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Biomedical subjects

E Otto

Publications and source records attributed to E Otto.

At least 37 records · Page 2Linked to original sources
PubMed1999

Efficacy of antiretroviral agents against murine replication-competent retrovirus infection in human cells.

Retroviral vectors for gene therapy are designed to minimize the occurrence of replication-competent retrovirus (RCR); nonetheless, it is possible that a vector-derived RCR could establish an infection in a patient. Since the efficacy of antiretroviral agents can be impacted by interactions between virus, host cell, and drug, five commonly used antiretroviral drugs were evaluated for their abilities to inhibit the replication of a murine leukemia virus (MLV)-derived RCR in human cells. The results obtained indicate that the combination of nucleoside analogs zidovudine and dideoxyinosine with the protease inhibitor indinavir effectively inhibits MLV-derived RCR replication in three human cell lines. In addition, MLV-derived RCR was found to be inherently resistant to the nucleoside analogs lamivudine and stavudine, suggesting that mutations conferring resistance to nucleoside analogs in human immunodeficiency virus type 1 have the same effect even in an alternative viral backbone.

Cell Line↗
PubMed1998

Biosafety monitoring of patients receiving intracerebral injections of murine retroviral vector producer cells.

Patients with recurrent malignant brain cancer, who were receiving gene therapy by intracerebral injection of murine retroviral vector producer cells (VPCs), were monitored for the presence of replication-competent retrovirus (RCR). RCR sequences were not detected by polymerase chain reaction (PCR) in any of the 608 peripheral blood leukocyte (PBL) samples analyzed. Vector DNA sequences were detected transiently in PBL samples from a subset of 34 patients. Humoral immune responses to a retroviral core protein p30 and murine VPC were detected in some patients, most frequently in patients receiving repeated administrations of VPC. RCR was not detected in biological assays of PBLs from 41 patients who had either anti-retroviral antibodies in sera and/or vector DNA in PBLs. Our data suggest that in situ generation of RCR was not detected following intracerebral inoculation of VPCs in any of the 128 patients evaluated.

Animals↗
PubMed1998

Analysis of orbital T cells in thyroid-associated ophthalmopathy.

Thyroid-associated ophthalmopathy (TAO) has a major effect on the two compartments of the retro-orbital (RO) space, leading to enlargement of the extraocular muscles and other RO tissues. T lymphocyte infiltration of RO tissue is a characteristic feature of TAO and there is current interest in whether these T cells are specifically and selectively reactive to RO tissue itself. We recently established 18 T cell lines (TCL) from RO adipose/connective tissue of six patients with severe TAO by using IL-2, anti-CD3 antibodies and irradiated autologous peripheral blood mononuclear cells (PBMC) to maintain the growth of T cells reactive to autologous RO tissue protein fractions. Here we report on the phenotype characteristics and cytokine gene expression profiles of these orbital TCL and on their immunoreactivity to the organ-specific thyroid antigens thyrotropin receptor (TSH-R), thyroidal peroxidase (TPO) and thyroglobulin (TG). Flow cytometry revealed that 10 TCL were predominantly of CD4+ phenotype, three being mostly CD8+ and five neither CD4+ nor CD8+. Analysis with reverse transcriptase-polymerase chain reaction (RT-PCR) of cytokine gene expression revealed both Th1- and Th2-like products in all TCL: IL-2 product (in 17 TCL), interferon-gamma (IFN-gamma) (n = 10), tumour necrosis factor-beta (TNF-beta) (n = 15), IL-4 (n = 12), IL-5 (n = 17), IL-6 (n = 13), TNF-alpha (n = 12) and IL-10 (n = 4). Reactivity to thyroid antigens was observed only in two TCL, the other 16 being uniformly unreactive. Although 10 out of 18 RO tissue-reactive TCL were predominantly CD4+ there were no significant relationships between TCL phenotype, cytokine gene profile, magnitude of reactivity to RO tissue protein or the (rare) occurrence of thyroid reactivity. The findings of both Th1- and Th2-like cytokine gene expression in all RO tissue-reactive TCL support the concept that TAO is a tissue-specific autoimmune disease, distinct immunologically from the thyroid, and involving both T cell and B cell autoimmune mechanisms in disease pathogenesis.

Adult↗
PubMed1997

A novel gene encoding an SH3 domain protein is mutated in nephronophthisis type 1.

Juvenile nephronophthisis (NPH), an autosomal recessive cystic kidney disease, is the primary genetic cause of chronic renal failure in children. About two thirds of patients with NPH carry a large homozygous deletion at the gene locus NPH1 on 2q13. We here identify a novel gene. NPHP1, which extends over most of this common deletion. The 4.5-kb transcript encodes a protein with an SH3 domain, which is highly conserved throughout evolution. The 11-kb interval between the 3' end of NPHP1 and an inverted repeat containing the distal deletion breakpoint was found to contain the first exon of a second gene, MALL. In patients with a hemizygous deletion of the NPH1 region, additional point mutations were found in NPHP1 but not in MALL.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Therapy of malignant brain tumors by intratumoral implantation of retroviral vector-producing cells.

Intratumoral implantation of murine cells modified to produce retroviral vectors containing the herpes simplex virus-thymidine kinase (HSV-TK) gene induces regression of experimental brain tumors in rodents after ganciclovir (GCV) administration. We evaluated this approach in 15 patients with progressive growth of recurrent malignant brain tumors. Antitumor activity was detected in five of the smaller tumors (1.4 +/- 0.5 ml). In situ hybridization for HSV-TK demonstrated survival of vector-producing cells (VPCs) at 7 days but indicated limited gene transfer to tumors, suggesting that indirect, "bystander," mechanisms provide local antitumor activity in human tumors. However, the response of only very small tumors in which a high density of vector-producing cells had been placed suggests that techniques to improve delivery and distribution of the therapeutic gene will need to be developed if clinical utility is to be achieved with this approach.

Adult↗
PubMed1997

HPLC glycosaminoglycan analysis in patients with Graves' disease.

1. Orbital accumulation of hydrophilic, interstitial glycosaminoglycans (GAGs) and subsequent expansion of retrobulbar tissue lead to the clinical manifestation of exophthalmos in patients with Graves' eye disease. 2. A highly specific method to determine the concentration and biochemical composition of different GAGs was developed in order to obtain a sensitive test system for the activity of the disease. By means of this method, GAG excretion in 24 h urine collections of 56 patients and 21 controls was analysed by precipitation with cetylpyridinium chloride and potassium acetate in ethanol, followed by sequential enzymic hydrolysis with chondroitin AC lyase, chondroitin ABC lyase and hyaluronate lyase, with HPLC analysis of the resulting alpha, beta-unsaturated disaccharides by anion-exchange chromatography. 3. Concentrations of GAG, chondroitin sulphate A (CA), dermatan sulphate (DS) and hyaluronic acid (HA) were determined in patients and controls, with high recovery rates [72.2 +/- 5.3%, mean +/- SEM; detection limit, 4.2 micrograms/l (0.01 mumol/l)], revealing marked differences in urinary concentrations of total GAG and HA, as well as an elevation of CA in patients compared with controls. 4. Method sensitivity was 0.86 for patients with active Graves' eye disease, and 0.87 for patients with untreated ophthalmopathy, whereas specificity was 1.0 for patients with inactive disease. Patients with increased GAG concentration responded well to steroids and/or orbital irradiation (before therapy: GAG, 111.49 +/- 40.32; CA, 59.58 +/- 21.34; DS, 25.05 +/- 8.12; HA, 26.88 +/- 11.63 mg/24 h; during therapy: GAG, 54.22 +/- 10.94; CA, 20.52 +/- 4.58; DS, 17.65 +/- 3.46; HA, 16.05 +/- 3.69 mg/24 h), whereas GAG excretion increased markedly 2-3 months after stopping prednisone therapy in patients with still active eye disease (GAG, 109.9 +/- 10.51; CA, 63.8 +/- 7.34; DS, 24.1 +/- 5.07; HA, 22.0 +/- 6.28 mg/24 h). 5. This sensitive method determines the nature of renally excreted GAGs, reflecting the aberrant synthesis pattern of fibroblasts in patients with Graves' disease.

Acute Disease↗
PubMed1997

Diabetic microangiopathy and urinary glycosaminoglycans.

Alterations in the metabolism of glycosaminoglycans (GAG) may play a role in the pathogenesis of diabetic-associated microangiopathy. Consequently, the relationship between diabetic nephropathy and retinopathy and urinary GAG distribution was assessed in 96 IDDM patients in comparison to 103 healthy controls. GAG concentration in 24h urine samples was determined by precipitation with cetylpyridinium chloride and potassium acetate in ethanol followed by a colorimetric test with carbazole. A marked difference (P = 0.0008) in urinary GAG excretion between patients (24.3 +/- 1.5 mg/24 h, mean +/- SEM) and controls (16.2 +/- 0.75 mg/24 h) could be detected. In patients with IDDM of longer duration, GAG excretion was increased (< or = 10 yr: 20.8 +/- 2.1 vs > 10 yr: 27.4 +/- 2.1 mg/24 h; P = 0.03). Furthermore, IDDM patients with class 4 nephropathy and retinopathy exhibited a markedly higher GAG excretion compared to those without nephropathy (33.1 +/- 3.0 vs 22.6 +/- 1.7 mg/24 h, P = 0.005) or retinopathy (29.7 +/- 2.8 vs 21.2 +/- 1.7 mg/24 h, P = 0.009). An increased urinary GAG concentration was detected in IDDM patients with albuminuria (> 300 mg/24 h: 29.9 +/- 3.3 vs < 30 mg/24 h: 23.0 +/- 1.7 mg/24 h; P = 0.048), proteinuria (> 0.5 g/24 h: 30.3 +/- 3.7 vs < 0.05 g/24 h: 22.7 +/- 1.6 mg/24 h) and in patients with augmented serum creatinine in comparison to those with normal values (> 0.12 mg/L: 34.9 +/- 2.3 vs < 0.12 mg/L: 22.4 +/- 1.6 mg/24 h; P = 0.01). The results demonstrate a close relationship renal GAG excretion and the presence of microangiopathy in IDDM patients.

Adolescent↗
PubMed1996

T cells and orbital connective tissue in endocrine orbitopathy.

In order to analyse the immunological changes in patients with endocrine orbitopathy (EO) the antigenic character of orbital connective tissue was studied. Counter-stimulation assays of patients' lymphocytes with autologous retrobulbar fibroblasts resulted in a markedly increased lymphocyte proliferation in comparison to incubation with retrobulbar control fibroblasts. Proliferation tests of retrobulbar T cell lines showed significant responses to autologous retro-orbital connective tissue proteins, with molecular weights of 6-10 kD and 19-26 kD. Phenotypic analysis of orbital T cell lines indicates that they consisted predominantly of CD4+ cells. Hyaluronic acid production of orbital fibro blasts following co-cultivation with lymphocytes of EO patients or controls revealed a threefold increased synthesis in patients with EO. Furthermore, distribution pattern of orbital extracellular matrix glycosaminoglycans (GAG) differs in EO patients in comparison to controls. The results suggest the presence of autoreactive T cells directed against antigens of orbital fibroblasts, whose stimulation results in an augmented GAG synthesis in patients with EO.

Connective Tissue↗
PubMed1995

Autoimmune endocrine ophthalmopathy and retrobulbar antigens.

Endocrine Ophthalmopathy (EO) is based on autoimmune processes that lead to lymphocyte infiltration of the retrobulbar space. In this study, antigenic character of retrobulbar adipose, connective and muscle tissue as well as of cultured fibroblasts and myoblasts were examined. Samples were obtained from EO patients (n = 13, 8 fem., age 26-82 years, median 47 years) undergoing orbital decompression surgery. Retrobulbar and abdominal tissue from 7 controls (4 fem., 48 - 74 y) was investigated, too. Tissues were homogenized and the proteins were separated by SDS-PAGE according to molecular weight. In order to recover the separated proteins in soluble form, an electroelution technique was employed. Twenty-two separated soluble protein fractions were used as antigenic stimuli for autologous peripheral blood mononuclear cells (PBMC) separated by Ficoll gradient centrifugation. Subsequently, the proliferation of T cells was measured by [3H]-thymidine uptake. A marked T cell response to protein fractions with molecular weight of 6 - 10 kD and 19 - 26 kD was detected (p < 0.001). These autoantigens were found readily reproducible in adipose tissue in 8 out of 9 EO patients, stimulation index (SI) to antigen 6 - 10 kD 29 +/- 4.6 (mean +/- SEM); 19 - 26 kD 5 +/- 1.4 and in 3 out of 4 patients using retrobulbar eye muscle tissue (SI: 6 - 10 kD 23 +/- 4.2; 19 - 26 kD 6 +/- 2). Using the proteins of cultured fibroblasts as antigen, the autologous PBMC from 2 out of 4 tested EO-patients also responded (SI: 7 +/- 2; 4 +/- 1.4). Testing cultured retrobulbar myoblasts of an EO patient, a response to the 19 - 26 kD antigen was found only (SI: 8.0). In response to retrobulbar or muscle proteins, PBMC of 2 controls showed also a higher proliferation rate (SI: 16 +/- 3.5; 13 +/- 2.8), whereas, a response to abdominal adipose or muscle proteins (4 controls) was never found. Thus, two orbital antigens reacting with autologous T cells could be demonstrated and may play an important role in the immunopathogenesis of EO. According to these findings, retrobulbar fibroblast antigens are most likely the main T cell targets.

Adipose Tissue↗
PubMed1995

Transduction of rIL-2 expanded CD4+ and CD8+ ovarian TIL-derived T cell lines with the G1Na (neor) replication-deficient retroviral vector.

We have expanded ovarian tumor-infiltrating lymphocytes (TIL) in low concentrations of recombinant interleukin-2 (rIL-2) to conduct intraperitoneal adoptive immunotherapy trials in patients with ovarian cancer. We have previously demonstrated that certain T cell lines and clones derived from ovarian TIL exhibit in vitro autologous tumor-specific cytotoxicity and/or cytokine production (interferon-gamma, tumor necrosis factor-alpha) preferentially in response to autologous tumor cells. Studies that utilize a marker gene introduced into the DNA of TIL can provide useful information on specific uptake or localization of TIL at tumor sites and on the survival of TIL in vivo. We have conducted a series of preclinical experiments in which we have successfully transfected TIL with G1Na, which encodes the gene for neomycin phosphotransferase (neoR). NeoR was detected in at least 10% of CD8+ cells (mean = 10.4%) and between 2.5 and 20% of CD4+ TIL (mean = 8.5%). Transduction of ovarian TIL with G1Na caused no substantial changes to the T cell phenotypes or in vitro cytotoxicities against ovarian and hematogenous tumor cell targets, or on the rIL-2 requirements of TIL for growth and proliferation. In addition, the intact G1Na provirus in transduced TIL cells was rescuable by replication-competent retrovirus and was transferred into the genome of NIH-3T3 fibroblasts, which were rendered resistant to G418. An enhanced polymerase chain reaction (PCR) procedure utilizing detection by ethidium bromide staining was developed. The enhanced PCR detected 1 in 100,000 neoR-labeled cells. Furthermore, detection of the G1Na genome in transduced TIL by in situ hybridization with an RNA probe provided evidence for expression of the neoR gene in transduced TIL. Results obtained from these studies suggest that ovarian TIL-derived T cell lines transduced with the neoR gene post infection with the G1Na retroviral vector can be utilized to examine the in vivo trafficking pattern of ovarian TIL-derived T cell lines expanded in low concentrations of rIL-2 and their survival.

3T3 Cells↗
PubMed1995

An improved retroviral vector encoding the herpes simplex virus thymidine kinase gene increases antitumor efficacy in vivo.

Brain tumors have been treated clinically by intratumoral injection of cells that produce retroviral vectors encoding the herpes simplex virus thymidine kinase (HSV-TK) gene followed by systemic administration of the antiviral drug ganciclovir. In vitro and in vivo comparisons of two different HSV-TK vector producer clones, which were made using standard transfection and transinfection techniques, were conducted. The two clones, PA317/G1TkSvNa.53 (TK.53) and PA317/G1Tk1SvNa.7 (TK1.7), both used in clinical trials, differ with respect to sequences 3' to the HSV-TK stop codon. The retroviral construct used to generate the TK.53 vector producer cell clone contains an open reading frame encoding a portion of the herpes simplex virus glycoprotein H (gH), a potential polyadenylation site and a putative splice site in this region. These sequences were removed from the retroviral construct used to create the TK1.7 vector producer cell clone. Supernatants obtained from TK1.7 vector producer cells had 100- to 1000-fold higher titers (G418 or HAT) than did corresponding supernatants from TK.53 vector producer cells. A murine subcutaneous tumor model was used to assess transduction efficiency and antitumor activity of each vector producer cell clone. In vivo tumor cell transduction was 13- to 18-fold more efficient with TK1.7 cells as compared with TK.53 cells at equivalent doses. Complete tumor ablation was achieved using a 10-fold lower dose of TK1.7 cells as compared with TK.53 cells. These results suggest that TK1.7 cells combined with ganciclovir may provide a more potent antitumor response in humans.

Animals↗
PubMed1994

Improved methods of retroviral vector transduction and production for gene therapy.

To facilitate clinical applications of retroviral-mediated human gene transfer, retroviral vectors must be of high titer and free of detectable replication-competent retroviruses. The purpose of this study was to optimize methods of retroviral vector production and transduction. Studies were conducted using 22 retroviral vector producer cell lines. Inactivation of retroviral vectors was greater at 37 degrees C than at 32 degrees C. A 5- to 15-fold increase of vectors was produced at 32 degrees C compared to 37 degrees C; the vector increase at 34 degrees C was intermediate. For example, PA317/G1Na.40 grew to a titer of 1.8 x 10(7) cfu/ml at 32 degrees C, compared to 5.0 x 10(5) cfu/ml at 37 degrees C. The production of retroviral vectors was scalable achieving similar results in flasks, roller bottles, or a CellCube Bioreactor. Retroviral vectors were concentrated 15-24 times with vector recovery ranging from 91 to 96% in a Pellicon tangential flow filtration system. Retroviral supernatants were successfully lyophilized. The combination of glucose or sorbitol with gelatin resulted in recovery rates of 64-83%. In studies on transduction by retroviral vectors, centrifugation of vector supernatants onto target cells significantly increased transduction efficiency as measured by vector titration for G418 resistance, fluorescence-activated cell sorting (FACS), and polymerase chain reaction (PCR) analyses. The combination of the above methods has significantly increased the growth and transduction by this vector system.

3T3 Cells↗
PubMed1994

Characterization of a replication-competent retrovirus resulting from recombination of packaging and vector sequences.

A replication-competent retrovirus (RCR) was detected by S+/L- assays in three lots of retroviral vector G1Na that were harvested on consecutive days from a single culture of PA317/G1Na producer cells. Using a number of retrovirus-specific primer pairs, it was shown that this RCR was a novel recombinant created by exchanges between G1Na and helper sequence pPAM3 and was not an existing RCR introduced by cross-contamination. Sequencing of clones of DNA amplified in six independent PCR reactions confirmed that the 3' portion of this RCR was composed of retroviral envelope sequences unique to pPAM3 joined to a 3' long terminal repeat (LTR) unique to G1Na. Comparison of pPAM3 and G1Na sequences at the site corresponding to this junction revealed a short segment of patchy nucleotide identity (8 out of 10 bp), suggesting that these helper and vector sequences were joined by homologous recombination. Generation of RCR by exchanges between helper and vector sequences underscores the necessity of testing by efficient methods all retroviral vectors for the presence of RCR before their use. Production of 171 lots (855 liters) of various retroviral vectors that were free of RCR, including 42 lots of G1Na, however, indicates that the combination of exchanges required to generate an RCR are infrequent in this system.

Animals↗
PubMed1994

Cellular immunity and retrobulbar fibroblasts in Graves' ophthalmopathy.

In Graves' ophthalmopathy (GO), retrobulbar connective tissue is infiltrated by T cells whose role in the pathogenesis of the disease was investigated in the present work. The aims included firstly to characterize subsets of blood lymphocytes and of sessile lymphocytes cloned from a retroorbital tissue specimen. Second, in counterstimulation assays, the ability of patients' T cells to influence cultivated retrobulbar fibroblasts and in turn the enhancement of lymphocyte proliferation by retrobulbar fibroblasts was investigated. Blood lymphocytes of 16 GO patients and 12 controls isolated by density gradient centrifugation and retrobulbar fibroblasts obtained from orbital decompression were alternately exposed to irradiation of 4000 rad (to suppress proliferation of either cell type), then cocultivated for 48 h. Subsequently, the cells (250,000 lymphocytes, 5000 retrobulbar fibroblasts) were incubated with [3H]thymidine for 24 h. A stimulation index representing the degree of proliferation in comparison with a control was determined. Screening of 62 retrobulbar lymphocyte clones by cell-ELISA revealed a CD4/CD8 ratio of 8.2, contrasting with a normal ratio of 2.1 in peripheral lymphocytes (as obtained by FACS analysis). Incubation of patient's lymphocytes with autologous retrobulbar fibroblasts resulted in a markedly elevated stimulation index (9.7) compared to incubation of lymphocytes with retrobulbar fibroblasts from controls (2.5). In another assay, the degree of stimulation amounted to 2.2 vs. -0.8. Retrobulbar fibroblasts were stimulated twice as much by lymphocytes of the same patient compared to retrobulbar fibroblasts from controls. The autologous reactions demonstrated suggest the presence of autoreactive T lymphocytes in GO patients directed against retrobulbar fibroblasts, which, in addition, are stimulated by these lymphocytes.

Adult↗
PubMed1993

Glycosaminoglycan antibodies in endocrine ophthalmopathy.

An increased accumulation of glycosaminoglycans (GAG) in the orbita has been reported in endocrine ophthalmopathy (EO). In this study we investigated whether antibodies directed against GAG are present in the sera of 52 EO patients and 47 healthy controls. Three out of 52 patients exhibited low titers of antinuclear antibodies and all patients were negative for antibodies against extractable nuclear antigens. Isotype IgG antibodies were detected by means of an ELISA using hyaluronic acid and dermatan sulfate as antigens. Values were expressed as optical density at 405 nm. In comparison to the control group (0.445, 0.364, 0.588; median, 25th, 75th percentile) significantly (p < 0.001) higher hyaluronic acid antibody levels were found in EO patients (0.626, 0.473, 0.801). Untreated patients had significantly higher hyaluronic acid antibody values compared to controls (0.770 vs 0.437, median, p < 0.005). Thyroid status or antithyroid treatment did not influence binding of hyaluronic acid antibodies. When screening for dermatan sulfate antibodies no such difference could be observed between patients and controls. Thus, the detection of GAG antibodies in EO patients emphasizes the important role of glycosaminoglycans in the pathogenesis of thyroid eye disease.

Adolescent↗