NI-1: a novel canine mastocytoma model for studying drug resistance and IgER-dependent mast cell activation.

BACKGROUND: Advanced mast cell (MC) disorders are characterized by uncontrolled growth of neoplastic MC in various organs, mediator-related symptoms, and a poor prognosis. Kit mutations supposedly contribute to abnormal growth and drug resistance in these patients. METHODS: We established a novel canine mastocytoma cell line, NI-1, from a patient suffering from MC leukemia. RESULTS: NI-1 cells were found to form mastocytoma lesions in NOD/SCID IL-2Rgamma(null) mice and to harbor several homozygous Kit mutations, including missense mutations at nucleotides 107(C→T) and 1187(A→G), a 12-bp duplication (nucleotide 1263), and a 12-bp deletion (nucleotide 1550). NI-1 cells expressed several MC differentiation antigens, including tryptase, Kit, and a functional IgE receptor. Compared to the C2 mastocytoma cell line harboring a Kit exon 11 mutation, NI-1 cells were found to be less responsive against the Kit tyrosine kinase inhibitors (TKI) masitinib and imatinib, but were even more sensitive against proliferation-inhibitory effects of the mammalian target of rapamycin (mTOR) blocker RAD001 and PI3-kinase/mTOR blocker NVP-BEZ235. The Kit-targeting multikinase inhibitors PKC412 and dasatinib were also found to override TKI resistance in NI-1 cells, and produced growth inhibition with reasonable IC(50) values (<0.1 µM). CONCLUSION: NI-1 may serve as a useful tool to investigate IgE-dependent reactions and mechanisms of abnormal growth and drug resistance in neoplastic MC in advanced mastocytosis.

[An unusual course of thoracic actinomycosis]. / Ungewöhnlicher Verlauf einer thorakalen Aktinomykose.

A 56-year-old farmer who had been suffering from exogenous allergic alveolitis developed, after a long prodromal period of illness, a granulocyte-rich pericardial effusion and bilateral pleural effusions in which Actinomyces aggregates were identified. Despite intensive treatment with clindamycin, erythromycin and tetracycline (he was allergic to penicillin) he died in septic shock.

Correlation between low CSA plasma concentration and severity of acute GvHD in bone marrow transplantation.

Between 1982 and 1986 51 patients were treated with ciclosporin a (CSA) to prevent graft versus host disease (GvHD) after bone marrow transplantation (BMT). Major side effects of the drug were tremor, hypertension, hepatotoxicity and nephrotoxicity. Acute GvHD 0 degree to II degree occurred in 80% of our patients, and GvHD III degree and IV degree in 20% despite the use of CSA. Two to four days before the onset of GvHD, CSA serum levels were significantly lower on the average in patients who developed GvHD III degree and IV degree compared to the others. Our data indicate that plasma CSA concentrations higher than 250 ng/ml should be achieved to reduce the severity of GvHD after BMT.

Prevention of CMV infection after BMT in high-risk patients using CMV hyperimmune globulin.

Prophylactic administration of an intravenous cytomegalovirus hyperimmune globulin in bone marrow transplant recipients provided protection against primary as well as reactivated CMV infection in patients considered to be at high risk for cytomegalovirus infections. Forty-one patients were divided in six subgroups according to factors considered to increase the incidence and severity of CMV infections following bone marrow transplantation. All of these patients received blood products from donors not screened for active or latent CMV infections. In spite of this, patients undergoing intensified conditioning therapy or mismatch transplantation, as well as those transplanted in relapse of their leukemia and even patients receiving granulocyte transfusions from donors unscreened for CMV serostatus, were found not to develop primary or secondary CMV infections. Only in the group of patients older than 35 years and among those suffering from severe GVHD six patients were found to have CMV infections, only two a symptomatic form. Intravenous administration of CMV hyperimmune globulin effectively protected even patients at high risk for CMV infection against severe complications of primary infection or reactivation of this virus.

[Oral prophylaxis of herpes infections using acyclovir following bone marrow transplantation: a clinical and clinico-pharmacological study]. / Orale Prophylaxe von Herpes-Infektionen mit Acyclovir nach Knochenmarktransplantation: Eine klinische und klinisch-pharmakologische Untersuchung.

Viral infections are one of the major complications after bone marrow transplantation, with high mortality and morbidity. Fourty-six patients between 3 and 48 years old (median 15 years) received orally 400 mg (under age 6, 200 mg) acyclovir 4 times daily from day -12 before to day 84 after BMT. All patients were isolated in laminar-airflow units for at least 23 days with total enteral decontamination. They were concomitantly treated with anti-CMV-hyperimmunoglobulin and cotrimoxazol. During acyclovir prophylaxis seven patients had herpes simplex virus infections, all of them were seropositive before BMT. Acyclovir plasma concentrations were measured by use of a new HPLC method. No acyclovir was present (detection limit 40 ng/ml) in the plasma of five out of six patients with HSV infections. Three of them had non-compliance, and a lack of acyclovir absorption developed in two patients under conditioning regimen. No drug-related side effects were observed. Laboratory tests did not show liver or renal toxicity. Take and hematologic reconstitution were unchanged. In our study, oral acyclovir reduced the incidence of herpes simplex infections after bone marrow transplantation. Herpes infections only occurred in patients with non-compliance or lack of acyclovir absorption.

[Bone marrow transplantation in acute leukemia, chronic myeloid leukemia, severe aplastic anemia and stage IV neuroblastoma. Effect of antiviral prevention with anti-CMV-hyperimmunoglobulin and acyclovir]. / Knochenmarktransplantation bei akuter Leukämie, chronisch myeloischer Leukämie, schwerer aplastischer Anämie und Neuroblastom Stadium IV. Einfluss antiviraler Prophylaxe mit Anti-CMV-Hyperimmunglobulin und Azyklovir.

Bone marrow transplantation was performed between IV/82 and X/85 in 64 patients with acute leukemia (n = 36), chronic myelogenous leukemia (CML; n = 13), severe aplastic anemia (n = 12), and neuroblastoma stage IV (n = 3). Of these patients 57 received allogeneic marrow from HLA-ABCDR identical, MLC-negative sibling donors. Six transplants were performed with syngenic marrow and one with autologous marrow. Of the 64 patients 48 survived 40-1,250 days after transplantation, resulting in a survival rate (SR) of 75% and a survival probability (SP) of 71%. Of the 36 patients suffering from acute leukemia (SR = 64%, SP = 51%), patients with acute myelogenous leukemia (AML) in first complete remission (n = 11; SR = 81%, SP = 76%), as well as patients with acute lymphatic leukemia (ALL) in 1st to 4th complete remission at the time of transplantation (n = 14; SR = 81%, SP = 76%) show a favorable prognosis. A poor survival rate was seen for patients with AML when transplanted in second or partial remission (1/5; SR = 20%), as well as for patients suffering from ALL and transplanted during relapse or partial remission (1/6; SR = 16%). Of 13 patients suffering from CML 12 survived the transplantation free of relapse (SR = 93%, SP = 92%), and one patient died from varicella zoster pneumonia. Of the transplanted patients with severe aplastic anemia, 12 of 13 are surviving with complete hematologic reconstitution; one patient, however, died on day 10 from a sepsis. In our patient group, the SR as well as the SP has been improved through changes in the irradiation protocol concomitant with prophylactic application of anti-CMV hypergammaglobulin, as well as through additional oral medication of Azyklovir. The 41 patients (BMT No. 7-47) with total body irradiation at one time show an SR of 44% and an SP of 41%. The following 46 patients (BMT No. 48-93) have reached an SR of 83% and an SP of 74% under the regimen of fractionated total body irradiation, plus prophylaxis with anti-CMV hypergammaglobulin and Azyklovir. Within this group, no fatal CMV pneumonia was encountered as opposed to six patients lost from CMV pneumonia in the first group.

[Bone marrow transplantation in severe aplastic anemia]. / Knochenmarktransplantation bei schwerer aplastischer Anämie.

Bone marrow transplantations were performed on 15 patients (aged 5-39 years) with severe aplastic anaemia. Twelve patients are alive 76-1930 days (median 668 d) after transplantation, with complete haematopoetic recovery. Total-body radiation with 3.6 Gy in four patients, cyclosporin A administration to ten patients and buffy-coat transfusion to nine patients entirely prevented early rejection. Two patients died of pneumonia (aspergillus; varicella-zoster virus), one patient died of bleeding from a splenic-artery aneurysm. In patients under the age of 40 years with severe aplastic anaemia bone marrow transplantation as early as possible after diagnosis is the treatment of choice if HLA-identical siblings are available as donors. In patients over 40 years treatment should at first be tried with antithymocyte globulin.

Natural killer cell activity against a thymoma cell line Thy 121 in bone marrow transplant recipients.

Twenty-one patients with acute and chronic leukemia or severe aplastic anemia were studied for NK activity against a thymoma cell line (Thy 121) before and after allogeneic bone marrow transplantation. The means of the pretransplant and post-transplant levels did not differ from the mean of 134 NK determinations in 67 healthy donors. There was no correlation between pretransplant NK levels and the appearance of graft-versus-host disease. Three weeks following bone marrow transplantation, pretransplant NK levels were observed. The sensitivity of NK cells to interferon was the same as in normal donors both before and after bone marrow transplantation. In contrast to methotrexate, cyclosporin A inhibited NK activity in patients and controls in vitro. In vivo cyclosporin A treatment, however, did not decrease NK levels in bone marrow recipients.

[Bone marrow transplantation in chronic myeloid leukemia. Influence of gnotobiotic measures, especially antiviral prophylaxis]. / Knochenmarktransplantation bei chronischer myeloischer Leukämie. Einfluss gnotobiotischer Massnahmen, insbesondere antiviraler Prophylaxe.

Bone marrow from HLA-identical siblings was transplanted in 14 patients with chronic myeloid leukaemia (CML), including one patient in acceleration phase and one in chronic phase following 2 blast crises. Restoration of the bone-marrow occurred in all cases and Philadelphia chromosome could not be detected in any of the patients after the transplantation. Two patients died due to lung complications. Twelve patients who received antiviral prophylaxis (aciclovir per os, anti-CMV-hyperimmunoglobulin) after the transplantation are in very good condition with 140 to 790 days complete clinical and cytogenetic remission. Bone-marrow transplantation, as a curative measure for patients with CML up to the age of 45 (50), should be included in therapy schemes when an HLA-identical sibling is available.

Bone marrow transplantation for acute leukemia in second or subsequent remission.

Twenty-one patients with acute leukemia in second to fifth remission were treated with bone marrow transplantation: 19 patients with transplants from HLA-matched siblings and two with transplants from identical twins. Twelve patients survived from 15 to 1,625 days after transplantation: six of 11 in the ALL group and six of 10 in the AML group. Recurrence of leukemia after marrow transplantation occurred in five patients. The cause of death in five patients was infection, in two patients combined with graft-versus-host disease. Long-term disease-free survival can probably be achieved in 30%-35% of all patients with acute leukemia who receive a marrow transplant in second or subsequent remission.

Immunohistological skin alterations in allogeneic bone marrow transplantation.

Skin biopsies of 26 patients with leukemia and seven patients with aplastic anemia were investigated before and at different stages after allogeneic bone marrow transplantation (BMT) to establish the immunological criteria which distinguish skin alterations during normal reconstitution from dermal lesions mediated by graft-versus-host disease (GvHD). Of the 33 patients studied 27 presented with clinically diagnosed acute and/or chronic GvHD, one patient died of bone marrow rejection. Immunohistological analysis of the respective skin biopsies with selected monoclonal antibodies against human leukocyte antigens (HLA) and differentiation antigens of the lympho-hematopoietic cells revealed low dermal mononuclear cell counts with phenotypically normal constituents in five cases with uncomplicated reconstitution post-grafting. In contrast, increased dermal cellular infiltrates predominantly consisting of Lyt 3+, OKT 8+ T-lymphocytes, as well as of a large number of Ia-like (immune response associated = HLA-D) determinant + monocytes/macrophages were observed in all patients with active acute/chronic GvH reactivity. As sign of activation simultaneous expression of HLA-D region products was also found on a subset of the invading OKT8+ T-lymphocytes. Progression of GvHD was associated with additional surface staining of keratinocytes for Ia-like determinants. Loss of Ia-like determinant+, OKT6+ dentritic epithelial cells in all leukemic patients, as well as in patients with aplastic anemia with or without GvHD suggested damage of Langerhans cells due to the previous radiotherapy and/or specific immunological destruction. In patients with fatal outcome of GvHD prolonged reduction of these dentritic epithelial cells seemed to be indicative of impaired immune reconstitution or bone marrow dysfunction. Thus immunopathological features of skin GvHR may enable early recognition and prognostic evaluation of this disease possibly allowing more effective therapy.

[Bone marrow transplantation in patients with leukemia]. / Knochenmarktransplantation bei Patienten mit Leukämien.

Between October 1979 and March 1982, bone marrow transplantations were performed by the Tübingen Group for BMT on 19 patients with acute leukemia in remission and on one patient with chronic myelocytic leukemia in chronic phase. The conditioning regimen consisted of 2 x 60 mg cyclophosphamide/kg and 10 Gy whole-body irradiation with the linear accelerator. The lung dose was limited by shielding to 8 Gy. In 15 patients, the bone marrow cell suspension of the donor was preincubated with antihuman T-cell globulin (AHTCG) for prophylaxis of graft-versus-host disease (GVHD). All patients showed prompt engraftment of donor cells with good hemopoietic function and complete chimerism. Under reverse isolation in sterile units, no severe bacterial or fungal infections were seen in the phase of bone marrow aplasia. Twelve in twenty patients survived between 25 and 900 days. A severe GVHD was seen only in two patients - one after preincubation with AHTCG. One patient died from relapse of his leukemia, another patient had a testicular relapse which was treated with local radiotherapy. Major problems were seen with chronic GVHD (six patients) and infectious complications, most importantly interstitial pneumonia, in the late post-transplant period.

Immunohistology of graft-versus-host disease mediated skin lesions and its correlation to a large granular lymphocyte surface phenotype and function.

Skin lesions of five patients presenting with acute and chronic Gvhd after bone marrow transplantation were analyzed on frozen tissue sections using selected monoclonal antibodies against various T-cell determinants and HLA-antigens in order to define immunological phenomena characteristic for cutaneous damage seen in various GvHD states. Four of these patients showed marked increase of a certain T-cell subpopulation positive for HLA-D region products in the upper dermis. A considerable number of these T-cells seemed to show cytotoxic reactivity on the basal cell layers of the epithelium and to correlate with the appearance of an OKT4-, OKT8+, HLA-DR+ T-cell subset in the peripheral blood presenting natural killer (NK) cell like activity on various targets. Ia antigen expression on keratinocytes observed in one patient with chronic GvHD could result from a rapid and irregular turnover of epidermal cells affected by continuous stimuli of these T-cells. Further immunological studies on skin biopsies of patients with different states of GvHD and autoimmune diseases may lead to valuable diagnostic criteria for an early and accurate assessment of various skin lesions in patients after bone marrow transplantation.