Young adult rabbits were used which were bred in our own la­

boratory or were obtained from the Centraal Proefdierenbedrijf T.N.O. The animals were randomly bred Gold Agouti, Californian or Alaska males and females. The irradiations were performed when the animals were approximately 4 months old; at this age their weight was approximately 3 Kgs.

B . X-IRRADIATIONS

All irradiations were done at the Department of Radiopathology (Head: Prof. H. B. Lamberts). A Philips-Muller machine - type MG 300-was used working at 200 kV and 1 5 rnA. The filters were 0.5 mm Cu and 0.6 mm Al; the resulting radiation had an H.V.L.

of around 1 .0 mm Cu. For total body irradiations the machine was used without a tube; local irradiations were delivered through a tube with a target (focus-skin) distance of 40 em and a target aperture of 6 X 8 em.

Local irradiation of the thymus was performed with the rabbit fixed on its back. The irradiation was delivered from above with the tube aperture carefully positioned on the thymus region: from 3 em above to 5 em below the clavicle. At the skin surface the ir­

radiation dose was 7 50 rads in air ( 60 rads f min), resulting in a dose of approximately 700 rads in the thymus, scatter included.

Local irradiation of a popliteal lymph node was performed in the same way, with the tube fixed on the popliteal region. The irradiation

dose actually adsorbed in the lymph node was estimated to be somewhat smaller than in the case of the thymus because of less extensive scatter.

Sublethal total body irradiation with or without thymus shielding was given in such a way that the irradiation was delivered as ho­

mogeneously as possible throughout the animal body. The rabbits were irradiated in a circular box of p::rforated plywood, diameter 40 em, width 1 4 em, standing upright, and with an upper and lower compartment each of which housing a rabbit. Half of the irradiation was given on the right, the other half on the left skin. At a target distance of 80 em 225 rads (dose rate 2 1 rads/min) were delivered from each side, remlting in an estimated over-all tissue dose of 450 rads, scatter included. Whenever scheduled the thymus was shielded by 6 mm lead plate moulded around the lower neck and upper thoracic region; to avoid disturbance of the shielding plates the animals were anesthetized during this latter type of irradiation.

Potentially lethal irradiation with bone marrow shielding was done in a similar way with one hind leg shielded by 6 mm lead plate. As in this case the popliteal nodes would also be protected these were removed surgically 24-48 hrs. before the irradiation or locally ir­

radiated (700 rads) immediately afterwards. Again the radiation was delivered from two sides and the total estimated tissue dose was increased to 900 rads. In some cases the animals were irradiated while spread-out on a dissecting table; in this case the target distance was 1 03 em. After half of the radiation dose was delivered, the animal was turned over and the other half was given.

C. ANTIGENS AND MODES OF ANTIGEN ADMINISTRATION

Salmonella parat_yphi-B (Java) vaccine was kindly prepared by dr.

A. Jansz, Regional Public Health Laboratory (Head: dr. R. K.

Koopmans). The organisms were so cultured as to have a high con­

tent of the flagellar antigen (H-antigen); upon harvesting they were killed in formol, washed and diluted to 6 . 1 08 organisms/mi.

The usual dosis was 0. 1 ml. It may be noted that the vaccme contained 0-antigen in addition to the flagellar antigen.

3 7

Horse gamma globulin (HGG) was obtained from Pentex Inc., Kankakee, Ill., U.S.A. (Horse gamma globulins, Fraction II) . The dose used was 5 mg, freshly dissolved in I ml sterile saline.

Horse spleen ferritin (2 X recrystallized) was purchased from Nutritional Biochemical Corporation (Cleveland, Ohio, U.S.A.). The dose used was 5 mg in 0.25 ml sterile saline.

All of these antigens were given subcutaneously either in the hind leg around the Achilles tendon, or in the scapular region to stimulate the popliteal or axillary lymph node re­

spectively. To check correct choice of axillary nodes 0.5- l .O ml of trypanblue I % in saline was occasionally injected into the same skin region 2-3 min before lymph node removal; only deep-blue nodes were considered appropriate.

Two chemical sensitizers known to elicit cell-mediated types of immunity were used.

2,4-Dinitrochlorobenz:,ene (DNCB) was obtained from the dispensary (Head: Prof. T. Huizinga) of the University Hospital. Approx­

imately 1 . 5 ml of a 50 % (w/v) solution in acetone was painted on a previously shaved skin area.

2-Phenyl-4-ethoxymethylene-5-oxazolone ( oxazolone) was synthesized by dr. H. G. Seijen (Dept. ofBiochemistry, Histological Laboratory) ; the prescription (BARBER and SLACK I 949) was kindly put at our disposal by Prof. J. L. Turk (St. John's Hospital for Diseases of the Skin, London E9, England). A dose of 50 mg dry powder was applied to a previously shaved skin area, and dissolved at the ap­

plication site by 2-3 ml of 70 % ethanol.

The application sites of both DNCB and oxazolone were either the skin of the hind leg just above the heel or the right or left scapular region, in order to stimulate antigenically either a popliteal or an axillary lymph node. The application sites were kept dressed during the course of the experiment. At the moment of lymph node biopsy the dressing was removed to inspect the application site. DNCB was found to cause severe skin lesions ; oxazolone never did. In addition DNCB application as described here caused sensitization of the investigator; again oxazolone did not.

Skin auto- and allografting was done in the right and left scapular region and performed according to BILLINGHAM and MEDA wAR ( 195 1 ) . Round, ful thickness pieces of skin, diameter 1-1 .5 em, were cut with a hollow-shaped surgical knife in such a way that the subcutaneous vascular network remained undamaged. The skin piece taken from the left scapular region was always autologous­

ly grafted to the corresponding graft bed on the right side, and served as a control. The left side graft bed was always used for the allograft. The grafts were only fixed by dressing with paraffin gauze ('optule'-OPTEX Ltd., Greenford, Middlesex, England) and with elastic bandages around the thorax.

In document University of Groningen Histophysiology and electron microscopy of the immune response Veldman, Jan Edze (Page 54-57)

Related documents