Transthyretin amyloidosis and two other aging-related amyloidoses in an aged vervet monkey.

An aged male vervet monkey showed severe cardiac arrhythmia for more than 3 years. A multifocal amyloid consisting of transthyretin was deposited in all areas of the heart wall, especially in the extracellular stroma among muscle fibers and external tunica of arterioles. Moreover, the amyloid was deposited in the stroma and arterioles of other systemic organs except the liver and spleen. These characteristics are consistent with senile systemic amyloidosis in humans. A second amyloid consisting of amyloid beta protein was in senile plaques and cerebral amyloid angiopathy in the cerebral cortex. A third amyloid consisting of islet amyloid polypeptide was deposited in islets of the pancreas. Apolipoprotein E and amyloid P component colocalized with the 3 amyloids. Thus, 3 different aging-related amyloids were found in an aged vervet monkey. In particular, to our knowledge, this is the first report on spontaneous transthyretin amyloidosis in animals.

Neuronal specificity of alpha-synuclein toxicity and effect of Parkin co-expression in primates.

Recombinant adeno-associated viral (rAAV) vector-mediated overexpression of alpha-synuclein (alphaSyn) protein has been shown to cause neurodegeneration of the nigrostriatal dopaminergic pathway in rodents and primates. Using serotype-2 rAAV vectors, we recently reported the protective effect of Parkin on alphaSyn-induced nigral dopaminergic neurodegeneration in a rat model. Here we investigated the neuronal specificity of alphaSyn toxicity and the effect of Parkin co-expression in a primate model. We used another serotype (type-1) of AAV vector that was confirmed to deliver genes of interest anterogradely and retrogradely to neurons in rats. The serotype-1 rAAV (rAAV1) carrying alphaSyn cDNA (rAAV1-alphaSyn), and a cocktail of rAAV1-alphaSyn and rAAV1 carrying parkin cDNA (rAAV1-parkin) were unilaterally injected into the striatum of macaque monkeys, resulting in protein expression in striatonigral GABAergic and nigrostriatal dopaminergic neurons. Injection of rAAV1-alphaSyn alone decreased tyrosine hydroxylase immunoreactivity in the striatum compared with the contralateral side injected with a cocktail of rAAV1-alphaSyn and rAAV1-parkin. Immunostaining of striatonigral GABAergic neurons was similar on both sides. Overexpression of Parkin in GABAergic neurons was associated with less accumulation of alphaSyn protein and/or phosphorylation at Ser129 residue. Our results suggest that the toxicity of accumulated alphaSyn is not induced in non-dopaminergic neurons and that the alphaSyn-ablating effect of Parkin is exerted in virtually all neurons in primates.

In vivo selective expansion of gene-modified hematopoietic cells in a nonhuman primate model.

A major problem limiting hematopoietic stem cell (HSC) gene therapy is the low efficiency of gene transfer into human HSCs using retroviral vectors. Strategies, which would allow in vivo expansion of gene-modified hematopoietic cells, could circumvent the problem. To this end, we developed a selective amplifier gene (SAG) consisting of a chimeric gene composed of the granulocyte colony-stimulating factor (G-CSF) receptor gene and the estrogen receptor gene hormone-binding domain. We have previously demonstrated that primary bone marrow progenitor cells transduced with the SAG could be expanded in response to estrogen in vitro. In the present study, we evaluated the efficacy of the SAG in the setting of a clinically applicable cynomolgus monkey transplantation protocol. Cynomolgus bone marrow CD34(+) cells were transduced with retroviral vectors encoding the SAG and reinfused into each myeloablated monkey. Three of the six monkeys that received SAG transduced HSCs showed an increase in the levels of circulating progeny containing the provirus in vivo following administration of estrogen or tamoxifen without any serious adverse effects. In one monkey examined in detail, transduced hematopoietic progenitor cells were increased by several-fold (from 5% to 30%). Retroviral integration site analysis revealed that this observed increase was polyclonal and no outgrowth of a dominant single clonal population was observed. These results demonstrate that the inclusion of our SAG in the retroviral construct allows selective in vivo expansion of genetically modified cells by a non-toxic hormone treatment.

Specific gravity of whole blood in cynomolgus monkeys (Macaca fascicularis), squirrel monkeys (Saimiri sciureus), and tamarins (Saguinus labiatus) and total blood volume in cynomolgus monkeys.

Blood collection is a common laboratory procedure in animal experiments. The purpose of this study is to establish baseline data for two essential hematologic parameters, total blood volume (TBV) and specific gravity of blood (SGB), of nonhuman primates. The SGB was determined by dropping samples of whole blood into cupric sulfate solution. The values for the mean SGB +/- 1 standard deviation are: cynomolgus monkeys, 1.0526 +/- 0.0019 [males (n = 39), 1.0531 +/- 0.0017; females (n = 48), 1.0522 +/- 0.001]; squirrel monkeys, 1.0555 +/- 0.0037 [males (n = 56), 1.0581 +/- 0.0027; females (n = 76), 1.0536 +/- 0.0032]; and tamarins, 1.0582 +/- 0.0020 [males (n = 13), 1.0582 +/- 0.0023; females (n = 17), 1.0581 +/- 0.0018]. To determine the TBV, blood was collected in tubes containing 1.5 mg EDTA after intravenous injection of Evans Blue solution. The TBV was obtained after correcting for the hematocrit and the dilution factor of the Evans Blue solution. The formulae were established to estimate TBV by referring to body weight (BW). There was no significance between TBV and BW in male monkeys weighing more than 6 kg.

A new method for bone marrow cell harvesting.

To minimize contamination of bone marrow cells (BMCs) with T cells from the peripheral blood, a new "perfusion method" for collecting BMCs is proposed using cynomolgus monkeys. Two BM puncture needles are inserted into a long bone such as the humerus, femur, or tibia. One needle is connected to an extension tube and the end of the tube is inserted into a culture flask to collect the BM fluid. The other needle is connected to a syringe containing 30 ml of phosphate-buffered saline. The solution is pushed gently from the syringe into the medullary cavity, and the medium containing the BM fluid is collected into the culture flask. There is significantly less contamination with peripheral blood, determined from the frequencies of CD4(+) and CD8(+) T cells, when using this method (<6%) than when using the conventional method (>20%) consisting of multiple BM aspirations from the iliac crest. Furthermore, the number and progenitor activities of the cells harvested using this "perfusion method" are greater than those harvested using the conventional aspiration method. This perfusion method was carried out 42 times using 15 cynomolgus monkeys, and no complications such as pulmonary infarction or paralysis were observed. These findings suggest that the "perfusion method" is safe and simple and would be of great advantage in obtaining pure BMCs, resulting in a less frequent occurrence of acute graft-versus-host-disease in allogeneic BM transplantation.