LEA29Y expression in transgenic neonatal porcine islet-like cluster promotes long-lasting xenograft survival in humanized mice without immunosuppressive therapy.

Genetically engineered pigs are a promising source for islet cell transplantation in type 1 diabetes, but the strong human anti-pig immune response prevents its successful clinical application. Here we studied the efficacy of neonatal porcine islet-like cell clusters (NPICCs) overexpressing LEA29Y, a high-affinity variant of the T cell co-stimulation inhibitor CTLA-4Ig, to engraft and restore normoglycemia after transplantation into streptozotocin-diabetic NOD-SCID IL2rγ-/- (NSG) mice stably reconstituted with a human immune system. Transplantation of INSLEA29Y expressing NPICCs resulted in development of normal glucose tolerance (70.4%) and long-term maintenance of normoglycemia without administration of immunosuppressive drugs. All animals transplanted with wild-type NPICCs remained diabetic. Immunohistological examinations revealed a strong peri- and intragraft infiltration of wild-type NPICCs with human CD45+ immune cells consisting of predominantly CD4+ and CD8+ lymphocytes and some CD68+ macrophages and FoxP3+ regulatory T cells. Significantly less infiltrating lymphocytes and only few macrophages were observed in animals transplanted with INSLEA29Y transgenic NPICCs. This is the first study providing evidence that beta cell-specific LEA29Y expression is effective for NPICC engraftment in the presence of a humanized immune system and it has a long-lasting protective effect on inhibition of human anti-pig xenoimmunity. Our findings may have important implications for the development of a low-toxic protocol for porcine islet transplantation in patients with type 1 diabetes.

A CD19-specific single-chain immunotoxin mediates potent apoptosis of B-lineage leukemic cells.

CD19 is a B-lineage-specific transmembrane signaling protein participating in the control of proliferation and differentiation. It is present at high surface density on chronic B-lymphocytic leukemia (B-CLL) cells and cells of other B-cell malignancies, and is a prime target for therapy with antibody-derived agents. Many attempts have been made to target malignant cells via CD19, but to date none of these agents have received drug approval. Here we report the design of a monovalent immunotoxin consisting of a CD19-specific single-chain Fv antibody fragment fused to a derivative of Pseudomonas Exotoxin A. This fusion protein induced efficient antigen-restricted apoptosis of several human leukemia- and lymphoma-derived cell lines including Nalm-6, which it eliminated at an effective concentration (EC(50)) of 2.5 nM. The agent displayed synergistic toxicity when used in combination with valproic acid and cyclosporin A in cell-culture assays. It induced apoptosis of primary malignant cells in 12/12 samples from B-CLL patients, including patients responding poorly to fludarabine, and of cells from one pediatric acute lymphoblastic leukemia patient. In NOD/SCID mice transplanted with Nalm-6 cells, the toxin prevented engraftment and significantly prolonged survival of treated mice. Owing to its efficient antigen-restricted antileukemic activity, the agent deserves further development towards clinical testing.

Attitudes on euthanasia, physician-assisted suicide and terminal sedation--a survey of the members of the German Association for Palliative Medicine.

BACKGROUND: Due to recent legislations on euthanasia and its current practice in the Netherlands and Belgium, issues of end-of-life medicine have become very vital in many European countries. In 2002, the Ethics Working Group of the German Association for Palliative Medicine (DGP) has conducted a survey among its physician members in order to evaluate their attitudes towards different end-of-life medical practices, such as euthanasia (EUT), physician-assisted suicide (PAS), and terminal sedation (TS). METHODS: An anonymous questionnaire was sent to the 411 DGP physicians, consisting of 14 multiple choice questions on positions that might be adopted in different hypothetical scenarios on situations of "intolerable suffering" in end-of-life care. For the sake of clarification, several definitions and legal judgements of different terms used in the German debate on premature termination of life were included. For statistical analysis t-tests and Pearson-correlations were used. RESULTS: The response rate was 61% (n = 251). The proportions of the respondents who were opposed to legalizing different forms of premature termination of life were: 90% opposed to EUT, 75% to PAS, 94% to PAS for psychiatric patients. Terminal sedation was accepted by 94% of the members. The main decisional bases drawn on for the answers were personal ethical values, professional experience with palliative care, knowledge of alternative approaches, knowledge of ethical guidelines and of the national legal frame. CONCLUSIONS: In sharp contrast to similar surveys conducted in other countries, only a minority of 9.6% of the DGP physicians supported the legalization of EUT. The misuse of medical knowledge for inhumane killing in the Nazi period did not play a relevant role for the respondents' negative attitude towards EUT. Palliative care needs to be stronger established and promoted within the German health care system in order to improve the quality of end-of-life situations which subsequently is expected to lead to decreasing requests for EUT by terminally ill patients.

Cancer in pregnancy: maternal-fetal conflict.

The occurrence of malignancies during pregnancy has increased over the last decades. They complicate approximately 1 per 1000 pregnancies. The most common malignancies associated with pregnancy include malignant melanoma, malignant lymphomas and leukemia, and cancer of the cervix, breast, ovary, colon and thyroid. Since it is impossible for prospective randomized clinical trials to be conducted in this field, relevant data have been generated from case reports and matched historical cohort studies in order to evaluate the treatment outcomes and the issues complicating the management of malignancy in the pregnant patient. There is almost always a conflict between optimal maternal therapy and fetal well-being. The maternal interest is for an immediate treatment of the recently diagnosed tumor. However, the optimal therapy, be it chemotherapy, radiotherapy or surgery, may impose great risks on the fetus. Consequently, either maternal or fetal health, or both, will be compromised. Therefore, both the pregnant patient and her physician are often in a dilemma as to the optimal course. On the basis of the medical facts, we discuss the issues raising potential ethical conflicts and present a practical ethical approach which may help to increase clarity in maternal-fetal conflicts. We review the available data informing the incidence and impact of the most common malignancies during pregnancy and their treatment on both the pregnant woman and her fetus. The optimal therapy for the tragic diagnosis of cancer in pregnancy requires a collaborative and interdisciplinary approach between gynecologists, oncologists, obstetricians, surgeons, neonatologists, psychologists, nursing staff and other disciplines. The purpose of this article is not to answer specific questions or to construct management schemes for specific tumors but to provide a framework for approaching some of these complex issues.

Peripheral blood monoclonal B-cells predict the event free survival in multiple myeloma.

Reinfusion of myeloma progenitor cells may contribute to relapse of multiple myeloma after autologous stem cell transplantation. The aim of our study was to investigate whether monoclonal B-cells are present in the apheresis product and to evaluate the clinical relevance of these cells. Leukapheresis products of 55 patients were purged with anti-B-cell-Monoclonal antibodies (MoAbs) and immunobeads. Monoclonal B-cells were found in 85% of patients within the B-cell population. In one third of all myeloma patients, the majority of B-cells was represented by monoclonal myeloma progenitor B-cells, whereas in two thirds of patients monoclonal cells only represented a small part of the entire B-cell population. As shown by sequence analysis, monoclonal precursor B-cells and malignant plasma cells had the identical genetic CDR III sequence. The purging efficacy, using a negative selection system, was a median of 3 logs (range 1,5-3,5). No statistical difference in the purging efficacy was found when 3, 4 or 5 MoAbs against B-cells antigens were used. However, a tumor specific signal could be detected in the purged harvest of all patients, when the highly sensitive ASO-PCR approach was used. Furthermore, we found a direct correlation between the amount of remaining monoclonal cells after negative selection and the event free survival of myeloma patients. 10/15 patients with a median of 20 x 10(3) monoclonal cells in the purged product relapsed at a median of 1,4 years, whereas only 6/24 patients with an oligoclonal pattern including a low number of remaining monoclonal cells relapsed at a median of 2,2 years. The event free survival (EFS) was statistically different between the two groups (p = 0,014).

Detection of K-ras and p53 mutations in bronchoscopically obtained malignant and non-malignant tissue from patients with non-small cell lung cancer.

Molecular screening may increase the likelihood to identify early malignant lesions in non-small cell lung cancer. However the presence of gene mutations in non-malignant bronchial tissue has remained controversial. The present study was carried out to investigate systematically the presence of mutations of the K-ras and p53 gene in bronchial biopsies taken during routine bronchoscopy of normal as well as tumour tissues from a series of 40 patients with histologically verified non-small cell lung cancer (NSCLC). K-ras mutations were analysed with specific detection oligonucleotides, p53 mutations were examined by SSCP analysis. In all biopsies the wildtype of both K-ras and p53 could be detected. The overall frequency of mutations was 14 (35%) with 2 K-ras mutations (5%) and 12 mutations of the p53 gene (30%). In 3 cases (1 ras mutation, 2 p53 mutations) the same mutation could be shown in the tumour biopsy and in the distant normal control. In another case only the normal appearing tissue had a mutation of the p53 gene. All other mutations could be detected in the tumour tissue only. Our data confirm that K-ras mutations and p53 can be detected not only in malignant but also in non-malignant bioptic samples from patients with NSCLC. The use of molecular screening for the early detection of lung cancer may be a promising new approach.

The relationship between monoclonal myeloma precursor B cells in the peripheral blood stem cell harvests and the clinical response of multiple myeloma patients.

The aim of this study was to determine the presence of monoclonal myeloma precursor B cells in peripheral blood stem cell harvests and to investigate their role in the clinical outcome of multiple myeloma patients. A total of 39 multiple myeloma patients were treated with a sequential therapy including double high-dose melphalan therapy followed by a double transplant procedure. The apheresis products for the second transplant were purged using a panel of four or five different mouse monoclonal antibodies against B-cell antigens (CD10, CD19, CD20, CD22 and CD37). In 19/39 patients a tumour-specific CDR III signal was identified in the diagnostic bone marrow. Gene scan analysis after CDR III PCR of the magnetic bead isolated B-cell fraction from the apheresis products in these 19 patients revealed three different patterns: 32% of patients had a predominantly monoclonal B-cell population; 63% of patients had an identifiable monoclonal signal within an oligoclonal B-cell population. In only 1/19 patients were no monoclonal B cells identified in the B-cell population of the apheresis product. A correlation between the clonal pattern and the clinical response after sequential chemotherapy was found. Patients with a predominance of monoclonal myeloma or myeloma precursor B cells had an early relapse or achieved a minimal response or a partial remission. Patients with an oligo- and/or polyclonal pattern achieved a high percentage of partial as well as complete remissions.

Applications of consensus polymerase chain reaction with subsequent electrophoretic distinction of amplificates.

Conserved sequences within gene families permit the design of consensus primers that match several members of a given class of homologous genes. Polymerase chain reaction (PCR) products obtained with such consensus primers were characterized by restriction mapping or single-strand conformation polymorphism (SSCP) analysis, using precast polyacrylamide minigels and automated silver staining. Examples for the electrophoretic distinction of consensus amplificates are presented in the fields of guanylyl cyclase expression studies and in the determination of B-cell clonality in human blood samples. Guanylyl cyclase expression in inner ear tissues of guinea pigs was investigated by reverse transcription PCR using consensus primers with specificity for the subclass of particulate guanylyl cyclases. The resulting PCR products were assigned to three representatives of this group by restriction mapping. The consensus PCR approach enabled the detection of an unexpected receptor type, namely guanylyl cyclase C, in the inner ear. The distinction by SSCP analysis of denatured consensus amplificates was appropriate for the identification of clone-specifically rearranged immunoglobulin heavy chain genes of B-lymphocytes. Genomic DNA isolated from blood samples of leukemia patients served as the template for the consensus amplification of clone-specific VDJ rearrangements. Rapid distinction and re-identification of consensus PCR products was achieved by SSCP analysis for regular antigen receptor rearrangements and for t(14; 18) translocations. The potential of these procedures for detecting leukemia or lymphoma clones when monitoring minimal residual disease was assessed.

Complementary anchor PCR of rearranged variable T-cell receptor beta-chain cDNA regions.

Sequencible amplificates comprising the variable cDNA sequences of the rearranged T-cell receptor (TCR) beta-chain were obtained from the T-leukemia cell line Jurkat using a single-sided PCR approach based on five synthetic oligonucleotides derived from the flanking constant sequence. Double-stranded cDNA was cleaved by a restriction enzyme creating cohesive ends, to which an anchor oligonucleotide was ligated. Since this anchor was complementary to the antisense strand of the known constant region, exclusively the desired ligation product folded into a stem-loop-structure that was enzymatically extended to yield a PCR template, now flanked at both ends by primer binding sites appropriate for nested PCR.