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Biomedical subjects

D A Vallera

Publications and source records attributed to D A Vallera.

At least 19 recordsLinked to original sources
PubMed1992

Promotion of murine marrow alloengraftment and hematopoietic recovery across the major histocompatibility barrier by administration of recombinant human interleukin-1 alpha.

Irradiated C57BL/6 (H-2b) recipients of T-cell-depleted (TCD) BALB/c (H-2d) bone marrow (BM) and recombinant interleukin-1 alpha (IL-1 alpha) (1 microgram/d) had a significantly (P less than or equal to .006) higher 100-day actuarial survival rate, accelerated hematopoietic recovery, and higher levels of alloengraftment than a group of transplanted control mice treated identically, but given phosphate-buffered saline (PBS). To elucidate the mechanisms involved with IL-1 alpha-induced promotion of alloengraftment and hematopoietic recovery, we performed sequential splenic FACS studies on transplanted mice and secondary transfer studies in syngeneic mice given IL-1 alpha or PBS. Splenic phenotyping showed that recipients of IL-1 alpha had a higher proportion of donor granulocytes (52% v 19%) as compared with PBS controls as early as 7 days after bone marrow transplantation (BMT). On day 11 post-BMT, recipients of IL-1 alpha had a more than fourfold increase in splenocyte number, which included a higher percentage (90% v 59%) of donor cells, especially donor granulocytes (52% vs 32%), and a sevenfold increase in donor T cells as compared with controls. Host T-cell numbers were not affected. Taken together, these data suggest that IL-1 alpha stimulated bipotential (myeloid and lymphoid) donor cell engraftment. In a syngeneic BMT system, administration of IL-1 alpha resulted in a higher incidence of survival when recipients were transplanted with BM cells, indicating that IL-1 alpha administration probably either expanded or potentiated engraftment of a committed progenitor cell pool. Secondary transfer experiments using marrow from IL-1 alpha-treated mice showed that the number of day 12 colony-forming unit-spleen (CFU-S) cells was unaltered compared with untreated control mice, suggesting that more primitive, albeit committed, hematopoietic progenitor cells were not affected. We also examined the potential additive effects of IL-1 alpha and granulocyte-macrophage colony-stimulating factor (GM-CSF) administered in combination (for 14 days). Mice receiving a suboptimal amount of IL-1 alpha along with GM-CSF had significantly higher levels of donor alloengraftment (92%) with superior hematopoietic recovery, as compared with mice receiving either IL-1 alpha (57%) or GM-CSF (18%) alone.

Animals↗
PubMed1992

Effect of recombinant human macrophage colony-stimulating factor in irradiated murine recipients of T-cell-depleted allogeneic or non-depleted syngeneic bone marrow transplants.

Recombinant macrophage colony-stimulating factor (rM-CSF), which reacts exclusively with cells of monocyte lineage, was evaluated in the murine bone marrow (BM) transplant setting for in vivo effects on recipient survival, hematologic recovery, and engraftment. Two types of fully allogeneic donors were selected based on the expression (BALB/c), or lack of expression (DBA/1), of hybrid hematopoietic histocompatibility (Hh1) antigens. These antigens are established targets for monocyte and/or natural killer (NK) cell-mediated graft rejection. Irradiated C57BL/6 mice were used as recipients for all experiments. Recipients of T-cell-depleted (TCD) BALB/c BM and a 14-day continuous subcutaneous infusion of 16.8 micrograms/d rM-CSF (n = 30) showed a significant decrease in donor cell engraftment as compared with recipients of donor BM administered pumps delivering saline. These mice administered rM-CSF also displayed significantly reduced levels of circulating leukocytes (predominantly lymphocytes) on day 14 posttransplant (compared with saline controls). Neither engraftment effects nor leukocyte effects were observed when C57BL/6 recipients were administered Hh1 nonexpressing TCD DBA/1 BM cells (n = 30), suggesting that the monocyte/macrophage population is important in long-term alloengraftment in certain donor-recipient strain combinations in which donor Hh1 antigens can serve as target antigens for host effector cells, but are not important in strain combinations in which they are not recognized. Circulating tumor necrosis factor alpha (TNF alpha) levels measured at two time periods during rM-CSF infusion were not elevated. Thus, the reduction in alloengraftment is not likely to be directly related to TNF alpha. However, in vivo elimination of NK cells in the BALB/c into C57BL/6 model prevented the impairment of engraftment mediated by rM-CSF. Thus, rM-CSF-mediated inhibition of alloengraftment is contingent on the presence of host NK cells with antidonor reactivity. Survival was unaffected when rM-CSF was administered in either allogeneic BM transplant model, but was significantly reduced when rM-CSF was administered to C57BL/6 recipients of syngeneic BM transplants. These data are the first analyzing the effects of rM-CSF in murine allogeneic BM transplantation and extend our previous studies using the BALB/c into C57BL/6 model in which in vivo infusions of recombinant granulocyte-macrophage CSF, but not recombinant granulocyte-CSF, lead to decreases in alloengraftment. These data show that rM-CSF-induced stimulation of monocytes may increase BM graft rejection in instances in which NK cells are involved in the rejection process. These data may have future clinical implications for the use of rM-CSF in allogeneic BM transplantation.

Animals↗
PubMed1992

Successful treatment of human acute T-cell leukemia in SCID mice using the anti-CD7-deglycosylated ricin A-chain immunotoxin DA7.

The study of new therapeutic approaches for refractory human leukemia has been hampered by the lack of relevant in vivo models with disseminated disease, particularly T acute lymphoblastic leukemia (T-ALL). In the present study we evaluated methods for establishing and therapy of a human T-ALL cell line (MT-ALL) in 73 SCID mice. MT-ALL is a T-cell receptor alpha/beta +, CD3+, and CD7+ leukemia cell line, derived from a patient with refractory disease and early death. Injection of 5 x 10(7) MT-ALL cells i.v. caused disseminated human leukemia in hematopoietic and nonhematopoietic organs in 100% of SCID mice (n = 9) leading to death or terminal disease at 65 to 70 days after a uniform clinical course. To study possible therapeutic approaches for disseminated leukemia we utilized an immunotoxin, DA7, constructed by chemically linking the mouse IgG2b anti-CD7(3A1E) monoclonal antibody which recognizes a pan-T-cell marker expressed on almost all T-cell leukemias to deglycosylated ricin A-chain, a catalytic plant toxin and inhibitor of protein synthesis. Administration of DA7 led to greater than 5 log kill of clonogenic MT-ALL cells in vitro and selectively inhibited protein synthesis. DA7 was administered to mice at a dose of 10 micrograms/mouse/day for 5 consecutive days starting 8 days after i.v. inoculation of leukemia. The immunotoxin therapy resulted in significant long term survival over 348 days compared to untreated or control mice treated with anti-CD7 antibody and deglycosylated ricin A-chain which were all dead by day 70 (P less than 0.001). Even after more than 11 months there was no evidence of disease in 82% of the DA7 treated animals. SCID mice given i.p. injections (n = 9) developed an i.p. tumor mass but demonstrated metastasis outside the peritoneum with disseminated leukemia in hematopoietic and nonhematopoietic organs, a finding different from most conventional nude mouse models. The leukemia was fatal in 100% and killed the animals at 68-95 days. SCID mice given i.p. injections of MT-ALL completely responded to therapy with DA7, resulting in survival of 100% of the animals (n = 10) at 216 days (P less than 0.001 compared to untreated animals). Anti-CD7 antibody, deglycosylated ricin A-chain, and a control anti-melanoma immunotoxin (IND1-RTA) showed no therapeutic effect. We conclude that DA7 is an effective in vivo therapeutic agent against human MT-ALL in the SCID mouse system, suggesting potential usefulness for therapy of humans with poor prognosis T-cell leukemia.

Animals↗
PubMed1992

Anti-CD3 immunotoxin prevents low-dose STZ/interferon-induced autoimmune diabetes in mouse.

Autoimmune diabetes was induced with an established model in which 3 daily injections of 95 mg/kg body wt/day streptozocin (STZ) and 2 x 10(4) U interferon-gamma (IFN-gamma) were administered to C57BL/6 mice. Diabetes onset was accompanied by precipitous increases in serum glucose levels and validated by immunoperoxidase studies showing diminished islets in pancreatic tissue sections. Administration of two to three doses of a monoclonal antibody (MoAb) or an immunotoxin (IT) directed against the CD3 epsilon-chain before STZ/IFN-gamma treatment prevented increases in serum glucose and protected islets from damage. IT was made by crosslinking anti-CD3 to a low oligosaccharide-containing fraction of purified ricin toxin A chain (RTA; a catalytic inhibitor of protein synthesis) with a stabilized derivative of 2-iminothiolane. Protection was complete, long-lived, and selective because two different control ITs did not prevent diabetes onset. A second pan T-cell-reactive IT was synthesized by linking the MoAb anti-Ly1 to the same RTA toxin. Anti-Ly1 reacts with the murine homologue of human CD5. Anti-Ly1 RTA also protected against diabetes onset in a dose-dependent manner requiring higher doses and a longer schedule than anti-CD3 or anti-CD3 RTA. These studies demonstrate for the first time the importance of CD3+ and CD5+ cells in diabetes onset in the low-dose STZ/IFN-gamma model and show that anti-CD3, anti-CD3 RTA, or anti-CD5 RTA may be useful in vivo for the treatment of diabetes or perhaps other T-cell-mediated autoimmune diseases. These data may have important therapeutic implications for early autoimmune diabetes in humans.

Animals↗
PubMed1991

Prevention of murine graft-versus-host disease and bone marrow alloengraftment across the major histocompatibility barrier after donor graft preincubation with anti-LFA1 immunotoxin.

We have investigated the effects of the in vitro depletion of LFA1 positive cytolytic T lymphocytes, natural killer (NK) cells, and monocytes on the afferent phase of graft-versus-host disease (GVHD). Lethal GVHD was induced across the murine major histocompatibility complex by injecting C57BL/6 (H-2b) bone marrow (BM) cells (a source of stem cells) and splenocytes (S) (a source of T cells) into lethally irradiated B10.BR (H-2k) recipients. Because anti-LFA1 does not bind complement (C') effectively, we conjugated anti-LFA1 alpha chain monoclonal antibody (MoAb) to ricin toxin A chain (RTA) as a means of facilitating target cell elimination. A 2-hour preincubation of C57BL/6 bone marrow/spleen (BMS) with anti-LFA1-RTA in the presence of ammonium chloride (a potentiator of immunotoxin toxicity), but not a control immunotoxin (IT), reduced CTL activity by greater than 2 logs, significantly reduced NK cell activity, and prevented B10.BR mice from developing GVHD. Depletion of target cells by toxin-labeled-MoAb and not the blockade of the LFA1 molecule by the anti-LFA1 MoAb accounted for our results, because incubating cells with IT in the absence of a potentiator had no effect on GVHD prevention. In contrast, C57BL/6 recipients of C3H BMS grafts only partially benefited from anti-LFA1-RTA preincubation, demonstrating that in this system, different cells not expressing LFA1 were involved in GVHD generation. The same findings observed with anti-LFA1-RTA preincubation were observed with preincubation with L-leucyl-L-leucine methyl ester, a chemical compound eliminating cytolytic cells, providing further support that GVHD induction in the C3H/HeJ into C57BL/6 system is not entirely mediated by classical cytolytic T cells. We next tested anti-LFA1-RTA in a model devised to measure its effect on alloengraftment (B10.BR recipients given lower doses of irradiation). Anti-LFA1-RTA BM preincubation selectively reduced alloengraftment in the model. This observation, combined with experiments showing that LFA1-RTA preincubation, but not anti-Thy 1.2 + C' or control IT preincubation, reduced colony-forming unit-spleen formation, indicates that anti-LFA1 alpha chain IT may remove accessory cells or stem cells critical to engraftment. Still, anti-LFA1-RTA may be useful for clinical GVHD prevention when combined with positive selection techniques designed to enrich for stem cells.

Animals↗
PubMed1991

In vivo administration of anti-CD3 monoclonal antibodies or immunotoxins in murine recipients of allogeneic T cell-depleted marrow for the promotion of engraftment.

The role of host anti-donor cells in rejection of fully allogeneic donor T cell-depleted marrow was investigated by using mAb or immunotoxins directed against T cell or NK cell determinants. Immunotoxins consisting of mAb conjugated to a low oligosaccharide-containing fraction of purified ricin toxin A chain (RTA) facilitated in vivo-depletion of target cell populations. BALB/c and DBA/1 donors were selected based upon their expression (BALB/c) or lack of (DBA/1) hemopoietic histocompatibility (Hh1) Ag, which may serve as targets for donor rejection in C57BL/6 hosts. When studies directed toward eliminating CD3+ cells were performed in both systems, injections of intact anti-CD3 mAb or anti-CD3-RTA reproducibly produced the highest engraftment values. The fact that engraftment values obtained with anti-CD3 or anti-CD3-RTA therapy in allogeneic systems were substantially higher than in syngeneic controls suggested that engraftment stimulatory proteins were released upon TCR engagement. Elevated levels of cytokines and a high mortality rate in allogeneic recipients confirmed that this was the case. Nonstimulatory preparations of anti-CD3F(ab')2 fragments and anti-CD3F(ab')2-RTA promoted engraftment of both types of allogeneic marrow, as measured by short term 125I-IUdR assays, suggesting that stimulation was not a prerequisite for engraftment. Recipients of anti-CD3F(ab')2 or anti-CD3F(ab')2-RTA showed a marked reduction of host CD3+ cells as measured by immunofluorescence and flow cytometry. In long term chimerism studies, recipients of Hh1-disparate marrow and anti-CD3F(ab')2 had a dramatic increase in donor cell engraftment as compared to controls, indicating that positive effects on engraftment were long lived. Studies further showed that BALB/c donor cells exhibiting an Hh1 disparity were rejected by host cells expressing NK1.1 or Ly-1 (NK cells and T cells). In contrast, DBA/1 donor cells that were not Hh1-disparate were rejected by cells expressing Ly-1, but not NK1.1 (T cells only). These studies provide definitive data that CD3+ cells participate in the rejection of either Hh1+ or Hh1null T cell-depleted allografts and offer new strategies for alloengraftment using regimens containing nonmitogenic anti-CD3.

Animals↗
PubMed1991

Radiotherapy in mice with yttrium-90-labeled anti-Ly1 monoclonal antibody: therapy of the T cell lymphoma EL4.

Yttrium-90 is a potent beta-emitting radionuclide with potential for therapy of lymphoma. A monoclonal antibody against Ly1, the murine homologue of human CD5, was labeled with 90Y and found to selectively bind to Ly1-positive, radiation-sensitive, EL4 mouse lymphoma cells. When tested in this aggressive model of T cell lymphoma, in vivo studies in C57BL/6 mice showed that a single 140-microCi i.p. dose of 90Y-anti-Ly1, given 1 day after i.v. injection of a lethal dose of EL4 cells, resulted in significant but transient improvement in survival. Protection was selective, since a 90Y-labeled irrelevant control antibody did not prolong survival. Biodistribution studies showed that protection was likely limited by inadequate localization of labeled antibody to tumor. Importantly, labeled anti-Ly1 specifically localized in the immunological tissue (spleen and thymus) and lowered the WBC count, perhaps limiting the tolerated dose. Myelosuppression, which is considered one of the major side effects associated with 90Y usage, was not a lethal complication, since WBC counts recovered in mice given a 140-microCi dose of 90Y-anti-Ly1 without EL4 cells and 100% of these animals survived. The maximum tolerated dose was less than 200 microCi. Despite the high localization of 90Y-anti-Ly1 in spleen, splenectomies of tumor-injected mice did not improve the antitumor efficacy of radiolabeled antibody. Further evidence for inadequate delivery of radionuclide to tumor was shown when external total-body irradiation was given to mice given injections of a lethal dose of EL4 tumor cells. Comparison of internal and external irradiation studies indicated that the partially protective effect of 140 microCi 90Y-Ly1 was equivalent to external radiation of only 100-200 cGy. Because this model reflects the current clinical limitations of radiolabeled antibodies for therapy, including partial antitumor efficacy, delivery of labeled anti-T cell antibodies to the immune system, and low maximum tolerated dose, the model may be useful for examining strategies which could increase the tolerated dose and therapeutic efficacy.

Animals↗
PubMed1991

Radiotherapy in mice with yttrium-90-labeled anti-Ly1 monoclonal antibody: therapy of established graft-versus-host disease induced across the major histocompatibility barrier.

A monoclonal antibody recognizing Ly1, the murine homologue of CD5, was labeled with 90Y. In vivo biodistribution studies showed that 90Y-anti-Ly1 selectively localized in lymphoid tissue. Groups of B10,BR mice (H-2k) were lethally irradiated and given major histocompatibility complex-disparate C57BL/6 (H-2b) bone marrow and spleen cells to induce graft-versus-host disease (GVHD). Eight days later, mice with active GVHD were administered a single i.p. injection of 50 microCi90Y-anti-Ly1. Fifty % of these mice were alive 2 months after treatment. Long term (greater than 4-month) survival was significantly higher than in phosphate-buffered saline-treated mice. Survival was slightly improved in groups of mice receiving control irrelevant antibody labeled with 90Y or mice receiving free 90Y. However, survival in these groups was not significantly different from the phosphate-buffered saline-treated control group. The improved survival was supported by data showing improved mean animal weight. An anti-GVHD effect was confirmed by histopathological analysis. Unlabeled anti-Ly1 monoclonal antibody at comparable doses to 90Y-anti-Ly1 was not effective. Animals that died following 50-microCi treatment did not die of radiation toxicity, since all mice receiving 50 microCi 90Y-anti-Ly1 plus syngeneic bone marrow survived. The window of therapy was narrow in our studies, since 100 microCi 90Y-anti-Ly1 did not confer any survival advantage. Animals that did survive long term were studied for evidence of alloengraftment and found to have high levels of circulating donor mononuclear cells. 90Y-Anti-Ly1 localized in the spleen, thymus, liver, kidney and bone marrow but not in the bowel, lung, muscle, or skin. Animals given similar doses of free 90Y, 90Y-anti-Ly1, or labeled irrelevant antibody eliminated free 90Y fastest, followed by 90Y-anti-Ly1 and then labeled irrelevant antibody. Hematological analysis of peripheral blood from 90Y-anti-Ly1-treated mice showed reduction in total WBC counts, absolute lymphocyte numbers, and absolute neutrophil numbers on day 24 after treatment. Myelosuppression recovered by day 38. These findings indicate that Ly1-positive cells are involved in the effector phase of GVHD and that radiolabeled antibodies may be useful as cell-specific probes for studying the GVHD network. 90Y-Anti-Ly1 protected recipients long term from lethal GVHD, and the fact that it had a rather remarkable inhibitory and selective effect on the lymphoid system of mice suggests that these agents may have broader application in the field of transplantation.

Animals↗
PubMed1991

Toxicity and efficacy of anti-T-cell ricin toxin A chain immunotoxins in a murine model of established graft-versus-host disease induced across the major histocompatibility barrier.

Graft-versus-host disease (GVHD) was induced across the murine major histocompatibility complex by injecting C57BL/6 (H-2b) bone marrow and splenocytes into lethally irradiated B10.BR (H-2k) murine recipients. An immunotoxin (IT) composed of a pan T-cell monoclonal antibody called anti-Ly1 (the murine homologue to human anti-CD5) was conjugated to ricin toxin A chain (anti-Ly1-RTA) and used to treat recipient mice. In vitro, IT was as active as free RTA, bound selectively, and inhibited T-cell proliferation even in the absence of potentiators. Mice administered anti-Ly1-RTA in vivo during ongoing GVHD, at a dose of 10 micrograms/d for 5 days, showed lower numbers of splenic Thy1.2+ T cells and significantly improved survival as compared with mice given phosphate-buffered saline (PBS) or irrelevant control RTA IT. Protection was transient because GVHD and weight loss occurred when injections ceased. Survival could not be enhanced by crosslinking RTA30, a low oligosaccharide-containing fraction of purified RTA. Treatment with anti-Ly1-RTA caused a significant elevation in neutrophils, and higher doses were associated with mild hepatotoxicity. In contrast, infusion of identical doses and schedules of another pan T-cell immunotoxin, anti-Thy1.2-RTA, caused a significant decrease in lymphocytes, but not neutrophils; a precipitous increase in weight; a decrease in total plasma protein (TPP); and an increase in pleural and peritoneal effusions reminiscent of vascular leak syndrome (VLS). Although the toxic effects of anti-Thy1.2-RTA were too severe to show a survival advantage in a GVHD model, histopathologic studies showed a definite anti-GVHD effect. The most significant decline in GVHD as compared with the PBS-treated controls was observed in skin, and to a lesser extent, in liver and lung. To investigate the cause of IT toxicity, anti-Thy1.2-RTA was administered intraperitoneally to lethally irradiated B10.BR (H-2k) recipients of syngeneic bone marrow. These recipients showed the same weight gain, hypoproteinuria, and VLS observed in the GVHD model. Death occurred at higher anti-Thy1.2-RTA doses (30 or 50 micrograms/daily injections administered days 8 through 12 posttransplant). Anti-Thy1.2-RTA had a negligible effect on renal function, but histologic studies showed patchy dropout of the renal tubules. Treatment resulted in pulmonary vascular congestion, but there was no pathologic evidence of liver, brain, or colon toxicity. Weight gain was enhanced by irradiation because nonirradiated normal mice did not undergo such a precipitous weight increase.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Accuracy of portable blood glucose monitoring. Effect of glucose level and prandial state.

Glucose was determined on capillary and venous blood samples from 274 adult diabetics by three different methodologies: the Glucoscan 2000 and Accu-Check II portable glucose meters (capillary) and the Kodak Ektachem 700 analyzer (venous). Both glucose meters correlated significantly with the Ektachem results. A significant positive bias was found for the Glucoscan compared with Ektachem, not found with the Accu-Check II. The Accu-Check performed better than the Glucoscan at venous plasma glucose levels less than 1 g/L. The mean error of Glucoscan determinations was significantly greater and biased positive when the measurement was performed within 4 hours of eating, whereas no such effect was seen with Accu-Check error. Multiple regression analysis revealed that the Glucoscan measurement was independently influenced by both venous plasma glucose and prandial state, whereas the Accu-Check II measurement was not dependent on either variable. The within-run precision for both glucose meters were comparable.

Adult↗
PubMed1991

Autologous bone marrow transplantation for progressive non-Hodgkin's lymphoma: clinical impact of immunophenotype and in vitro purging.

From 1983 to 1989 we performed a prospective trial of 70 consecutive, in vitro purged autologous bone marrow transplants (BMT) for patients with progressive non-Hodgkin's lymphoma. Forty-nine patients had responsive disease at the time of transplantation while 21 others had refractory high risk lymphoma. Forty-two patients with B-lineage lymphoma received autologous marrow purged in vitro with monoclonal antibody (anti CD9, CD10, CD24) plus complement, 12 with T-lineage lymphoma received monoclonal antibody immunotoxins (anti CD5, CD7-ricin conjugates) along with 4-hydroperoxycyclophosphamide purging and 16 received unpurged marrow. All received cyclophosphamide, 57 with fractionated total body irradiation, and 13 with BCNU and cytarabine. Hematologic engraftment was prompt and unaffected by phenotype (B vs. T) or by in vitro purging used (B vs. T vs. none) although nine of 16 non-relapse deaths were related to poor graft function. Fifty-one patients (73%) were alive in complete remission (CR) 1 month following transplantation while 15 patients (12 with initially refractory disease) had persistent disease. Subsequently, 41 +/- 18% (by Kaplan-Meier estimate; +/- 95% confidence limits) of those who achieved CR remained relapse free 1-6.4 (median 3) years post-BMT. Neither risk group, purging, nor immunophenotype predicted subsequent post-transplant relapse. Among those 51 who achieved CR, 13 of 43 (27 +/- 14%) with responsive disease survive disease free while three of eight (38 +/- 34%) refractory patients survive disease free (p = 0.96). Overall, 24 patients survive, 16 in continuous complete remission 1-6.5 years following transplantation.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1990

Autologous bone marrow transplantation in high-risk remission T-lineage acute lymphoblastic leukemia using immunotoxins plus 4-hydroperoxycyclophosphamide for marrow purging.

Fourteen patients with high-risk T-lineage acute lymphoblastic leukemia (ALL) in complete remission underwent autologous bone marrow transplantation (BMT) in an attempt to eradicate their residual disease burden. A combined immunochemotherapy protocol using a cocktail of two immunotoxins directed against CD5/Tp67 and CD7/Tp41 T-lineage differentiation antigens in combination with the in vitro active cyclophosphamide congener 4-hydroperoxy-cyclophosphamide (4-HC) was used to purge autografts. Despite high dose pretransplant radiochemotherapy and effective purging of autografts, 9 of 14 patients relapsed at a median of 2.5 months (range, 1.2 to 16.8 months) post BMT. Two patients remain alive and disease free at 26 and 28 months post BMT. We used a novel quantitative minimal residual disease (MRD) detection assay, which combines fluorescence activated multiparameter flow cytometry and cell sorting with leukemic progenitor cell (LPC) assays, to analyze remission bone marrow (BM) samples from T-lineage ALL patients for the presence of residual LPCs. Notably, high numbers of residual LPC detected in remission BM before BMT constituted a poor prognostic indicator, providing the first evidence for the biologic significance and clinical value of in vitro T-lineage ALL LPC assays. The median value for the residual leukemia burden before BMT, was approximately 8.6 x 10(3) LPC/10(8) mononuclear cells (MNC) (approximately 0.0086% LPC). Patients with a residual leukemia burden less than this median value appeared to have a better outlook for remaining free of relapse after autologous BMT than patients with a greater leukemia burden (53 +/- 25% v 14 +/- 13%, P = .006, Mantel-Cox). By comparison, the log kill efficacy of purging, the remaining numbers of LPC in purged autografts, or the estimated numbers of reinfused LPC, did not correlate with the probability of disease-free survival (DFS). These results indicate that the primary reason for the recurrence of leukemia was inefficient pretransplant radiochemotherapy rather than inefficient purging of autografts.

Adolescent↗
PubMed1990

Antitumor activity of L6-ricin immunotoxin against the H2981-T3 lung adenocarcinoma cell line in vitro and in vivo.

The monoclonal antibody L6 recognizes a determinant that is expressed on lung, breast, colon, and ovarian carcinomas and is present only at trace levels in normal tissues. L6 was covalently linked to intact ricin by a thioether bond to produce an immunotoxin (IT). Gel analysis revealed that this IT was heterogeneous, but mostly one monoclonal antibody molecule linked to one ricin molecule. The L6-ricin IT selectively bound and was selectively toxic to L6-positive H2981-T3 adenocarcinoma cells in protein synthesis inhibition assays in which lactose was added to block the native ricin binding site. Clonogenic studies showed that 1 microgram/ml L6-ricin could inhibit about 99.99% of H2981-T3 growth in a limiting dilution assay, even in the presence of a 20-fold excess of human bone marrow cells. Treatment of bone marrow cells with the same dose of L6-ricin resulted in the growth of ample numbers of bone marrow progenitor cells (colony-forming units-mixed, colony-forming units granulocyte/macrophage, and blast-forming units erythroid) after 14 days. We also evaluated the antitumor effect of L6-ricin administered intratumorally with lactose against established H2981-T3 tumors in a nude mouse model. Thirty % of the tumor-bearing animals responded completely to single-dose treatment, while 60% gave partial responses. The in vivo effects were not absolutely specific, since irrelevant anti-CD5 IT also induced tumor regression in this model (10% responded completely, while 30% gave partial responses). However, irrelevant IT gave higher systemic toxicity (50% mortality) than L6-ricin (23% mortality). The nonspecific activity of IT was possibly due to Fc binding, which was demonstrated in vitro, or due to ricin B-chain binding. Ricin alone was too toxic for sustained tumor protection. Unconjugated L6 had no antitumor effect. The data suggest that L6-ricin may be useful for in vitro purging of autologous bone marrow from patients with solid tumors and marrow involvement and for in vivo regional therapy of L6-positive carcinomas.

Adenocarcinoma↗
PubMed1990

Pretreatment of murine donor grafts with L-leucyl-L-leucine methyl ester: elimination of graft-versus-host disease without detrimental effects on engraftment.

Incubation of murine bone marrow and splenocytes with the dipeptide methyl ester, L-leucyl-L-leucine methyl ester (Leu-Leu-OMe), which results in the selective depletion of cytotoxic T cells and their precursors, natural killer cells, and monocytes, completely protected 30 recipients of fully allogeneic donor grafts from lethal graft-versus-host disease (GVHD). These results were comparable with those obtained in 30 recipients of anti-Thy 1.2 plus complement (C')-treated donor marrow. However, in contrast to antibody- and C'-dependent T-cell depletion, which reduces the level of donor cell engraftment in our model system, we did not observe such effects using Leu-Leu-OMe marrow pretreatment. As compared with the 24 H-2 typed recipients of anti-Thy 1.2 + C'-treated donor grafts, the 29 H-2 typed recipients of Leu-Leu-OMe-treated donor grafts had significantly (P less than .001) higher percentages of donor cells (mean = 93% v 74%) and significantly (P less than .001) lower percentages of host cells (mean = 6% v 15%) posttransplantation. In vitro limiting dilution assay (LDA) was performed to assess the comparative efficacy of cytolytic T-lymphocyte (CTL) precursor depletion by Leu-Leu-OMe or anti-Thy 1.2 + C' pretreatment. We observed greater levels of CTL precursor depletion in Leu-Leu-OMe treated as compared with anti-Thy 1.2 + C'-treated bone marrow plus spleen cells (BMS) obtained from nontransplanted mice. This suggests that the in vivo results cannot simply be attributed to a less efficacious functional inactivation of cytolytic T-cell precursors by Leu-Leu-OMe treatment as compared with anti-Thy 1.2 + C' treatment. Immunoreconstitution was similar in recipients of Leu-Leu-OMe-treated grafts and anti-Thy 1.2 + C'-treated grafts 100 days posttransplant. In our opinion, Leu-Leu-OMe marrow pretreatment deserves further investigation as a methodology to achieve GVHD prevention without significantly reducing the propensity toward host cell repopulation.

Animals↗
PubMed1990

Kupffer cell engraftment across the major histocompatibility barrier in mice: bone marrow origin, class II antigen expression, and antigen-presenting capacity.

The source of population renewal for Kupffer cells (KC), the major antigen-presenting cells of the liver, remains controversial. Using a well-described murine bone marrow transplantation (BMT) model in which the donor and recipient are disparate at the major histocompatibility complex (MHC), we have studied (a) the source of KC renewal by genotypic analysis, cell surface (class II or Ia) antigens, and immune function assays; (b) the level of KC Ia expression post-BMT in transplant recipients; and (c) the capacity of newly repopulating KC to present antigen to an Ia-restricted T cell clone of donor Ia type. Kupffer cell engraftment, as assessed by each of these three methods, was noted to be predominantly of donor marrow origin by day 21 post-BMT. Cell surface Ia expression was comparable to that of nontransplanted controls of the same strain as donor mice. Within 7 days post-BMT, KC were mature antigen-presenting cells. We conclude that KC rapidly repopulate the liver from donor bone marrow post-BMT, and these macrophages are able to interact with T lymphocytes in an immunocompetent manner.

Animals↗
PubMed1990

Bone marrow transplantation for acute lymphocytic leukemia utilizing total body irradiation followed by high doses of cytosine arabinoside: lack of superiority over cyclophosphamide-containing conditioning regimens.

The efficacy of a pre-bone marrow transplantation (BMT) preparative regimen consisting of single, high dose rate total body irradiation followed by 12 courses of high dose cytosine arabinoside (Ara-C) over 6 days with allogeneic or autologous rescue was studied in 68 consecutive patients with poor risk acute lymphocytic leukemia (ALL). Toxicity associated with the above preparative regimen included conjunctivitis in the majority of patients, severe gastrointestinal symptoms in seven patients (10%), skin desquamation in four patients (6%), and interstitial pneumonitis of unknown etiology in two patients (3%). One patient died from a 'capillary leak' syndrome that may have been attributable to Ara-C toxicity. With a follow-up of 1.1-4.3 years, the estimated survival is 21% (95% confidence interval, 10-32%), with a disease-free survival of 18% (8-28%) and an estimated relapse rate of 75% (62-88%) at 3 years. These results are not significantly different from overall outcome in 105 patients treated previously at this same institution with cyclophosphamide-containing preparative regimens (31% (22-40%) estimated survival, 29% (20-38%) disease-free survival, and an estimated relapse rate of 63% (52-74%) at 3 years), especially when results are stratified based on allogeneic versus autologous BMTs. Although efficacious for a minority of patients with poor risk ALL, a pre-BMT preparative regimen consisting of total body irradiation followed by high doses of Ara-C did not improve long-term survival over more conventional preparative regimens consisting of cyclophosphamide and total body irradiation. Future strategies may need to investigate novel approaches for reducing leukemic relapse rates in patients with ALL undergoing bone marrow transplantation.

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