Pre-clinical toxicity & immunobiological evaluation of DNA rabies vaccine & combination rabies vaccine in rhesus monkeys (Macaca mulatta)

Background & objectives: Pre-clinical toxicology evaluation of biotechnology products is a challenge to the toxicologist. The present investigation is an attempt to evaluate the safety profile of the first indigenously developed recombinant DNA anti-rabies vaccine DRV (100 mu g)] and combination rabies vaccine CRV (100 mu g DRV and 1.25 IU of cell culture-derived inactivated rabies virus vaccine)], which are intended for clinical use by intramuscular route in Rhesus monkeys. Methods: As per the regulatory requirements, the study was designed for acute (single dose - 14 days), sub-chronic (repeat dose - 28 days) and chronic (intended clinical dose - 120 days) toxicity tests using three dose levels, viz. therapeutic, average (2x therapeutic dose) and highest dose (10 x therapeutic dose) exposure in monkeys. The selection of the model i.e. monkey was based on affinity and rapid higher antibody response during the efficacy studies. An attempt was made to evaluate all parameters which included physical, physiological, clinical, haematological and histopathological profiles of all target organs, as well as Tiers I, II, III immunotoxicity parameters. Results: In acute toxicity there was no mortality in spite of exposing the monkeys to 10XDRV. In sub chronic and chronic toxicity studies there were no abnormalities in physical, physiological, neurological, clinical parameters, after administration of test compound in intended and 10 times of clinical dosage schedule of DRV and CRV under the experimental conditions. Clinical chemistry, haematology, organ weights and histopathology studies were essentially unremarkable except the presence of residual DNA in femtogram level at site of injection in animal which received 10X DRV in chronic toxicity study. No Observational Adverse Effects Level (NOAEL) of DRV is 1000 ug/dose (10 times of therapeutic dose) if administered on 0, 4, 7, 14, 28th day. Interpretation & conclusions: The information generated by this study not only draws attention to the need for national and international regulatory agencies in formulating guidelines for pre-clinical safety evaluation of biotech products but also facilitates the development of biopharmaceuticals as safe potential therapeutic agents.

Effect of age and breeding season on the developmental capacity of oocytes from unstimulated and follicle-stimulating hormone-stimulated rhesus monkeys

Effects of age and season on the developmental capacity of oocytes from unstimulated and FSH-stimulated rhesus monkeys were examined, Immature cumulus-oocyte complexes were matured in vitro in modified CMRL-1066 medium containing 20% bovine calf serum and

Energy substrate requirement for in vitro maturation of oocytes from unstimulated adult rhesus monkeys

The energy substrates lactate, pyruvate, and glucose were evaluated for supporting in vitro cytoplasmic maturation of rhesus monkey oocytes. A total of 321 cumulus-oocyte complexes (COCs) aspirated from greater than or equal to 1000 mum diameter follicles

17 beta-Estradiol and progesterone improve in-vitro cytoplasmic maturation of oocytes from unstimulated prepubertal and adult rhesus monkeys

BACKGROUND: Effects of 17beta-estradiol and progesterone on rhesus monkey oocyte maturation in vitro were evaluated by embryo development subsequent to IVF. METHODS AND RESULTS: In experiment 1, immature cumulus-oocyte complexes collected from unstimulated adult females during the non-breeding season were matured in modified medium CMRL-1066 containing various combinations of gonadotrophins (FSH + LH), estradiol and/or progesterone. Formation of morulae and blastocysts was greatest in oocytes matured in medium containing estradiol and/or progesterone, with or without gonadotrophins (morula 38-46%, blastocyst 14-20%) than in control oocytes matured without estradiol or progesterone (morula 14%, blastocyst 0%). In experiment 2, cumulus-oocyte complexes from unstimulated prepubertal female monkeys were matured in medium with gonadotrophins, estradiol or progesterone. The best development to the morula stage was obtained with oocytes matured with gonadotrophins and estradiol or gonadotrophins and progesterone (43 and 25 morulae, respectively), while control oocytes matured with gonadotrophins but without steroid hormones gave the poorest morula developmental response (12%). However, there was no difference in blastocyst development across all groups (0-3%). CONCLUSIONS: These results demonstrate that during rhesus monkey oocyte maturation in vitro: (i) estradiol or progesterone can improve oocyte developmental competence; (ii) immature oocytes from prepubertal versus adult females have differential responses to challenge with estradiol or progesterone.

PHYLOGENY OF RHESUS-MONKEYS (MACACA-MULATTA) AS REVEALED BY MITOCHONDRIAL-DNA RESTRICTION ENZYME ANALYSIS

Mitochondrial DNA, purified from 36 samples of 23 local populations which are widely distributed in Vietnam, Burma, and 10 provinces of China, has been analyzed to model the phylogeny of rhesus monkeys. The 20 local populations of China may represent nearly all major populations in China. Using 20 restriction endonucleases of 6-bp recognition, we observed a total of 50-61 sites in the various samples. By combining the cleavage patterns for each enzyme, the 36 samples were classified into 23 restriction types, each of which was found exclusively in the respective population from which samples were obtained By combining the earlier study of Indian rhesus monkeys, phylogenetic trees, which have been constructed on the basis of genetic distance, indicate that rhesus monkeys in China, Vietnam, India, and Burma can be divided into seven groups. Integrating morphological and geographical data, we suggest that rhesus monkeys in China, Vietnam, and Burma may be classified into six subspecies-M. m. mulatta, M. m. brevicaudus, M. m. lasiotis, M. m. littoralis, M. m. vestita, and M. m. tcheliensis-and rhesus monkeys in India may be another valid subspecies. M. m. tcheliensis is the most endangered subspecies in China. Divergence among subspecies may have begun 0.9-1.6 Ma. The radiation of rhesus monkeys in China may have spread from the southwest toward the east. The taxonomic status of the Hainan monkey and the Taiwan monkey require further investigation.

Superovulatory response to a low dose single-daily treatment of rhFSH dissolved in polyvinylpyrrolidone in rhesus monkeys

To simplify the procedure for superovulation in the rhesus monkey, this study was designed using polyvinylpyrrolidone (PVP) solution as a solvent for gonadotropins. Thirty-five cycling females (aged 5-8 years old) were divided into six groups during the b

Effects of rhFSH regimen and time interval on ovarian responses to repeated stimulation cycles in rhesus monkeys during a physiologic breeding season

We studied the effects of repeated stimulation by recombinant human FSH (rhFSH) at various time intervals during a physiologic breeding season in rhesus monkeys. Ovarian recovery and responses were assessed by ultrasonography, serum steroid concentrations

Dynamic changes in ovarian follicles measured by ultrasonography during gonadotropin stimulation in rhesus monkeys

The objective was to study dynamic changes of ovaries in rhesus macaques stimulated by gonadotropins to identify an indicator for predicting ovarian response to stimulation. Twenty-one cycling monkeys were given 36 IU/d recombinant human follicle-stimulat

Ovarian response to gonadotropin stimulation in juvenile rhesus monkeys

The objective of this study was to investigate juvenile rhesus monkeys responding to various gonadotropin regimen Stimulations. Thirty-two prepubertal rhesus monkeys were randomly allocated into five groups for ovarian stimulation as follows: Groups I, II

A STUDY ON THE EFFECT OF LESIONS OF AREA-7 OF THE PARIETAL CORTEX ON THE SHORT-TERM VISUAL SPATIAL MEMORY OF RHESUS-MONKEYS (MACACA-MULATTA)

This research is focused on the contribution of area 7 to the short-term visual spatial memory. Three rhesus monkeys (Macaca mulatta) were trained in the direct delayed response task in which 5 delay intervals were used in each session. When each monkey reached the criterion of 90% correct responses in 5 successive sessions, two monkeys underwent a surgery while the other one received a sham operation as a control. In the first stage of the surgery, bilateral areas 7a, 7b and 7ip of the parietal cortex of two monkeys were precisely lesioned. After 7 days of recuperation, the monkeys were required to do the same task. The average percentage of correct responses in the lesioned animals decreased from 94.7% to 89.3% and 93.3% to 82.0% respectively (no significance, P > 0.05, n = 2). In addition, the monkeys' complex movements were mildly impaired. The lesioned monkeys were found to have difficulty picking up food from the wells. In the second stage, bilateral area 7m was lesioned. In the 5 postoperative sessions, the average percentage of correct responses in one monkey, with a relatively precise 7m lesion, decreased from 94.7% to 92.2% (no significance, P > 0.05), while the other monkey, with widely spread necrosis of lateral parietal cortex, showed an. obvious decline in performance, but still over the chance level. After 240 trials this monkey reattained the normal criterion. The results of this research suggest that the lesions of area 7 of the parietal cortex did not significantly affect the short-term visual spatial memory, which has been shown to be sensitive to lesions of the prefrontal cortex; they also support the notion of dissociation of spatial functions in the prefrontal and parietal cortices.

Age-related effects of bilateral frontal eye fields lesions on rapid eye movements during REM sleep in rhesus monkeys

Rapid eye movement (REM) is one of the most characteristic features of REM sleep, but the mechanisms underlying its regulation remain unclear. The present study aims to investigate whether the frontal eye field (FEF) is involved in the regulation of the r

EEG activities in the orbitofrontal cortex and dorsolateral prefrontal cortex during the development of morphine dependence, tolerance and withdrawal in rhesus monkeys

Investigating the activities of the prefrontal cortex (PFC) in the process of addiction is valuable for understanding the neural mechanism underlying the impairments of the PFC after drug abuse. However, limited data are obtained from primate animals and few studies analyze Electroencephalogram (EEG) in the gamma band, which plays an important role in cognitive functions. In addition, it is yet unclear whether drug abuse affects the orbitofrontal cortex (OFC) and dorsolateral PFC (DLPFC) - the two most important subregions of the PFC - in similar ways or not. The aim of this study is to address these issues. We recorded EEG in the OFC and DLPFC in three rhesus monkeys. All animals received a course of saline (NaCl 0.9%, 2 ml) injection (5 days) followed by 10 days of morphine injection (every 12 h), and then a further series of saline injection (7 days). A main finding in the present study was that morphine decreased EEG power in all frequency bands in a short period after injection in both the OFC and DLPFC in monkeys. And gamma power decreased not just in short period after morphine injection but lasted to 12 h after injection. Moreover, we found that although the changes in EEG activities in the OFC and DLPFC at 30-35 min after injection were similar, the DLPFC was more sensitive to the effect of morphine than the OFC. (c) 2005 Elsevier B.V. All rights reserved.