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Summary. A polymorphic determinant on the surface of canine peripheral blood mononuclear cells (PBMC) has been identified using the murine monoclonal antibody W3G10. The determinant, which has a molecular weight of approximately 39 Kd, is associated with DLA-Dwl (w = workshop designation), -Dw3, -Dw4, -Dw10, -Dsea1 (sea = Seattle designation) and -Dsea9, but not -Dw8, -Dw9, -Dsea4, -Dsea10, -Dsea 16 or -Dsea17, and segregates in Mendelian fashion. There does not appear to be any linkage disequilibrium with currently recognized DLA-A, -B or -C alleles. Cytotoxic effector function was eliminated when W3G10 positive alloantigen primed PBMC were treated with W3G10. However, cytotoxicity was not affected when W3G10 negative PBMC were used, thus further confirming polymorphism. The data suggest that the antigen identified by McAb W3G10 is not a typical class I or class II MHC antigen, but may be encoded by a closely linked gene.

The frequencies of 12 DLA-D alleles in a random canine population were determined in one-way mixed lymphocyte cultures using a panel of homozygous typing cells established in this laboratory. The homozygous typing cells served as stimulators for responder lymphocytes obtained from 160 random dogs. The results of these studies were compared to those with lymphocytes from 75 dogs in our research laboratory. DLA-D allelic frequencies were estimated by maximum likelihood techniques. The use of a relative response (RR) less than or equal to 5% as a definition of a typing response resulted in the recognition of a total allele frequency of 59% in dogs from the research laboratory. Three of the 12 DLA-D alleles were not detected. Typing responses of cells from random dogs to the 12 DLA-D alleles were determined using RRs less than or equal to 5%, less than or equal to 10%, less than or equal to 15%, and less than or equal to 20%. With RRs of less than or equal to 5%, less than or equal to 10%, and less than or equal to 15%, the total allele frequencies recognized were 39%, 47%, and 55%, respectively. Within each of these % RR ranges all but one of the DLA-D alleles were detected. With an RR less than or equal to 20% the total allele frequency recognized was 58% and all 12 alleles were detected. Our results indicate that an RR of less than or equal to 10% could be used to define a phenotypic DLA-D typing response in the dog. The level of allelic frequencies detected in both the research and random canine populations indicates the need to identify additional DLA-D alleles through expanded family studies using mixed lymphocyte culture and homozygous cell typing.

Allogeneic bone marrow transplantation was carried out in an 81-day-old Portuguese water dog with GM1 gangliosidosis using a DLA identical sibling as donor. Engraftment was complete and beta-galactosidase activity in leukocytes of the transplanted dog were similar to those in the donor. Over the next 2.5 months neurological deterioration in the transplanted dog was similar to that in untreated dogs with GM1 gangliosidosis. Cerebral ganglioside GM1 concentrations were not diminished by bone marrow transplantation and cerebral beta-galactosidase activity was negligible. We conclude that allogeneic bone marrow transplantation early in life is ineffective in canine GM1 gangliosidosis.

Background. Previous studies have demonstrated that most marrow grafts from dog leukocyte antigen (DLA)-mismatched unrelated donors were rejected after 9.2 Gy total body irradiation (TBI), and that graft resistance could be overcome by infusing viable peripheral blood mononuclear cells (PBMCs) in addition to marrow. Methods. To investigate the donor cell populations that facilitate engraftment, we determined the minimal dose of PBMCs required to ensure stable engraftment. Nineteen dogs underwent transplantation with DLA-mismatched unrelated marrow and PBMCs in a dose de-escalation study. In subsequent studies, 12 dogs were given selected CD4 or CD8 cells in addition to marrow. Results. When 3×10 8 PBMC/kg were given in addition to a median of 4×10 8 marrow cells/kg, five of six animals engrafted. At a dose of 1 × 10 8 PBMC/kg, four of eight animals engrafted, and none of five dogs engrafted at a dose of 3×10 7 PBMC/kg. Accordingly, 12 dogs were given 9.2 Gy TBI, marrow grafts from DLA-mismatched unrelated dogs, and a median of 5.2×10 7 selected CD8 cells/kg or 10.4 x 10 7 selected CD4 cells/kg corresponding to the number of CD8 or CD4 cells contained in 3×10 8 PBMC/kg. Five of six dogs given CD8 cells and five of six dogs given CD4 cells engrafted. Conclusion. Results indicate that at least 3×10 8 unmodified PBMC/kg are needed for stable engraftment of DLA-mismatched unrelated marrow, and that both CD4 and CD8 cell subpopulations are capable of facilitating engraftment.

Purpose: We compared gastrointestinal toxicity of single vs. fractionated total body irradiation (TBI) administered at dose rates ranging from 0.021 to 0.75 Gy/min in a canine model of marrow transplantation. Methods and Materials: Dogs were given otherwise marrow-lethal single or fractionated TBI from dual 60 Co sources at total doses ranging from 8–18 Gy and delivered at dose rates of 0.021, 0.05, 0.10, 0.20, 0.40, and 0.75 Gy/min, respectively. They were protected from marrow death by infusion of previously stored autologous marrow cells and they were given intensive supportive care posttransplant. The study endpoint was 10-day mortality from gastrointestinal toxicity. Logistic regression analyses were used to jointly evaluate the effects of dose rate, total dose, and delivery regimen on toxicity. Results and Conclusion: With increasing dose rates, mortality increased for either mode of delivery of TBI. With dose rates through 0.10 Gy/min, mortality among dogs given single vs. fractionated TBI appeared comparable. Beginning at 0.20 Gy/min, fractionation appeared protective for the gastrointestinal tract. Results in dogs given TBI at 0.40 and 0.75 Gy/min, respectively, were comparable, and dose fractionation permitted the administration of considerably higher total doses of TBI than were possible after single doses, an increment that was on the order of 4.00 Gy. The data indicate that the impact of fractionating the total dose at high dose rates differs from the effect of fractionation at low dose rates.

Previous studies found that bone marrow (BM) allografts from DLA- identical littermates resulted in survival of two thirds of recipient dogs after otherwise lethal doses of 450 to 600 cGy of total body irradiation (TBI) because of successful allografts or autologous recovery after rejection of the allografts. The current study asked whether survival could be further improved by treating allograft recipients with recombinant canine granulocyte colony-stimulating factor (G-CSF), stem cell factor (SCF), or G-CSF/SCF. Of 21 dogs, 14 (67%) receiving allografts but no growth factors survived, 10 with successful allografts (including 5 mixed chimeras) and 4 with autologous recovery; whereas 7 animals died, 5 from infections during BM aplasia and 2 from acute graft-versus-host disease. By comparison, 30 of 34 dogs (88%) receiving hematopoietic growth factors in addition to the BM graft survived, 17 with successful allografts (including 10 mixed chimeras) and 13 with autologous recovery; whereas 4 died, all with infection related to BM aplasia after rejection of the allograft. Survival was similar for recipients of G-CSF, SCF, or the combination of G-CSF and SCF. Logistic regression analyses, which accounted for possible effects of TBI dose, showed a trend for improved survival in dogs receiving growth factors (P = .09), no change in allogeneic engraftment (P = .74), and a slight increase in autologous recovery (P = .22). In agreement with previous data, we found that grafts of BM from DLA-identical littermates improved survival of recipient dogs exposed to low but otherwise lethal doses of TBI. A further improvement in survival could be achieved by additional treatment with G-CSF, SCF, or G-CSF/SCF. Results suggest that treatment by hematopoietic growth factors along with BM grafts should be considered for victims of radiation accidents.

We explored in dogs the immunosuppressive properties of 450 cGy total body irradiation (TBI) delivered from two opposing 60Co sources, as assessed by the criterion of successful engraftment of allogeneic genotypically DLA-identical littermate marrow. Two questions were asked in this study. Firstly, does dose rate affect the immunosuppressive effect of TBI when administered in a single dose? Secondly, does fractionation alter the immunosuppression of TBI when delivered at a very fast dose rate? Dose rates studied included 7 and 70 cGy/min, and fractionation involved four fractions of 112.5 cGy each, with 6-hour minimum interfraction intervals. Six of 7 dogs receiving 450 cGy single- dose TBI at 70 cGy/min showed sustained engraftment of the allogeneic marrow, compared with 1 of 7 dogs receiving single-dose TBI at 7 cGy/min (P = .01). Fractionated TBI at 70 cGy/min resulted in sustained allogeneic engraftment in 3 of 10 dogs, a result that was statistically significantly worse than that with single-dose TBI at 70 cGy/min (P = .03) and not statistically different (P = .24) from that with fractionated TBI delivered at 7 cGy/min (0 of 5 dogs engrafted). A single dose of 450 cGy of TBI delivered at a rate of 70 cGy/min is significantly more immunosuppressive than the same total dose delivered at 7 cGy/min. Fractionated TBI at 70 cGy/min is significantly less immunosuppressive than single-dose TBI at 70 cGy/min and not significantly different from fractionated TBI administered at 7 cGy/min. Results are consistent with the notion that significant DNA repair in lymphoid cells is possible during interfraction intervals at the relatively high dose rate of 70 cGy/min.

Dogs given total-body irradiation and marrow transplants from DLA-identical littermates exhibit prompt and sustained hematopoietic engraftment. However, animals given three preceding blood transfusions from the marrow donor before transplant become sensitized and reject the marrow graft. Rejection is due to exposure to polymorphic minor non-DLA histocompatibility antigens expressed on blood mononuclear cells. We sought to determine whether heat treatment would prevent blood from sensitizing recipients in this model since heating blood to 45 degrees C for 45 min abrogates the ability of blood mononuclear cells to stimulate in mixed lymphocyte culture. Three of 4 evaluable dogs given heat-treated blood before transplant rejected their marrow grafts. To prevent possible reexpression/reacquisition of mononuclear cell functional activity in vivo after transfusion, subsequent dogs were given heated blood that was additionally exposed to 2000 cGy gamma irradiation. Eight of 10 evaluable dogs given blood treated in this fashion engrafted. Unexpectedly, 9 out of 10 evaluable dogs transfused with blood treated only with gamma irradiation also engrafted. These results demonstrate that treatment of blood with gamma irradiation alone or in combination with heat prevents transfusion-induced sensitization to minor histocompatibility antigens. Results from this canine model suggest that blood products be gamma irradiated before transfusion in patients who are transplant candidates in order to prevent sensitization to minor histocompatibility antigens and reduce the risk of marrow graft rejection.

Silastic catheters were fabricated and aseptically implanted through the skin into the jugular vein of 64 dogs with the intravascular tip located in the right atrium. Solutions were infused through the catheter at 2 to 2.5 mL/h by a portable pump worn by the dog. Following 9.2 Gy total body irradiation (TBI) and allogeneic bone marrow transplantation (BMT), succinyl acetone, an experimental chemotherapeutic agent, was infused into 34 dogs. Hematopoietic growth factors were infused into an additional 30 dogs, two of which had 9.2 Gy TBI and an autologous BMT, and four of which had 4.0 Gy TBI and no BMT. All dogs received continuous oral and parenteral antibiotics while the catheters were in place. All catheters functioned well until electively removed (n = 28) or until the dogs died or were euthanized (n = 36) at 12 to 68 days after implantation. Mean length of catheter function was 30.3 +/- 1.5 (SEM) days. No catheters were dislodged and there was no evidence of catheter-related blood loss or sepsis. Semiquantitative cultures of 5 catheters were negative, but Staphylococcus epidermidis was isolated from 3 of 7 catheters cultured in broth. Six dogs had thrombosis adjacent to the intravascular catheter tip. The catheters were well tolerated and facilitated successful long-term infusion of solutions into dogs.

L-leucyl-L-leucine methyl ester (Leu-Leu-OMe) is a lysosomotropic agent that selectively kills cytotoxic T cells and their precursors, natural killer cells, and monocytes but not helper T cells or other cells of hematopoietic origin. In this study, the effects of treatment of bone marrow and peripheral blood buffy coat with Leu-Leu-OMe on the outcome of allogeneic marrow transplantation were studied in several canine models. Whereas incubation of autologous marrow with Leu-Leu-OMe had no adverse effects on subsequent engraftment, incubation of marrow from dog leukocyte antigen (DLA)-identical littermates resulted in a high rate of graft failure. Previous studies have demonstrated that the addition of peripheral blood buffy coat allows engraftment of unrelated DLA-nonidentical marrow, and in this study we found that incubation of buffy coat with Leu-Leu-OMe did not alter this graft promoting effect. In a final experiment it was demonstrated that incubation of both marrow and peripheral blood buffy coat did not prevent the development of graft-versus-host disease in recipients of marrow from DLA-haploidentical littermates. In considering the eventual application of Leu-Leu-OMe in the clinic, these results are less encouraging than those previously reported using murine models.

The exquisite sensitivity of lymphohematopoietic tissues to ionizing radiation has been known since shortly after the discovery of x rays by Roentgen. The most prominent features of the hematopoietic syndrome in experimental animals are hemorrhagic complications and susceptibility to infections. Since the late 1940’s it has been known that the hematopoietic radiation injury could be modified by subsequent infusion of bone marrow. By the mid-1950’s, three independent groups of investigators showed that the lifesaving effect of marrow infusions was due to the presence of pluripotent stem cells in the transplant, from which regrowth of the damaged hematopoietic system occurred.

Four murine monoclonal antibodies were used in cytolytic assays to identify cell populations involved in canine T cell effector functions. Antibody DT-2, directed at T cells, and antibody DLy-6, a panlymphocyte antibody, inhibited mixed lymphocyte culture (MLC) responses and cytotoxic lymphocyte (CTL) activity only when lymphocytes were treated before culture (day 0), but they had no significant effect on these functions when cells were treated after 6 days in culture. Antibody DLy-1, reacting with canine lymphocytes and monocytes, abrogated MLC responses and CTL activity when responder cells were treated on day 0. When cells were treated after 6 days in culture, MLC responses were reduced to 47% of control, whereas CTL activity increased slightly. In contrast, the anti-Ia antibody 7.2 significantly reduced MLC responses and CTL activity of cells treated on either day 0 or day 6 of culture, suggesting that canine CTL express Ia antigens.

The Third International Workshop on Canine Immunogenetics involved 80 potentially DLA-D homozygous typing cells obtained from dogs of various breeds and submitted from five laboratories in Europe and the United States. Mutual reactivity of all cells was studied in mixed leukocyte cultures, and stabilized relative responses were used for analysis. Intralaboratory and interlaboratory comparisons of results suggest that a stabilized relative response of 30% represents an acceptable parameter for "typing responses" indicating phenotypic DLA-D identity of stimulator and responder cells. Using this criterion, 10 clusters of homozygous typing cells were defined and accepted on an international level, and they were assigned the specificities Dw1 to Dw10. At least six additional (provisional) specificities were recognized that were well characterized within individual laboratories but require additional testing before workshop specificities can be assigned. These data show that DLA-D typing is feasible and represents a useful tool in the genetic analysis of the canine major histocompatibility complex. Much work is needed to confirm the present results in family studies, to determine gene frequencies, and to analyze at a molecular level the antigens responsible for mixed leukocyte culture reactivity.

Recipient dogs were conditioned with 9.2 Gy of total-body irradiation followed by the infusion of bone marrow and peripheral blood leukocytes from a DLA-haploidentical littermate (N = 10) or a completely allogeneic unrelated donor (n = 9). Graft-vs.-host disease (GVHD) prophylaxis consisted of methotrexate (MTX) and cyclosporine (CsA). Postgrafting all dogs were complete lymphohemopoietic chimeras. Lymphocytes of haploidentical chimeras without GVHD were unresponsive to stimulation by host lymphocytes cryopreserved pregrafting. Lymphocytes of haploidentical chimeras with GVHD proliferated in response to host cells, albeit less than donor cells pregrafting. In completely allogeneic chimeras, neither lymphocytes from dogs with GVHD, nor those from dogs without the disease showed responses to host lymphocytes. In addition, cells from haploidentical chimeras obtained early after transplantation nonspecifically suppressed donor cell proliferation. Later on, lymphocytes from dogs without GVHD showed specific suppression of donor cells, while lymphocytes from chimeras with GVHD continued to show nonspecific suppression. Cells from completely allogeneic chimeras both with and without GVHD never suppressed donor cells specifically. Both specific and nonspecific suppressor cells were enriched by nylon wool adherence, expressed T cell markers, and were not affected by the addition of indomethacin. Even after removing nylon wool-adherent cells, however, chimera cells were unresponsive to stimulation by host cells. By one year after transplant, chimera lymphocytes no longer showed suppression. In cell-mediated lympholysis assays, lymphocytes from all chimeras, regardless of GVHD, failed to generate cytotoxic cells against host cell targets. However, while haploidentical chimeras showed cytotoxicity against third-party targets, completely allogeneic chimeras did not. This deficiency was not overcome by the addition of mixed leukocyte culture supernatant or donor lymphocytes. All chimeras had basically normal antibody responses to keyhole limpet hemocyanin and phage X174. However, while haploidentical chimeras had normal responses to bacillus Calnette-Guerin (BCG) sensitization and rejected DLA-incompatible skin grafts within the normal time frame, completely allogeneic chimeras were not sensitized by BCG and showed delayed skin graft rejection. Histopathological studies revealed slow thymic reconstitution in all chimeras, particularly in the presence of GVHD. However, while healthy haploidentical chimeras eventually showed thymic histology normal for age, completely allogeneic chimeras did not.(ABSTRACT TRUNCATED AT 400 WORDS)

Bone marrow transplants with low marrow cell doses (less than or equal to4 X 10/sup 8/ cells/kg) from unrelated donors were carried out in 16 dogs conditioned with 9 Gy (900 rad) of total body irradiation. No immunosuppression was given after grafting. Eleven donor-recipient pairs were phenotypically identical (group 1) for the known antigens of the canine major histocompatibility complex (DLA) and in five the donor was homozygous and the recipient heterozygous for DLA (group 2), as determined by serological histocompatibility typing and mixed leukocyte cultures including homozygous cell typing. In addition, lymphocytes from donors and recipients in group 1 were mutually nonreactive in cell-mediated lympholysis; lymphocytes from recipients in group 2 were not cytotoxic against donor cells. Eight dogs rejected their grafts and eight showed sustained engraftment; of these, four died from graft-versus-host disease. The incidence of rejection was higher than in DLA-identical littermates but lower than in DLA-nonidentical unrelated or littermate dogs. These results indicate that antigens different from the recognized alleles at DLA are involved in the control of engraftment. These antigens most likely represent the expression of unrecognized differences within DLA or are coded for by a locus different from but linked to DLA-A, B, Cmore » or D; they are not recognized in the cell-mediated lympholysis assay.« less

Incubation of canine marrow and peripheral blood mononuclear cells with L-leucyl-L-leucine methyl ester resulted in the inhibition of mitogen- and alloantigen-induced blastogenesis, the elimination of allosensitized CTL and NK activity, and prevented the development of CTL from pCTL. The effects of these incubations were similar to those described in mice and humans. Additionally, in vitro CFU-GM growth from treated canine marrow was reduced, but could be regained when the Leu-Leu-OMe-treated marrow was cocultured with either untreated autologous peripheral blood mononuclear cells or monocyte-enriched PBMC but not with untreated monocyte-depleted PBMC. Six of seven dogs conditioned with 920 cGy total-body irradiation engrafted successfully after receiving autologous marrow that was incubated with Leu-Leu-OMe prior to infusion. These cumulative results indicate that incubation with Leu-Leu-OMe is a feasible method to deplete canine marrows of alloreactive and cytotoxic T cells prior to transplantation.

Cytotoxic lymphocytes (CTL) were generated in bulk mixed leukocyte culture (MLC) using peripheral blood mononuclear cells from homozygous dogs identical to each other for DLA-A and -B but different for DLA-D loci. These CTL were tested against 51Cr-labeled targets (Con-A-stimulated PBMC) derived from the MLC sensitizing dogs and from other dogs homozygous for various DLA-D locus alleles. Indirect cell-mediated lympholysis (CML) assays, with and without cold target blocking and pretreatment of 51Cr-labeled targets with DLA alloantisera were used to study the target cell determinants recognized by these CTL. The resultant target cell lysis and cold target blocking of specific target cell lysis indicated target determinants associated with DLA-D-locus-encoded specificities, and different from those encoded by DLA-A and -B loci. The pattern of shared target determinants among different DLA-D HTC may be explained by: (A) two separate previously unrecognized MHC associated loci, each with 2 alleles defined so far; or (B) epitopes shared among certain DLA-D alleles but not present in others. This study demonstrates that DLA-D associated determinants different from previously described DLA-D specificities may serve as CML target cell determinants when there are no apparent DLA-A and -B loci encoded antigen differences between responder and stimulator cells used to generated MLC-derived CTL.

A canine model of marrow transplantation was used to further define the host cells mediating resistance to marrow engraftment. Recipient dogs were given 9.2 Gy of total body irradiation followed by marrow infusion from unrelated DLA-nonidentical donors. No postgrafting immunosuppression was given. At three and ten days posttransplantation recipient marrow and peripheral blood cells were obtained and characterized by the following in vitro studies: morphologic analysis; phenotypic analysis with monoclonal antibodies; assays for natural killer cell (NK) activity; and cocultures with donor marrow to study the effect on donor CFU-GM growth. Daily differential cell counts revealed a proliferation of peripheral blood mononuclear cells approximately eight days posttransplant. By day 10 surviving host cells were uniformly large granular lymphocytes which were phenotypically of T cell lineage, had NK activity, and were capable of suppressing donor marrow CFU-GM growth. Mononuclear cells from dogs given total body irradiation only and no marrow infusion (radiation control group), did not suppress CFU-GM growth when cocultured with marrow from unrelated DLA-mismatched dogs. These results suggest that radioresistant host cells with the morphology of large granular lymphocytes and NK activity and which proliferate in response to the infused donor marrow cells mediate resistance to DLA-nonidentical marrow grafts. It remains to be determined, however, whether in vitro functional studies reflect the mechanisms involved in vivo.

We examined the effect of methotrexate (MTX) and cyclosporin A (Cy A) on engraftment, graft-versus-host disease (GVHD), and the induction of tolerance in dogs prepared for marrow transplantation by 9 Gy of total body irradiation and grafted with bone marrow and buffy coat cells. Nineteen dogs were given grafts from DLA-identical littermates followed by immunosuppression with Cy A for 25 or 100 days. All had sustained engraftment, and 12 became healthy long-term chimeras. Sixty dogs were given grafts from DLA-nonidentical unrelated donors. Among nine given MTX only postgrafting, one rejected the graft nd eight died with GVHD. Among 18 dogs given Cy A only postgrafting, eight failed to achieve engraftment, seven died of various causes, and three died with GVHD. Thirty-four dogs were given both MTX and Cy A in various regimens postgrafting. The only long-term survivors were 4 of 10 dogs given MTX on days 1, 3, 6, and 11 and Cy A from days 0 through 100. Two have chronic GVHD. We conclude that Cy A can induce graft-host tolerance across minor, but not major, histocompatibility differences. The combination of MTX early after transplantation with Cy A prevents failure of engraftment of histoincompatible marrow and some recipients become long-term survivors.

We explored the ability of fractionated total body irradiation (TBI) given at a rate of 7 cGy/min from opposing dual 60Co sources at otherwise lethal doses of 450, 600, 700, 800, and 920 cGy to condition dogs for marrow grafts from DLA-identical littermates. Results were compared with those of a previously reported study using single-dose TBI administered under otherwise identical conditions. Fractionated TBI was less immunosuppressive than single-dose TBI, as evidenced by a significantly higher rate of graft rejection (P = .001). Specifically, sustained allogeneic engraftment was observed in only two of 18 (11%) dogs that received 600 to 800 cGy fractionated TBI as compared with 11 of 17 (65%) dogs that received comparable doses of single-dose TBI. Only at 450 cGy (none of the ten dogs studied had sustained engraftment) and at 920 cGy (four of five dogs that received fractionated and 20 of 21 dogs that received single-dose TBI engrafted) were we unable to find differences between the two modes of radiation. Most dogs that rejected their graft survived with autologous recovery (13 of 22 that received fractionated and eight of 12 that received single-dose TBI; P = .49), presumably the result of extended support provided by the transient allogeneic grafts. We conclude that at equivalent doses fractionated TBI is significantly less effective than single-dose TBI to condition DLA-identical littermate dogs for marrow transplantation. These findings have implications for the design of conditioning programs in clinical transplantation, especially when T- cell-depleted marrow grafts are used.

We evaluated the marrow toxicity of 200 and 300 cGy total-body irradiation (TBI) delivered at 10 and 60 cGy/min, respectively, in dogs not rescued by marrow transplant. Additionally, we compared toxicities after 300 cGy fractionated TBI (100 cGy fractions) to that after single-dose TBI at 10 and 60 cGy/min. Marrow toxicities were assessed on the basis of peripheral blood cell count changes and mortality from radiation-induced pancytopenia. TBI doses studied were just below the dose at which all dogs die despite optimal support. Specifically, 18 dogs were given single doses of 200 cGy TBI, delivered at either 10 (n=13) or 60 (n=5) cGy/min. Thirty-one dogs received 300 cGy TBI at 10 cGy/min, delivered as either single doses (n=21) or three fractions of 100 cGy each (n=10). Seventeen dogs were given 300 cGy TBI at 60 cGy/min, administered either as single doses (n=5) or three fractions of 100 cGy each (n=10). Within the limitations of the experimental design, three conclusions were drawn: 1) with 200 and 300 cGy single-dose TBI, an increase of dose rate from 10 to 60 cGy/min, respectively, caused significant increases in marrow toxicity; 2) at 60 cGy/min, dose fractionation resulted in a significant decrease in marrow toxicities, whereas such a protective effect was not seen at 10 cGy/min; and 3) with fractionated TBI, no significant differences in marrow toxicity were seen between dogs irradiated at 60 and 10 cGy/min. The reduced effectiveness of TBI when a dose of 300 cGy was divided into three fractions of 100 cGy or when dose rate was reduced from 60 cGy/min to 10 cGy/min was consistent with models of radiation toxicity that allow for repair of sublethal injury in DNA. Biol Blood Marrow Transplant 1999;5(3):155-61.

The major histocompatibility complex (MHC) of the dog, DLA, consists of several polymorphic allelic systems similar to those of most other species studied (Templemn and Thomas 1971, van Dam 1981). Two loci (A and B), and possibly a third (C), which code for class I antigens, have been identified (Vriesendorp et al. 1976). Class II antigens, which have been defined in mouse and man (Klein 1979, van Rood et al. 1981), are assumed to represent the antigenic expression of genes within the DLA-D region. Recently, 12 DLA-D alleles have been described (Storb et al. 1977, Raft et al. 1983). A polymorphic system corresponding to DR has not been reported, although Ia-like antigens are expressed on canine lympho-hemopoietic cells (Deeg et al. 1982). Since the dog is used as a model for experimental marrow transplantation (Storb et al. 1980), further clarification of the DLA complex is important for a better understanding of the mechanisms of engraftment, rejection, and graft-versus-host disease (GVHD). In the present study, we used the primed lymphocyte test (PLT) as described (Weiden et al. 1979) to search for additional specificities encoded for by previously recognized or new loci. PLT cells were generated by priming with appropriate homozygous typing cells (HTC) and tested by restimulation with a panel of HTC (Table 1, Raft et al. 1983). This approach allowed the recognition of a supertypic determinant shared by three typing cells that otherwise expressed distinct specificities as recognized in mixed lymphocyte culture (MLC). Sharing of other PLT defined determinants was observed in additional HTC that also were unique DLA-D types. An initial problem encountered in preliminary experiments was that of discrimination of negative and positive PLT responses. The normalized median response (Morling et al. 1980a) was not a workable system because of the small number of PLT cells used for each experiment. However, when percent relative response (% RR) and stimulation index (SI) values were plotted, it became apparent

Experiences sur chiens. Le traitement par anticorps anti-Ia ne peut pas prevenir le rejet provoque par des transfusions; le seul moyen pour prevenir ce rejet semble actuellement l'irradiation totale prealable et le traitement «anti-cellules T» (procarbazine, serum anti-thymocytes et cyclosporine)