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Biomedical subjects

E Weidmann

Publications and source records attributed to E Weidmann.

At least 55 records · Page 3Linked to original sources
PubMed1997

Establishment and characterization of a new, factor-independent acute myeloid leukemia line designated Ei501.

We established a factor-independent acute myeloid leukemia cell line, designated Ei501. The line has been growing in RPMI 1640 media for 18 months and can be maintained without addition of growth factors. Ei501 is positive for myeloperoxidase and negative for esterase and PAS. Cytogenetic analysis revealed the FAB M3 associated t(15;17) translocation and a translocation of the chromosomes 7 and 8: 46 XX, -7, +t(7;8)(q32;q13), t(15;17)(q22;q12). This karyotype was confirmed by fluorescence in situ hybridization. Ei501 cells express AML-associated surface markers such as CD13, CD33 and CD38. Although 42% of the patient's blast cells were CD34-positive, the line lacks surface expression of CD34. Furthermore the line has a number of characteristics which are detectable in blasts from AML patients, such as surface adhesion molecules, cytokines such as TGF-beta, cytokine receptors such as the IL-2 receptor beta and gamma chains or the IL-4 receptor and the genes for the transcription factor wt-1 (Wilms' tumor gene) and for the proto-oncogene bcl-2, both shown to be present in the majority of patients with AML. Additionally the line can be used as target in cytotoxicity assays using IL-2 activated cytotoxic lymphocytes as effector cells. In conclusion, besides a rare karyotype the Ei501 cell line has several features common in AML, and may therefore be used as a model to study pathogenetic mechanisms in acute myeloid leukemia.

Adolescent↗
PubMed1997

Wilms tumor gene expression in acute myeloid leukemias.

The Wilms' tumor gene product (WT-1) is suggested to act as a tumor suppressor in childhood malignancies of the kidney and as a transcription factor with regulating activity on a number of growth and differentiation factors. Wt-1 has been shown to be expressed in blast cells of the vast majority of patients with acute myeloid and lymphoblastic leukemias (AL) by a number of workers. High levels of wt-1 mRNA expression in blast cells of newly diagnosed AML patients predict worse prognosis when compared to patients with no or low wt-1 mRNA expression. Patients achieving complete responses after chemotherapy usually lose detectable signals of wt-1. In relapse of the disease reoccurrence of wt-1 mRNA can be determined in almost all patients with initially detectable wt-1 mRNA. Using sensitive techniques such as reverse transcription polymerase chain reaction (RT-PCR) relapses are preceded by wt-1 expression in some cases. Although a subpopulation of normal hematopoietic precursor cells has also been shown to express message for wt-1, detectable levels of wt-1 during follow-ups in AML patients have been shown to be useful as a marker for residual blast populations or even to predict relapse of AML. Whether the high level of wt- expression is a non-specific phenomenon resulting from malignant transformation or whether it has an impact on the pathophysiology of AML or the uncontrolled growth of AML blasts is still controversial. However, there are indicators for an involvement of wt-1 in malignant events of AML blasts such as the downregulation of wt-1 in chemically induced differentiation of AML blast cell lines or the interactions of wt-1 with the protooncogene bcl-2 and the tumor suppressor gene p53. In conclusion, its possible relevance as an AML marker and its role in pathophysiological mechanisms in AML will still have to be defined in the future.

Acute Disease↗
PubMed1997

The Wilms' tumor gene is expressed in a subset of CD34+ progenitors and downregulated early in the course of differentiation in vitro.

The Wilms' tumor gene (wt1) is strongly expressed in malignant blasts of acute myeloid leukemia (AML) in approximately 80% of all cases. However, the role of wt1 expression in non malignant hematopoietic cells remains unclear. To characterize the expression of wt1 in differentiating hematopoietic progenitors, we isolated and cultured CD34+ progenitor cells from four healthy bone marrow donors with stem cell factor (SCF) and granulocyte colony stimulating-factor (G-CSF) to induce differentiation into granulocytes. Four different cultures were carried out for 12 days. During culture, wt1 mRNA expression was analyzed by defining its ratio relative to beta-actin using reverse transcriptase polymerase chain reaction (RT-PCR). To monitor the stage of differentiation, expression of cell surface markers and peroxidase was analyzed daily. The initial purity of CD34+ cells ranged between 80% and 90%; after 12 days, the frequency of neutrophil bands and segmented neutrophils was approximately 60%. Using RT-PCR to determine the ratio of wt1 to beta-actin expression, we reproducibly detected maximum expression of wt1 mRNA at day 0 in two cultures and at day 1 in two other CD34+ cell cultures; at both these time points nearly all cells fulfilled the morphological and immunephenotypical criteria of early hematopoietic blast cells. Wt1 expression dropped rapidly at day 1 and 2, respectively, in these two pairs of cultures, and was accompanied by an increase of cells expressing CD33 surface antigen. Our data suggest that wt1 expression is restricted to a subset of CD34+ progenitors and downregulated in later stages of differentiation in vitro.

Antigens, CD34↗
PubMed1996

Peripheral T-cell lymphomas respond well to vincristine, adriamycin, cyclophosphamide, prednisone and etoposide (VACPE) and have a similar outcome as high-grade B-cell lymphomas.

Peripheral T-cell lymphomas (PTCL) represent a heterogeneous group of T-cell malignancies including subentities with favourable (large cell anaplastic) or unfavourable (pleomorphic) prognosis. The clinical outcome of PTCL has been controversially discussed, but a worse prognosis than high-grade B-cell Non-Hodgkin's lymphomas (NHL) has been postulated by most authors. In this report we summarize the results of a prospective comparative study investigating the therapy outcome of 27 patients (pts) with PTCL and 55 pts. with high grade B-cell NHL and give an overview of therapy studies in PTCL. The histological subtypes were 14 pleomorphic, 8 large-cell anaplastic (Ki-1+), 2 angioimmunoblastic (AILD) and 3 other PTCL. In three patients the PTCL was associated with non-tropical sprue (11%). Nineteen patients presented with an advanced stage of disease (stage III and IV, 70%), 17 (63%) pts. had B-symptoms. The patients were treated with vincristine 2 mg d1, adriamycin 25 mg/m2 d1-3, cyclophosphamide 800 mg/m2 d1, prednisone 60 mg/m2 d1-7 and etoposide 120 mg/m2 d1-3 (VACPE). In 77% of pts. with PTCL and 84% of patients with high-grade B-cell NHL a complete remission (CR) was achieved. 75% of the complete responders with PTCL and 70% with B-NHL are still in ongoing CR. The subgroup of large-cell anaplastic attained a CR in 88%. The median observation time is 44 months (1(+)-77+). The probability of 1-, 3- and 5-year overall and disease-free survival for the T-cell group were 76%, 54%, 48% and 76%, 62%, 62%, respectively according to Kaplan-Meier. There was no significant difference regarding the remission rate, the overall-, event-free or disease-free survival compared to high-grade B-cell lymphomas. In conclusion, the VACPE regimen is an effective and feasible regimen in the management of PTCL achieving complete remissions in a large proportion of patients.

Adolescent↗
PubMed1995

Lactate dehydrogenase-release assay: a reliable, nonradioactive technique for analysis of cytotoxic lymphocyte-mediated lytic activity against blasts from acute myelocytic leukemia.

Treatment of patients in remission of acute myelocytic leukemia using immunotherapy with interleukin 2 is a new approach to prolonging remission duration in this disease. As an important mechanism for the pathophysiology of eradication of residual myelocytic blast populations, activation of cytotoxic effector lymphocytes has frequently been discussed. However, the associated immunological research has been complicated to some extent, because in conventional chromium 51-release assays, blast cells frequently fail to incorporate sufficient amounts of 51Cr and/or spontaneously release high amounts of 51Cr. Recently, we established a culture system which promotes the outgrowth of cytotoxic T lymphocytes in bone marrow-derived mononuclear cells cultured in IL-2. To study cytotoxicity and the responsible mechanisms of the obtained T-cell lines and clones, we modified a previously described cytotoxicity assay, based on the release of lactate dehydrogenase (LDH-release assay) for use in cryopreserved blasts obtained from the bone marrow of patients with acute myelocytic leukemia. Using this assay, we were able to detect cytotoxicity of IL-2-activated peripheral blood lymphocytes from three healthy controls against a number of blast samples obtained from the bone marrow of patients with AML (up to more than 40% lysis at an effector target cell ratio of 20:1). However, a minority of AML blasts seem to be resistant to lysis by IL-2-activated lymphocytes. In bone marrow-derived T-cell lines from patients with AML we detected lytic activity against autologous blasts in three of seven cases tested by LDH release, ranging from 29 to 63% at an effector target ratio of 10:1. Additionally, T-cell clones with different phenotypes were established which were able to mediate cytotoxicity against autologous blast cells. Thus, cytotoxicity against freshly isolated blasts from patients with acute myelocytic leukemia can be analyzed reliably, reproducibly, and without the use of isotopes by the LDH-release assay.

Blast Crisis↗
PubMed1995

Bone marrow-derived T-cell clones obtained from untreated acute myelocytic leukemia exhibit blast directed autologous cytotoxicity.

The eradication of minimal residual blast populations by activation of autologous cytotoxic cells with interleukin 2 (IL-2) is a new promising tool in the treatment of acute myelocytic leukemia (AML). However, the immunological effector cells are not yet clearly defined. The present study was designed to investigate the presence of cytotoxic precursor cells in active AML and to identify phenotypical and functional characteristics of autologous anti-leukemic cytotoxic effector cells. For this purpose, mononuclear cells (MNC) containing at least 70% leukemic blasts were isolated from bone marrow of untreated AML and cultured in the presence of 3000 IU/ml recombinant IL-2 (rIL-2) for 6-8 weeks. Under these conditions, T-cells were selected in the bone marrow cultures and overgrew the leukemic blasts. The resulting T-cell populations were cloned by limiting dilution and the clones obtained were characterized for their phenotypical and functional patterns. Totally, cloning resulted in 68 clones and a few cell lines. The clonality was verified by RT PCR analysis of TCR V beta gene expression. All clones obtained stained positive for CD2, CD3, DR and CD56. The vast majority (68%) of T-cell clones/lines was CD4+, a few clones expressed CD8 (19%) or CD4 and CD8, and four clones were of TCR gamma delta origin. Seven of 15 clones tested, including three CD4+, two CD8+ and two TCR gamma delta(+)-clones were found to be cytotoxic against autologous leukemic blast cells. All except one clone expressed oncolytic activities against allogeneic blasts too. One of the TCR gamma delta(+)-clones demonstrated NK activity by lysis of K562 targets. The majority of the T-cell-clones released IL-2, IL-8, TNF-alpha, GM-CSF but only a few IFN gamma and expressed high levels of mRNA for IL-2, TGF-beta and IL-10. None of the clones was found to produce IL-3, IL-4, IL-7 and TNF-beta. The data provide evidence of the existence of T-cell precursors in untreated AML bone marrow differentiating to cytotoxic cells with activity against autologous and allogeneic AML blast cells.

Bone Marrow Cells↗
PubMed1995

AML blasts variably express interleukin 2 receptor alpha, beta or gamma chains without measurable effects on proliferation, cytokine message expression or surface expression of adhesion molecules upon stimulation with interleukin 2.

Preliminary clinical studies including interleukin 2 (IL-2) in chemotherapy strategies for treatment of acute myeloblastic leukemia (AML) suggest that IL-2 may improve the disease-free survival of the patients. Because of reports showing interleukin 2 receptor expression on AML blasts, it is important to know whether IL-2 may directly influence these leukemic cells. In initial studies using flow cytometry to analyze surface expression of interleukin 2 receptors (IL-2R), we found expression of the IL-2R alpha chain on blast cells in 26% and of the IL-2R beta-chain in 81% of the patients. To confirm these results at the transcriptional level, we studied the expression of RNA of the IL-2R alpha, beta, and gamma chains by RT-PCR in the bone marrow of 38 newly diagnosed patients with AML and in three AML-derived cell lines. RNA of the alpha chain was detectable in 11/38 patients, the beta chain in 10/38 patients and the gamma chain in 30/38 patients with AML. Blast cells obtained from colonies growing in semisolid media expressed mRNA for IL-2R. RNA for all three IL-2R chains was expressed in lines KG1, HEL 92.1.7 and K562. In comparison with unstimulated cell lines, incubation of the three lines with various amounts of IL-2 over 3 and 14 days did not increase their growth or change message expression of IL-2R, IL-10, and TGF-beta and surface expression of the adhesion molecules CD 11, CD 18, CD 29 and CD 54. In conclusion, despite expression of IL-2 receptors, AML blasts do not respond to IL-2 by proliferation, message expression for various cytokine genes and surface expression of cellular adhesion molecules.

Base Sequence↗
PubMed1995

The Wilms' tumor gene is frequently expressed in acute myeloblastic leukemias and may provide a marker for residual blast cells detectable by PCR.

BACKGROUND: The tumor suppressor gene wt-1 was isolated by cytogenetic deletion analysis of patients with Wilms' tumor (wt-1). This gene encodes for a zinc finger DNA-binding protein with transcription-repressing properties. During normal ontogenesis it is expressed in a time- and tissue-dependent manner mainly in the kidneys and gonads. Recently, the expression of wt-1 in acute leukemias (AL) was reported. Here we investigated the prognostic potential of wt-1 mRNA expression during the course of the disease using the PCR technique. PATIENTS AND METHODS: Blast cells from 83 patients with newly diagnosed AML and 20 AML patients during follow-up in complete remission were analyzed for wt-1 mRNA expression. Peripheral blood mononuclear cells (PBMNC) and bone marrow (BM) from healthy persons (n = 13) and sorted CD34-positive cells from normal donors (n = 4) were used as negative controls. RESULTS: Wt-1-specific m-RNA was detectable in 67/83 (81%) patients with AML. Normal donors did not express wt-1 m-RNA but in 1/4 sorted CD34+ cell samples a weak amplified product was observed. After achieving cytological CR 14/20 studied patients lost wt-1 expression. In 7/8 patients in morphological CR the reappearance of wt-1 expression preceded relapse of the disease, in 1/8 patients wt-1 remained positive in CR. Response to therapy, disease-free survival, overall survival and FAB-subtype did not correlate with wt-1 m-RNA expression in newly diagnosed AML before therapy. CONCLUSIONS: In the majority of acute leukemias wt-1 is expressed and probably blast cell-associated, at least in levels detectable by PCR. Wt-1 mRNA was detectable in bone marrow cells of AML patients in clinical CR. The results strongly suggest that the persistence or reappearance of wt-1 predicts relapse of the disease prior to morphological relapse.

Adult↗
PubMed1995

The inhibition of lymphokine-activated killer cells in acute myeloblastic leukemia is mediated by transforming growth factor-beta 1.

In acute myeloblastic leukemia (AML), the T cell response and cytotoxic activity are impaired at time of diagnosis due to not-yet-identified soluble immunosuppressing factors. The inhibition of autologous antileukemic immune response by these factors may support immunosurveillance of AML. A well-known inhibitor of lymphokine-activated killer (LAK) cell activity is transforming growth factor-beta 1 (TGF-beta 1). To evaluate the possible significance of TGF-beta 1 for the impaired cytotoxic activity in AML at time of diagnosis, we looked for the TGF-beta 1-specific mRNA, for the production and release of TGF-beta 1, and for its relevance for immunosuppressing activities in AML. In the culture supernatants of 18 investigated AMLs, we detected various amounts of TGF-beta protein. The TGF-beta 1 and TGF-beta 2 protein concentrations were 105 pg/mL (< 50-240 pg/mL) and 32 pg/mL (< 2-91 pg/mL), respectively. In 13 of 15 patients, the leukemic blasts expressed TGF-beta 1 mRNA. To exclude possible interferences with contaminating mononuclear cells (MNC), the data were confirmed by analysis of sorted blast cells and leukemic cell lines. All investigated leukemic cell lines expressed TGF-beta 1 protein and mRNA. The culture supernatants of AMLs inhibited LAK activity strongly in a dose-dependent manner. The inhibition of cytotoxicity could be restored by the addition of neutralizing TGF-beta 1 antibodies. The data suggest TGF-beta 1 to be a relevant factor for the inhibition of cytotoxic activities in AMLs.

Adult↗
PubMed1994

Expression of interleukin 2 receptors on human carcinoma cell lines and tumor growth inhibition by interleukin 2.

We have previously shown that human squamous cell carcinomas (SCC) express the interleukin 2 receptor (IL2R)-alpha and -beta chains, and that the ligand, IL2, directly inhibits growth of the tumor in vitro and in vivo in the tumor xenograft-nude mice model. We now show that the alpha and beta chains of IL2R are expressed on a variety of human carcinoma cell lines and on normal human keratinocytes in early-stage cultures. While all carcinoma cells in a population expressed IL2R-alpha and -beta proteins, in keratinocytes obtained from different normal donors, variable proportions of cells were positive, as measured by flow cytometry. The carcinoma lines and 2/5 keratinocyte lines studied were also found to contain transcripts for the IL2R-gamma chain detectable by combined reverse transcription-PCR (RT-PCR) and hybridization with the specific cDNA probe. Incubation of the gastric (HR) or renal cell carcinoma (RCC) cell lines, but not of other IL2R+ carcinoma cell lines or normal keratinocytes, in the presence of IL2 resulted in dose-dependent inhibition of tumor cell growth. Monoclonal antibodies (MAbs) specific for IL2R-beta chain completely reversed this growth inhibitory effect of IL2. The ligand, IL2, also down-regulated surface expression of its own receptor and of intercellular adhesion molecule-I (ICAM-I) or class I major histocompatibility complex (MHC) antigens on IL2R+ tumor cells. All carcinoma cells studied incubated in the presence of IL2 exhibited significantly increased sensitivity to growth-inhibitory effects of other cytokines such as interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha or transforming growth factor (TGF)-beta. IL2 inhibited growth of the HR cells by arresting a significant proportion of tumor cells in the G0/G1 phase of the cell cycle. Thus, IL2 can have direct effects on IL2R+ carcinoma cells, leading to changes in growth or to increases in sensitivity of tumor cells to cytostatic activities of other cytokines.

Antigens, Neoplasm↗
PubMed1994

Evidence for superantigen involvement in insulin-dependent diabetes mellitus aetiology.

Insulin-dependent diabetes mellitus (IDDM) is a T-cell-mediated autoimmune disease whose onset is believed to be triggered by unknown environmental factors acting on a predisposing genetic background. Islet-infiltrating T (IIT) cells from two IDDM patients, who had died at the onset of the disease from brain swelling as a complication of ketoacidosis, were analysed. The results provided evidence for the involvement of a pancreatic islet cell membrane-bound superantigen as a diabetes aetiopathogenetic factor. There was a selective expansion of a T-cell receptor (TCR) variable segment of the beta-chain (V beta 7) in these IIT cells in association with unselected V alpha-chain segments; extensive junctional diversity of the TCR V beta 7 chains; and evidence of positive selection, after exposure to diabetic islet cell membrane preparations, of V beta 7+ T-cell clones among peripheral blood lymphocytes from non-diabetic individuals.

Amino Acid Sequence↗
PubMed1994

HLA restriction and T-cell-receptor V beta gene expression of cytotoxic T lymphocytes reactive with human squamous-cell carcinoma of the head and neck.

A human cytotoxic-T-lymphocyte (CTL) line capable of killing autologous tumor (AuTu) cell targets was established from peripheral-blood lymphocytes of a patient with squamous-cell carcinoma of the tongue. The cultured CTL were CD3+CD8+CD11b-HLA-DR+T cell receptor (TCR) alpha/beta+. When tested in 4-hr 51Cr-release assays against various lines of squamous-cell carcinoma of the head and neck (SCCHN) and a variety of non-squamous human tumor and normal cell targets, the CTL were found to lyse the autologous SCCHN cell line (PCI-50) and 7 allogeneic SCCHN lines: PCI-1, -2, -4A, -4B, -13, -30 and -38. Of these tumor cell lines, PCI-13, -30 and -38 shared HLA-A2 locus with the AuTu, PCI-50, while PCI-4A and -4B shared HLA-B44 with AuTu. Lysis of AuTu (A2+B44+), PCI-13 (A2+B44-) and PCI-4B (A2- B44+) by the CTL was efficiently inhibited by monoclonal antibodies (MAbs) to CD3, CD8, TCR alpha/beta or the major-histocompatibility-complex (MHC)-class-I antigens. MAbs to HLA-A2 antigens inhibited lysis of PCI-50 or PCI-13 targets by the CTL. In cold-target inhibition assays, unlabeled PCI-4B or PCI-13 cells inhibited CTL lysis of AuTu targets. The CTL incubated in the presence of the HLA-A2+ SCCHN PCI-50 or -13, but not an HLA-A2+ gastric carcinoma, produced TNF-alpha, IFN-gamma and GM-CSF. The CTL were tested for their TCR V beta gene expression by polymerase chain reaction (PCR). At week 10 in culture, the time of the highest AuTu cytotoxicity mediated by the CTL line, V beta 6 was expressed by 26% of T cells. Three clones, obtained by limiting dilution from 10-week CTL and selected for high cytotoxicity against AuTu, were found to be V beta6+. Further analysis of the specificity of these clones indicated lytic activity against PCI-13 (A2+B44-), but not PCI-4B (A2-B44+) targets. In 16-week cultures, which retained AuTu cytotoxicity as well as V beta 6 expression, TCR V beta 2 was also expressed at high frequency (29%), and AuTu-reactive clones were found to be V beta 2+. Our results indicate that at least 2 different CTL populations (V beta 6+ and V beta 2+) are able to recognize SCCHN-associated antigen(s) and that the V beta 6+ T cells are HLA-A2 restricted, while V beta 2+ T cells may be HLA-B44 restricted.

Antibodies, Monoclonal↗
PubMed1994

The expression of the Wilms' tumor gene in acute myelocytic leukemias as a possible marker for leukemic blast cells.

The Wilms' tumor gene (wt-1) is expressed in the developing fetal kidney, gonads and in Wilms' tumors. Recently, the expression of wt-1 in leukemia-derived cell lines and cases of acute leukemias (AL) was reported. The present study was designed to investigate the potential of wt-1 as genetic marker for acute myelocytic leukemias (AML). Blast cells from 52 patients with AML, 14 patients in complete remission (CR) and four leukemic cell lines were examined for expression of wt-1 mRNA. Peripheral blood mononuclear cells (PBMNC) and bone marrow (BM) from 13 healthy persons were used as negative controls. RNA of the wt-1 gene was expressed in 41/52 (79%) patients with previously untreated AML. The majority of the 14 patients studied in CR lost wt-1 expression. In three out of the four patients in CR reappearance of wt-1 expression preceded relapse of the disease. In three of the four tested cell lines wt-1 specific transcription was demonstrated. No correlation to FAB classification, immunophenotype or response to treatment was found. Our experiments indicate wt-1 expression in the majority of AML, but not in bone marrow or PBMNC of healthy controls. Therefore, wt-1 expression may be associated with the presence of malignant blast cells and the analysis of wt-1 gene expression via PCR may be a sensitive method for the detection of leukemic blast cells.

Adult↗
PubMed1993

Evidence for oligoclonal T-cell response in a metastasis of renal cell carcinoma responding to vaccination with autologous tumor cells and transfer of in vitro-sensitized vaccine-draining lymph node lymphocytes.

Peripheral blood lymphocytes (PBL) and tumor-infiltrating lymphocytes (TIL) of a patient with von Hippel-Lindau disease and renal cell carcinoma were studied for the T-cell receptor beta chain variable region (TCR-V beta) repertoire. The patient was vaccinated with irradiated autologous tumor cells from a renal tumor mass, a vaccine-draining lymph node was removed, and lymphocytes were cultured in the presence of autologous tumor cells and low-dose interleukin 2 (IL2). These lymphocytes were adoptively transferred to the patient together with systemic IL2 (30,000 IU/kg every 8 h). Analysis of TCR-V beta expression was performed by polymerase chain reaction in PBL before, during, and after therapy, in vaccine-draining lymph node lymphocytes, and in TIL obtained from moderately infiltrated, nonresponding renal tumor mass and from a more intensely infiltrated lung metastasis, which was responding to treatment. Significant differences in the expression of TCR-V beta 13.1 by T-cells recovered from these various sites were observed. Also, TIL recovered from the responding lung metastasis and cultured in the presence of IL2 gave rise to autologous tumor-reactive CD4+ T-cells, whereas the nonresponsive renal tumor yielded a mixture of T- and natural killer cells. In PBL obtained prior to treatment and during IL2 therapy, expression of V beta 13.1 was 0.7 and 1.8%, respectively, of the total V beta gene repertoire. Fresh vaccine-draining lymph node lymphocytes contained 5.9% of V beta 13.1-expressing T-cells. After IL2 therapy, V beta 13.1 gene expression increased to 5.4% in PBL. In the nonresponding tumor mass, the frequency of V beta 13.1 gene expression among TIL was 12%, whereas in the responding, highly infiltrated nodule, it was 28%, with a striking loss of expression of other V beta gene families. Sequencing of the amplified product of V beta 13.1 complementary DNA from the responding pulmonary metastasis showed restrictions in the complementarity-determining region 3. Thus, in vivo expansion of V beta 13.1-expressing CD4+ T-cells, possibly in response to a tumor-associated antigen, occurred in the responding tumor mass following this form of therapy and correlated with tumor course.

Amino Acid Sequence↗
PubMed1993

Daily alternating administration of high-dose alpha-2b-interferon and interleukin-2 bolus infusion in metastatic renal cell cancer. A phase II study.

BACKGROUND: Both interleukin-2 (IL-2) and alpha-interferon (alpha-IFN) have some efficacy in renal cell cancer (RCC) as single agents. Additionally, there is some evidence for additive or synergistic antitumoral activity of IL-2 and alpha-IFN in vitro and possibly in vivo. Based on these data, the authors initiated a Phase II trial with a combination of recombinant IL-2 (rIL-2) and recombinant alpha-IFN (alpha-rIFN) in advanced RCC. METHODS: Thirty-six assessable patients with metastatic RCC were entered in this Phase II trial using a daily alternating schedule of alpha-rIFN and rIL-2. Over a period of 14 days, the patients received daily alternating treatment with 10 x 10(6) IU/m2 of recombinant alpha-2b-interferon subcutaneously and 18 x 10(6) IU/m2 of rIL-2 as a 1-hour intravenous infusion. This treatment schedule was repeated every sixth week up to a maximum of four cycles. After the second cycle, patients were examined for response. Patients with stable disease or better received two additional cycles of therapy. Patients with progressive disease were available for other strategies. RESULTS: Thirty-six patients entered the trial and were assessable for toxic effects. Thirty of 36 patients completed at least two cycles and were assessable for response. Nine patients achieved an objective response: 2 had complete responses (CR) and 7 had partial responses (PR). Three patients had a minor response. No effect was observed in patients with local relapse or bone metastases. A relapse-free survival length of 6 months or longer was seen in both patients with CR (12, 23 + months) and in four of seven patients with PR (6, 7, 12, 12 months). The toxicity was moderate and included fever and nausea in most patients, and hypotension, fatigue, skin rash, and arthralgia in a minority of the patients. No Grade 4 and only occasionally Grade 3 toxicity was observed. Fluid retention was negligible. The monitoring of immunologic parameters showed a significant rebound lymphocytosis including cytotoxic (CD56+) cells; in responders the peak of lymphocytosis occurred up to 1 week later than in nonresponders. Peripheral lymphocytes obtained after therapy showed only a slight increase of natural killer cell and lymphokine-activated killer cell activity. During therapy, there was a great release of secondary cytokines as tumor necrosis factor-alpha, gamma-interferon, and interleukin-6, with a peak level 2-4 hours after rIL-2 infusion. CONCLUSIONS: In conclusion, daily alternating administration of alpha-rIFN and rIL-2 is effective in RCC with less toxicity, and the response rate is comparable to those of other immunotherapeutic schedules, including adoptive immunotherapeutic schedules, including adoptive immunotherapy and combinations of high-dose IL-2 and alpha-IFN.

Aged↗
PubMed1993

Interferon-alpha and interleukin-2 in the treatment of metastatic melanoma. Comparison of two phase II trials.

BACKGROUND: Interferon-alpha (IFN alpha) and interleukin-2 (IL-2) are active agents against malignant melanoma. There is, however, no consensus on the optimal dosing schedule of both drugs. This is a report of two sequential immunotherapy trials in patients with metastatic melanoma using two different IL-2 dosing schedules. METHODS: Schedule A consists of IFN alpha, 10 million U/m2/day subcutaneously for 5 days, followed by continuous intravenous infusion of IL-2, 1 mg/m2/24 hours for 5 days. Schedule B consists of the same dose of IFN alpha, but a modified regimen of IL-2. To improve the induction of high-affinity IL-2 receptors, the initial IL-2 dose was increased (1 mg/m2/6 hours, followed by 1 mg/m2/12 hours, and 1 mg/m2/24 hours). To reduce toxicity, the dose was reduced thereafter to 0.25 mg/m2/24 hours for the following 3 days. Both regimens were repeated after 4 weeks. RESULTS: 27 patients were treated with schedule A with a response rate of 18% (1 complete response [CR], 4 partial responses [PR]), 95% confidence interval, 6-36%. The response rate in 27 patients treated with schedule B was 41% (3 CR, 8 PR), 95% confidence interval, 22-61%. Severe, often dose-limiting toxicity was associated with IL-2 in schedule A, particularly hypotension and fluid retention. Toxicity was reduced significantly in schedule B. Maximal serum levels of soluble CD25 were 17,022 +/- 13,070 U/ml in schedule A, and 31,148 +/- 4227 U/ml in schedule B (P < or = 0.01). Serum levels of TNF alpha were significantly lower in schedule B than in schedule A, as were the side effects. CONCLUSIONS: Toxicity of IL-2 is reduced by modifying the schedule of administration, which also enhances the immunologic response and appears to increase the response rate.

Adult↗
PubMed1993

Usage of T-cell receptor V beta chain genes in fresh and cultured tumor-infiltrating lymphocytes from human melanoma.

Tumor-infiltrating lymphocytes (TIL) freshly obtained from human malignant melanomas as well as the same TIL grown in the presence of interleukin 2 (IL2) were studied for gene expression of the T-cell receptor (TCR) variable beta regions (V beta). To perform the TCR-V beta analysis, total RNA was isolated from TIL and reverse-transcribed into cDNA, which was then amplified by PCR using 22 different 5' primers specifically recognizing the sequences of 20 V beta gene families and a 3' primer annealing to the constant region of the beta chain. The TCR-alpha constant region (C alpha) gene was co-amplified as a standard for the calculation of the percentage of each TCR-V beta gene expressed. The frequency of individual V beta regions expressed on TIL was computed from the ratio of cpm V beta to cpm C alpha for each V beta region in relation to the total of all 22 ratios. With fresh TIL obtained from 8 different melanomas, oligoclonal distribution of V beta genes expressed on TIL was observed, in comparison with a broader and unrestricted distribution seen with peripheral-blood T cells of 8 normal individuals. The oligoclonal patterns of V beta-gene expression in fresh melanoma TIL were distinct in every tumor. Several of the V beta-genes usually expressed in normal PBL were not expressed in fresh TIL in melanoma TIL cultured in the presence of IL2 and IL4 and in the absence of autologous tumor (AuTu) or antigen-presenting cells for 23 to 65 days, selection of T-cell lines expressing a restricted number of V beta genes occurred. Although in 4/5 TIL cultures this selection involved the V beta 7 gene, no relationship could be established between V beta gene expression in fresh TIL and that in T-cell lines outgrowing in long-term cultures. Selection in culture of CD3+CD8+ T-cell lines with V beta-gene expression restricted to 1 or 2 V beta families did not correlate with the presence or level of AuTu cytotoxicity mediated by these T cells. The results indicate that in TIL cultures random selection of T-cell lines with reactivity not relevant to AuTu may account for poor expression or loss of AuTu cytotoxicity by most TIL cultured long-term in the presence of cytokines and in the absence of specific antigenic stimulation.

CD3 Complex↗