Intensified postgrafting immunosuppression failed to assure long-term engraftment of dog leukocyte antigen-identical canine marrow grafts after 1 gray total body irradiation

BACKGROUND: Late graft rejection after conditioning with 1 Gy of total body irradiation (TBI) was consistently seen in historical dogs given two postgrafting immunosuppressive drugs. METHODS: Here, 16 dogs were given four different three-drug combinations of cyclosporine, mycophenolate mofetil, sirolimus, or methotrexate after 1 Gy TBI and dog leukocyte antigen-identical marrow grafts. In addition, we assessed the effects of TBI doses of 0.5, 1.0, 2.0, or 3.0 Gy, respectively, on immune functions in six dogs not given marrow grafts. RESULTS: All dogs showed initial engraftment, 13 rejected, and three had sustained grafts beyond 26 weeks. The dogs with durable grafts had received greater median numbers of nucleated marrow cells compared with the 13 dogs that rejected their grafts (6.14 vs. 3.6 x 10(8) per kg; P=0.03). In a Cox proportional hazard model, which included data from 16 historical dogs, each increase in transplanted marrow cell numbers by 1 x 10(8) per kg decreased the hazard ratio of rejection by 0.5. Decreasing percents of remaining CD3, CD4, and CD8 cells in peripheral blood and lymph nodes were observed with increasing TBI doses. Further, greater suppressions of B-cell- and T-cell-dependent production of IgM and IgG antibodies in response to sheep red blood cell injections were observed after 2 Gy compared with 1 Gy TBI. CONCLUSION: Overall, triple postgrafting immunosuppression after 1 Gy TBI was well tolerated but failed to prevent graft rejection in this model. In vivo radiation studies have shown higher numbers of remaining host lymphocytes and better T-cell-dependent antibody production after 1 Gy compared with 2 Gy TBI.     

Prolonged allogeneic marrow engraftment following nonmyeloablative conditioning using 100 cGy total body irradiation and pentostatin before and pharmacological immunosuppression after transplantation

In a canine model of dog leukocyte antigen (DLA)-identical nonmyeloablative marrow transplantation including postgrafting immunosuppression with cyclosporine (CSP) and mycophenolate mofetil (MMF), engraftment was only transient with 100 cGy total body irradiation (TBI) conditioning, indicating suboptimal pretransplant immunosuppression. In contrast, grafts after 200 cGy TBI were durable in 11/12 recipients. We hypothesized that addition of pentostatin before transplantation could, in part, substitute for 100 cGy TBI. Pharmacokinetic studies showed pentostatin significantly inhibited adenosine deaminase in canine lymphocytes. Eight dogs were conditioned with 6x4 mg/m pentostatin and 100 cGy TBI, whereas two dogs received 3x4 mg/m pentostatin plus 100 cGy TBI. All were given MMF/CSP posttransplant. All showed initial engraftment; four remained stable mixed chimeras for >32 weeks. The median duration of engraftment was 13 (range 9 to >39) weeks, which was significantly longer than in six historical controls conditioned with 100 cGy TBI alone (median 10, range 3-12 weeks) (P=0.01).     

The use of radiolabeled anti-CD33 antibody to augment marrow irradiation prior to marrow transplantation for acute myelogenous leukemia.

Disease recurrence remains a major limitation to the use of marrow transplantation to treat leukemia. Previous transplant studies have demonstrated that higher doses of total-body irradiation result in less disease recurrence, but more toxicity. In this study, the possibility of delivering radiotherapy specifically to marrow using a radiolabeled anti-CD33 antibody (p67) was explored. Biodistribution studies were performed in nine patients using .05-.5 mg/kg p67 trace-labeled with 131I. In most patients initial specific uptake of 131I-p67 in the marrow was seen, but the half-life of the radiolabel in the marrow space was relatively brief, ranging from 9-41 hr, presumably due to modulation of the 131I-p67-CD33 complex with subsequent digestion and release of 131I from the marrow space. In four of nine patients these biodistribution studies demonstrated that with 131I-p67 marrow and spleen would receive more radiation than any normal nonhematopoietic organ, and therefore these four patients were treated with 110-330 mCi 131I conjugated to p67 followed by a standard transplant regimen of cyclophosphamide plus 12 Gy TBI. All four patients tolerated the procedure well and three of the four are alive in remission 195-477 days posttransplant. This study demonstrates the feasibility of using a radiolabeled antimyeloid antibody as part of a marrow transplant preparative regimen and also highlights a major limitation of using conventionally labeled anti-CD33--namely, the short residence time in marrow. Strategies to overcome this limitation include the use of alternative labeling techniques or the selection of cell surface stable antigens as targets.     

Isoamylase levels in bone marrow transplant patients are affected by total body irradiation and not by graft-versus-host disease

The mean total serum amylase levels in patients was 3.2±0.5 kat/l (±SE) before total body irradiation (TBI) prior to bone marrow transplantation of which 50% was due to pancreatic isoamylase and 50% salivary isoamylase. Total serum amylase increased to a maximum of 100.3±12.3 kat/l on the first day after TBI and most of this increase was due to an increase in salivary isoamylase (90.0±12.1 kat/l). In association with this, all patients had clinical symptoms of parotitis. An increase in pancreatic isoamylase was found in 27% of the patients; however; none of them had clinical symptoms of pancreatitis. Serum amylase levels returned to normal within 5 days after TBI but then decreased to subnormal values, remaining below the normal range for 3 weeks. Pancreatic isoamylase returned to pre-irradiation levels 1.5 months after TBI, while salivary isoamylase remained low for the rest of the observation time. TBI of 7.5 Gy at 26 cGy/min gave significantly lower salivary amylase at 2 days after TBI compared with 10 Gy at 4 cGy/min: 32±4 versus 76±13 kat/l (P<0.05). At 2.5 and 6 months after TBI significantly higher total amylase levels were recorded for patients treated with 7.5 Gy of TBI compared with 10 Gy: 2.5±0.4 and 2.7±0.3 versus 2.0±0.5 and 0.8±0.3 kat/l, respectively (P<0.01, P<0.05, respectively). Acute or chronic GVHD did not affect acinar cells in this investigation.     

Isoamylase levels in bone marrow transplant patients are affected by total body irradiation and not by graft-versus-host disease

Abstract. The mean total serum amylase levels in patients was 3.2 ± 0.5 μkat/l (± SE) before total body irradiation (TBI) prior to bone marrow transplantation of which 50% was due to pancreatic isoamylase and 50% salivary isoamylase. Total serum amylase increased to a maximum of 100.3 ± 12.3 μkat/l on the first dayafter TB Iandmostofthis increase was due to an increase in salivary isoamylase (90.0 ± 12.1 μkat/l). In association with this, all patients had clinical symptoms of parotitis. An increase in pancreatic isoamylase was found in 27% of the patients; however, none of them had clinical symptoms of pancreatitis. Serum amylase levels returned to normal within 5 days after TBI but then decreased to subnormal values, remaining below the normal range for 3 weeks. Pancreatic isoamylase returned to pre-irradiation levels 1.5 months after TBI, while salivary isoamylase remained low for the rest of the observation time. TBI of 7.5 Gyat 26 cGy/min gave significantly lower salivary amylase at 2 days after TBI compared with 10 Gy at 4 cGy/min: 32 ± 4 versus 76 ± 13 μkat/l ( P < 0.05). At 2.5 and 6 months after TBI significantly higher total amylase levels were recorded for patients treated with 7.5 Gy of TBI compared with 10 Gy: 2.5 ± 0.4 and 2.7 ± 0.3 versus 2.0 ± 0.5 and 0.8 ± 0.3 μkat/1, respectively ( P < 0.01, P < 0.05, respectively). Acute or chronic GVHD did not affect acinar cells in this investigation.     

Effect of trimethoprim-sulfamethoxazole on hematological recovery after total body irradiation and autologous marrow infusion in dogs.

To test the effect of the combination of trimethoprim-sulfamethoxazole (TMP-SMX) on hematological recovery after bone marrow transplantation, dogs were conditioned with 1,200 R total body irradiation and then infused with autologous marrow. Twenty dogs were given TMP-SMX at doses equivalent to 10, 20, or 40 mg TMP/kg/day beginning on the day of marrow infusion and continued until the granulocyte count reached 10(3)/mul; in addition, one-half of the dogs received methotrexate, 0.4 mg/kg on days 1, 3, 6, and 11 and then weekly until termination of the study. Granulocyte and platelet changes were compared to those of dogs given 1,200 R, autologous marrow infusion, and no TMP-SMX. Dogs given 10 and 20 mg TMP/kg/day had normal granulocyte and platelet recovery after irradiation. Dogs given 40 mg TMP/kg/day showed a significant delay in granulocyte recovery and mildly delayed platelet recovery. This suggests that TMP-SMX in prophylactic doses (5 mg TMP/kg/day) can be given safely to human patients immediately after allogeneic marrow transplantation and thus probably prevent even very early cases of Pneumocystis carinii pneumonia, and possibly bacterial infections as well.     

Graft versus host disease after stem cell allotransplantation with low-dose total body irradiation, fludarabine, and antithymocyte globulin

BACKGROUND: We previously showed that antithymocyte globulin (ATG) given with total body irradiation (TBI) 200 cGy and fludarabine results in high rate of donor engraftment. Its influence on acute and chronic graft versus host disease (GVHD) and on graft versus tumor effect is less known. METHODS: Sixty-five patients underwent nonmyeloablative stem cell transplant with ATG, TBI 200 cGy, and fludarabine. GVHD prophylaxis was mycophenolate mofetil and cyclosporine. Forty-two patients (pts) (65%) had match related donors, 18 (27%) match unrelated, 1 (1.5%) mismatch related, and 4 (6%) mismatch unrelated donors. RESULTS: At a median follow-up of 862 days, 24 patients (37%) developed GVHD. The median age of the patients with and without GVHD was 56 years respectively. Acute GVHD grade II-IV developed in 19 pts (29%). Fatal GVHD of liver and/or gut occurred in nine pts (14%). Forty-one pts survived more than 100 days. Five pts (12%) had chronic GVHD, two had extensive, and three had limited involvement. Relapsed disease was observed in 22 pts (34%). Infections occurred in 15 pts (23%) and were fatal in 13 (20%). CONCLUSIONS: The addition of ATG to TBI 200cGy and fludarabine resulted in a modest incidence of GVHD. The best transplant outcomes were observed in pts with lymphoid malignancies.     

Effects of BWH-4 anti-CD4 monoclonal antibody on rat vascularized cardiac allografts before and after engraftment.

We studied the effects and mechanism of action of BWH-4, an IgG2a mouse antirat CD4 monoclonal antibody that recognizes a distinct epitope on the CD4 molecule, in LEW recipients of (LEW x BN)F1 vascularized heterotopic cardiac allografts. Ten animals received daily injections of 700 micrograms BWH-4 for ten days after engraftment. Median actuarial allograft survival was 37 days for the treated animals compared with 7.5 days for controls (n = 6). There was depletion of peripheral blood CD4+ lymphocytes to 4 +/- 1% one week posttransplant (normal = 48 +/- 4%, n = 6). Six additional animals were treated with a lower dose (100 micrograms) of BWH-4 daily for ten days. Median actuarial allograft survival was 10 days and the circulating CD4+ cells decreased to 30 +/- 6% at one week posttransplant. All six low-dose-treated animals mounted an anti-BN alloantibody response by 3 weeks, while only one of the ten high-dose-treated animals had positive alloantibodies two weeks posttransplant. Lymphocyte-mediated cytotoxicity against donor lymphoblasts was completely abolished in the high-dose-treated animals when compared with acutely rejecting controls. We also used low-dose (100 micrograms) BWH-4 to pretreat eleven experimental animals for 7 days prior to engraftment. The circulating CD4+ cells decreased to only 12 +/- 2% on the day of transplantation and 26 +/- 9% one week posttransplant. However, six (55%) pretreated animals survived more than 55 days. There was a 66% decrease in lymphocyte-mediated cytotoxicity against donor lymphoblasts when compared with acutely rejecting controls. We conclude that the anti-CD4 mAb, BWH-4, prevents acute rejection of vascularized heterotopic rat cardiac allografts; this effect is mediated by depletion of the CD4+ T cell subset, suppression of alloantibody production, and inhibition of lymphocyte-mediated cytotoxicity.     

Comorbidity does not predict long-term mortality after total hip arthroplasty

Background and purpose — In-hospital death following total hip arthroplasty (THA) is related to comorbidity. The long-term effect of comorbidity on all-cause mortality is, however, unknown for this group of patients and it was investigated in this study.

Patients and methods — We used data from the Swedish Hip Arthroplasty Register, linked to the National Patient Register from the National Board of Health and Welfare, for patients operated on with THA in 1999–2012. We identified 120,836 THAs that could be included in the study. We evaluated the predictive power of the Charlson and Elixhauser comorbidity indices on mortality, using concordance indices calculated after 5, 8, and 14 years after THA.

Results — All comorbidity indices performed poorly as predictors, in fact worse than a base model with age and sex only. Elixhauser was, however, the least bad choice and it predicted mortality with concordance indices 0.59, 0.58, and 0.56 for 5, 8, and 14 years after THA.

Interpretation — Comorbidity indices are poor predictors of long-term mortality after THA.     

Hematopoietic cell transplant outcomes after myeloablative conditioning with fludarabine,busulfan, low-dose total body irradiation,and rabbit antithymocyte globulin

Optimal conditioning and graft-vs-host disease (GVHD) prophylaxis for hematopoietic cell transplantation (HCT) are unknown. Here, we report on outcomes after low toxicity, myeloablative conditioning consisting of fludarabine, busulfan, and 4 Gy total body irradiation, in combination with thymoglobulin and post-transplant methotrexate and cyclosporine. We retrospectively studied 700 patients with hematologic malignancies who received blood stem cells from 7 to 8/8 HLA-matched unrelated or related donors. Median follow-up of surviving patients was 5 years. At 5 years, overall survival (OS), relapse-free survival (RFS), and chronic GVHD/relapse-free survival (cGRFS) were 58%, 55%, and 40%. Risk factors for poor OS, RFS, and cGRFS were (1). high to very high disease risk index (DRI), (2). high recipient age, and (3). cytomegalovirus (CMV)-seropositive recipient with seronegative donor (D−R+). The latter risk factor applied particularly to patients with lymphoid malignancies. Neither donor other than HLA-matched sibling (7-8/8 unrelated) nor one HLA allele mismatch was risk factors for poor OS, RFS, or cGRFS. In conclusion, the above regimen results in excellent long-term outcomes. The outcomes are negatively impacted by older age, high or very high DRI, and CMV D−R+ serostatus, but not by donor unrelatedness or one HLA allele mismatch.     

A background infusion of morphine does not enhance postoperative analgesia after cardiac surgery

PURPOSE: To compare the effects of patient-controlled analgesia (PCA), with or without a background infusion of morphine on postoperative pain relief and stress response after cardiac anesthesia. METHODS: With University Ethics approval, 35 consenting adults undergoing elective open-heart surgery were randomly assigned preoperatively in a double-blind fashion to receive either morphine PCA alone (Group I, n = 15) or morphine PCA plus a continuous basal infusion (Group II, n = 14) for 44 hr postoperatively. Pain scores with visual analogue scale (VAS) at rest, deep inspiration and with cough, sedation scores, stress hormone levels [cortisol, adrenocorticotropin (ACTH) and growth hormone (GH)] and morphine consumption were assessed, and serum morphine levels were measured at four, 20, 28 and 44 hr after surgery. Adverse effects including nausea, vomiting, constipation, urinary retention and pruritus were noted. Total blood, fluid requirements, drainage and urinary output were recorded. RESULTS: Postoperative morphine consumption at 44 hr was less in Group I (29.43 +/- 12.57 mg) than in Group II (50.14 +/- 16.44 mg), P = 0.0006. There was no significant difference between groups in VAS scores, GH levels, blood levels of morphine and adverse effects. While VAS scores, ACTH and GH levels decreased significantly in both groups, plasma cortisol levels increased significantly in Group I only at four hours. In Group II, ACTH and cortisol were higher at four and 44 hr respectively. CONCLUSION: PCA with morphine effectively controlled postoperative pain after cardiac surgery. The addition of a background infusion of morphine did not enhance analgesia and increased morphine consumption.     

Changes in total body calcium after renal transplantation: effect of low-dose steroid regime

Total body calcium was measured in 8 men and 4 women, aged 20-51 years, undergoing kidney transplantation. The initial measurement was made within 8 weeks of operation and subsequent measurements up to 33 months postoperatively. Transplant rejection was prevented by low-dose prednisolone therapy (20 mg/day). 2 patients underwent parathyroidectomy for hypercalcaemia, and their total body calcium increased by 29 g (3%) and 66 g (8%). In the remainder the mean annual change was -0.9% (3.7, SD) over an average follow-up period of 17 months. This fall in total body calcium was statistically insignificant and was smaller than that previously described in patients treated with higher doses of steroids.     

The use of an anti-TCRalphabeta monoclonal antibody to control host-versus-graft reactions in canine marrow allograft recipients conditioned with low dose total body irradiation.

BACKGROUND: A limitation in the application of marrow transplantation has been complications related to the conditioning regimens that have been intensified to the point where organ toxicities have been common, resulting in morbidity and mortality. METHODS: A conditioning regimen consisting of low-dose total body irradiation (TBI*) was used to test whether postgrafting therapy with a monoclonal antibody (mAb) against the T cell receptor (TCR)alphabeta facilitated sustained engraftment of marrow from dog leukocyte antigen (DLA)-identical canine littermates. The anti-TCRalphabeta mAb 15.9D5 was selected for in vivo studies because it induced hyporesponsiveness to allogeneic stimulator cells in mixed leukocyte culture and was not mitogenic in vitro. RESULTS: When recipients of genotypically DLA-identical marrow were conditioned by the barely "lethal" dose of 450 cGy TBI alone, almost 60% of grafts failed (n=39). The remainder engrafted, either in the form of stable mixed donor/host or all donor hematopoietic chimerism. In contrast to results in controls, 5 of 6 dogs that were given, in addition, a loading dose of mAb 15.9D5 of 1 mg/kg on day -1, 450 cGy TBI on day 0, followed by mAb at 0.3 mg/kg/day until day +7, showed sustained engraftment (P=.058). To accomplish a comparable rate of engraftment in the absence of anti-TCRalphabeta antibody, 920 cGy TBI were needed for pretransplant conditioning. CONCLUSIONS: Results strongly suggested that in vivo administration of a mAb against TCRalphabeta prevented rejection of allogeneic marrow grafts in the setting of conditioning with a relatively nontoxic but otherwise suboptimal dose of 450 cGy TBI. In vivo administration of m Ab 15.9D5 was well tolerated without any noticeable side effects. The exact mechanism by which the mAb works in vivo is as yet poorly understood, but it does not involve CD3/TCR complex modulation or elimination of T cells from the circulation.