and Imm

some other conditions. Conclusions: The rboGM-CSF pro-

260 Abstracts / Veterinary Immunology

Dynamics of B-cell repertoire in sheep jejunal and ilealPeyer’s patch single follicles

Masahiro Yasuda 1,2, Craig N. Jenne 1, Laurie J. Kennedy 1,John D. Reynolds 1

1 Immunology Research Group, Department of Cell Biology andAnatomy, University of Calgary, Canada2 Department of Veterinary Anatomy, University of Miyazaki,JapanKeywords: Ileal Peyer’s patch; Jejunal Peyer’s patch; B-cellrepertorie; Prenatal and postnatal

Species: RuminantsIn the ruminant’s intestine, there are two types of gut-

associated lymphoid organ: jejunal Peyer’s patch (PP) andileal PP. Ileal PP is thought to be the primary lymphoidorgan of B-cell, whose repertoire is diversified by gene con-version and/or somatic hypermutation. On the other hand,jejunal PP is thought to be the secondary lymphoid organfor local mucosal immunity and the functions of this lym-phoid organ keep throughout the animal’s life. The prenataldevelopment of follicles in the PP begins first in the jejunumduring the middle of gestation and then in the ileum duringlate gestation. Therefore it can be considered that jejunal PPfollicle also contributes making primary B-cell repertoireas well as ileal PP follicle at fetal development. Then afterbirth, jejunal PP may form the character for local mucosalimmunity. We attempt to analyze B-cell repertoire of ilealand jejunal PP single follicles during ontogeny. Both PP sin-gle follicles at prenatal and postnatal development wereisolated under stereoscopic microscopy. Ig light chain isdominant light chain in sheep. V�-J�1 was amplified by PCRor PT-PCR. At the postnatal stage, ileal PP follicles containedoligoclonal B-cell, but jejunal PP follicles contained muchmore polyclonal B-cell. Similar tendency observed at prena-tal stage. Hence clonality of both PP follicles was markedlydifferent. At prenatal stage, point mutation accumulated inCDR region in V� gene of both PP follicular B-cell (14–19point mutations/kb were in ileal PP and 3–13 mutations/kbwere in jejunal PP). B-cell diversity is observed in not onlyileal PP follicle but also jejunal PP follicle. The data showthat both PP follicles contribute making B-cell repertoireduring prenatal development. At the postnatal develop-ment, much more mutations observed in CDR regions in V�gene of both PP follicular B-cell (32–64 point mutations/kbwere in ileal PP and 39–54 mutations/kb were in jejunal PP).Especially many replacement mutations observe in CDR3region of ileal PP follicles. Therefore ileal PP follicles con-

tribute making very wide diversity of B-cell after birth. Thisevent probably causes the appearance many self-reactiveB-cell.

doi:10.1016/j.vetimm.2008.10.107

unopathology 128 (2009) 211–347

Stability of the recombinant GM-CSF produced by bac-ulovirus gene expression system

Shigeki Inumaru ∗, Hideyuki Takahashi, Satoko Watanabe,Masato Ohta, Takayuki Kubota

NARO National Institute of Animal Health, JapanKeywords: GM-CSF; Stability; Therapeutics; Mastitis

E-mail address: inumaru@affrc.go.jp (S. Inumaru).

Species: RuminantsGM-CSF is known as a cytokine that affects the var-

ious haematopoietic cells. Bovine GM-CSF is expectedto use a therapeutic agent for diseases caused by com-plex and opportunistic infection such as mastitis in cows.Since natural GM-CSF is produced only trace amount byparticular cells, we established the efficient method toproduce recombinant bovine GM-CSF (rboGM-CSF) by bac-ulovirus/cell culture gene expression system and reportedthat this rboGM-CSF was a potential therapeutic agentfor subclinical mastitis of dairy cows caused by S. aureusinfection. Though the information about stability of therboGM-CSF is important to develop rboGM-CSF agent, it isnot cleared up. Therefore we are studying the stability of therboGM-CSF under several conditions. Methods: Bovine GM-CSF cDNA sequence was inserted to a baculovirus (AcNPV)genome. The rboGM-CSF was prepared with this recom-binant virus infected insect cell (TN5 cells). The culturefluid, containing rboGM-CSF was ultra-filtrated to removevirus particle and diluted with PBS with 10 % FBS (PBS+)or without FBS (PBS−). It is then stored at 4 C or −20 C.The biological activities were measured with rboGM-CSFadopted TF-1 cells. Results and Discussion: To study the sta-bility of rboGM-CSF at 4 C, rboGM-CSF in PBS+ was storedat 4 C, and the biological activity was measured. The activ-ity was not reduced at least 5 month. Similarly, the activityof rboGM-CSF in PBS− was not reduced at least 5 month.To study the stability of rboGM-CSF on the freeze and thaw,rboGM-CSF in PBS+ was frozen at −20 C and thawed repeat-edly. At least three times repetition, the biological activitywas not reduced. The activity of rboGM-CSF in PBS− wasalso not decreased by the freeze and thaw at least threetimes repetition. These results clearly showed that rboGM-CSF produced by baculovirus/cell culture gene expressionsystem is very stable at 4 C and by freeze and thaw. It isan advantageous factor to develop rboGM-CSF therapeuticagent for mastitis etc. We are studying the stability under

duced by baculovirus/cell culture gene expression systemis stable for storage at 4C and freeze and thaw.

doi:10.1016/j.vetimm.2008.10.108