Effect of cyclophosphamide or x-rays on spontaneously occurring metastases from tumors transplanted into the tails of mice.

A spontaneous metastases model in mice is being used to test the efficiency of various treatments in eliminating metastases. Solid tumors were transplanted into the tails of mice and removed by tail transection when they had grown to a 4- to 5- or 6- to 7-mm mean diameter. Subsequently, 70 to 95% of mice not given other treatment developed metastases in the lungs or in regional lymph nodes (lumbar sacral region), or in both sites. The present paper reports the effects of whole-body or partial-body treatment on these metastases. The treatments, which started at the time of surgical transection of the tail, included a range of single or fractionated doses of cyclophosphamide (CTX) or X-rays given either to the whole body or locally to the lungs only. CTX reduced the incidence of metastases in both sites although the incidence of lung metastases was reduced by smaller doses of CTX than that of the lumbar sacral metastases. Whole-body irradiation of 6 grays (600 rads) had no effect on the incidence of metastases, whereas local irradiation of the lungs with single doses of 14.5 or 20 grays reduced the number substantially, as did 95 mg or more of CTX per kg. Thus, CTX or radiation reduced the incidence of lung metastases in a system where metastases developed from cells seeded from a primary tumor rather than from a cell suspension injected into the tail vein.

Comparison of different busulfan analogues for depletion of hematopoietic stem cells and promotion of donor-type chimerism in murine bone marrow transplant recipients.

Busulfan (1,4-butanediol dimethanesulfonate, BU) is relatively unique among other standard chemotherapy compounds in its ability to deplete noncycling primitive stem cells in the host and consequently to allow for high levels of long-term, donor-type engraftment after bone marrow transplantation (BMT). Such a property explains why this drug can be used as an alternative to total body irradiation in preparative regimes for BMT. However, as with radiation, BU conditioning is still troubled by severe toxicities that limit its applications to suboptimal drug doses. These problems stress the need for other BMT-conditioning drugs that are better tolerated and more selectively targeted toward normal and malignant hematopoietic stem cells. We have therefore compared the effects of various novel dimethanesulfonate compounds (related to BU) in terms of their toxicity to different stem cell subsets in vivo and in vitro and their ability to provide for long-term donor bone marrow engraftment using the congenic glucose-6-phosphate isomerase type 1 marker. Introduction of a benzene or cyclohexane ring in some of these drugs affords rigidity to the molecule and restricts the spatial positioning of the alkylating groups. Among 25 different compounds thus far tested at single doses, PL63 [cis-1,2-(2-hydroxyethyl) cyclohexane dimethanesulfonate] proved to be the most effective in providing for hematopoietic engraftment. The transisomer of the same compound gave significantly less engraftment and was comparable with the effects of dimethylbusulfan and Hepsulfam. The engraftment data correlated well with the depletion of different bone marrow stem cell subsets in the host as measured using the cobblestone area forming cell assay. The extent of stem cell depletion could not be explained on the basis of the distance and orientation of the two alkylating groups. Pharmacokinetic data, however, indicate that there is a correlation between biological activity and plasma levels reached. The diverse cytotoxic effects shown by these novel analogues of BU have provided a basis for relating biological activity with pharmacokinetic properties rather than with structural properties such as distance and orientation of the two alkylating groups. The identification of highly active compounds such as PL63 offers an opportunity for further developing other closely related drugs for potential application in clinical BMT conditioning therapy.

Increased rejection of murine allogeneic bone marrow in presensitized recipients.

The role of presensitizing murine recipients with donor spleen cells prior to T cell-depleted or -repleted H-2 compatible allogeneic bone marrow transplantation (BMT) was investigated at two different doses of total body irradiation (TBI). Recipients that were presensitized with 2 x 10(7) irradiated donor spleen cells at 1 week before a sublethal dose of 6 Gy TBI and BMT showed no evidence of donor blood chimerism while unsensitized recipients showed about 80% donor engraftment as determined by blood Gpi phenotyping. After raising the TBI dose to 9.5 Gy an increase in mortality from marrow failure was observed in presensitized animals. No significant engraftment-promoting effect of up to 2 x 10(6) T cells (20% of total marrow dose) was seen either in presensitized or unsensitized mice. It can be concluded that presensitized recipients are more susceptible to acute marrow rejection and that T cells added to the bone marrow did not influence the level of donor engraftment in these recipients.

Transient and permanent engraftment potential of murine hematopoietic stem cell subsets: differential effects of host conditioning with gamma radiation and cytotoxic drugs.

Transplant of sorted donor (B6-Gpi-1a) hematopoietic stem cell subsets and host (B6-Gpi-1b) treatment with total body irradiation (TBI) or cytotoxic drugs were compared for induction of short- and long-term engraftment in a murine chimera model of congenic bone marrow transplantation (BMT). Parallel studies on donor and host marrow were performed in vitro in long-term bone marrow cultures to determine early and late cobblestone area forming cell (CAFC) frequencies in the grafts or in the transplant recipients 1 day after conditioning. Bone marrow cells (BMC) sorted for high wheat germ agglutinin affinity (WGA ) were enriched about 30-fold for early developing CAFC (colony-forming unit-spleen [CFU-S]) but not for primitive late CAFC (pre-CFU-S). This fraction showed only temporary engraftment when transplanted in irradiated recipients. In contrast, the low affinity (WGA+) fraction were preferentially enriched (200- to 300-fold) for late developing CAFC and were very effective for providing stable long-term engraftment following transplantation. Substituting radiation for chemotherapy in the host conditioning treatment also had diverse effects on the development of bone marrow engraftment. Pretreatment with 5-fluorouracil (5-FU, 200 mg/kg) allowed a discrete wave of donor engraftment that peaked at 2 to 4 weeks and then subsided to leave mostly host cells at 10 weeks and beyond. Busulfan preparation gave over 50% engraftment at 1 month after BMT but this continued to increase to reach stable donor chimerism of 80 to 90% beyond 10 weeks. The level of long-term engraftment given by 50 mg/kg busulfan appeared similar to that induced by doses of 6 to 8 Gy TBI. The changing patterns of erythroid chimerism for each preparative agent showed a remarkable correlation with depletion of defined hematopoietic stem cell subsets as quantified using the CAFC assay at 1 day after recipient treatment. These findings collectively show that the level of depletion of host CFU-S (CAFC-10) determines the extent of early and transient repopulation, whereas the degree of pre-CFU-S (CAFC-35) depletion determines the percentage of stable chimerism. Preliminary data on bone marrow CAFC content at 9 months after busulfan and BMT revealed a long-term reduction of the stem cell pool by about 50% but with relatively minor effects on supporting bone marrow stromas. The differential cytotoxic effects on stem cell subsets in relation to subsequent donor marrow engraftment offer a systematic and mechanistic approach toward identifying more effective chemotherapeutic compounds for use in clinical BMT conditioning regimens.

Studies on the hyperthermic sensitivity of the murine hematopoietic stem cell compartment. II. Heat effect on donor stem cells with long-term repopulating ability.

Variations in hyperthermic sensitivity among different hematopoietic progenitor and stem cell populations of the bone marrow have been previously described for clonogenic subsets responsible for short-term hematopoiesis. However, less is known of the heat sensitivity of more primitive stem cells capable of long-term repopulation in irradiated recipients. In the present study, control and heat-treated (60 minutes at 43 degrees C) donor bone marrow cells from congenic B6-Gpi-1a mice were transplanted at different cell doses (10(4), 10(5), 10(6), and 10(7) nucleated cells) in pre-irradiated (6 Gy) B6-Gpi-1b mice. The development and levels of donor marrow engraftment were determined from blood Gpi phenotyping, and the bone marrow dose required for equivalent long-term engraftment at 20 weeks provided an estimate of the surviving fraction corresponding to primitive stem cells of long-term repopulating ability (LTRA). Comparison with previous bone marrow cell survival values demonstrates that LTRA cells are less sensitive to hyperthermic treatment than other hematopoietic subsets, confirming a relationship between the heat sensitivity and the hierarchical structure of the hematopoietic stem cell compartment.

O6-Benzylguanine potentiates BCNU but not busulfan toxicity in hematopoietic stem cells.

OBJECTIVE: Busulfan (BU) is often used in conditioning regimens prior to bone marrow transplantation, but its mechanism of action remains to be resolved. We have examined the possibility that BU may exert part of its toxic effects via DNA alkylation at the O6 position of guanine as this might provide an approach to improving the conditioning regimen. METHODS: Survival of LAMA-84 and RJKO cells was assessed by colony-forming assay and cell counting, respectively. O6-alkylguanine-DNA alkyltransferase (ATase) activity was assayed by transfer of radioactivity from [3H]-methylated DNA. Colony-forming potential of normal human bone marrow cells (BMC) was measured in the presence of appropriate growth factors as the formation of both granulocyte-macrophage colony-forming units (CFU-GM) or burst-forming unit erythroids (BFU-E) within the same assay. Murine hematopoietic precursors were grown under a bone marrow stromal cell line to allow measurement of the frequency of cobblestone area-forming cells (CAFC) that correspond to CFU-GM, spleen colony-forming units (CFU-S), and the primitive stem cells with long-term repopulating ability. RESULTS: Inactivation of ATase by O6-benzylguanine (O6-BeG) sensitized a human erythromegakaryocytic cell line (LAMA-84) and normal human bone marrow progenitors to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) but not to BU toxicity. BCNU, but not BU, inactivated ATase in LAMA-84 cells. Overexpression of human ATase in cDNA transfected Chinese hamster cells attenuated the toxicity of BCNU but not BU. Finally, the in vivo treatment of mice showed that the depletion of primitive stem cells by BU as measured in the CAFC assay was not affected by addition of O6-BeG. O6-BeG did, however, dramatically potentiate BCNU toxicity in all CAFC subsets, leading to depletion of more than 99% stem cells. CONCLUSION: These data suggest that BU does not elicit toxicity via alkylation at the O6 position of guanine in DNA in a way that can be influenced by ATase modulation.

Dose-limiting complications from upper half body irradiation in C3H mice.

This investigation evaluates the usefulness of an experimental technique in mice that has been used to study lung tolerance as a major dose-limiting tissue in clinical radiotherapy. The pathological sequalae of upper half body irradiation using a range of single and fractionated (2 Gy per fraction once or twice daily) doses was characterized in C3H/HeJ mice. Four phases of potentially lethal syndromes were revealed starting with the very acute effects of oral inflammation within 1 month. Incisor damage occurred between 1 and 3 months when the supplying of powdered food appeared to prevent lethality from starvation. Single radiation doses then produced a predictable incidence of pneumonitis (3 to 6 months) followed by pleural effusions (6 to 12 months). These later two syndromes were absent in mice that survived the acute effects of fractionated UHBI. In accordance with other rapidly proliferating tissues, the estimated alpha/beta ratios for oral epithelial and incisor damage were notably larger than that previously reported for lung. This denotes the smaller capacity of the acute responding target cells to repair sublethal damage. The consequent predominance of acute reactions in the fractionated courses therefore confined the maximal tolerated dose to that which produced an unmeasurable level of pulmonary injury. Our discussion of these results warn against the simple extrapolation of fractionated or low dose rate UHBI lethality data from mouse to man without due consideration of extrapulmonary radiation effects.

The effect of low dose-rate and cyclophosphamide on the radiation tolerance of the mouse lung.

Mice were given cyclophosphamide 30 mg/kg intraperitoneally before thoracic irradiation at high dose-rate (HDR, 100 cGy/min) or low dose-rate (LDR, 5 cGy/min). The development of pneumonitis was monitored by regular measurement of breathing rate. Peak rises in breathing rate occurred around 4-9 weeks in those given cyclophosphamide before irradiation, and around 14-16 weeks in those given radiation alone, regardless of dose-rate. The dose reduction factor (DRF = LDR/HDR ratio) for LDR irradiation was congruent to 1.8 but LDR sparing was abolished (DRF congruent to 1.0) when cyclophosphamide was given before irradiation.

Fractionation and dose rate effects in mice: a model for bone marrow transplantation in man.

This study was designed to compare several fractionation and dose rate schedules to optimize the therapeutic ratio for total body irradiation (TBI). C3H/HeJ mice were given TBI and the bone marrow survival fraction was calculated using the CFUS assay. Irradiation was given at two dose rates: low dose rate (LDR) at 5 cGy/min or high dose rate (HDR) at 80 cGy/min in single fraction and fractionated regimens. The fractionated regimens were given as either 120 cGy three times daily, 200 cGy twice daily, or 200 cGy daily. The Do was 80 cGy for the single fraction, HDR group and 85 for the LDR group. For the fractionated regimens, the apparent Do's ranged from 55-65 indicating no sparing effect of fractionation for the normal bone marrow stem cells. Indeed, the Do's were smaller suggesting an increased sensitivity to irradiation with fractionation. Low dose rate (LDR) and fractionation were also studied for their influence on normal tissue toxicity following upper half body irradiation (UHBI). All the fractionated regimens had higher LD50/30 and LD50/30-180 values than those achieved by single fraction LDR alone. There was no significant dose rate effect for LD50/30 when 120 or 200 cGy fractions were used. However, dose rate was important for LD50/30-180 with 200 cGy but not with 120 cGy fractions. These results demonstrate protection of non-hematopoietic tissues with fractionation and low dose rate without protecting hematopoietic stem cells and may have implications for human bone marrow transplantation.

WR-2721 protection of pneumonitis and fibrosis in mouse lung after single doses of x rays.

The radioprotective effect of WR-2721 has been studied in mouse lung after single doses of radiation. Using the breathing rate assay and lethality, radioprotection was assessed at monthly intervals between 3 and 18 months after irradiation during both pneumonitis and chronic fibrosis. The degree of radioprotection was greater for fibrosis than for pneumonitis using both assays. In replicate experiments, dose modifying factors (DMF's) ranging from 1.2 to 1.4 were obtained for pneumonitis and 1.5 and 1.6 for fibrosis. The differences in DMF's for the two phases of lung damage were significant. A difference in the time course of expression of damage was seen in both the breathing rate and lethality assays between mice irradiated with and without WR-2721: the damage ended sooner in the drug-treated mice. This difference is best explained by protection of all damage after 5 months by WR-2721. No evidence of drug toxicity was found. We conclude that WR-2721 protects against chronic lung fibrosis caused by radiation at least as well as against the earlier appearing pneumonitis after single doses of radiation. Thus, if WR-2721 is dose modifying and if late tissue complications are dose limiting in clinical radiotherapy, then a therapeutic benefit would be obtained by the use of this drug in clinical radiotherapy, provided that the radioprotection of tumors did not exceed a factor of 1.5-1.6.

Is there a loss of repair capacity in mouse lungs with increasing numbers of dose fractions?

The capability of mouse lung to repair sublethal damage after up to 10 fractions of X rays was assessed by an in situ breathing rate assay and lethality. The whole thorax of mice was irradiated "daily" with 4, 7 or 10 fractions of 2.75 or 3.0 Gy of X rays followed at 24 hours by graded "test" doses of X rays or neutrons. Repair capability was measured by determining the difference in test dose between 4 and 7 fractions or 7 and 10 fractions at a given isoeffect. Damage was assessed monthly up to 76 weeks after irradiation, during pneumonitis and chronic fibrosis. The data from both assays for the pneumonitis phase suggested that there may be some loss of repair between 7 and 10 fractions, although it was not large enough in only 10 fractions to be clearly demonstrated. In contrast, there was no suggestion of loss of repair for late damage after up to 10 fractions of X rays using either assay.

The effects of anesthetics and misonidazole on the development of radiation-induced lung damage in mice.

The measurement of breathing frequency as a functional end-point of radiation-induced lung injury in mice allowed two phases of damage to be discerned; the first was manifest at 12-20 weeks after irradiation, the second beyond 28 weeks. Anesthesia by pentobarbitone sodium or steroids gave significant radioprotection of the lung during the early pneumonitic phase. Addition of the hypoxic cell sensitizer misonidazole removed the protective influence of the anesthetics but did not sensitize the lungs of unanesthetized mice. No anesthetic protection was detected for the late response, showing evidence for dissociation between early and late lung damage. The degree of epilation was measured on the dorsal thoracic region of the same mice. Protection by anesthetics and its reversal by misonidazole was also demonstrated. These results provide a warning of potential hazards in the laboratory evaluation of chemical radiosensitizers. The use of anesthetics at the time of irradiation could lead to an exaggerated enhancement of normal tissue damage.

Late tissue-specific toxicity of total body irradiation and busulfan in a murine bone marrow transplant model.

Total body irradiation (TBI) and busulfan were compared for late effects in a murine model of bone marrow transplantation (BMT). Male C57BL/6 mice were given fractionated TBI or busulfan given in 4 equal daily doses followed by infusion of 10(7) syngeneic bone marrow cells. Total doses of 16.4 Gy TBI and 3.4 mg busulfan were chosen for their equivalence in inducing near complete engraftment of allogeneic marrow from donor mice of the LP strain. The two treatment groups had a late wave of mortality starting at about 80 weeks after transplantation. Specific tissue damage was manifested in bone marrow stem cells, splenic T-cell precursors, hair greying and cataract formation for both TBI and busulfan but to varying degrees. Severe nephrotoxicity and anemia were observed only after TBI. Although both busulfan and TBI kill early marrow stem cells and are effective preparative agents in bone marrow transplantation, their effects on other stem cell and organ systems are not similar. In addition, many of the injuries seen are late to occur. The delayed expression of injury deserves careful long-term evaluation of BMT recipients before the therapeutic potential of effective preparative regimens can be fully appreciated.

Recipient preparation for bone marrow transplantation. I. Efficacy of total-body irradiation and busulfan.

The efficacy of total body irradiation and busulfan were studied in recipient preparation for bone marrow transplantation. Male C57BL/6 (B6) mice were prepared for BMT with fractionated TBI or busulfan given in 4 equal doses over 3 days. Both TBI and busulfan are potent stem-cell killers. Both agents resulted in an exponential decrease in CFUs survival with increasing dose down to a 1 x 10(-4) CFUs survival. There appeared to be no break in the curve for either agent. Extrapolated fractional CFUs survival, as related to equivalent donor marrow engraftment or to equivalent 30-day survival without marrow transplantation, appeared lower for TBI as compared to busulfan. This may be due to the effect of busulfan and TBI on different stem-cell populations. Erythroid engraftment was tested after transplanting H-2 compatible LP marrow cells into treated B6 recipients. Greater than 80% of animals demonstrated complete engraftment with 3.4 mg busulfan or 1640 cGy TBI. At these 2 doses, the rate of recovery of donor marrow cellularity and CFUs content in treated recipients were identical for both preparative agents such that a selective effect on the host hematopoietic microenvironment harmful to engraftment was not seen. Complete engraftment in 100% of busulfan-prepared animals could not be achieved as such doses resulted in severe and fatal pulmonary vascular injury at 7-12 weeks posttransplant.

The effect of donor T lymphocytes and total-body irradiation on hemopoietic engraftment and pulmonary toxicity following experimental allogeneic bone marrow transplantation.

To study the effects of donor T lymphocytes on engraftment and graft-versus-host disease in relation to recipient total-body irradiation, we have returned small numbers of T cells to T-cell-depleted bone marrow transplanted across a minor histocompatibility barrier in mice (B10.BR-->CBA). T-cell-depleted B10.BR marrow (10(7) cells) was transplanted into CBA recipients prepared with TBI doses ranging from 4 to 14 Gy. Selected animals also received 10(4) (0.1%) and 10(5) (1.0%) measured B10.BR T lymphocytes. The extent of donor marrow engraftment was determined from hemoglobin and carbonic anhydrase phenotyping of peripheral blood at 3 months posttransplant. Toxicity was assessed from breathing-rate measurements, histopathology, and animal survival. Addition of T cells had a profound effect on survival related to radiation dose. The TBI doses resulting in an LD50 at 12 weeks were 6.9 Gy, 9.3 Gy, and 13.0 Gy for animals receiving 10(5), 10(4), and no T cells, respectively. Mortality was associated with pulmonary dysfunction as measured by an elevation of breathing rates. Autopsy and histological analysis revealed extensive damage to the lung parenchyma. In contrast to the toxicity data, addition of T cells to the donor marrow had no effect on the TBI dose required for equivalent erythroid engraftment. These results demonstrate that in combination with TBI small numbers of T cells in the transplanted marrow do not aid engraftment but do significantly increase the risk of pulmonary toxicity.

Depletion of anti-gal antibodies in baboons by intravenous therapy with bovine serum albumin conjugated to gal oligosaccharides.

BACKGROUND: Anti-Galalpha 1-3Gal (Gal) antibodies (Ab) play a key role in the rejection of pig cells or organs transplanted into primates. A course of extracorporeal immunoadsorption (EIA) of anti-Gal Ab using an immunoaffinity column of a Gal type 6 oligosaccharide depletes Ab successfully, but Ab returns during the next few days. Although therapy with an anti-CD154 monoclonal antibody (mAb) prevents an induced Ab response to Gal or non-Gal epitopes, T cell-independent natural anti-Gal IgM and IgG return to baseline (pretransplant) levels. We have investigated the capacity of continuous i.v. infusion of bovine serum albumin conjugated to Gal type 6 oligosaccharide (BSA-Gal) to deplete or maintain depletion of circulating anti-Gal Ab. METHODS: Porcine peripheral blood mobilized progenitor cells (PBPC) obtained by leukapheresis from MHC-inbred miniature swine (n=6) were transplanted into baboons. Group 1 baboons (n=4) underwent whole body (300 cGy) and thymic (700 cGy) irradiation, T cell depletion with antithymocyte globulin, complement depletion with cobra venom factor, short courses of anti-CD154 mAb therapy (20 mg/kg i.v. on alternate days), cyclosporine (CyA) (in two baboons only), mycophenolate mofetil, and porcine hematopoietic growth factors. Anti-Gal Ab depletion by EIA was carried out before transplantation of high doses (2-4x 1010 cells/kg) of PBPC. Group 2 baboons (n=3) received the group 1 regimen (including CyA) plus a continuous i.v. infusion of BSA-Gal. To prevent sensitization to BSA, anti-CD154 mAb therapy was continued until BSA-Gal administration was discontinued. RESULTS: In group 1, Gal-reactive Ab returned to pre-PBPC transplant levels within 15-21 days, but no induced Ab to Gal or non-Gal determinants developed while anti-CD154 mAb therapy was being administered. In group 2, anti-Gal Ab was either not measurable or minimally measurable while BSA-Gal was being administered. After discontinuation of BSA-Gal, Ab did not return to pre-PBPC transplant level for more than 40-60 days, and no sensitization developed even when all therapy was discontinued. In one baboon, however, Ab to Gal type 2, but not type 6, returned during BSA-Gal therapy. CONCLUSIONS: Prevention of the induced humoral response to Gal and non-Gal epitopes by anti-CD154 mAb therapy has been reported previously by our group, but our studies are the first to demonstrate a therapy that resulted in an absence of natural anti-Gal Ab for a prolonged period. The combination of BSA-Gal and T cell costimulatory blockade may facilitate survival of pig cells and organs transplanted into primates. The return in one baboon of Ab reactive with the Gal type 2 oligosaccharide, but not type 6, indicates some polymorphism of anti-Gal Ab and suggests that, to be effective in all cases, the infusion of a combination of type 6 and type 2 BSA-Gal may be required.

High-dose porcine hematopoietic cell transplantation combined with CD40 ligand blockade in baboons prevents an induced anti-pig humoral response.

BACKGROUND: In pig-to-primate organ transplantation, hyperacute rejection can be prevented, but the organ is rejected within days by acute vascular rejection, in which induced high-affinity anti-Gal alpha1-3Gal (alphaGal) IgG and possibly antibodies directed against new porcine (non-alphaGal) antigenic determinants are considered to play a major role. We have explored the role of an anti-CD40L monoclonal antibody in modifying the humoral response to porcine hematopoietic cells in baboons pretreated with a nonmyeloablative regimen. METHODS: Porcine peripheral blood mobilized progenitor cells obtained by leukapheresis from both major histocompatibility complex-inbred miniature swine (n=7) and human decay-accelerating factor pigs (n=3) were transplanted into baboons. Group 1 baboons (n=3) underwent whole body (300 cGy) and thymic (700 cGy) irradiation, T cell depletion with ATG, complement depletion with cobra venom factor, short courses of cyclosporine, mycophenolate mofetil, porcine hematopoietic growth factors, and anti-alphaGal antibody depletion by immunoadsorption before transplantation of high doses (2-4 x 10(10)/cells/kg) of peripheral blood mobilized progenitor cells. In group 2 (n=5), cyclosporine was replaced by eight doses of anti-CD40L monoclonal antibodies over 14 days. The group 3 baboons (n=2) received the group 1 regimen plus 2 doses of anti-CD40L monoclonal antibodies (on days 0 and 2). RESULTS: In group 1, sensitization to alphaGal (with increases in IgM and IgG of 3- to 6-fold and 100-fold, respectively) and the development of antibodies to new non-alphaGal porcine antigens occurred within 20 days. In group 2, no sensitization to alphaGal or non-alphaGal determinants was seen, but alphaGal-reactive antibodies did return to their pre- peripheral blood mobilized progenitor cells transplant levels. In group 3, attenuated sensitization to alphaGal antigens was seen after cessation of cyclosporine and mycophenolate mofetil therapy at 30 days (IgM 4-fold, IgG 8-30-fold), but no antibodies developed against new porcine determinants. In no baboon did anti-CD40L monoclonal antibodies prevent sensitization to its own murine antigens. CONCLUSIONS: We believe these studies are the first to consistently demonstrate prevention of a secondary humoral response after cell or organ transplantation in a pig-to-primate model. The development of sensitization to the murine elements of the anti-CD40L monoclonal antibodies suggests that nonresponsiveness to cell membrane-bound antigen (e.g., alphaGal) is a specific phenomenon and not a general manifestation of immunological unresponsiveness. T cell costimulatory blockade may facilitate induction of mixed hematopoietic chimerism and, consequently, of tolerance to pig organs and tissues.

Pig hematopoietic cell chimerism in baboons conditioned with a nonmyeloablative regimen and CD154 blockade.

BACKGROUND: In an attempt to induce mixed hematopoietic chimerism and transplantation tolerance in the pig-to-primate model, we have infused high-dose porcine peripheral blood progenitor cells (PBPC) into baboons pretreated with a nonmyeloablative regimen and anti-CD154 monoclonal antibody (mAb). METHODS: Group 1 baboons (n=2) received a nonmyeloablative regimen including whole body irradiation, pharmacological immunosuppression, porcine hematopoietic growth factors, and immunoadsorption of anti-Galalpha1,3Gal (Gal) antibody before infusion of high doses of PBPC (2.7-4.6x10(10) cells/kg). In group 2 (n=5), cyclosporine was replaced by anti-CD154 mAb. Group 3 (n=3) received the group 1 regimen plus anti-CD154 mAb. RESULTS: In group 1, pig chimerism was detected in the blood by flow cytometry (FACS) for 5 days (with a maximum of 14%), and continuously up to 13 days by polymerase chain reaction (PCR). In group 2, pig chimerism was detectable for 5 days by FACS (maximum 33%) and continuously up to 28 days by PCR. In group 3, initial pig chimerism was detectable for 5 days by FACS (maximum 73%). Two of three baboons showed reappearance of pig cells on days 11 and 16, respectively. In one, in which no anti-Gal IgG could be detected for 30 days, pig cells were documented in the blood by FACS on days 16-22 (maximum 6% on day 19) and pig colony-forming cells were present in the blood on days 19-33, which we interpreted as evidence of engraftment. Microchimerism was continuous by PCR up to 33 days. CONCLUSIONS: These results suggest that there is no absolute barrier to pig hematopoietic cell engraftment in primates, and that this may be facilitated if the return of anti-Gal IgG can be prevented.

The assessment of early and late radiation injury to the mouse lung using X-ray computerised tomography.

X-ray computerised tomography (CT) was performed on the lungs of CBA and C57Bl mice at varying time intervals after 13 and 16 Gy irradiation to the whole thorax. With careful consideration of artefacts associated with lung cross-sectional area and breathing rate, the mean density of the lung was evaluated in Hounsfield units (CT number). In CBA mice, this parameter showed a biphasic increase in lung density with time from irradiation, corresponding to an early phase of radiation pneumonitis and a late phase dominated by pleural effusions. Reduced lung volumes were also seen during the late response and lung compression due to accumulations of pleural fluid is considered a major factor in these observations. C57Bl mice did not develop radiation pneumonitis but appeared to be equally responsive to later radiation-induced increases in lung density. The results obtained from CT-derived densitometry compared well with measurements gained from functional and survival endpoints. X-ray CT provides a sensitive and informative technique for assessing the extent of radiation injury to the mouse lung and is potentially useful for quantifying the counterpart in patients.

Lung damage after hemithoracic irradiation: dependence on mouse strain.

A comparative study was performed on CBA, C57Bl mice and their F1 hybrid cross (CBBF1) after right hemithorax irradiation using doses ranging from 15 to 35 Gy. A dose-dependent increase in breathing rate was observed, corresponding to the expression of radiation pneumonitis in the irradiated lung. The use of hemithoracic irradiation enabled this to be observed without any lethality. Results obtained from X-ray computerized tomography (CT), lung weight and histology complemented the breathing rate studies. CBA mice showed a peak response at 16 weeks followed by a decline in breathing rate coincident with a compensatory hypertrophy of the shielded left lung. The manifestation of radiation pneumonitis in C57Bl mice was considerably delayed, supporting previous findings on whole-thorax irradiated animals. The latent period in CBBF1 hybrid mice was intermediate between the parent strains.