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Biomedical subjects

V Diehl

Publications and source records attributed to V Diehl.

At least 181 records · Page 10Linked to original sources
PubMed1999

CD3X anti-nitrophenyl bispecific diabodies: universal immunotherapeutic tools for retargeting T cells to tumors.

We developed a universal recombinant bispecific molecule (BiMol) that is capable of redirecting cytotoxic T cells to tumor cells via tagged anti-tumor ligands such as antibody fragments or cytokines. A recombinant bispecific diabody with binding specificities for the CD3 molecule on T cells as well as for the hapten nitrophenyl (NIP) was produced. This bispecific molecule is capable of redirecting cytotoxic T cells to kill a series of malignant cells, including B cell lymphoma, Hodgkin's lymphoma, and colon carcinoma via NIP-conjugated ligands to tumor-associated antigens. Cytotoxic activity of the diabody was found to be comparable to tetradoma-derived bispecific antibodies with similar specificities. Our findings demonstrate that universal CD3xanti-NIP diabodies could be used for T cell based cellular immunotherapy in a variety of human malignancies. Additionally, these bispecific molecules allow fast and economic testing of tumor-associated antigens on malignant cells for their potential use as immunotherapeutic target structures if corresponding hapten-conjugated antibodies or ligands are available.

Antibodies, Bispecific↗
PubMed1999

Salvage therapy with regimens containing ritonavir and saquinavir in extensively pretreated HIV-infected patients.

OBJECTIVE: To evaluate the efficacy and toxicity of salvage regimens containing ritonavir and saquinavir in patients failing highly active antiretroviral therapy (HAART), and to correlate outcome with plasma concentrations of protease inhibitors. DESIGN: Prospective, non-randomized interventional study. SUBJECTS AND METHODS: Thirty extensively pretreated HIV-infected patients with virological failure under HAART were treated with ritonavir (400 mg twice daily) and saquinavir (600 mg twice daily) and at least one reverse transcriptase inhibitor. HIV-RNA, CD4 cell counts and plasma concentrations of protease inhibitors were determined, and patients were monitored for toxicity at monthly intervals. RESULTS: Six patients showed complete virological success (HIV-RNA <200 copies/ml at week 12) which was sustained for a median follow-up of 6.3 months. Partial virological response (decrease of HIV-RNA of >1 log10 at week 12) was achieved by a further three patients. Patients with a virological response had significantly higher CD4 cell increases than patients without virological response (mean increase at week 12: 66x10(6) cells/l versus 6x10(6) cells/l; P = 0.01). No clinical events were observed during 6 months of follow-up. Neither the use of a non-nucleoside reverse transcriptase inhibitor (NNRTI) nor the number of newly introduced drugs influenced the virological response. Plasma concentrations of protease inhibitors did not statistically differ between patients with and without success. Toxicity included gastrointestinal disturbances, lipid abnormalities and liver dysfunction. CONCLUSIONS: In extensively pretreated patients, salvage regimens containing ritonavir and saquinavir had only limited and short-term anti-HIV activity and were associated with substantial toxicity. Plasma concentrations of saquinavir were not predictive for virological response.

Adult↗
PubMed1999

[Role of high-dose chemotherapy in hematology and internal medicine/ oncology].

BACKGROUND: High-dose chemotherapy (HDCT) with stem cell rescue is increasingly being used as a salvage or consolidation therapy for patients with a variety of malignant diseases. The authors give an overview of the current role of HDCT in lympho-hematopoietic malignancies and solid tumors. METHODS: The use of allogenic or autologous hematopoietic stem cells allows an increase of the dose of chemotherapeutic drugs by a factor of between 4- and 30-fold compared to conventional chemotherapy protocols. In recent years mobilized peripheral blood stem cells (PBSC) have largely replaced the use of autologous bone marrow due to more rapid hematopoietic reconstitution and are increasingly used in the allogenic setting. This article reviews the data of high-dose chemotherapy in non-Hodgkin's lymphoma, Hodgkin's lymphoma, multiple myeloma, acute leukemias, chronic myelogenous leukemia, chronic lymphatic leukemia, myelodysplastic syndrome, breast cancer, ovarian cancer, germ cell cancer, and lung cancer. Current clinical trials further evaluating the role of HDCT are described. RESULTS: Data on results of HDCT are available for a variety of malignancies showing the feasibility and efficacy of the procedure with tolerable toxicity. Although numerous clinical trials of HDCT have been performed, only few randomized trials have demonstrated that such strategies are statistically significantly better than conventional forms of therapy. CONCLUSION: HDCT has been shown to be useful in the treatment of certain patients with lymphomas, leukemias, myeloma, breast cancer, and testicular cancer. Most of these findings still have to be confirmed by randomized clinical trials. Therefore, HDCT should only be given in controlled studies and at suitable centers.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1999

Carcinoembryonic antigen (CEA)-specific T-cell activation in colon carcinoma induced by anti-CD3 x anti-CEA bispecific diabodies and B7 x anti-CEA bispecific fusion proteins.

Two bispecific recombinant molecules, an anti-CD3 x anti-carcinoembryogenic antigen (CEA) diabody and a B7 x anti-CEA fusion protein, were tested for their capacity to specifically activate T cells in the presence of CEA-expressing colon carcinoma cells. T-cell activation by the anti-CD3 x anti-CEA diabody required close contact to CEA-positive cells and resulted in diabody-mediated cytotoxicity against the target cells. Additionally, CD28-mediated costimulation in combination with anti-CD3 x anti-CEA diabodies induced activation of autologous T cells in CEA-positive primary colon carcinoma specimens, as determined by flow cytometry. The high specificity of the bispecific diabody approach could be further enhanced by the use of B7 x anti-CEA fusion proteins because the costimulatory CD28-signaling to the T cells strictly depended on the expression of CEA on the target cells. We demonstrate that displaying engagement sites for the T-cell antigens CD3 and CD28 on the surface of colon carcinoma cells is a suitable way to activate and retarget T cells in a highly tumor-specific manner. For clinical purposes, B7 x anti-tumor-associated antigen (TAA) fusion proteins, which are equally effective but more specific compared with anti-CD28 monoclonal anti-bodies, thus may improve the tumor specificity of anti-CD3 x anti-TAA bispecific antibodies. Furthermore, B7-negative tumors can be converted into B7-positive tumors by B7 x anti-TAA fusion proteins without the need for B7 gene transfer to the malignant cells.

Amino Acid Sequence↗
PubMed1999

Somatic mutations within the untranslated regions of rearranged Ig genes in a case of classical Hodgkin's disease as a potential cause for the absence of Ig in the lymphoma cells.

Hodgkin-Reed-Sternberg (H-RS) cells are clonal B cells carrying Ig gene rearrangements. However, in situ hybridization methods failed to demonstrate Ig gene expression in H-RS cells of classical Hodgkin's disease (HD). Because somatic mutations rendering potentially functional Ig gene rearrangements nonfunctional were detected in some cases of the disease, it was speculated that H-RS cells in classical HD may have lost the ability to express antigen receptor as a rule. Recently, we established a novel cell line (L1236) from H-RS cells of a patient with mixed cellularity subtype of HD. L1236 cells harbor a potentially functional VH1 and a potentially functional Vkappa3 gene rearrangement. However, no antibody expression was detected. To show potential reasons for this lack of Ig expression, we analyzed the genomic organization of the Ig genes and their transcription in the primary and cultivated H-RS cells of this patient. The H-RS cells were found to have switched their isotype to IgG4, confirming their mature B-cell nature. By amplifying cDNA from L1236 cells as well as from frozen biopsy material transcripts of the Vkappa3 and the VH1 gene rearrangement were detected for both sources of cDNA. However, Northern blot hybridization of L1236 RNA failed to demonstrate VH1 and Vkappa3 transcripts, indicating only a low level of transcription. Sequence analysis of the promoter and leader regions of the VH1 gene rearrangement from L1236 cells as well as from lymphoma-affected tissue showed a somatic mutation in the conserved octamer motif of the promoter region. Somatic mutations were also detected within the 3' splice site of the leader intron and adjacent nucleotides in the rearranged Vkappa light chain gene, leading to aberrant splicing. These mutations might prevent the generation of adequate amounts of functional Ig gene transcripts as template for translation into protein. Thus, mutations in H-RS cells that prevent Ig gene expression might also be located outside the coding region of the Ig genes.

Base Sequence↗
PubMed1999

[Meningococcal sepsis in 3 young men].

HISTORY AND CLINICAL FINDINGS: Three young men became ill one after the other with fever, headaches, vomiting, arthralgias and muscle pain. One day after beginning of symptoms all three patients developed a haemorrhagic rash with petechial and ecchymotic lesions most intense on distal extremities. 24 hours later patient no. 1 and 3 were in septic shock. INVESTIGATIONS: Laboratory tests showed signs of systemic infection, disseminated intravascular coagulation and renal failure. On the day of admission to the hospital blood cultures showed Neisseria meningitidis in patient no. 1 and 3. In patient no. 2 blood cultures were negative. TREATMENT AND COURSE: Intravenous antibiotic therapy was started immediately after admission. In patient no. 1 and 3 purpura fulminans with multiple organ failure demanded intensive care treatment. Patient no. 1 recovered. Necrotic toes made amputations necessary. Patient no. 2 was never critically ill. Patient no. 3, whose course was complicated by a long lasting disseminated intravascular coagulation, died from massive cerebral bleeding 6 days after admission. Patient no. 2, who was treated with ciprofloxacin after symptoms began was never critically ill. CONCLUSION: Neisseria meningitidis sepsis has a high mortality rate. Rapid admission to the hospital and beginning of an antibiotic therapy with penicillin G or a third-generation cephalosporin is a priority when meningococcal disease is suspected. Chemoprophylaxis should be offered to close contacts of patients.

Acute Disease↗
PubMed1999

A new series of pET-derived vectors for high efficiency expression of Pseudomonas exotoxin-based fusion proteins.

Recombinant immunotoxins (rITs) are highly specific anti-tumor agents composed of monoclonal antibody fragments or other specific carriers coupled to plant or bacterial toxins. A major problem in the purification of rITs is the low periplasmic yield in currently available expression systems. Thus, the aim of this study was the development of a new bacterial expression system for high-level production of rITs. We constructed a series of pET-based vectors for pelB-directed periplasmic secretion or cytoplasmic production under the control of the T7lac promoter. Expression in Escherichia coli BL21(DE3)pLysS allowed a tightly regulated isopropyl beta-d-thiogalactopyranoside (IPTG) induction of protein synthesis. An enterokinase-cleavable poly-histidine cluster was introduced into this setup for purification by affinity chromatography. A major modification resulted from the insertion of a specifically designed multiple cloning site. It contains only rare restriction enzyme recognition sites used for cloning of immunoglobulin variable region genes, as well as unique SfiI and NotI restriction sites for directed insertion of single-chain variable fragments (scFv) available from established bacteriophage systems. For this purpose, we deleted two naturally occurring internal SfiI consensus sites in a deletion mutant of Pseudomonas aeruginosa exotoxin A (ETA'). Each single structural element of the new vector (promoter, leader sequence, purification tag, scFv sequence, selectable marker, and toxin gene) was flanked by unique restriction sites allowing simple directional substitution. The fidelity of IPTG induction and high-level expression were demonstrated using an anti-CD30 scFv (Ki-4) fused to ETA'. These data confirm a bacterial vector system especially designed for efficient periplasmic expression of ETA'-based fusion toxins.

ADP Ribose Transferases↗
PubMed1999

CD3xCD19 bispecific antibodies and CD28 bivalent antibodies enhance T-cell reactivity against autologous leukemic cells in pediatric B-ALL bone marrow.

Bispecific CD3xCD19 antibodies and CD28 co-stimulating antibodies were used to activate T cells in bone marrow aspirates (n = 8) of children with B cell-derived acute lymphoblastic leukemia. Bone marrow specimens were incubated for 10 days with CD3xCD19 bispecific and CD28 antibodies. Changes in the numbers of T lymphocytes and tumoral B cells as well as surface expression of T cell-activation markers were determined by flow cytometry, and cytokines (human IFN-gamma, IL-2, IL-4 and IL-12) were measured in the cell culture supernatant. In 7 of 8 bone marrow samples, an increase in the number of CD4- and CD8-positive T lymphocytes was found, which correlated with an up-regulation of T cell-activation markers. Additionally, we demonstrated a decrease of tumoral B cells in 3 samples and enhanced cytotoxic T-cell activity against autologous malignant B cells. ELISpot analyses in an autologous Epstein-Barr virus model showed that bispecific antibodies (CD3xCD19+CD28) were more potent at generating T-cell responses against autologous and allogeneic tumoral targets than a combination of monospecific antibodies (CD3+CD28). Thus, T-cell targeting by CD3xCD19 bispecific and CD28 antibodies may be used to eliminate leukemic B cells ex vivo and reconstitute immunological control of residual malignant disease by the induction of anti-tumoral T-cell responses.

Antibodies, Bispecific↗
PubMed1999

Expression and a role of functionally coupled P2Y receptors in human dendritic cells.

We investigated the physiology and function of P2Y receptors expressed in human dendritic cells (DCs) differentiated in vitro from CD14+ cells (DC-14). These were obtained after a 10 day stimulation period in GM-CSF, IL-4 and monocyte conditioned medium. DC-14 were found to express high amounts of MHC class II, B7, CD40 as well as CD83. The functional analysis, using single cell Ca2+ imaging, demonstrated the expression of at least three subtypes of P2Y receptors. We further found using patch-clamp measurements that ATP evoked a pertussis toxin insensitive non-selective cation current with a peak current amplitude of -276+/-43 pA (holding potential -80 mV, n = 23). This current was not Ca(2+)-activated, since it was still observed under conditions of high intracellular Ca2+ buffering and could be blocked by Gd3+ (0.5 mM). In addition, intracellular application of GTP-gamma-S (0.3 mM) also activated the current. Interestingly, DC-14 redirected the orientation of their dendrites as well as cell shape towards a pipette containing ATP as observed with time lapse microscopy. These data suggest that in human DCs, ATP acts via P2Y receptors and induces chemokine effects.

Adenosine Triphosphate↗
PubMed1999

Virological efficacy and plasma drug concentrations of nelfinavir plus saquinavir as salvage therapy in HIV-infected patients refractory to standard triple therapy.

Therapeutic options are limited for patients refractory to triple therapy with two reverse transcriptase inhibitors and one protease inhibitor. Preliminary results showing favorable effects of protease inhibitor combination therapy with nelfinavir and saquinavir due to inhibition of metabolism by cytochrome P450 (CYP450) prompted us to study this combination. - Thirteen patients with incomplete suppression of plasma HIV-RNA were enrolled and treatment was started with nelfinavir 750 mg tid and saquinavir 400 mg bid. Saquinavir-dosage was escalated in weekly intervals to 400 mg tid and 600 mg tid, respectively. All doses were given with food, and plasma levels of saquinavir and nelfinavir were assessed 4 hours post-dosing after 1, 2, 3, 4 and 8 weeks. Treatment was considered virologically efficacious if HIV-viral load was reduced by at least 0.5 log10 from baseline. - Double protease inhibitor-treatment with nelfinavir and saquinavir was virologically efficacious in 5/13 (39%) patients after 4 weeks but only in 4/13 (31%) patients after 8 and 1/13 (8%) after 16 and 24 weeks, respectively. No statistical difference in plasma concentrations was observed when saquinavir was administered in increasing doses of 400 mg bid, 400 mg tid or 600 mg tid in combination with nelfinavir. 5/13 (39%) patients developed diarrhea (>4/d), no other serious side-effects were observed. By eight weeks, the mean CD4 count for all patients was significantly higher when compared to baseline. - In patients refractory to standard triple therapy the combination of nelfinavir and saquinavir showed significant elevation of CD4-count, but only short term virological efficacy in a minority of patients. Plasma concentrations of saquinavir could not be increased by weekly dose escalation of the drug from 400 mg bid to 600 mg tid. Saquinavir drug concentrations of 600 mg saquinavir tid and nelfinavir showed rather non-significant lower values when compared to historical controls treated with a double-dose 1200 mg saquinavir tid regimen alone. We conclude that in these patients the combination of nelfinavir plus saquinavir has no advantage in terms of increasing bioavailibility of saquinavir or virological efficacy. During short observation time a beneficial effect on CD4-count was observed.

Adult↗
PubMed1999

Prognostic impact of apoptosis and proliferation in idiopathic (primary) myelofibrosis.

A retrospective study of 120 patients with the clinically and histologically established diagnosis of idiopathic (primary) myelofibrosis (IMF) was performed to determine prognostic factors of predictive value, including parameters characterizing the dynamics of hematopoietic cell kinetics. In contrast to previous studies, our cohort comprised the full spectrum of the disease, from initial prefibrotic to advanced osteosclerotic stages. The in situ end-labeling (ISEL) technique was used to demonstrate apoptosis, in order to determine dynamic parameters of predictive value. Cell proliferation was evaluated by employing the monoclonal antibody PC10 directed against proliferating cell nuclear antigen (PCNA). Proliferative activity (PCNA index) and frequency of apoptosis showed significant differences between early and advanced fibrosclerotic stages of disease. Decrease in proliferation indicated a significantly shorter survival, whereas a higher frequency of apoptotic cells was associated with a better prognosis. It may be speculated that a normal or enhanced proliferation rate expressed by PCNA positivity (late G1- and S-phase of the cell cycle) that is accompanied by a higher incidence of apoptosis reflects the regenerative (turnover) capacity of hematopoiesis. This may apply especially to early hypercellular stages without relevant myelofibrosis. In consideration of a recently published multivariate risk model, a simplified synthesis score for stratification of a patient's prognosis was constructed. Age, degree of anemia, leukocytes, and platelet count were regarded as the most important parameters. A substantial improvement of prognostic efficiency was further achieved by including PCNA index and frequency of apoptosis. Our results are in keeping with the assumption that generalization, indicated by myeloid metaplasia, has a prodigious impact on prognosis in IMF. Furthermore, in this context dynamic features such as proliferative activity and frequency of apoptosis exert an additional predictive value.

Age Factors↗