David Lee-Parritz

In healthy primates, circulating autoreactive T cells mediate autoimmune disease.

A T cell response against myelin basic protein (MBP) is thought to contribute to the central nervous system (CNS) inflammation that occurs in the human demyelinating disease multiple sclerosis. To test whether MBP-reactive T cells that are normally retrieved from the circulation are capable of inducing CNS disease, MBP-reactive T cell clones were isolated from the peripheral blood of healthy, unimmunized Callithrix jacchus (C. jacchus) marmosets. This primate species is characterized by a natural chimerism of bone marrow elements between siblings that should make possible adoptive transfer of MBP-reactive T cells. We report that MBP-reactive T cell clones efficiently and reproducibly transfer CNS inflammatory disease between members of C. jacchus chimeric sets. The demyelination that is characteristic of experimental allergic encephalomyelitis induced in C. jacchus by immunization against human white matter did not occur after adoptive transfer of the MBP-reactive clones. It was noteworthy that encephalitogenic T cell clones were diverse in terms of their recognition of different epitopes of MBP, distinguishing the response in C. jacchus from that in some inbred rodents in which restricted recognition of MBP occurs. These findings are the first direct evidence that natural populations of circulating T cells directed against a CNS antigen can mediate an inflammatory autoimmune disease.

Experimental allergic encephalomyelitis in cynomolgus monkeys. Quantitation of T cell responses in peripheral blood.

Chronic relapsing-remitting experimental allergic encephalomyelitis (EAE) was induced in cynomolgus monkeys by a single immunization with a homogenate of human brain white matter (BH) in adjuvant. Proliferative T lymphocyte responses to BH, to myelin basic protein (MBP), but not to proteolipid protein, were detected in peripheral blood mononuclear cells (PBMC) of all animals and persisted until their death or, in surviving animals, for greater than 10 mo postimmunization. Responses of higher magnitude tended to be associated with fatal, compared with nonfatal, episodes of clinical EAE. The frequency of MBP-reactive T cells in PBMC of animals with acute EAE was quantitated with a soft agar colony system; the ratio of T cells that proliferated specifically to MBP was estimated at between 5 and 20 per 10(6) PBMC. A similar frequency of peptide-specific T cells was estimated from PBMC of monkeys immunized with a synthetic 14-mer peptide corresponding to a region near the carboxy terminus of MBP. Thus, autoantigen-reactive T cells can be detected in the circulation throughout the course of chronic EAE, are predictive of disease severity, and occur at a frequency similar to that estimated to be present in humans with multiple sclerosis.

In Vivo T-Lymphocyte Activation and Transient Reduction of Viral Replication in Macaques Infected with Simian Immunodeficiency Virus

ABSTRACT While it is well established that cellular activation can increase human immunodeficiency virus (HIV) replication in T lymphocytes, it is also clear that both activated CD8+ and CD4+ T lymphocytes mediate anti-HIV activity. To assess the relative importance of these contrary effects on HIV replication in vivo, we evaluated the consequences of Mycobacterium bovis BCG and staphylococcal enterotoxin B (SEB) inoculation in vivo in rhesus monkeys chronically infected with simian immunodeficiency virus of macaques (SIVmac). BCG inoculation induced as much as a 2.5-log reduction of plasma and intracellular SIV RNA in SIVmac-infected monkeys. This down-regulation of virus replication persisted as long as 4 weeks after BCG inoculation. Similarly, SEB injection resulted in up to a 3-log decrease in plasma and intracellular SIV RNA in SIVmac-infected macaques. Interestingly, the short-term reduction of viremia in these monkeys correlated with the peak in vivo production of SEB- and BCG-induced cytokine responses. However, no long-term clinical benefit was observed in the SIVmac-infected macaques. These studies provide in vivo evidence that potent T-cell stimulation driven by antigens other than the virus itself can, under some circumstances, mediate short-term reduction of viremia in AIDS virus-infected individuals.

The disruption of macaque CD4+ T-cell repertoires during the early simian immunodeficiency virus infection.

Abstract: T‐cell receptor (TCR) complementarity determining region 3 (CDR3) spetratyping analysis was employed to assess the ability of an AIDS virus to disrupt CD4 + T‐cell repertoires during the primary infection. Rhesus and pig‐tailed macaques infected with simian immunodeficiency virus (SIV)mac 251 and SIVsmmFGb, respectively, were evaluated. Following SIV infection, the macaques exhibited an apparent decline of CD4 + peripheral blood lymphocyte (PBL) counts, which was associated with a change in CDR3 profiles from multiple‐length distribution to one‐ or two‐length dominance in the selected TCR Vβ‐expressing CD4 + PBL subpopulations. Molecular analysis of the perturbed cell subpopulations suggested that the CD4 + T cells bearing the dominant CDR3 length were clonally expanded. These results indicate that SIV infection can induce a disruption of macaque CD4 + T‐cell repertoires during the primary infection. The finding in this study, therefore, suggests that the virus‐induced clonal dominance can contribute to the disruption of CD4 + T‐cell repertoires.

Anaesthetic Management of Baboons Undergoing Heterotopic Porcine Cardiac Xenotransplantation

A detailed anaesthetic technique for baboons (Papio anubis) undergoing heterotopic abdominal cardiac xenotransplantation is described. Twenty-two baboons served as transplant recipients. Donors were either crossbred farm pigs (Sus scrofa) (n = 4) or transgenic pigs (Sus scrofa) (n = 18) expressing human complement regulatory proteins on the endothelium. Intra-operative management was complicated by the physiological consequences of infrarenal, abdominal aortic cross-clamping, in addition to the immunological sequelae related to cross-species transplantation. In choosing anaesthetics for this procedure, we considered the need for maximal cardiac stability throughout a long surgical procedure that required abdominal aortic cross-clamping to facilitate the implantation of an oversized porcine cardiac graft. Baboons received a balanced anaesthetic consisting of inhaled isoflurane in oxygen, intravenous fentanyl and intravenous pancuronium. The pharmacological techniques employed were found to be safe and reliable and were well tolerated by our recipients without any significant side-effects.

7 – Animal Care and Maintenance

Investigators and research institutions have an ethical and legal responsibility to minimize pain and distress in research animals. Experimental use of animals is a vital part of biomedical research. Appropriate use of animal models allows prospective, controlled disease investigation that cannot be conducted in human patients or volunteers. Investigators must consider scientific, practical, and humane issues when developing studies that use animals. High-quality research requires close collaboration between veterinary and research professionals to provide humane care and use of laboratory animals, reducing animal pain and distress to the absolute minimum while maintaining the scientific goals of the project. The chapter briefly reviews ethical and regulatory issues and lists out available information resources. Remaining sections address specific technical issues related to animal surgery, including design of surgical facilities, animal selection, anesthesia, and analgesia. Russell and Burch formulated the concept of the “three Rs” as a strategy to minimize experimental animal pain and distress. The first principle, replacement , states that non-animal models should be used instead of animals to the maximum extent possible. Although inanimate models rarely allow examination of complex physiological processes often encountered in surgical research, suitable alternatives to the use of animals in surgical education may be appropriate. The principle of reduction states that investigators should use the minimum number of animals consistent with statistical power. The most important of the three Rs is the principle of refinement , which states that investigators should use the least invasive technique possible to minimize animal pain and distress.

Animal Surgery and Care of Animals

The appropriate use of animal models in biomaterials research allows prospective, controlled evaluation of disease processes and candidate therapeutics in a manner that is impossible in human patients or volunteers. Investigators must consider scientific, practical, and humane issues when developing studies that use animals. High-quality research requires close collaboration between veterinary and research professionals to guide model selection and development, minimize animal pain and distress, and to advance the scientific goals of the project. This chapter briefly reviews ethical and regulatory issues, describes available information resources and discusses the design of surgical facilities, animal selection, anesthesia, and analgesia.

Response to Protocol Review Scenario: within his rights.

1. Office of Laboratory Animal Welfare. Guidance to IACUCs Regarding Use of Designated Member Review (DMR) for Animal Study Proposal Review Subsequent to Full Committee Review (FCR). Notice NOT-OD-09-035. (National Institutes of Health, Washington, DC, 8 January 2009). <http://grants.nih.gov/grants/guide/noticefiles/NOT-OD-09-035.html> 2. Silverman, J., Suckow, M.A. & Murthy, S. The IACUC Handbook 2nd edn. (CRS Press, Boca Raton, FL, 2007).