Multidrug resistance protein 4 (MRP4) polymorphisms impact the 6-mercaptopurine dose tolerance during maintenance therapy in Japanese childhood acute lymphoblastic leukemia.

Multidrug resistance protein 4 (MRP4) is involved in the efflux of nucleoside derivatives and has a role in the determination of drug sensitivity. We investigated the relationship between MRP4 genetic polymorphisms and doses of the 6-mercaptopurine (6-MP) and methotrexate. Further, we evaluated the frequency of therapeutic interruption during maintenance therapy in Japanese children with acute lymphoblastic leukemia (ALL). Ninety-four patients received an initial 6-MP dose in the range of 30-50 mg m(-2) in this analysis. Patients with homozygous variant allele in any of MRP4 G2269A, C912A and G559T required high frequency of 6-MP dose reduction compared with non-homozygous individuals. Average 6-MP dose for patients with homozygous variant allele on either MRP4 or inosine triphosphate pyrophosphatase was significantly lower than that for patients with non-homozygous variant allele during maintenance therapy (30.5 versus 40.0 mg m(-2), P=0.024). Therefore, MRP4 genotyping may be useful for personalizing the therapeutic dose of 6-MP during the ALL maintenance therapy in Japanese.

Fatal tracheal aspergillosis during rituximab combined chemotherapy for diffuse large B-cell lymphoma that developed after lung transplantation.

Invasive tracheal aspergillosis (ITA) is an infection that is unique to patients who have undergone lung transplantation (LT). Although the activity of this disease often appears on imaging, we encountered a case of ITA that became exacerbated, despite few computed tomography (CT) findings, during rituximab combined chemotherapy for diffuse large B-cell lymphoma. ITA developed during immunosuppressive therapy after LT. Because CT findings may show false-negative results, bronchoscopy is recommended for such cases.

[Tracheal resection and reconstruction for tracheal stenosis].

Five patients underwent surgery for tracheal stenosis. The cause of stenosis was congenital tracheal stenosis in 1 case, post-intubation tracheal stenosis in 1 case, and tracheal stenosis due to thyroid cancer invasion in 3 cases. All 5 patients required circumferential tracheal resection and end-to-end anastomosis using 4-0 or 5-0 absorbable sutures. The number of tracheal rings removed ranged from 3 to 6. There was no anastomotic complication. Technical points of this procedure were summarized as follows : 1) the circumferential dissection of the trachea should be made only at the level of the lesion that is to be excised, 2) preserve at least one side of recurrent nerve, 3) the traction sutures facilitate tensionless knot of the sutures, 4) prevention of excessive extension of the neck in the immediate postoperative period.

Epstein-Barr virus-infected T lymphocytes in Epstein-Barr virus-associated hemophagocytic syndrome.

The clonal composition of EBV-infected cells was examined in three cases of EBV-associated hemophagocytic syndrome by analysis of the heterogeneity of terminal repetitive sequences in the EBV genome, indicating monoclonal expansion of EBV-infected cells in all cases. Involvement of T lymphoid cells was determined by in situ hybridization using 35S-labeled RNA probes specific for the small EBV-encoded nuclear RNAs, EBER1 and EBER2, in combination with immunostaining for the TCR-beta chain, CD45RO, CD20, CD30 and CD68 antigens in these three cases. The majority of lymphoid cells showing EBER transcripts were stained by antibodies against CD45RO and T cell receptor-beta. In contrast, EBER-specific signals were not detectable on B cells or hemophagocytic cells. These data support the concept that EBV-associated T cell proliferation is a primary feature of EBV-AHS.

[Castleman disease arising from cardiophrenic angle; report of a case].

We report the first case of Castleman disease arising from cardiophrenic angle. The patient was referred to our hospital to treat his mediastinal tumor. Computed tomography (CT) showed a well-enhanced mass arising from the right cardiophrenic angle. We speculated the tumor to be a Castleman disease or hemangioma. Right thoracotomy was performed, and the tumor was removed after the ligation of the feeding artery and drainage vein. The histological findings of the tumor led to a diagnosis of Castleman disease hyaline vascular type.

Long-term outcome of 6-month maintenance chemotherapy for acute lymphoblastic leukemia in children.

In the treatment of childhood acute lymphoblastic leukemia (ALL), excess shortening of maintenance therapy resulted in high relapse rate, as shown by our previous trial, TCCSG L92-13, in which maintenance therapy was terminated at 1 year from initiation of treatment. In this study, we aimed to confirm the long-term outcome of L92-13, and to identify who can or cannot be cured by shorter duration of maintenance therapy. To obtain sentinel cytogenetics information that had been missed before, we performed genetic analysis with genomic microarray and target intron-capture sequencing from diagnostic bone marrow smear. Disease-free survival (DFS) at 10 years from the end of therapy was 66.0±2.8%. Females (n=138) had better DFS (74.6±3.7%) than males (n=142, 57.5±4.2%, P=0.002). Patients with TCF3-PBX1 (n=11) and ETV6-RUNX1 (n=16) had excellent DFS (90.9±8.7% and 93.8±6.1%, respectively), whereas high hyperdiploidy (n=23) was the most unfavorable subgroup, with 56.6±10.3% of DFS. Short duration of therapy can cure more than half of pediatric ALL, especially females, TCF3-PBX1 and ETV6-RUNX1. Our retrospective observations suggest a gender/karyotype inhomogeneity on the impact of brief therapy.

[Descending necrotizing mediastinitis treated by mediastinal drainage through bilateral thorax; report of a case].

Descending necrotizing mediastinitis is a severe infection spreading from the cervical region to the mediastinal connective tissue. The mortality rate was high and appropriate treatment is necessary to obtain favorable results. The present study describes a case of a 69-year-old man with severe descending necrotizing mediastinitis due to parapharyngeal abscess. The patient was successfully treated with cervical drainage, surgical debridement of the mediastinum via small thoracotomy. Postoperatively, continuous mediastinal and pleural irrigation with saline was performed. Swallowing disturbance due to dissection of cervical muscles prolonged duration of hospital stay. Eventually, the patient recovered and was able to eat by muscle rehabilitation.

Fluorescent substances in mouse and human sera as a parameter of in vivo lipid peroxidation.

Water-soluble fluorescent substances like lipofuscin were found in the protein fraction of mouse and human sera and had fluorescence characteristics with excitation and emission maxima at 335-340 and 435-440 nm for mice, and at 355-360 and 430-435 nm for human, respectively. When oxidized [U-14 C]linoleic acid was given intraperitoneally to mice, or added to the serum in vitro, both the fluorescence intensity and radioactivity of serum protein increased dose-dependently in the two tests. Also, the fluorescent substances responded well to acceleration of in vivo lipid peroxidation caused by carbon tetrachloride. These results indicate that the substances were some binding compounds between the degraded lipids and serum proteins, and that they could be taken as a parameter of in vivo lipid peroxidation. The distribution of the pigments in the serum proteins, albumin and globulins, was shown to depend upon the number of free amino groups in each protein which appear to be binding sites of degraded lipids. Spectral characteristics and some chemical properties of the substances suggest that they might not be conjugated Schiff bases formed from protein and malondialdehyde but might be due to some other stable compounds. Significantly high levels of the substances were observed in sera of patients with diabetes and hypertension.

Six months of maintenance chemotherapy after intensified treatment for acute lymphoblastic leukemia of childhood.

PURPOSE: We postulated that intensification of chemotherapy immediately after remission induction might reduce the leukemic cell burden sufficiently to allow an abbreviated period of antimetabolite therapy. PATIENTS AND METHODS: Three hundred forty-seven children (ages 1 to 15 years) with previously untreated acute lymphoblastic leukemia (ALL) were enrolled onto the Tokyo L92-13 study, which excluded patients with mature B-cell ALL and patients less than 1 year old. One hundred twenty-four patients were classified as standard risk, 122 as high risk, and 101 as extremely high risk, according to age, peripheral-blood leukocyte count, selected genetic abnormalities, and immunophenotype. All subjects received four drugs for remission induction, followed by a risk-directed multidrug intensification phase and therapy for presymptomatic leukemia in the CNS. Maintenance chemotherapy with oral mercaptopurine and methotrexate was administered for 6 months, with all treatment stopped by 1 year after diagnosis. RESULTS: The mean (+/- SD) event-free survival (EFS) and overall survival rates for all patients were 59.5% +/- 3.4% and 81.5% +/- 2.2%, respectively, at 5. 5 years after diagnosis. EFS rates by risk category were similar (60. 2% +/- 6.0% for standard risk, 57.7% +/- 5.6% for high risk, and 62. 5% +/- 5.7% for extremely high risk), whereas overall survival rates differed significantly (91.2% +/- 2.7%, 80.0% +/- 4.1%, and 72.1% +/- 4.5%, respectively, P <.0001 by the log-rank test). There were 107 relapses. Eighty-five (79.4%) of these 107 patients achieved second complete remissions, with subsequent EFS rates of 61.5% +/- 7. 9% (standard risk), 42.6% +/- 8.1% (high risk), and 9.6% +/- 6.4% (extremely high risk). Of the five risk factors analyzed, only the response to prednisolone monotherapy among extremely high-risk patients proved important. CONCLUSION: Early treatment intensification did not compensate for a truncated phase of maintenance chemotherapy in children with standard- or high-risk ALL. However, 6 months of antimetabolite treatment seemed adequate for extremely high-risk patients who were good responders to prednisolone and received intensified chemotherapy that included high-dose cytarabine early in the clinical course.

Delay of the diagnostic lumbar puncture and intrathecal chemotherapy in children with acute lymphoblastic leukemia who undergo routine corticosteroid testing: Tokyo Children's Cancer Study Group study L89-12.

PURPOSE: To determine the effects of eliminating initial lumbar punctures in 418 consecutively treated children with acute lymphoblastic leukemia (ALL). PATIENTS AND METHODS: Patients were enrolled onto a trial conducted in central Japan between 1989 and 1992. Treatment consisted of standard four-drug induction therapy followed by a risk-based intensification phase, reinduction therapy, late intensification, and remission maintenance therapy (total of 104 weeks). The initial lumbar puncture, with an intrathecal injection of chemotherapy, was performed after 1 week of prednisolone sensitivity testing (day 8). End points included response to prednisolone, CNS status at the time of the day 8 lumbar puncture, subsequent adverse events in CNS and bone marrow, and event-free survival (EFS). RESULTS: The remission induction rate was 93.1% with a 6-year EFS rate (+/- SE) of 68.7% +/- 2.4%, which is similar to historical results for patients who received their diagnostic lumbar puncture and first instillation of intrathecal chemotherapy on day 0. Overall, 84.5% of the patients had good responses to prednisolone, whereas 15.5% had poor responses. Clinical outcome was strikingly better for the good responders (6-year EFS, 74.1% +/- 2.5% compared with 40.1% +/- 6.4% for patients with poor responses), suggesting that omission of intrathecal chemotherapy did not alter the predictive value of drug sensitivity testing. Eighteen patients experienced CNS relapse as their first adverse event (cumulative risk, 5.1%; 95% confidence interval, 2.7% to 7.4%), coincident with reports from groups using conventional strategies of CNS clinical management. Bleeding into the CSF at the time of the day 8 lumbar puncture was apparent in 29 cases (8.1%), but leukemic blasts were identified in only two. CONCLUSION: Delay of the initial lumbar puncture and intrathecal injection of chemotherapy seems to be feasible in children with ALL. Further controlled evaluations are needed to establish the validity of this conclusion.

Analysis of the target cell for Epstein-Barr virus infection in Epstein-Barr virus associated hemophagocytic syndrome (EBV-AHS).

Epstein-Barr virus (EBV) infected cells were examined in three cases of EBV-associated hemophagocytic syndrome (EBV-AHS) by analysis of the heterogeneity of terminal repetitive sequences in the EBV genome, indicating monoclonal expansion of EBV-infected cells in all cases. Involvement of T lymphoid cells was determined by the finding of in situ hybridization using [35S]-labeled RNA probes specific for the small EBV-encoded nuclear RNAs, EBER1 and EBER2, in combination with immunostaining for the TCR-beta chain, CD45RO, CD20, CD30 and CD68 antigens in these three cases. The majority of lymphoid cells showing EBER transcripts were stained by antibodies against CD45RO and TCR-beta. In contrast, EBER-specific signals were not detectable on B cells or hemophagocytic cells. These data support the concept that subclinical EBV-associated T cell proliferation is the primary characteristic of EBV-AHS, rather than proliferations of hemophagocytosing histiocytes.

Frequency and clinical significance of the MLL gene rearrangements in infant acute leukemia.

We have analyzed the frequency and clinical significance of the MLL gene rearrangements in 42 cases of infant acute leukemias; including 37 cases of acute lymphoblastic leukemia (ALL) and five cases of acute myeloid leukemia (AML). MLL gene rearrangements were found in 27 of the 37 ALL cases (73 percent), and in all five AML cases. Cytogenetic studies showed 11q23 abnormalities in 24 of 27 ALL cases with MLL gene rearrangements. MLL gene rearrangements were significantly correlated with absence of CD10 expression and poor prognosis, but not with age under 6 months, hyperleukocytosis, myeloid-associated antigen expression, or CNS leukemia. The 3-year overall survival rate for ALL cases with MLL gene rearrangements was 5.3 +/- 5.2 percent, compared with 88.9 +/- 10.5 percent for cases with germline MLL (P=0.0001). Absence of CD10 expression was also associated with poor prognosis (9.9 +/- 6.6 percent vs 85.7 +/- 13.2 percent, P = 0.0003). Of the five AML cases, three have remained alive for 27 months to 67 months. These findings suggest that infant ALL with MLL gene rearrangement is strongly associated with poor prognosis. We consider that infant ALL should be treated on different chemotherapy protocols according to the presence or absence of MLL gene rearrangement.

Myelodysplastic syndrome in childhood: a retrospective study of 189 patients in Japan.

We report a retrospective analysis of children with myelodysplastic syndrome (MDS) diagnosed between 1990 and 1997 in Japan. In total, 189 patients were enrolled: 122 cases of primary MDS (26 RA, 18 RAEB, 25 RAEBt, 53 CMML/JMML), 24 cases with constitutional predisposition to MDS, and 43 cases of therapy-related MDS (t-MDS). The frequency of pediatric MDS was estimated to be 7.7% of all leukemias. Cytogenetic abnormalities were observed in 41% of primary MDS and 90% of t-MDS cases. The 4-year survival rate, estimated by Kaplan-Meier analysis, for primary RA was 78.9%, while other types of MDS and JMML had rates lower than 40%, and t-MDS showed an even more unfavorable prognosis. In primary MDS, the survival rate of patients with cytogenetic abnormalities was significantly lower. Among prognostic variables by IPSS, only the cytogenetic pattern was useful for predicting outcome in childhood MDS. There was no apparent advantage to chemotherapy for RA, and the survival rate in patients with primary RA, JMML, or t-MDS receiving stem cell transplantation was significantly higher. More precise designs of our diagnostic and classification systems, as well as therapeutic trials in large-scale prospective studies, are necessary for further improvements in MDS outcome.

Long-term follow-up of childhood acute lymphoblastic leukemia in Tokyo Children's Cancer Study Group 1981-1995.

The objectives were as follows: Firstly, to estimate the overall probability of event-free survival (EFS) and isolated CNS relapse in the studies for children with acute lymphoblastic leukemia (ALL) during the 1980s and 1990s. Secondly, to report the EFS according to presenting features and lineage. Thirdly, to evaluate the treatment results re-classified by the risks of NCI criteria. Four consecutive protocol studies were performed in the Tokyo Children's Cancer Study Group: L81-10 protocol (1981-1984, 189 patients), L84-11 (1984-1989, 484 patents), L89-12 (1989-1992, 418 patients) and L92-13 (1992-1995, 347 patients). Overall EFS at 5 years in each protocol was 56.5 +/- 3.8(1 s.e.)%, 71.0 +/- 2.1%, 67.8 +/- 2.3%, and 63.4 +/- 2.7%, respectively. The cumulative isolated CNS relapse rate at 5 years was 8.1 +/- 2.1%, 3.5 +/- 0.9%, 3.6 +/- 1.0%, 1.0 +/- 0.6. The EFS in SR/HR (standard risk/high risk) according to the NCI criteria in B-precursor ALL at 5 years was 61.9 +/- 4.3%/41.4 +/- 7.4% (lineage was not confirmed.), 72.5 +/- 2.6%/63.4 +/- 5.0%, 77.4 +/- 2.7%/56.3 +/- 4.7%, and 67.8 +/- 3.4%/56.7 +/- 5.4% in each protocol. Also EFSs according to NCI SR/HR at 5 years of T-ALL in protocols L84-11, L89-12 and L92-13 were 55.6 +/- 16.6%/60.9 +/- 10.1%, 72.7 +/- 13.4%/51.6 +/- 9.1%, and 77.1 +/- 14.4%/53.6/10.1%, respectively. The truncation of maintenance therapy to 6 months resulted in a decreased EFS in L92-13, particularly due to an increase of bone marrow relapse after cessation of therapy in SR and HR. The NCI risk criteria work properly even in the patients treated by different intensities, so that it makes the comparison possible among the patients in various groups. The overall EFSs in childhood ALL improved in 1980s, but it seemed stable or decreased in 1990s. The short maintenance therapy resulted in poor outcome in SR on the L92-13 protocol. Many of these late relapsers were effectively rescued and overall survival remained at a high level. The proportion of patients who received cranial irradiation reduced without any increase of the CNS events.

Delaying transplantation after total body irradiation is a simple and effective way to reduce acute graft-versus-host disease mortality after major H2 incompatible transplantation.

BACKGROUND: We have previously reported that delaying histoincompatible transplantation after total body irradiation (TBI) conditioning markedly decreased the mortality of acute graft-versus-host disease (GVHD) in severe combined immunodeficiency mice. However, it was not clear whether the delayed transplantation would affect the final engraftment and acute GVHD mortality in normal hosts. METHODS: BALB/c mice (H2d) were lethally irradiated with 8.5 Gy TBI and transplanted with C57BL/6 (H2d) bone marrow plus spleen cells on the same day (TBI+day 0) or 4 days after TBI conditioning (TBI+day 4). RESULTS: We again demonstrated that delaying transplantation by 4 days after TBI conditioning markedly reduced acute GVHD mortality in normal hosts after major histoincompatible transplantation. The survival rates were 66% in TBI+day 4 vs. 0% in TBI+day 0 allogeneic transplanted animals by day +60 (P<0.001). Further analysis demonstrated that the 4-day rest between the TBI and allogeneic transplantation broke the interaction of cell/inflammatory tumor necrosis factor-alpha, interleukin (IL)-1beta, and IL-6 cytokine reactions stimulated by TBI and incompatible transplantation. Flow cytometry revealed 97% donor cells in host marrow by 2 weeks in TBI+day 0 transplantation versus 57% in TBI+day 4 transplantation. There was no difference in percentage of donor CD3+ T-cell engraftment between the TBI+day 0 and TBI+day 4 allogeneic transplanted animals. In TBI+day 4 transplantation, the percentage of donor cells in host marrow steadily increased to 74% by day +60 and 93% by day +100. CONCLUSIONS: This 2- to 3-month early mixed chimerism in TBI+day 4 transplanted animals might be related to lower levels of tumor necrosis factor-alpha and IL-6 both of which have been shown to stimulate lymphohematopoiesis and was associated with lower acute GVHD mortality. The data again demonstrated in immunologically normal BALB/c mice that delaying allogeneic transplantation after TBI is a simple and effective way to reduce acute GVHD mortality, achieve satisfactory engraftment and significantly increase overall survival.

Induction of specific unresponsiveness to cardiac allografts by short-term administration of anti-T cell receptor alpha beta antibody.

Organ graft rejection is a T cell-dependent process in which activation of alloreactive T cells via the T cell receptor/CD3 complex is a critical step. Although treatment with anti-CD3 has been shown to prevent and reverse allograft rejection, there is little information available regarding the effects of immunotherapy using anti-TCR alpha beta mAb for rejection. In the present study, short-term preoperative treatment of rats with a mAb against alpha beta TCR (R73) completely prevented the rejection of cardiac allografts. These rats accepted second cardiac allografts from the same donor strain, but not from a third-party strain, without additional treatment. In mixed lymphocyte cultures, T cells from rats that had received cardiac grafts did not respond to donor-strain heart cells, but did respond to donor-strain spleen cells and third-party heart cells. These findings suggest that specific unresponsiveness to cardiac tissue was induced in R73-treated rats. Such unresponsiveness was induced only when rats were pretreated with the mAb and subsequently received a transplant. It is likely that administration of a small dose of R73 induced transient immunomodulation of TCR molecules, resulting in unresponsiveness to a subsequent cardiac allograft. Immunotherapy with mAb against TCR alpha beta is very effective, without apparent side effects, and may provide a new method for preventing graft rejection.

Long-term survival of cardiac allografts in rats treated before and after surgery with monoclonal antibody to CD2.

The rejection of a transplanted allograft is dependent on T cell activation, which requires T cell receptor engagement by antigen and costimulatory signals delivered by T cell surface molecules such as CD2. Anti-CD2 mAbs have been shown to suppress cell-mediated immunity. The effects of anti-CD2 mAbs OX34 and OX54 on rejection of BN (RT1n) rat hearts transplanted heterotopically to LEW (RT1l) rats were investigated. Administration of OX34 (7 mg/kg/day i.p.), either for 3 consecutive days immediately before or 8 consecutive days immediately after transplantation induced indefinite allograft survival (median survival time: 7, > 150, and > 150 days for control, preoperative treatment, and postoperative treatment, respectively). In contrast, pre- or postoperative treatment with OX54 (40 mg/kg/day) prolonged median survival time to only 28 and 11 days, respectively. Administration of OX34 or OX54 to naive rats induced a transient depletion of T cells in the peripheral immune organs. In vitro studies revealed that whereas OX54 had no effect on the allogeneic mixed lymphocyte reaction, OX34 partially inhibited both the allogeneic mixed lymphocyte reaction, in an IL-2-reversible manner, and T cell proliferation in response to immobilized mAb to either the T cell receptor or CD3. OX34-treated rats in which the cardiac allograft had survived > 100 days accepted a second heart from the donor strain. Treatment with OX34 induced an alloantigen-unresponsive state in T cells. These results suggest that treatment with an appropriate anti-CD2 mAb, especially postoperatively, may prove an effective approach for preventing cardiac allograft rejection.

Activation of T lymphocytes for adhesion and cytokine expression by toxin-conjugated anti-CD3 monoclonal antibodies.

BACKGROUND: Immunosuppressive drugs that target T cells are useful for prolonging allograft survival. The anti-CD3 immunotoxin FN18-CRM9 has been shown to effectively prolong renal allograft survival in a rhesus monkey model of transplantation. However, immunotoxin-treated monkeys showed increased levels of inflammatory cytokines and produced antibodies to donor proteins. To better understand the role of FN18-CRM9 in the production of cytokines and anti-donor antibodies in the monkey model, we examined whether this immunotoxin elicits functional responses in T cells. METHODS: Purified normal rhesus monkey T cells (>98% purity) were incubated with immunotoxin FN18-CRM9 or the unconjugated anti-CD3 monoclonal antibodies and then examined for changes in protein tyrosine phosphorylation, adhesion to fibronectin, gene expression, and proliferation in the presence or absence of anti-CD28 monoclonal antibodies (mAb) and interleukin-2. RESULTS: Immunotoxin treatment of T cells in vitro increased protein tyrosine phosphorylation, cell adhesion to the extracellular matrix, and expression of the inflammatory cytokines interferon-gamma and tumor necrosis factor-alpha. These immunotoxin effects were similar in magnitude to those induced by the unconjugated mAb. In contrast, immunotoxin-induced T cell proliferation was markedly less than that induced by the unconjugated mAb. Interestingly, the mitogenic molecules IL-2 and anti-CD28 mAb did not prevent immunotoxin-induced inhibition of cell proliferation. CONCLUSIONS: The activation of T cells for protein phosphorylation, adhesion, and cytokine expression strongly suggests that the actions of FN18-CRM9 in vivo are not limited to the inhibition of protein synthesis.

Synergistic effect of anti-T cell receptor monoclonal antibody and 15-deoxyspergualin on cardiac xenograft survival in a mouse-to-rat model.

BACKGROUND: Successful xenograft transplantation faces several obstacles including the presence of xenoantibodies, natural killer cell- and macrophage-mediated rejection, and T lymphocyte activation. METHODS: A mouse-to-rat cardiac xenograft model was used to examine the synergistic effect of anti-T cell receptor (TCR) monoclonal antibodies (mAb) and 15-deoxyspergualin (DSG) on graft survival. RESULTS: Pretransplantation injections (days -5, -3, and -1) of anti-TCR mAb (500 microg/kg/day) combined with continuous i.p. infusion of DSG (5 mg/kg/day) from day -7 to 28 significantly prolonged graft survival compared to untreated controls (3.3+/-0.5 vs. 44.2+/-5.6 days, P<0.001). Postoperative splenectomy combined with discontinuation of all other treatment on day 28 enhanced graft survival in rats treated with anti-TCR mAb and DSG to 71.0+/-2.5 days. Histological examination of grafts showed characteristic signs of vascular rejection: interstitial edema and hemorrhage, and polymorphonuclear cell infiltration. Antimouse antibody titers in recipients were increased upon rejection in each group that received a xenograft. Flow cytometry analysis showed a markedly decreased T cell population and a relatively increased mature B cell population (IgM(bright)/IgD(dull)) in spleens of rats treated with anti-TCR mAb and DSG on day 28. CONCLUSIONS: The mechanism of prolonged xenograft survival in this model may include inhibition of antibody production by arrest of B-cell maturation during development from IgM(dull)/IgD(bright) mature B cells to antibody producing cells, and inhibition of T cell activation. The rejection seen in our model may be caused by xenoreactive antibodies and may be associated with T cells, natural killer cells, and macrophages.

Immunotoxin FN18-CRM9 induces stronger T cell signaling than unconjugated monoclonal antibody FN18.

BACKGROUND: The T-cell receptor (TCR)/CD3 complex is the target of therapeutic strategies aimed at prolonging allograft survival. The immunotoxin FN18-CRM9, composed of the anti-CD3 monoclonal antibody FN18 and the mutated diphtheria toxin CRM9, is useful for prolonging allograft survival in preclinical models of transplantation. To explore the influence of conjugation of the mutated diphtheria toxin on functional activation of the TCR/CD3 complex, we compared the effects of FN18-CRM9 and unconjugated FN18 on protein tyrosine phosphorylation and ligand/receptor internalization in purified monkey peripheral blood T cells. METHODS: Purified normal rhesus monkey T cells were incubated with unconjugated FN18 or conjugated FN18-CRM9 and examined for differences in antibody binding, tyrosine phosphorylation, and CD3 internalization. RESULTS: Binding cross-inhibition studies demonstrated that both compounds were able to inhibit fluorescein isothiocyanate-FN18 binding to CD3 with similar efficacy and potency. However, FN18-CRM9 was more potent than FN18 in triggering the phosphorylation of several proteins on tyrosine residues and in inducing CD3 internalization. The tyrosine kinase inhibitor genistein blocked FN18-CRM9-induced protein tyrosine phosphorylation and CD3 internalization, suggesting that tyrosine phosphorylation is involved in the internalization of the immunotoxin. Interestingly, in FN18-CRM9- but not FN18-treated cells, there was a gradual decrease in cellular CD3 protein levels within 24 and 48 hr; such a decrease was not observed with the control protein Csk. CONCLUSIONS: Our findings suggest that the conjugation of the mutated diphtheria toxin CRM9 to FN18 modulates the monoclonal antibody-mediated cross-linking of the TCR/CD3 complex, leading to a stronger protein tyrosine phosphorylation and CD3 internalization. This may in turn contribute to the greater efficacy of the immunotoxin in prolonging allograft survival.