Biomedical subjects

U Junker

Publications and source records attributed to U Junker.

At least 37 records · Page 2Linked to original sources

PubMed1998

[Lymphocyte transformation test (LTT) as a model for evaluating cytokine-induced immunomodulation in renal cell carcinoma].

We investigated the immunomodulatory capacity of cytokines produced by renal cell carcinoma in vitro by analyzing their effects on mitogen-induced T-lymphocyte blast cell transformation. All of the tested 70 cell cultures, derived from 70 tumor areas in 33 patients, had immunomodulatory capacity. In addition to suppression in the lymphocyte transformation test (max. 44/70; 63%) there was also superinduction (max. 37/70; 53%). We found no significant correlation with the stage and grade of primary tumors. However, the suppression of mitogen-induced T-lymphocyte blast cell transformation was significant in multifocal tumors (0.08% TCM, P < 0.001) and non-significant in metastatic tumors. The production of the assayed cytokines IL-6, IL-10, IL-11, and TGF beta 1 was variable and there was no significant correlation to the immunomodulatory capacity of the tumors.

Carcinoma, Renal Cell↗

PubMed1998

Comparative analyses of intracellularly expressed antisense RNAs as inhibitors of human immunodeficiency virus type 1 replication.

The antiviral activities of intracellularly expressed antisense RNAs complementary to the human immunodeficiency virus type 1 (HIV-1) pol, vif, and env genes and the 3' long terminal repeat (LTR) sequence were evaluated in this comparative study. Retroviral vectors expressing the antisense RNAs as part of the Moloney murine leukemia virus LTR promoter-directed retroviral transcript were constructed. The CD4+ T-cell line CEM-SS was transduced with retroviral constructs, and Northern blot analyses showed high steady-state antisense RNA expression levels. The most efficient inhibition of HIV-1 replication was observed with the env antisense RNA, followed by the pol complementary sequence, leading to 2- to 3-log10 reductions in p24 antigen production even at high inoculation doses (4 x 10(4) 50% tissue culture infective doses) of the HIV-1 strain HXB3. The strong antiviral effect correlated with a reduction of HIV-1 steady-state RNA levels, and with intracellular Tat protein production, suggesting that antisense transcripts act at an early step of HIV-1 replication. A lower steady-state antisense RNA level was detected in transduced primary CD4+ lymphocytes than in CEM-SS cells. Nevertheless, replication of the HIV-1 JR-CSF isolate was reduced with both the pol and env antisense RNA. Intracellularly expressed antisense sequences demonstrated more pronounced antiviral efficacy than the transdominant RevM10 protein, making these antisense RNAs a promising gene therapy strategy for HIV-1.

3T3 Cells↗

PubMed1998

In situ hybridisation and direct fluorescence antibodies for the detection of Chlamydia trachomatis in synovial tissue from patients with reactive arthritis.

BACKGROUND: Chlamydia trachomatis is associated with Reiter's syndrome and reactive arthritis but the form in which the organism survives in synovial cells is unclear. AIM: To compare in situ hybridisation with direct fluorescence in the detection of inapparent chlamydial infection in synovial tissue. METHODS: Synovial tissue from four patients with reactive arthritis patients was examined using biotin labelled probes for chlamydial DNA and fluorescein isothiocyanate (FITC) labelled monoclonal antibodies against the major outer membrane protein. RESULTS: In two of the four patients, evidence of chlamydial infections was detected by in situ hybridisation in parallel sections but not with FITC labelled monoclonal antibodies. CONCLUSIONS: Detection of chlamydial DNA by in situ DNA hybridisation may be a better way to identify chlamydial infection in synovial tissue than phenotype targeting with FITC conjugated antibodies, which is used as a standard procedure for screening clinical specimens for chlamydia.

Adult↗

PubMed1998

Increased transforming growth factor beta1 plasma level in patients with renal cell carcinoma: a tumor-specific marker?

PURPOSE: The most worrying problem with renal cell carcinoma (RCC) seems to be the prediction of metastases by means of tumor-specific markers. Therefore, much effort is committed to the development of new markers. MATERIALS AND METHODS: The level of latent transforming growth factor beta1 (TGF-beta1) was measured in plasma samples by ELISA. These samples were collected from patients with RCC before they underwent radical nephrectomy, from patients 1 h after extracorporeal lithotripsy, from patients with pyelonephritis, and from healthy controls. RESULTS: In all cases of RCC the levels of latent TGF-beta1 in plasma were much higher (n = 20, 41.0 +/- 13.9 ng/ml, range 19.3-78.1 ng/ml) than in healthy controls (n = 20, 3.8 +/- 2.9 ng/ml, range 0.6-9.9 ng/ml, p < 0.0001). The TGF-beta1 levels in plasma after extracorporeal lithotripsy (n = 20, 7.4 +/- 4. 64 ng/ml, range 2.9-21.7 ng/ml, p < 0.01) and in patients suffering from pyelonephritis (n = 20, 18.93 +/- 14.2 ng/ml, range 4.2-46.7 ng/ml, p < 0.001) were also higher than in healthy controls. CONCLUSION: We conclude that increased levels of latent TGF-beta1 are common in the plasma of RCC patients. The TGF-beta1 plasma level in RCC was found to be significantly higher than in cases of inflammation. Thus, TGF-beta1 is a possible tumor-prognostic marker in RCC.

Biomarkers, Tumor↗

PubMed1997

A detailed protocol for the measurement of TGF-beta1 in human blood samples.

Quantification of the multifunctional cytokine Transforming Growth Factor-beta1 (TGF-beta1) in blood samples has aroused increasing interest in recent years, since an abnormal regulation of this cytokine appears to play a key role in the pathogenesis of different diseases, such as autoimmundiseases or malignant tumors. The measurement of TGF-beta1 is complicated by a lot of problems concerning the collection, preparation and handling of blood samples, the platelet contamination, and the TGF-beta1 activation procedure. Here, we recommend detailed instructions for measurement of TGF-beta1 in blood plasma samples which should be followed to exclude the determination of false positive or negative results.

Humans↗

PubMed1997

Antiviral potency of drug-gene therapy combinations against human immunodeficiency virus type 1.

Gene therapy for the treatment of human immunodeficiency virus type 1 (HIV-1) infection using intracellular immunization strategies is currently being tested in clinical trials. With the continuing development of potent antiretroviral drugs (e.g., reverse transcriptase [RT] and protease [PR] inhibitors), it is likely that HIV-1 gene therapy will be applied to humans concurrently receiving such antiretroviral medication. In this study, we assessed the in vitro antiviral efficacy of two gene therapy strategies (trans-dominant RevM10, Gag antisense RNA) in combination with clinically relevant RT (AZT, ddC) or PR (indinavir) inhibitors. Retrovirally transduced, human T cell lines expressing antiviral gene constructs were inoculated with high doses of HIV-1HXB3 in the presence or absence of inhibitors. The combination of RevM10 or Gag antisense RNA with antiviral drugs inhibited HIV-1 replication 10-fold more effectively than the single antiviral drug regimen alone. More importantly, we also addressed whether gene therapy strategies are effective against drug-resistant HIV-1 isolates. Both the RevM10 and Gag antisense RNA strategies showed antiviral efficacy against several RT inhibitor-resistant HIV-1 isolates equivalent to their inhibition of HIV-1HXB3 replication. In summary, our data demonstrate the greater than additive antiviral efficacy of gene therapy strategies and RT or PR inhibitors, and that gene therapy approaches are effective against drug-resistant HIV-1 viral isolates.

Acquired Immunodeficiency Syndrome↗

PubMed1997

Hematopoietic potential and retroviral transduction of CD34+ Thy-1+ peripheral blood stem cells from asymptomatic human immunodeficiency virus type-1-infected individuals mobilized with granulocyte colony-stimulating factor.

The potential of hematopoietic stem cells (HSCs) from human immunodeficiency virus type-1 (HIV-1)-infected individuals, eg, self-renewal and multilineage differentiative capacity, might be perturbed due to the underlying disease. In this study, we assessed the HSC activity in the CD34+ Thy-1+ cell population of peripheral blood stem cells (PBSCs) of three asymptomatic HIV-1-infected individuals after granulocyte colony-stimulating factor (G-CSF; 10 microg/kg/d) mobilization. On day 4 of G-CSF treatment, 0.8% to 1% of the total blood mononuclear cells were CD34+. Leukapheresis followed by a two-step cell isolation process yielded a CD34+ Thy-1+ cell population of high purity (76% to 92% CD34+ Thy-1+ cells). This cell population showed no evidence of HIV-1-containing cells based on a semiquantitative HIV-1 DNA polymerase chain reaction. Furthermore, the purified cells showed normal hematopoietic potential in in vitro clonogenic assays. Successful gene transfer into committed progenitor cells (colony-forming units-cells) and more primitive stem/progenitor cells (long-term culture colony-forming cells) could be shown after amphotropic retroviral transduction. These data provide evidence that the CD34+ Thy-1+ stem cell compartment can be mobilized and enriched in early stage HIV-1-infected patients. Furthermore, successful transduction of this cell population as a prerequisite for stem cell-based clinical gene therapy protocols was demonstrated.

Adult↗

PubMed1997

Cytogenetic, histopathologic, and immunologic studies of multifocal renal cell carcinoma.

BACKGROUND: Multifocal tumor areas occurred in 12-22% of patients with renal cell carcinoma. It is unknown whether these tumors have malignant potential and characterize a higher risk for metastases. The performance of nephron-sparing surgery in patients with low grade or low stage tumors is controversial. METHODS: Primary and secondary tumors were analyzed by conventional cytogenetics and fluorescence in situ hybridization. Production of interleukin (IL)-6, IL-10, IL-11, and transforming growth factor (TGF)-beta1 were determined using standard enzyme-linked immunosorbent assay and bioassays. RESULTS: In 15.2% of the renal cell carcinoma cases evaluated, multifocal tumors were detected. Cytogenetics revealed a concordance of primary and secondary tumors in 9 of 14 cases (64%). In 11 of 12 multifocal tumors (94%), the same immunologic activity status was observed in both primary and secondary tumors. CONCLUSIONS: Secondary tumors must be expected to have malignant potential similar to that of the primary tumors. This was underscored by the high concordance of cytogenetic, histopathologic, and immunologic data in this study.

Carcinoma, Renal Cell↗

PubMed1997

Flow cytometric analysis of band 3 protein of human erythrocytes.

Flow cytometry was used to quantify the transmembrane anion exchanger (band 3 protein) of human erythrocytes by covalently bound eosin-5-maleimide. In vitro and in vivo vesiculated red blood cells were investigated. The fluorescence and light scatter signals of cells after heat induced vesiculation, in vivo ageing, and in patients with hereditary spherocytosis were decreased. These results reflect a deficiency of band 3 protein which is presumably caused by membrane surface area loss. It was possible to distinguish control erythrocytes, erythrocytes from patients with hereditary spherocytosis, and from other forms of haemolytic anaemias on the basis of their light scatter and fluorescence signals characteristics.

Anion Exchange Protein 1, Erythrocyte↗

PubMed1997

Macrophages from rat livers with micronodular and macronodular cirrhosis differ with respect to mediator release and DNA-synthesis.

BACKGROUND/AIMS: Liver macrophages play an essential role in necro-inflammatory liver damage which leads to fibrosis and cirrhosis. The aim of the present study was to compare the mediator release and the DNA synthesis of macrophages at an early and at a later stage of liver cirrhosis induced by thioacetamide. METHODS: Liver macrophages were isolated by an enzymic digestion method, followed by elutriation. The release of reactive oxygen species and cytokines, and the synthesis of DNA were measured in cultivated cells. RESULTS: The vitality of isolated macrophages from cirrhotic livers was always higher than 98%. The total yield of macrophages was less in micronodular cirrhotic livers and was markedly higher in macronodular cirrhotic livers when compared with age-matched controls. The cellular granules measured by sideward light scattering showed a shift to larger sizes in macrophages from micronodular cirrhotic livers when compared with the controls and the other experimental group. Macrophages from both cirrhosis groups exhibited a markedly higher unstimulated and lipopolysaccharide-stimulated IL-6 production than the controls. The release of TNF-alpha did not differ between controls and the experimental groups. Macrophages from macronodular cirrhotic livers produced higher amounts of nitric oxide but less superoxide anion radicals than the controls. DNA synthesis was 10-12-fold and 3-10-fold higher in macrophages from micronodular and macronodular cirrhotic livers, respectively, when compared with the age-matched controls. CONCLUSIONS: The data presented provide evidence that it is possible to isolate and to cultivate macrophages from livers with high yield and vitality at different stages of cirrhogenesis. Our results clearly demonstrate functional differences between macrophages from livers with micro- or macronodular cirrhosis; this finding may be important for the pathogenesis or perpetuation of the cirrhogenetic process.

Animals↗

PubMed1997

Renal cell carcinomas produce IL-6, IL-10, IL-11, and TGF-beta 1 in primary cultures and modulate T lymphocyte blast transformation.

We investigated the immunomodulatory capacity of primary cultures of renal cell carcinomas (RCC) by assessing production of cytokines and modulation of mitogen-induced T lymphocyte blast transformation. The results clearly show that immunomodulatory capacity is a common feature of RCC and that in vitro these tumors can produce interleukin-10 (IL-10) up to 20 ng/ml, IL-6 up to 35 micrograms/ml (> 250 kU/ml in the B9 system), IL-11 up to 15 micrograms/ml, and transforming growth factor-beta 1 (TGF-beta 1) up to 22 ng/ml. Furthermore, these tumors have the capacity to modulate T cell blast transformation over two orders of magnitude in each direction. The correlations of the immunologic properties of tumor cell cultures with the conventional classification of tumors (histology, cytology, staging, grading, presence of metastases, and secondary tumors) are analyzed. The significance of these findings for modulation of local immunity by RCC as well as for patient outcome is discussed.

Adjuvants, Immunologic↗

PubMed1997

Serum transforming growth factor-beta1 in patients with renal cell carcinoma.

PURPOSE: A major problem of renal cell carcinoma is the prediction of metastases via tumor prognostic markers. Therefore, much effort has been committed to the development of new prognostic markers. MATERIALS AND METHODS: The level of transforming growth factor-beta1 was measured by enzyme-linked immunosorbent assay in serum samples collected from patients with renal cell carcinoma before radical nephrectomy. RESULTS: In serum samples from 21 patients with renal cell carcinoma and 21 healthy controls mean transforming growth factor-beta1 levels were 177 +/- 54.1 versus 65.6 +/- 15.8 ng./ml., respectively. This difference was statistically significant (Mann-Whitney U test p <0.001). CONCLUSIONS: Increased levels of transforming growth factor-beta1 are common in serum of patients with renal cell carcinoma.

Carcinoma, Renal Cell↗

PubMed1996

Intracellular expression of cellular eIF-5A mutants inhibits HIV-1 replication in human T cells: a feasibility study.

Previously, we described two mutants of the cellular Rev co-factor, eukaryotic initiation factor 5A (eIF-5A M13 and M14), which suppress human immunodeficiency virus type 1 (HIV-1) SF2 replication in clonal T cell lines. This study introduced the notion that it is possible to develop gene therapies against infectious agents on the basis of mutant host factors required for viral replication. In this report, we provide further evidence to support this new paradigm and describe murine leukemia virus (MLV)-based retroviral vectors expressing three different eIF-5A mutants from the viral long terminal repeat (LTR). HIV-1 replication (SF2, HXB-3) was reduced up to 2 orders of magnitude in transduced, polyclonal T cell populations. All eIF-5A mutants also showed antiviral activity (approximately seven-fold reduction) in a chronic HIV-1 infection model. Expression of eIF-5A mutant M13 delta in peripheral blood lymphocytes (PBLs) showed no difference in proliferation and metabolic activity as determined in a 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT)-assay, suggesting that expression of this type of mutant protein is not associated with cellular toxicity. In summary, these data suggest that gene therapy for HIV-1 infection can be developed on the basis of mutants of the Rev co-factor eIF-5A.

Blotting, Northern↗

PubMed1996

Retroviral vectors designed for targeted expression of RNA polymerase III-driven transcripts: a comparative study.

Retroviral gene delivery systems for RNA polymerase II (RNA pol II)-based promoters have been developed and are widely used in gene transfer studies. In contrast, gene delivery systems with RNA pol III-based expression cassettes have not been studied comprehensively, although therapeutic applications (e.g., ribozymes, antisense, triplex RNA and RNA decoys) have been proposed. In this report, we describe retroviral vectors designed to optimize expression of short chimeric RNAs transcribed from a number of RNA pol III promoters. Our results show that all analysed RNA pol III expression cassettes (tRNA, U6, Ad VA1), regardless of orientation, do not transcribe efficiently when located between the retroviral long terminal repeats (LTRs). In contrast, high steady-state expression levels can be achieved by inserting the RNA pol III expression cassette into the U3 region of the LTR (double-copy design). Compared to human tRNA gene promoters (tRNA(Met), tRNA(Val)), the human small nuclear RNA U6 gene (U6) and the adenovirus virus-associated RNA 1 (Ad VA1) gene promoters yielded higher expression levels. The majority of the chimeric U6-derived transcripts were detected in the nuclear RNA fraction, and the VA1 and tRNA-driven transcripts were predominantly detected in the cytoplasmic compartments. This report is the first comparative study of RNA pol III-driven promoters expressing short chimeric transcripts leading to an optimized retroviral-vector design.

Adenoviridae↗

PubMed1996

Transforming growth factor beta 1 is significantly elevated in plasma of patients suffering from renal cell carcinoma.

The levels of active and latent transforming growth factor beta 1 (aTGF-beta 1, ITGF-beta 1, respectively) in plasma samples were measured by enzyme-linked immunoabsorbent assay (ELISA). Samples were collected from patients suffering from renal cell carcinoma (RCC) before they underwent tumour resection. In all cases tested, the levels of latent TGF-beta 1 were much higher (n = 23, 41.0 +/- 13.9, range 19.3-78.1 ng/ml) than in healthy controls (n = 21, 3.8 +/- 2.9, range 0.6-9.9, P < < 0.0001), while active TGF-beta 1 did not differ that impressively (n = 38, 1.2 +/- 1.3, range 0.0-4.5 for RCC, n = 21, 0.1 +/- 0.2, range 0.0-1.1 in controls, P < 0.001). As for TGF-beta 1 production by proximal tubulus cells has been shown, it was speculated that these high TGF-beta 1 levels might be due to secretion by the tumour, which usually originates from proximal tubuli. Indeed, production of TGF-beta 1 was found in culture supernatants, and it was possible to show TGF-beta 1 mRNA expression in tumour samples. This TGF-beta 1 was predominantly secreted in the latent form. This is in contrast to data from other authors, who found TGF-beta 1 to be secreted mainly in the active form, but need not mean that it is inactive locally as the low pH encountered within tumours is in the range necessary for its activation. It is concluded that elevated latent TGF-beta 1 is common in RCC, is at least partially produced by the tumour and might be a cause for local immunosuppressive effects within the tumour.

Carcinoma, Renal Cell↗

PubMed1996

Differential expression of cytokine genes in CD27-positive and -negative CD4 lymphocyte subsets from healthy humans and rheumatoid arthritis patients.

Immunofluorescence analysis of CD27 expression by CD4 lymphocytes from the peripheral blood of healthy humans or rheumatoid arthritis (RA) patients and from the synovial fluid (SF) of RA patients was carried out, along with the estimation of cytokine gene [interleukin (IL) 2, IL-3, IL-4, IL-5, IL-6, IL-6R, IL-10 and interferon-gamma (IFN-gamma)] expression in these lymphocyte subsets by RT-PCR. Although no differences in CD27-positive and -negative peripheral blood CD4 cell subset distribution were revealed, marked differences in IL-3, IL-4, IL-5 and IFN-gamma mRNA expression were detected between these lymphocyte subsets and between control and disease states. These results showed that phenotyping of different cell subsets in disease cannot provide adequate information about lymphocyte functional status. To estimate differences in cytokine gene expression, CD4 lymphocytes from the peripheral blood and SF of RA patients were compared. In both cases, mRNAs for IL-2, IL-4, IL-10 and IFN-gamma were detected, but CD4 cells from SF failed to express detectable levels of IL-5 mRNA despite our findings of a CD27-cell accumulation within the synovial population of CD4 lymphocytes. These are the first data to demonstrate that expression of the IL-5 gene in RA SF CD27- lymphocytes is down-regulated and that IL-5 disregulation in RA cannot be ruled out.

Arthritis, Rheumatoid↗

PubMed1996

Intracellular expression of human immunodeficiency virus type 1 (HIV-1) protease variants inhibits replication of wild-type and protease inhibitor-resistant HIV-1 strains in human T-cell lines.

The enzymatic activity of the human immunodeficiency type 1 (HIV-1) protease (PR) is crucial to render HIV-1 virions mature and infectious. Hence, genetic intervention strategies based on trans-dominant (td) variants of the HIV-1 PR might be an alternative to current pharmacological and gene therapy regimens for AIDS. CD4-positive human CEM-SS T-cell lines were generated which constitutively expressed HIV-1 td PR variants. HIV-1 infection experiments demonstrated severely reduced HIV-1 replication in these td PR CEM-SS cell lines compared with control T cells expressing wild-type PR. Furthermore, replication of an HIV-1 isolate bearing a PR inhibitor-resistant PR was blocked, showing that genetic intervention strategies based on td PRs can be effective against HIV-1 isolates containing PR inhibitor-resistant mutants.

CD4-Positive T-Lymphocytes↗

PubMed1995

Functional role of CD26 on human B lymphocytes.

CD26 is a well-known activation marker on T cells and natural killer (NK) cells [1]. It is identical with the ectopeptidase dipeptidyl peptidase IV (DP IV). The expression of CD26 on B cells has been discussed controversially [2,3]. We have studied the expression of this enzyme on B cells from the peripheral blood of healthy donors and of CVID patients, on cells of the Daudi Burkitt line and the EBV-transformed B-cell lines Jojo and Laz509. DP IV was detected by using anti-CD26 monoclonal antibodies and with help of specific enzyme substrates. Further the influence of specific synthetic DP IV inhibitors on mitogenic activation of purified B cells and DNA synthesis of cell lines was studied. We could show that in both groups 0-5% of freshly isolated CD20-positive B cells do express the CD26 antigen. After stimulation with pokeweed mitogen or St. aureus protein, the fraction of CD26-positive cells was enhanced up to 51% and 36%, respectively. Interestingly, induction of CD26 expression on B cells from CVID patients occurs in a manner similar to the B cells from healthy donors. Treatment of peripheral blood B cells and B-cell lines with highly specific competitive DP IV inhibitors leads to a significant inhibition of DNA synthesis in a dose-dependent manner. These data show that CD26 can be considered to be an activation marker not only of T- and NK cells but also of a main population of B cells, suggesting an involvement of CD26 in B-cell activation.

B-Lymphocytes↗