Apoptotic cell death and fertility in three unilateral cryptorchid rat models.

Three rat strains have been studied, using a sensitive apoptotic detection method for germ-cell degeneration, to resolve the controversy regarding the effect of cryptorchidism on the contralateral descended testis (CDT). Sprague Dawley and Buffalo rats were made cryptorchid by operation at 20-22 days of age, while trans-scrotal (T-S) rats were a congenitally unilateral cryptorchid strain. Sham operated rats or normal T-S littermates were used as controls. Experiments were performed over a period ranging from 2 weeks to 18 months. Testis weight was assayed, as was the detection of apoptosis by agarose gel laddering and immunohistochemistry by using the TUNEL method. Labeled cells in 150 cross-sectioned testis tubules were counted on the TUNEL stained slides and the mean number of labeled cells per tubule was calculated. Paternity studies on Sprague Dawley and T-S rats were carried out at 12 and 24 weeks of age to assess fertility by the resultant number of pregnancies and litter sizes. Both Sprague Dawley and T-S rat models showed a biphasic distribution of apoptosis levels. This biphasic distribution was not observed in Buffalo rats as they were only studied at later time points (12-20 weeks). A significant effect on either testis weight or apoptosis in the CDT compared with the control descended testis (P > or = 0.1) has not been found in these three cryptorchid models, and the present results are discussed with reference to observations of other researchers in rodents and humans. While the cryptorchid testis showed a high level of labeled apoptotic cells per tubule in all rat strains, fertility was not affected and remained the same as controls at 12 and 24 weeks. There was, however, a marked strain difference in fertility in T-S as compared with Sprague Dawley rats. After 24 weeks of cryptorchidism, both control and cryptorchid T-S rats had a 44% pregnancy incidence compared with a 90% pregnancy incidence in Sprague Dawley rats. In addition, litter size in T-S control and cryptorchid rats were small compared with those of Sprague Dawley rats at 12 and 24 weeks.

In vitro fusion of human inguinal hernia with associated epithelial transformation.

The processus vaginalis (PV) is a peritoneal diverticulum which forms to allow descent of the fetal testis to the scrotum. During human development fusion and obliteration of the PV often fails to occur with the result that inguinal hernias are the most prevalent congenital abnormality requiring surgery in childhood. Androgen is proposed to regulate testicular descent via the genitofemoral nerve which releases the neuropeptide calcitonin gene-related peptide (CGRP). It is possible that subsequent fusion of the PV and tissue remodelling following descent is indirectly controlled by androgen via CGRP action. An organ culture assay was developed to assess fusion of the PV taken from inguinal herniotomy in infants. Fusion was induced in vitro by CGRP but not by CGRP 8-37, CGRP 27-37 or dihydrotestosterone in equimolar concentrations. Fusion was accompanied by transformation of the epithelium, as shown by staining of intermediate filament proteins, cytokeratin and vimentin. Localization studies for CGRP receptors on 25 specimens indicated CGRP acts on mesenchymal fibroblasts but not directly on PV epithelium suggesting an indirect pathway. Hepatocyte growth factor/scatter factor was found to induce fusion of PV and may be involved as an intermediate molecule in the fusion cascade. This study represents the first approach to understanding the humoral control and underlying mechanism by which the PV fuses.

Localization of calcitonin gene-related peptide within the genitofemoral nerve in immature rats.

BACKGROUND/PURPOSE: Calcitonin gene-related peptide (CGRP) has been proposed to influence migration and testicular descent by release from the genitofemoral nerve. The site of CGRP within the nerve has been controversial, with conflicting views on whether CGRP is synthesised and released from the motor nerves. METHODS: The genitofemoral nerve (GFN) was retrogradely labelled by fluorescent dye (DAPI) in 25 Sprague-Dawley rats (days 5, 16, and 31, n = 8 in each group; day 35, n = 1). Spinal cords and dorsal root ganglia (DRG) were removed two to three days later and sectioned for immunofluorescence. Substance P and CGRP-containing cells were labelled with fluorescein-linked antibodies. Specimens were examined by double fluorescence to identify cells with both markers. RESULTS: The motor nucleus of the GFN contained 119 cells on day 7 and 284 cells by days 19 through 34. A prominent band of CGRP-containing fibers, arising from the dorsal horn, synapsed with the GFN motor nucleus itself. CGRP-labelled GFN cells were found in the DRG by double labelling. CONCLUSIONS: CGRP from the GFN may affect gubernacular migration by release from the sensory nerves, rather than motor nerves as previously thought. The GFN motor nucleus receives CGRP-containing innervation from the dorsal horn, which may form part of the cremasteric reflex.

Congenital undescended testes in neonatal pigs and the effect of exogenous calcitonin gene-related peptide.

PURPOSE: We investigated the neonatal piglet as a possible animal model for cryptorchidism and to determine whether calcitonin gene-related peptide (CGRP), which has been proposed to regulate inguinoscrotal testicular descent, could induce testicular descent in piglets with congenital cryptorchidism. MATERIALS AND METHODS: We examined 38 cryptorchid piglets to document the anatomy in 8 and to investigate the role of CGRP in 30. The 2-week-old piglets were allocated randomly to receive a mini-osmotic pump containing CGRP at various concentrations or phosphate buffered saline. The pump was inserted surgically into the ipsilateral scrotum, with the contents blinded to the surgeon. The positions of the testes, pump and anatomical landmarks were measured and photographed. The pigs were sacrificed and dissected 2 weeks later, and the positions were remeasured and photographed. The testes were examined histologically. RESULTS: The 3 variants of cryptorchidism observed were intra-abdominal in 20 cases, inguinal in 9 and lateral inguinal ectopic in 9. CGRP had no effect on intra-abdominal or ectopic testes. In contrast, for inguinal testes exogenous CGRP caused a slight but significant 10 +/- 7.9 mm. descent towards the pump in 5 cases compared to -2.9 +/- 5.8 mm. in 4 controls. CONCLUSIONS: Exogenous CGRP stimulated migration of inguinal testes that had been arrested in the line of descent while ectopic testes did not respond. These results support a role for CGRP in testicular descent and suggest that a slow release depot preparation might be useful as a possible treatment in some forms of cryptorchidism.

Does testosterone diffuse down the wolffian duct during sexual differentiation?

PURPOSE: Sexual differentiation in gonadal dysgenesis is commonly asymmetrical. In patients with true hermaphroditism there may be an ovary and müllerian duct on 1 side, and a testis and wolffian duct on the other side. Such asymmetry suggests that testicular hormones only act locally at this early stage of sexual differentiation. We tested the hypothesis that testosterone reaches the wolffian duct by transport down the duct rather than by simple diffusion. MATERIALS AND METHODS: Mouse 14-day urogenital ridges were placed in organ culture and microinjected with testosterone-albumin-fluorescein isothiocyanate. RESULTS: At 17 hours fluorescence was found throughout the wolffian duct and by 48 hours it was maximal in the dilated caudal end. CONCLUSIONS: Our results support the hypothesis that androgens may be transported along the wolffian duct. Secretion of testicular hormones into the wolffian duct may maintain hormone levels in the biologically active range.

Characterization of the gubernacular contractile response to calcitonin gene-related peptide in the mouse.

Calcitonin gene-related peptide (CGRP) has no effect on quiescent skeletal muscle, but cultured neonatal mice gubernacula show rhythmic contractions in response to CGRP. This study investigated whether these contractions (1) require calcium ions, (2) are activated via acetylcholine receptors, or (3) require dihydropyridine receptors. Gubernacula (n = 20 for each experiment) from male mice aged 7 days old were preincubated for 30 minutes in aerated culture medium (Krebs-Henseleit solution) with (1) up to 1.8 mmol/L of calcium with or without CGRP (714 nmol/L), (2) up to 5.0 mumol/L of curare with CGRP and calcium (1.8 mmol/L), or (3) up to 1 mumol/L of nifedipine with CGRP and calcium. Then they were placed on agar-coated grids over the same compound solutions, and observed for 2 days by video camera to see contractions. Of gubernacula cultured with calcium (1.8 mmol/L) and CGRP, 90% showed contractions, which decreased to 20% without CGRP (P < .001) and 15% without calcium (P < .001). Only 5% of gubernacula cultured without both CGRP and calcium showed contractions. Also these contractions in response to CGRP depended on calcium concentrations in a dose-dependent manner. Curare did not suppress contractions. With increasing dose of nifedipine, the percentage of gubernacula contracting decreased from 95% to 0%. These results suggest that developing mouse gubernacular contractions in response to CGRP require influx of calcium ions to the sarcoplasm via dihydropyridine receptors in a similar manner to contractions of immature cardiac or smooth muscles, and these contractions are not activated via acetylcholine receptors.

The importance of DR4Dw4 beta chain residues 70, 71, and 86 in peptide binding and T cell recognition.

The expression of specific alleles of the human HLA-DR locus is associated with increased risk for the development of rheumatoid arthritis. Examination of the amino acid sequence of the DR beta chain has revealed that risk for RA correlates with a cluster of polymorphic residues located between positions 67 and 86, and in particular with the identity of residues 70, 71, and 86. To examine the contributions of these HLA-DR polymorphic residues to antigen-specific T cell responses, the DRB1*0401 gene was subjected to site-directed mutagenesis and forms possessing alanine in place of the naturally occurring amino acid at positions 70, 71, 86, and 70/71 were generated. The mutated genes were coexpressed with the DRA gene in Chinese hamster ovary cells and the transfectants were tested as stimulator cells for a panel of three human influenza virus hemagglutinin-specific T cell clones. Additionally, soluble forms of the mutant DR molecules were examined for their ability to bind peptide. All of the mutants had a modest loss of affinity for the peptide relative to the wild type, but there were no significant differences in peptide binding ability among the substituted molecules. In contrast to the relatively uniform influence on peptide binding, the impact of these mutations on T cell stimulation was heterogeneous. Specifically, these studies indicate that residue 71 plays a critical role in T cell stimulation either through direct contact with the T cell receptor or by changing the orientation or conformation of the peptide-MHC complex. Replacement of residue 71 with alanine abrogated stimulation of all of the T cell clones. Two of three clones were affected by changes at residue 70 while none lost recognition when amino acid 86 was converted from Val to Ala. These data emphasize that subtle alterations in structure can have a profound impact on T cell recognition.

Measurement of radiographic changes in adjuvant-induced arthritis in rats by quantitative image analysis.

OBJECTIVE: To apply quantitative analytical methods to the evaluation of radiographic images in experimental arthritis. METHODS: Adjuvant was used to induce arthritis in rats. Arthritis progression was followed by conventional methods. In addition, digitized images of radiographs of the calcaneus were examined for changes in the mean and in the distribution pattern of gray values. Periosteal new bone formation was measured as an increase in image area of the calcaneus. RESULTS: Significant changes in the gray value profile and increases in periosteal bone formation occurred in arthritic rats. More extensive changes occurred in Lewis rats than in Sprague-Dawley rats. Analysis of serial radiographs revealed an initial decrease in the density of juxtaarticular bone, followed by progressive increases in gray value variation due to concurrent bone loss and bone formation. Eventually, bone formation in arthritic rats resulted in increased gray values above those in nonarthritic rats. CONCLUSION: Image analysis represents a sensitive, quantitative method for detecting radiographic changes in experimental arthritis.

Effect of the carbocyclic nucleoside analogue MDL 201,112 on inhibition of interferon-gamma-induced priming of Lewis (LEW/N) rat macrophages for enhanced respiratory burst and MHC class II Ia+ antigen expression.

The effects of the carbocyclic nucleoside MDL 201,112 and the purine nucleoside adenosine on the interferon-gamma (IFN-gamma)-induced priming of macrophages (m phi s) for the respiratory burst and major histocompatibility class II (MHC class II) Ia+ antigen expression were compared. Priming of purified, peritoneal m phi s from Lewis (LEW/N) rats for 18 h with recombinant rat IFN-gamma (rRaIFN-gamma) in the presence of either adenosine (100 microM) or MDL 201,112 (10 microM) resulted in a fourfold decrease in superoxide anion (O2-) production after stimulation with opsonized zymosan. Both agents were effective even when added 2 or 4 h after rRaIFN-gamma treatment. Peritoneal m phi s from LEW/N rats stimulated with LPS/rRaIFN-gamma were observed to secrete immunoreactive and bioactive TNF-alpha over 18 h in vitro and this cytokine could be dose-dependently inhibited by MDL 201,112. MDL 201,112 did not bind to classical A1 or A2 receptors on rat brain homogenates. Physiological levels of adenosine deaminase, or treatment with the nucleoside transport inhibitor dipyridamole, reversed the effects of adenosine; however, these agents at physiological concentrations had little or no effect on the inhibition of O2- release mediated by MDL 201,112. Furthermore, incubation of LEW/N m phi s for 18 h in vitro with rRaIFN-gamma resulted in significant enhancement of MHC class II Ia+ antigen expression, and these levels could be blocked by nearly 50% by either MDL 201,112 (10 microM) or adenosine (100 microM). MDL 201,112 and adenosine were also effective in decreasing m phi opsonized zymosan-stimulated O2- levels and MHC class II Ia+ antigen expression in vivo. The effects of MDL 201,112 on the down-regulation of heat-killed M. tuberculosis-activated LEW/N m phi MHC class II Ia+ antigen expression in vitro appear to be mediated by a novel pathway, because there was no rank order of potency of ADO A1 or A2 agonist/antagonists (CCPA, NECA, XAC, or CPT) in our in vitro system. In summary, our data provide compelling evidence that immunoregulatory carbocyclic nucleoside analogues such as MDL 201,112 or adenosine appear to regulate LEW/N rat m phi activation through novel molecular mechanisms and may have important therapeutic implications for acute and chronic inflammatory diseases.

Cysteine proteinase inhibitors decrease articular cartilage and bone destruction in chronic inflammatory arthritis.

OBJECTIVE: To determine the effects of peptidyl fluoromethyl ketones on the in vitro activity of purified cathepsins B and L, on tissue cysteine proteinase activity, and on cartilage and bone destruction in experimental arthritis. METHODS: The effects of the fluoroketones on cathepsins B and L in vitro and the effects of oral administration of fluoroketones on ex vivo cysteine proteinase activity in tissue homogenates were determined by measuring the inhibition of fluorogenic substrate cleavage. To determine the effects on arthritis, animals were injected with adjuvant or type II collagen, treated orally with the fluoroketones, and the severity of arthritis was assessed by clinical, histologic, and radiologic methods. RESULTS: All of the fluoroketones tested were potent inhibitors of purified cathepsins B and L activity. Oral administration of the fluoroketones reduced tissue cysteine proteinase activity by up to 77%. In addition, fluoroketone treatment significantly reduced the severity of clinical joint disease and decreased the destruction of articular cartilage and bone. Quantitative analysis of radiographic images indicated that treatment significantly reduced soft tissue changes, periosteal proliferation, and bone erosion, but only partially reduced juxtaarticular osteoporosis. CONCLUSION: These studies suggest that cysteine proteinase inhibitors may limit tissue destruction in diseases such as rheumatoid arthritis.

The tammar wallaby (Macropus eugenii) and the Sprague-Dawley rat: comparative anatomy and physiology of inguinoscrotal testicular descent.

Inguinoscrotal testicular descent in the tammar wallaby (Macropus eugenii) and the Sprague-Dawley rat was studied by macroscopic dissection, histological evaluation and organ culture bioassay. In 3 or 4 d Sprague-Dawley rats (n = 10) the gubernacular tip bulged free from the surrounding tissues, particularly with the application of abdominal pressure. Microscopic examination revealed that only the body of the gubernaculum is connected posteriorly to the pubic region. In contrast, macroscopic dissection of male tammar wallabies (n = 17) revealed a densely adherent distal gubernacular attachment to the inside of the fibrous scrotal bulge while the body of the gubernaculum was less firmly attached. These attachments were present throughout the process of testicular descent, illustrating an important anatomical difference between these species. The gubernaculum from the tammar wallaby pouch young was studied in organ culture with rat calcitonin gene-related peptide for 4 d. Rhythmic gubernacular contractions similar to those documented previously in the rat were not observed. The hypothesis proposed in the rat for the control of inguinoscrotal gubernacular migration via the genitofemoral nerve and its neurotransmitters may not be applicable in marsupial mammals.

Germ cell development in neonatal mouse testes in vitro requires müllerian inhibiting substance.

To study the effect of müllerian inhibiting substance on testicular germ cell development, especially on gonocytes, whole testes (156) from newborn mice were cultured for 1 to 7 days in vitro. The synthetic medium contained either 10% fetal calf serum, which itself contains endogenous müllerian inhibiting substance, or transferrin, insulin and retinoic acid. Human recombinant müllerian inhibiting substance, rabbit antiserum against müllerian inhibiting substance and/or normal rabbit serum was added to some cultures. The cultured testes were fixed in Stieve's fixative and stained with hematoxylin and eosin, and the numbers and types of germ cells per tubule were counted under a light microscope. Preliminary studies showed that germ cell development in newborn mouse testes was similar in vitro to that observed in vivo, except for delay in vitro. Normal germ cell maturation from gonocytes to primary spermatocytes occurred in testes cultured with 10% fetal calf serum only (i), 10% fetal calf serum plus müllerian inhibiting substance plus anti-müllerian inhibiting substance antibody (ii), 10% fetal calf serum plus normal rabbit serum (iii) and transferrin, insulin and retinoic acid plus müllerian inhibiting substance (iv). Maturation from gonocytes to A-type spermatogonia was arrested in testes cultured with 10% fetal calf serum plus anti-müllerian inhibiting substance antibody (p < 0.01), transferrin, insulin and retinoic acid alone (p < 0.001) and transferrin, insulin and retinoic acid plus müllerian inhibiting substance plus anti-müllerian inhibiting substance antibody (p < 0.001). The results are consistent with the hypothesis that müllerian inhibiting substance may be involved in postnatal gonocyte development and suggest that it may be useful to treat infertility associated with undescended testes.

The effects of fluoromethyl ketone inhibitors of cathepsin B on adjuvant induced arthritis.

Peptidyl fluoromethyl ketones with the structure carbobenzoxy (Z)-L-phenylalanine-L-alanine-CH2F (MDL 201,053), and its diastereomer Z-L-phenylalanine-D-alanine-CH2F (MDL 201,117), were synthesized and evaluated in vitro for inhibition of purified human cathepsin B. MDL 201,053 was shown to be a potent inhibitor of cathepsin B activity, whereas MDL 201,117 was more than 100-fold less active. In rats with adjuvant induced arthritis, oral administration of MDL 201,053 (13 or 34 mg/kg/day), but not MDL, 201,117 (28 mg/kg/day), significantly decreased the severity of gross clinical arthritis and reduced histologically graded articular cartilage and bone destruction by 76 to 100%. Quantitative image analysis of radiographs indicated that MDL 201,053 treatment significantly reduced bone density changes and inhibited focal bone erosion that normally occur during the course of adjuvant disease. MDL 201,117 had no significant effect on cartilage or bone destruction by any of the evaluation methods used. The effects of MDL 201,053 treatment were dose dependent and treatment was at least partially effective when initiated after the onset of disease. Our studies suggest that inhibitors of cathepsin B may be useful for the treatment of chronic inflammatory joint disease.

Do donor cells function as veto cells in the induction and maintenance of tolerance across an MHC disparity in mixed lymphoid radiation chimeras?

Mixed lymphoid chimeras can be established across H-2b-->H-2d by injection of C57BL/6J (B-6) donor bone marrow cells into BALB/c hosts conditioned by sublethal irradiation, 235 cGy x3. These chimeras are specifically tolerant to both donor and host alloantigens. Tolerance cannot be broken even by injection of 4 x 10(8) normal BALB/c spleen cells (SC), suggesting a suppressor mechanism. In contrast to conventional suppression, however, in which suppressors are syngeneic to the cells they suppress, tolerance can be transferred only by cells of the allogeneic donor allotype (1,2). Thy 1+ cell depletion eliminates the capacity of the donor population to transfer tolerance and markedly reduces the capacity of B-6 BMC to induce tolerance (3). In the present studies spleen cells from tolerant chimeras (CSC), when coinjected in a 1:1 ratio, reduced the high GVHD mortality induced in irradiated H-2d/b F1 hybrids by injection of 2 x 10(7) normal BALB/c SC alone (P < .01). When coinjected at a 5:1 ratio CSC eliminated the GVHD mortality and weight loss induced by 5 x 10(6) BALB/c SC (P < .01). Depletion of B-6 cells from CSC removed their capacity to inhibit the GVHD induced by normal BALB/c SC. In contrast to conventional suppression, which requires the continued presence of suppressor cells, the BALB/c cells isolated from CSC, although unable to inhibit the GVHD of normal BALB/c SC, remained nonreactive against the H-2b allotype for a prolonged period following depletion of the B-6 cells. This prolonged response reduction dependent upon some interaction with allogeneic donor cells--which, in turn, are specifically nonreactive to host antigens--is compatible with a veto mechanism.

Peptidyl fluoromethyl ketones as inhibitors of cathepsin B. Implication for treatment of rheumatoid arthritis.

Peptidyl fluoromethyl ketones (FMKs), with the amino acid sequence Phe-Ala held constant but with variable N-terminal groups, were synthesized and tested for inhibition of the cysteine proteinase cathepsin B. The FMKs were effective in inhibiting cathepsin B activity in vitro. The inhibition was time dependent and was not reversed by dialysis, suggesting covalent modification of the enzyme. Cathepsin B activity present in livers and kidneys of rats treated with FMKs was reduced by 22-91% 4 hr after a single oral dose of 25 mg/kg. The FMKs inhibited the severity of inflammation and the extent of cartilage and bone damage in adjuvant-induced arthritis. These effects were seen during the late-stage of the disease with no effect on onset or incidence of disease. This is consistent with inhibition of protease-mediated damage. These FMKs or derivatives may be of clinical value in the treatment of arthritis.

Effects of Thy-1+ cell depletion on the capacity of donor lymphoid cells to induce tolerance across an entire MHC disparity in sublethally irradiated adult hosts.

Thy-1+ cell depletion with anti-Thy-1.2 mAb and complement markedly reduced the capacity of C57BL/6J, H-2b bone marrow to establish mixed lymphoid chimerism and induce tolerance to C57BL/6J skin grafts across an entire MHC disparity in BALB/c, H-2d hosts conditioned with sublethal, fractionated 7.5 Gy total-body irradiation. In this model tolerance can be transferred to secondary irradiated BALB/c hosts only by cells of C57BL/6J donor, not host, genotype isolated from the spleens of tolerant hosts. Thy-1+ cell depletion abolished the capacity of C57BL/6J donor cells from tolerant BALB/c host spleens to transfer tolerance. The capacity of semiallogeneic BALB/c x C57BL/6J F1, H-2d/b donor BM and spleen cells to induce chimerism and tolerance to C57BL/6J skin grafts in BALB/c parental hosts was also reduced by Thy-1+ cell depletion. Thus the requirement for donor Thy-1+ cells cannot be explained simply on the basis of alloaggression. It is unlikely that the requisite Thy-1+ cells are nonspecific suppressor cells: Thy-1+ cell depletion had no effect on the slight but significant prolongation of third-party C3H/HeJ, H-2k skin grafts in irradiated BALB/c hosts injected with allogeneic C57BL/6J or semiallogeneic BALB/c x C57BL/6J F1 BM compared to irradiated controls injected with medium only. Furthermore, injections of semiallogeneic F1 spleen cells had no significant effect on the survival of the third-party grafts, although these cells were fully capable of inducing tolerance, and their capacity to induce tolerance was significantly reduced by Thy-1+ cell depletion. The requirement for a specific population of lymphoid cells, i.e. Thy-1+, remains unexplained but suggests that donor cells might play a role in the induction or maintenance of tolerance in this model other than merely providing a circulating source of donor antigens.

The role of donor lymphoid cells in the transfer of allograft tolerance.

Tolerance to murine skin allografts across a MHC disparity was induced by conditioning primary hosts with sublethal fractionated total-body irradiation (FTBI) and transfusion of allogeneic bone marrow (BM). Tolerance could be adoptively transferred to secondary hosts conditioned by FTBI with infusion of spleen cells from hosts bearing intact skin allografts greater than 60 days. Tolerance could not be transferred by tolerant host spleen (THS) preparations from which cells of the donor genotype had been deleted by cytotoxic alloantisera. Deletion of host genotype cells, however, did not diminish the capability of THS to transfer tolerance. All of the tolerizing activity of THS appeared to reside within cells of the donor genotype. Small numbers of normal donor spleen cells could induce tolerance in FTBI hosts but only at the expense of very high mortality, in contrast to the low mortality observed with tolerizing injections of allogeneic donor cells from THS or injections of normal semiallogeneic F1 hybrid spleen cells. If an active immune response is responsible for tolerance induction/transfer in this model, allogeneic donor lymphoid cells derived from BM, in contrast to donor spleen cells, must be capable of mounting this response without concomitant severe GVHD. In future experiments, cells of donor genotype can be isolated from THS and purified in sufficient numbers to compare their tolerizing efficiency vs. that of normal donor cells, detect possible suppression of normal host cell alloreactivity in vitro and identify the donor cell phenotypes involved.

Sublethal fractionated total-body irradiation and donor bone marrow infusion for induction of allograft tolerance.

Tolerance to skin allografts across the strong histocompatibility barrier H-2b to H-2d was achieved with sublethal fractionated total-body irradiation, FTBI, delivered to H-2d mice in 3 doses of 250 rads within 24 hr, followed by transfusion of 3 X 10(7) H-2b donor bone marrow (BM) cells. H-2b skin allografts were applied within 48 hr after the initial radiation. 70% of the mice became long-term (greater than 180-day) survivors with fur-bearing grafts. Marked interexperiment variability in survival rates suggested that infection was the major cause of death in this model and lower weight gain and survival rates for allogenic BM vs. media-treated controls suggested that graft-versus-host disease (GVHD) was also a factor. The observation, however, that long-term survivors (70% of all mice) gained weight and appeared healthy suggested that the GVHD might be self-limiting. Chimeric analysis revealed that approximately 25% of spleen cells were of donor origin, both at short-term (6 weeks) and long-term (greater than 1 year) intervals after tolerance induction. In spite of hematopoietic chimerism, a low incidence of spontaneous tumors, less than 1%, occurred in the long-term survivors.