countries, ranging from regulations by multiple levels of government in China to self-regulation under multiple government guidelines in Japan. Common to all is incorporation of the internationally recognized principles of the 3Rs: replacement, reduction and refinement. This paper reviews how the framework of laws, regulations, and guidelines evolved in each of these countries, their current status, and the expectation that they will continue to evolve.

Angiotensin II type 1 and 2 receptors in conduit arteries of normal developing microswine.

OBJECTIVE: To identify vascular cells capable of responding to angiotensin II (Ang II) generated in conduit arteries, we examined the Ang II type 1 receptor (AT1R) and Ang II type 2 receptor (AT2R) in the thoracic aorta (TA) and abdominal aorta (AA) and branches in 90-day fetal, 3-week postnatal, and 6-month adult microswine. METHODS AND RESULTS: By autoradiography ((125)I-[Sar(1)Ile(8)]-Ang II with or without AT1R- or AT2R-selective analogues or (125)I-CGP 42112), there were striking rostrocaudal differences in (1) AT2R binding at all ages (prominent in AA wall and branches, sparse in TA wall and branches) and (2) a non-AT2R binding site for CGP 42112 (consistently evident in postnatal TA and branches but absent in AA and branches). Furthermore, patterns of AT2R distribution in infradiaphragmatic arteries were developmentally distinct. In fetal AAs, high-density AT2Rs occupied the inner 60% of the medial-endothelial wall. In postnatal AAs, AT2Rs were sparse in the medial-endothelial wall but prominent in a circumferential smooth muscle alpha-actin-negative cell layer at the medial-adventitial border, occupying approximately 20% to 25% of the AA cross-sectional area. AT1R density in the TA and AA medial-endothelial wall increased with age, whereas AT2R density decreased after birth. CONCLUSIONS: A novel AT2R-positive cell layer confined to postnatal infradiaphragmatic arteries physically links adventitial and medial layers, appears optimally positioned to transduce AT2R-dependent functions of local Ang II, and suggests that adventitial Ang II may elicit regionally distinct vascular responses.

A simple device for humidification of inspired gases during volatile anesthesia in rats.

The typical setup for administering volatile anesthetics to laboratory rats does not provide for humidification of the inspired gases, although it is known that failing to provide humidification can lead to airway inflammation and impaired pulmonary function during prolonged experimental protocols. We developed a simple humidification system in which a nebulizer was inserted into the nonrebreathing circuit used with a standard isoflurane vaporizer. We show here that the nebulizer system resulted in anesthetic stability, as measured by withdrawal reflex latency. Although the isoflurane concentration in the delivered gases was reduced, the reduction was a consistent percentage of the vaporizer output throughout the anesthetic range, thereby permitting straightforward adjustment of the vaporizer output.

Stepwise training for reconstructive microsurgery: the journey to becoming a confident microsurgeon in singapore.

Microsurgery training in Singapore began in 1980 with the opening of the Experimental Surgical Unit. Since then, the unit has continued to grow and have held microsurgical training courses biannually. The road to becoming a full-fledged reconstructive surgeon requires the mastering of both microvascular as well as flap raising techniques and requires time, patience and good training facilities. In Singapore, over the past 2 decades, we have had the opportunity to develop good training facilities and to refine our surgical education programmes in reconstructive microsurgery. In this article, we share our experience with training in reconstructive microsurgery.

Animal ethics in SIRS research.

It is well recognized that animals play a vital role and are indispensable to scientific and medical research. Over the years, a number of non-animal procedures have been developed. However, despite all the advances in science, as yet, no system has been evolved which can completely replace a living system to conduct basic research. There is still a need to test food, drugs, medical devices, treatment regimes etc. on some animals before they can be tested and used (if found suitable) in human beings. Even the most sophisticated technology models have failed to mimic completely the complex cellular interactions occurring in a living system. The search for a complete alternative to animal research is still on and in the mean time we can all help play our part by conducting animal research in a humane and responsible fashion. This chapter discusses the ethical issues in animal research highlighting the need to use animals conscientiously.

Morphometrics and pelage characterization of longtailed macaques (Macaca fascicularis) from Pulau Bintan, Indonesia; Singapore; and Southern Vietnam.

Cynomolgus (or longtailed) macaques (Macaca fascicularis) are used extensively as laboratory animals in biomedical research. Their use in Singapore, an emerging biomedical hub in Southeast Asia, is now increasing widely, with research subjects currently originating from Singapore, Vietnam, and Pulau Bintan, Indonesia. Limited data exist on the genetic and phenotypic polymorphisms and phylogenetic relationships of these groups, and the animals are used as research subjects without regard to potential differences or homogeneity. Here we characterize their phenotypes by using established primatology tools to detail morphometrics and pelage erythrism and saturation. Pelage analyses supported the Gloger rule, in which heavily pigmented forms predominate near the equator, with Singaporean and Bintan macaques having darker pelage than Vietnamese macaques. Morphometric variation patterns suggest a tendency toward insular dwarfism and correlate generally with the Bergmann rule, in which body mass increases with latitude and colder climate. Although the 3 populations all belong to the nominotypical subspecies M. f. fascicularis, phenotypic differences are evident and are valuable tools to analyze their phylogeographic history and phylogenetic relationships.

Cerebellar abscess in a cynomolgus macaque (Macaca fascicularis).

BACKGROUND: A cynomolgus macaque (Macaca fascicularis) presented with decreased appetite, lethargy, ataxia, disorientation and visual impairment. It was used in a hepatitis B (HBV) study involving injections of HBV plasmid construct (450 microg) and aflatoxin B1 (25 microg/kg) in an effort to develop a model of liver cirrhosis and hepatocellular carcinoma. METHODS: The case work-up included physical examination, including assessment of hydration, thoracic and abdominal radiographs and abdominal ultrasound. Clinical pathology included complete blood counts and tests for levels of plasma ammonia (NH(3)) and serum electrolytes, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, total bilirubin, and gamma-glutamyl transferase. Serum samples had also been serially tested for the presence of HBsAg, anti-HBc antibodies, HBV e-antigen, and HBV DNA. With a tentative diagnosis of hepatic encephalopathy, treatment with lactulose, antibiotics, and fluid therapy was initiated. RESULTS: Clinical pathology and diagnostic imaging performed on the animal revealed no abnormalities except for the hyperammoniemia. Absence of HBV markers in the serum indicated unsuccessful inoculation. Not fully responding to therapeutic intervention, the animal was euthanized. Necropsy revealed fibrous peritoneal adhesions and an abscess in the cerebellopontine angle. Exudate culture indicated the presence of alpha-hemolytic streptococcus, Eubacterium lentum, and Bacteroides stercoris. CONCLUSIONS: Brain abscesses are uncommon in non-human primates. This case of cerebellar abscess is characterized by important features similar to that found in humans, including the presenting signs and the presence of the above-cultured bacteria.

Pregnancy alters hemodynamic responses to hemorrhage in conscious rabbits.

Pregnant animals are less able to maintain mean arterial pressure (MAP) during hemorrhage compared with nonpregnant animals, but the hemodynamic basis of this difference is unknown. The hypothesis that pregnancy attenuates responses of cardiac output, as well as total peripheral resistance (TPR) and femoral conductance, to hemorrhage was tested in conscious rabbits in both the pregnant and nonpregnant state (n = 10). During continuous slow blood loss (2% of the initial blood volume per minute), MAP was maintained initially in both groups. However, MAP then abruptly decreased to <45 mmHg in all animals after a smaller percentage of the initial blood volume was removed in pregnant compared with nonpregnant rabbits (43.6 +/- 1.7%, nonpregnant; 29.6 +/- 2.2%, pregnant; P < 0.005). The more rapid transition to hypotension exhibited by pregnant rabbits was associated with greater initial falls in cardiac output (-56 +/- 10 ml/min, nonpregnant; -216 +/- 33 ml/min, pregnant; P < 0.005) and stroke volume (0.8 +/- 0.1 ml/beat, nonpregnant; -1.3 +/- 0.1 ml/beat, pregnant; P < 0.05). In addition, the increase in TPR as a function of the decrease in cardiac output was markedly attenuated (P < 0.0001) during pregnancy. Whereas femoral conductance decreased in nonpregnant rabbits, it did not change significantly in pregnant animals. In conclusion, the lesser ability of conscious pregnant rabbits to maintain MAP during hemorrhage is due largely to a greater decrease in cardiac output but also to inadequate reflex increases in TPR, possibly in part in the femoral vascular bed.

ANG II AT(1) and AT(2) receptors in developing kidney of normal microswine.

To identify an appropriate model of human renin-angiotensin system (RAS) involvement in fetal origins of adult disease, we quantitated renal ANG II AT(1) and AT(2) receptors (AT1R and AT2R, respectively) in fetal (90-day gestation, n = 14), neonatal (3-wk, n = 5), and adult (6-mo, n = 8) microswine by autoradiography ((125)I-labeled [Sar(1)Ile(8)]ANG II+cold CGP-42112 for AT1R, (125)I-CGP-42112 for AT2R) and by whole kidney radioligand binding. The developmental pattern of renal AT1R in microswine, like many species, exhibited a 10-fold increase postnatally (P < 0.001), with maximal postnatal density in glomeruli and lower density AT1R in extraglomerular cortical and outer medullary sites. With aging, postnatal AT1R glomerular profiles increased in size (P < 0.001) and fractional area occupied (P < 0.04), with no change in the number per unit area. Cortical levels of AT2R by autoradiography fell with age from congruent with 5,000 fmol/g in fetal kidneys to congruent with 60 and 20% of fetal levels in neonatal and adult cortex, respectively (P < 0.0001). The pattern of AT2R binding in postnatal pig kidney mimicked that described in human and simian, but not rodent, species: dense AT2R confined to discrete cortical structures, including pre- and juxtaglomerular, but not intraglomerular, vasculature. Our results provide a quantitative assessment of ANG II receptors in developing pig kidney and document the concordance of pigs and primates in developmental regulation of renal AT1R and AT2R.