PubMed Health⌕ Search

Biomedical subjects

R Repp

Publications and source records attributed to R Repp.

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

Clinical relevance of Fc gamma receptor polymorphisms.

Human IgG receptors are very heterogeneous and we currently distinguish three Fc gamma receptor classes specifying at least 12 receptor isoforms. On top of this complexity, Fc gamma R are further found to differ between different individuals. Polymorphisms have been identified for all three Fc gamma R classes. The best-studied ones represent allelic variation of Fc gamma RIIa (CD32) and Fc gamma RIIIb (CD16). The Fc gamma RIIa polymorphism is now considered to be a heritable risk factor for autoimmune and infectious diseases, and support for a relevant role of the IIIb polymorphism has also been obtained. A detailed analysis of the exact contribution of each of these Fc gamma R polymorphisms in relation to previously implicated risk factors should unravel the pathophysiological importance of Fc gamma R polymorphisms in the near future.

Animals↗
PubMed1997

Preclinical studies with Fc(gamma)R bispecific antibodies and granulocyte colony-stimulating factor-primed neutrophils as effector cells against HER-2/neu overexpressing breast cancer.

Immunotherapies directed to the proto-oncogene product HER-2/neu, which is overexpressed on a subset of breast and other carcinomas, currently receive considerable attention. We have investigated cell-mediated effector mechanisms of HER-2/neu antibodies against breast cancer cell lines. Compared to unfractionated control blood, whole blood from patients during granulocyte colony-stimulating factor (G-CSF) treatment exhibits significantly enhanced lysis (P < 0.001) of SK-BR-3 cells in the presence of HER-2/neu antibody 520C9. The extent of tumor cell killing correlated positively (r = 0.74) to polymorphonuclear neutrophil (PMN) blood counts. Fractionation of whole blood into plasma, mononuclear cells, and PMNs showed major killing capacity to reside in the granulocyte fraction. PMNs were efficiently cytolytic with a panel of HER-2/neu antibodies and against various breast cancer cell lines. Experiments with blocking antibodies to Fc(gamma)R documented Fc(gamma)RII (CD32) as the major trigger molecule for monoclonal antibody 502C9-mediated cytotoxicity. Killing via 520C9 was significantly influenced by an allotypic polymorphism of Fc(gamma)RIIa, the CD32 molecule expressed on PMNs. In reverse antibody-dependent cell-mediated cytotoxicity experiments with a panel of HER-2/neu-directed bispecific antibodies, Fc(gamma)RIII (CD16) proved to be an efficient trigger molecule in blood from healthy volunteers. During G-CSF treatment, however, Fc(gamma)RI (CD64)-expressed on monocytes and G-CSF primed, but not on healthy donor PMNs-became the predominant cytotoxic trigger molecule. Thus, G-CSF application increased effector cell numbers for HER-2/neu-directed immunotherapy, and G-CSF primed PMNs proved particularly effective with a [HER-2/neu x Fc(gamma)RI] bispecific antibody. These findings support clinical trials with HER-2/neu-directed antibodies in combination with G-CSF in breast cancer patients overexpressing HER-2/neu.

Antibodies, Bispecific↗
PubMed1997

Cloning and characterization of AFX, the gene that fuses to MLL in acute leukemias with a t(X;11)(q13;q23).

We report the cloning and characterization of the entire AFX gene which fuses to MLL in acute leukemias with a t(X;ll)(q13;q23). AFX consists of two exons and encodes for a protein of 501 amino acids. We found that normal B- and T-cells contain similar levels of AFX mRNA and that both the MLL/AFX as well as the AFX/MLL fusion transcripts are present in the cell line and the ANLL sample with a t(X;11)(q13;q23). The single intron of the AFX gene consists of 3706 nucleotides. It contains five simple sequence repeats with lengths of at least 12 bps, a chi-like octamer sequence (GCA/TGGA/TGG) and several immunoglobulin heptamer-like sequences (GATAGTG) that are distributed throughout the entire AFX intron sequence. In the KARPAS 45 cell line the breakpoints occur at nucleotides 2913/2914 of the AFX intron and at nucleotides 4900/4901 of the breakpoint cluster region of the MLL gene. The AFX protein belongs to the forkhead protein family. It is highly homologous to the human FKHR protein, the gene of which is disrupted by the t(2;13)(q35;q14), a chromosome rearrangement characteristic of alveolar rhabdomyosarcomas. It is noteworthy that the t(X;11)(q13;q23) in the KARPAS 45 cell line and in one acute nonlymphoblastic leukemia (ANLL) disrupts the forkhead domain of the AFX protein exactly at the same amino acids as does the t(2;13)(q35;q14) in case of the FKHR protein. In addition, the 5'-part of the AFX protein contains a conserved hexapeptide motif (QIYEWM) that is homologous to the functionally important conserved hexapeptide QIYPWM upstream of the homeobox domain in Hox proteins. This motif mediates the co-operative DNA binding of Pbx family members and Hox proteins and, therefore, plays an important role in physiologic and oncogenic processes. In acute leukemias with a t(X;11)(q13;q23), this hexapeptide motif is separated from the remaining forkhead domain within the AFX protein. The predicted amino acid sequence of AFX differs significantly from the partial AFX protein sequence published previously (Genes, Chromosomes and Cancer, 1994, 11, 79-84). This discrepancy can be explained by the occurrence of two sequencing errors in the earlier work at nucleotide number 783 and 844 (loss of a cytosine residue or guanosine residue, respectively) that lead to two reading frame shifts.

Acute Disease↗
PubMed1997

Clinical significance of IgG Fc receptors and Fc gamma R-directed immunotherapies.

Fc receptors for IgG (Fc gamma Rs) can trigger the inflammatory, cytotoxic and hypersensitivity functions of immune effector cells. Activation or deactivation of effector cells via Fc gamma Rs can be exploited to develop novel therapies for cancer, infectious diseases and autoimmune disorders. Initial results of clinical trials for several Fc gamma R-directed immunotherapies show the potential promise of this approach.

Animals↗
PubMed1997

Transcription of hepatitis B virus in peripheral blood mononuclear cells from persistently infected patients.

Hepatitis B virus (HBV) has been reported to exist in peripheral blood mononuclear cells (PBMC), but it is not clear whether it replicates there. A precondition for replication should be the formation of covalently closed viral DNA and transcription of all essential viral mRNAs. The mRNAs of HBV form a nested box with common 3' ends. In order to detect even low levels of potential replication, we developed a quantitative reverse transcription-PCR method for detection of a smaller HBV mRNA species in the presence of the larger ones. All three highly viremic patients tested so far had mRNAs for the large and the small surface proteins and the X protein of the virus within PBMC but not in the virus from their sera. Furthermore, we detected by PCR covalently closed viral DNA in their PBMC. These data suggest that HBV may be not only taken up but also replicated by mononuclear blood cells and that these cells may be an extrahepatic site of viral persistence. X mRNA was detected in the largest amount. Possibly, X protein interferes with functions of the mononuclear cells during the immune response against the virus.

Adult↗
PubMed1997

HLA class II antibodies recruit G-CSF activated neutrophils for treatment of B cell malignancies.

Monoclonal antibodies offer the potential to improve specificity of oncological therapy. However, future success in the clinic depends on enhancing antibody effector functions. Here, we suggest that target antigen selection may influence recruitment of effector cells for antibody therapy, and may improve the outcome of antibody treatment in patients. Comparing a wide range of antibodies to different B cell antigens, we found most were able to mediate antibody dependent cellular cytotoxicity (ADCC) with blood mononuclear cells (MNC). In direct contrast, however, polymorphonuclear granulocytes (PMN) from the same donors showed ADCC only with HLA class II antibodies. Based on this observation, we propose a therapeutic strategy with a combination of HLA class antibodies and G-CSF, the latter being required to increase number and activational state of neutrophils. In particular, we suggest using bispecific antibodies (BsAb) in which one arm binds to HLA class II on tumor cells, and the second to FcgammaRI on activated effector cells. The clinical potential of this approach for the treatment of B cell malignancies looks most attractive.

Animals↗
PubMed1996

HLA class II as potential target antigen on malignant B cells for therapy with bispecific antibodies in combination with granulocyte colony-stimulating factor.

We have investigated the capacity of polymorphonuclear phagocytes (PMN) to lyse malignant B-cell lines using antibodies and antibody derivates to a range of different B-cell antigens. PMN were found to mediate lysis of all tested B-cell lines in the presence of HLA class II antibodies L227, L243, F3.3, and CR3/43. Target cell lysis was significantly enhanced when PMN isolated during granulocyte colony-stimulating factor (G-CSF) treatment were compared with PMN from healthy donors. Only G-CSF primed PMN, expressing Fc gamma RI (CD64), lysed B cells in the presence of monoclonal antibody (MoAb) 1D10 or Lym-1 to HLA class II related epitopes. Remarkably, PMN were consistently unable to kill malignant B cells with antibodies to the B-cell related antigens CD19, CD20, CD21, CD37, and CD38. These target antigen restriction was not observed with mononuclear effector cells, which mediated cytotoxicity with antibodies to HLA class II, but also with mouse/human chimeric constructs to CD19, CD37, and CD38. Blocking studies with Fc gamma RI antibodies and reverse antibody-dependent cellular cytotoxicity (ADCC) experiments against Fc gamma R antibody expressing hybridoma targets confirmed the pivotal role of Fc gamma RI in enhanced killing by G-CSF primed neutrophils. Bispecific antibodies (BsAb) with one specificity for Fc gamma RI, and another for a tumor associated antigen, offer an interesting approach to improve effector cell recruitment for immunotherapy. In our studies, very effective lysis was observed with G-CSF primed PMN and an [HLA class II x Fc gamma RI] BsAb. The therapeutic implications of these findings and the possible use of BsAb in combination with G-CSF are discussed.

Antibodies, Bispecific↗
PubMed1996

In vivo blockade of TNF-alpha by intravenous infusion of a chimeric monoclonal TNF-alpha antibody in patients with rheumatoid arthritis. Short term cellular and molecular effects.

Due to the unknown etiology of RA, specific treatment is not available. Recently, in a double-blinded, placebo-controlled clinical trial, in vivo blockade of TNF-alpha by a single infusion of a chimeric TNF-alpha-blocking mAb, cA2, has proven to be highly effective in the treatment of RA. In parallel to this trial, we tested the consequences of cA2 infusion in ex vivo and in vitro experiments. In this paper, we describe an increase in CD4+ and CD8+ T lymphocyte counts on day 1 and a marked decrease in monocyte counts preferentially on day 7 after cA2 treatment, without major changes in B lymphocyte or NK cell counts. In addition, we found an increased responsiveness of PBMC to CD28 mAb/PMA, but not to CD3 mAb, superantigen staphylococcus enterotoxin B, or PHA on day 1 after infusion. The increase in DNA synthesis of PBMC was paralleled by increased IL-2 mRNA and IL-4 mRNA expression and IL-2 protein secretion in culture supernatants after in vitro stimulation of PBMC with CD28 mAb/PMA. In PBMC, we did not find any significant changes in mRNA or protein expression of CD28 Ag or CD28 ligands, B7-1 and B7-2. Serum concentrations of IL-1 beta, IL-6, and soluble CD14 were significantly diminished after in vivo TNF-alpha blockade. We did not see relevant changes in granulocyte function in vitro after cA2 infusion. Finally, we observed a statistically significant decrease in slCAM-1 molecules in the serum of patients treated with verum compared with that in the serum of subjects given placebo. This change in slCAM-1 concentration was evident on days 1 and 7 after the infusion of 10 mg/kg cA2, whereas it occurred only on day 7 in the serum of patients treated with the low dose (1 mg/kg) of cA2.

Adult↗
PubMed1996

Bifunctional NHS-BAT ester for antibody conjugation and stable technetium-99m labeling: conjugation chemistry, immunoreactivity and kit formulation.

UNLABELLED: Conjugation chemistry and kit formulated binding of the NHS ester of 6-(4'-(4"-carboxyphenoxy)butyl)-2, 10-dimercapto-2,10-dimethyl-4,8-diazaundecane (NHS-BAT ester) to monoclonal antibodies (MAbs) was investigated. The functionalities of the resulting BAT conjugated and 99mTc-labeled MAbs BW 431/26, MAb 425 and bispecific MDX210 (fragment construct) were tested by immunoreactivity and immunoscintigraphy. METHODS: The kinetics and chemistry of the conjugation reaction were monitored by high-performance liquid chromatography, size-exclusion chromatography and positive fast-atom-bombardment mass spectra (FAB-MS). The 99mTc BAT-MAbs were tested with various immunoreactivity assays. The biodistribution of 99mTc-BAT-BW 431/26 in rats was compared with directly labeled BW 431/26. RESULTS: At pH 8.5 and 25 degrees C, the reactivity of the NHS-BAT ester was high with 90% completion after 30 min. The conjugation yield of 19 microM MAb and 228 microM NHS-BAT ester amounted to 30%. Higher NHS-BAT ester concentrations afforded higher BAT-to-MAb ratios. According to FAB-MS, the conjugation competing hydrolysis surprisingly occurred at the NHS ring. Almost quantitative 99mTc labeling was achieved after 5 min at 25 degrees C. Immunoreactivity of the 99mTc-BAT antibodies showed > 90% recovery and proved to be insensitive to BAT-to-MAb ratios of up to 10. The 99mTc-BAT-BW 431/26 showed similar organ distribution but revealed less urinary excretion compared with the directly labeled BW 431/26. Immunoscintigraphy with 99mTc-labeled and BAT-BW 431/26 and BAT-MAb 425 showed the respective biological function in vivo. CONCLUSION: According to straightforward conjugation chemistry, the ease of 99mTc labeling and the application of a simple ultrafiltration technique, the NHS-BAT ester represents a nondestructive, universally applicable biofunctional ligand to introduce stable 99mTc protein binding sites. Kit formulated conjugation/labeling can be performed with little time requirements and laboratory experience.

Animals↗
PubMed1996

Incidence and clinical outcome of children with BCR/ABL-positive acute lymphoblastic leukemia (ALL). A prospective RT-PCR study based on 673 patients enrolled in the German pediatric multicenter therapy trials ALL-BFM-90 and CoALL-05-92.

A variety of oncogenes are activated by specific chromosomal translocations, which are associated with distinct subtypes of leukemia. The identification of these rearrangements provides critical diagnostic and prognostic information, which may contribute to the selection of specific anti-leukemic therapy. The translocation t(9;22), the equivalent of the BCR/ABL rearrangement, is associated with a poor prognosis. We therefore used RT-PCR to detect this molecular event in a prospective study including 890 children. 673 of them suffered from acute lymphoblastic leukemia (ALL) at primary diagnosis and a transcription of the chimeric gene was detected in 21 of 648 with a successful analysis (3.2%). All children were treated by one of the two German multicenter childhood ALL therapy studies ALL-BFM-90 or COALL-05-92, respectively. Comparison of clinical features between BCR/ABL-positive and -negative children showed no significant differences regarding WBC, percentage of blasts, splenomegaly, hepatomegaly and age. Immunophenotypic studies at diagnosis in 21 BCR/ABL-positive children identified common ALL in 16 patients (76.2%), pre-B-ALL in four (19.0%), and an early T-lineage ALL in one (4.8%). Coexpression of myeloid antigens (CD13 and/or CD33) was observed in six of 16 common ALL patients as well as in the one child with early T-lineage ALL phenotype. The type of breakpoint (m-BCR/ABL: n = 14; M-BCR/ABL: n = 7) showed no correlation with clinical parameters. A comparison of cytogenetic and molecular data was performed in 16 positive patients and was concordant in all of them. We analyzed the response to the prednisone pretreatment and found a higher incidence of poor responders among the BCR/ABL-positive children. Regarding the event-free survival (EFS) of BCR/ABL-positive (0.53) and -negative patients (0.79) after a follow-up of 2 years, significant differences (P < 0.05) between both groups could be demonstrated.

Adolescent↗
PubMed1996

Single somatic ras gene point mutation in soft tissue malignant fibrous histiocytomas.

The frequency of ras gene mutations in human soft tissue malignant fibrous histiocytomas within and around the hot spot codons (12, 13, and 61) of all ras genes, (H-ras-1, K-ras-2, and N-ras) was studied by nested polymerase chain reaction and direct DNA sequencing from archival formalin-fixed, paraffin-embedded tissue. Light microscopy and immunohistochemistry served to define malignant fibrous histiocytoma. All of the four differentiation subtypes (storiform-pleomorphic, inflammatory, myxoid, and giant cell) were investigated. Nine of thirty-two malignant fibrous histiocytomas (28%) contained ras gene point mutations. The highest incidence was found in the myxoid subtype (four of nine). H-ras-1 gene codon 12.2 was the only codon affected and contained in all mutated cases a GGC-->GTC exchange. Seven of the nine mutations were homozygous and probably affected more than 80% of the tumor DNA. The flanking regions of all hotspot codons did not contain any point mutation. The presence of a single and often homozygous point mutation of the H-ras-1 gene, especially in myxoid malignant fibrous histiocytoma could serve as a basis for further genomic discrimination of myxoid sarcomas.

Aged↗
PubMed1995

Secondary acute myeloid leukemia with translocation (4;11) and MLL/AF4 rearrangement in a 15-year-old boy treated for common acute lymphoblastic leukemia 11 years earlier.

Secondary acute myeloid leukemia occurring in a 15-year-old boy 11 years after initial treatment of a common lymphoblastic leukemia (c-ALL) is described. Initial complete remission was terminated after 4 years by an isolated testicular relapse, followed by first bone marrow relapse within 18 months. After he achieved remission again, an allogeneic bone marrow transplantation from his HLA-identical brother was performed. Five years and 9 months later, the patient developed thrombocytopenia, leukopenia, and anemia, but bone marrow biopsies at this time demonstrated only myelofibrosis, with no blast cell population present. A polymerase chain reaction assay of a peripheral blood sample recognized the mRNA fusion region for the MLL/AF4 rearrangement, i.e., the molecular equivalent of the translocation (4;11)(q21,q23). Four weeks later, a blast cell population with AML-M1 morphology according to the FAB classification appeared in the bone marrow, and translocation (4;11) was detected by cytogenetics. Thus, secondary leukemias with chromosomal 11q23 rearrangement can develop after a long latency period and can be diagnosed earlier with the PCR technique.

Acute Disease↗
PubMed1995

Characterization of chromosome 8 abnormalities by fluorescence in situ hybridization in childhood B-acute lymphoblastic leukemia/non-Hodgkin lymphoma.

Using fluorescence in situ hybridization (FISH), we studied chromosome 8 abnormalities in 30 children with mature B-cell acute lymphoblastic leukemia (B-ALL) or B-cell non-Hodgkin lymphoma (B-NHL). FISH was performed on metaphase spreads and interphase nuclei with a whole chromosome 8 painting probe. Fifteen patients were studied retrospectively after metaphases from the malignant cell specimen had already been G-banded. When interphase nuclei were examined, FISH was able to confirm t(8;14)(q24;q32) in all nine patients positive by previous G-banding. FISH, however, was positive in metaphase spreads from only seven patients. Another 15 patients were included in a prospective study. In six of them (40%), a translocation involving chromosome 8 was shown by a split small segment (8q24-8qter) on interphase nuclei. Analysis of metaphase spreads showed only three positive cases each by FISH or G-banding, respectively, with corresponding results in two patients. By interphase FISH, trisomy of chromosome 8 also was detectable. In three patients shown by G-banding to have trisomy, interphase FISH study showed high scores of three chromosome 8 signal positive cells. There was no cross-hybridization to other chromosomes interfering with FISH analysis. FISH analysis on interphase nuclei using a whole chromosome 8 hybridization probe will supplement and can be more sensitive than metaphase cytogenetic techniques for detection of chromosome 8 rearrangements in B-ALL/NHL.

Adolescent↗
PubMed1995

G-CSF-stimulated PMN in immunotherapy of breast cancer with a bispecific antibody to Fc gamma RI and to HER-2/neu (MDX-210).

Myeloid cells can mediate tumor cell cytotoxicity via certain receptors for immunoglobulins. Among the different Fc receptors, the high-affinity IgG receptor (Fc gamma RI, CD64) is a promising trigger molecule because it is selectively expressed on effector cells, including monocytes/macrophages and granulocyte colony-stimulating factor (G-CSF)-primed neutrophils. In vitro, a bispecific antibody (BsAb) (MDX-210, constructed by chemically cross-linking F(ab') fragments of monoclonal antibody (mAb) 520C9 to HER-2/neu and F(ab') fragments of mAb 22 to Fc gamma RI) mediated effective lysis of HER-2/neu overexpressing breast cancer cell lines. HER-2/neu (c-erbB2) is overexpressed in approximately 30% of breast and ovarian carcinomas and is a target for immunotherapy in clinical trials. In vitro assays showed Fc gamma RI-positive neutrophils to constitute a major effector cell population during G-CSF therapy. Based on these preclinical data and a preceding study at Dartmouth (New Hampshire) with a single dose of MDX-210 alone, a combination of G-CSF and MDX-210 is tested in a phase I study in breast cancer patients. In this study, patients receiving G-CSF are treated with escalating single doses of MDX-210. This therapy was generally well tolerated by the treated patients, some of whom reacted with fever and short periods of chills, which were temporally related to elevated plasma levels of IL-6 and TNF-alpha. After MDX-210 application, a transient decrease in the total white blood count and absolute neutrophil count (ANC) was observed. During G-CSF application, isolated neutrophils were highly cytotoxic in the presence of MDX-210 in vitro. These data indicate a potential role for G-CSF and BsAb in immunotherapy.

Antibodies, Bispecific↗
PubMed1995

Construction of RNA standards for high-resolution automatic product analysis in quantitative competitive RT-PCR.

The exponential character of PCR amplification may compromise quantitative assays because it multiplies minor sample-to-sample variations. To overcome these problems, several authors have used recombinant standard DNA or RNA molecules to be spiked into the samples in a dilution series of known copy numbers before co-amplification by PCR. To obtain an equal efficacy of reverse transcription and PCR amplification, standard and template molecules should be highly homologous. However, the limited resolution of commonly used agarose gel electrophoresis requires rather large differences in size and nucleotide sequence to separate both molecules from each other after PCR. Due to a much higher resolution, automatic post-PCR analyzing systems based on laser-induced fluorescence may help to overcome these difficulties. For using the capabilities of these systems in quantitative competitive RT-PCR, we developed a protocol to construct recombinant RNA standard molecules that only differ from the target sequence by a small deletion of 8 nucleotides. It is based on PCR-induced mutagenesis and solid-phase in vitro transcription. This protocol was applied to quantify multidrug resistance gene (MDRI) mRNA in malignant cells, but it can easily be adapted to any gene of interest.

Base Sequence↗