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Biomedical subjects

M Heuer

Publications and source records attributed to M Heuer.

12 recordsLinked to original sources
PubMed1999

Generation of large numbers of fully mature and stable dendritic cells from leukapheresis products for clinical application.

Dendritic Cell (DC)-based vaccination approaches in man require a reproducible DC generation method that can be performed in conformity with GMP (Good Manufacturing Practice) guidelines and that circumvents the need for multiple blood drawings to generate DC. To this end we modified our previously described method to generate mature DC from CD14 + monocytes by a two step method (priming in GM-SF + IL-4 followed by maturation in monocyte conditioned medium) for use with leukapheresis products as a starting population. Several adaptations were necessary. We established, for example, a modified adherence step to reliably enrich CD14 + DC precursors from apheresis mononuclear cells. The addition of GM-CSF + IL-4 at the onset of culture proved disadvantageous and was, therefore, delayed for 24 h. DC development from apheresis cells occurred faster than from fresh blood or buffy coat, and was complete after 7 days. Monocyte conditioned medium when added on day 6 resulted in fully mature and stable DC (veiled, highly migratory and T cell sensitizing cells with a characteristic phenotype such as 85% CD83 + , p55/fascin + , CD115/M-CSF-R - , CD86 + ) already after 24 h. The mature DC progeny were shown to remain stable and viable if cultured for another 1-2 days in the absence of cytokines, and to be resistant to inhibitory effects of IL-10. Freezing conditions were established to generate DC from frozen aliquots of PBMC or to freeze mature DC themselves for later use. The approach yields large numbers of standardized DC (5-10 x 10(8) mature CD83 + DC/leukapheresis) that are suitable for performing sound DC-based vaccination trials that can be compared with each other.

Cell Adhesion↗
PubMed1996

Generation of mature dendritic cells from human blood. An improved method with special regard to clinical applicability.

Two methods to generate human dendritic cells from hematopoietic precursor cells in peripheral blood have recently been published. One approach utilizes the rare CD34+ precursors and GM-CSF plus TNF-alpha. The other method makes use of the more abundant CD34- precursor population and GM-CSF plus IL-4. Here we report a method that is based on the latter approach. However, the GM-CSF and IL-4 treated cells are not stable mature dendritic cells, e.g., the characteristic morphology and nonadherence of dendritic cells is lost if the cytokines are removed. We describe the need for a monocyte-conditioned medium to generate fully mature and stable dendritic cells. This is achieved by adding a 3 day 'maturation culture' to the initial 6-7 day culture in the presence of GM-CSF and IL-4. Macrophage-conditioned medium contains the critical maturation factors. Mature dendritic cells are defined by their pronounced display of motile cytoplasmic processes ('veils'), their high capacity to induce proliferative responses in resting T cells, particularly in naive umbilical cord T cells, their down-regulated antigen processing ability, and their characteristic phenotype: expression of CD83, high levels of MHC molecules and CD86, lack of CD115 and perinuclear dot-like CD68 staining. These features are stable for at least 3 days upon withdrawal of cytokines and conditioned media. IL-4 can be replaced by IL-13. When CD34+ progenitors are depleted from blood, there is only a minor reduction in the yield of dendritic cells by this method. We have adapted the method to consider several variables that are pertinent to clinical use, including a change from fetal calf serum to human plasma and to media approved for clinical use like X-VIVO or AIM-V. 1% plasma and RPMI 1640 are currently optimal. Additional reagents used for cell culture (Ig. cytokines) and cell separation (immunomagnetic beads) are approved for or already used in clinical applications. For 40 ml blood, the yield is 0.8-3.3 x 10(6) mature dendritic cells as defined by the expression of the new dendritic cell-restricted marker CD83. CD83+ cells constitute between 30 and 80% of all cells recovered at the end of the culture period. Yields can be enhanced up to six-fold if the blood donors are pretreated with G-CSF. Stable, mature dendritic cells generated by this method should be a powerful tool for active immunotherapy.

Cell Culture Techniques↗
PubMed1996

Expression of tumour necrosis factor-alpha (TNF-alpha) mRNA and protein in pathological thyroid tissue and carcinoma cell lines.

There has been much controversy about the presence of TNF-alpha within thyroid tissue. We therefore conducted a study to determine if TNF-alpha mRNA is present in thyroid tissue and thyroid-derived cells. Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was employed with a heterologous competitor fragment. Significantly lower levels of TNF-alpha mRNA were found in the autonomous nodules from patients with thyroid autonomy (TA; n = 4; 5.7 +/- 1.3 arbitrary units (AU) (mean +/- s.e.m.); P < 0.03) and in normal thyroid tissue (n = 2, 7.0 +/- 3.1 AU) compared with tissue from patients with Graves' disease (GD; n = 13; 27.9 +/- 10.3 AU), non-toxic multinodular goitre (NTG; n = 5; 20.9 +/- 5.8 AU) and perinodular tissue from TA patients (20.3 +/- 4.0 AU). Higher levels were detected in tissues from patients with Hashimoto's thyroiditis (HT; n = 2; 51.3 +/- 10.3 AU). Cultures of pure thyroid-derived fibroblasts (46 +/- 18 AU thyrocytes (33 +/- 8 AU), and the anaplastic thyroid carcinoma cell lines 8505 C (39 +/- 11 AU), SW 1736 (214 +/- 16 AU) and C643 (3 +/- 1 AU) showed significantly lower TNF-alpha mRNA levels than thyroid-derived lymphocytes (1650 +/- 32 AU). TNF-alpha was detected in the supernatants of unstimulated lymphocytes (22.1 +/- 1.1 pg/ml) and SW 1736 cells (3.5 +/- 0.9 pg/ml), but not in unstimulated fibroblasts and thyrocytes. Using an intracellular labelling technique in flow cytometry, the immunophenotype of stimulated TNF-alpha-positive lymphocytes was determined as predominantly CD3+CD45RO+. Our results suggest that TNF-alpha is present in the thyroid tissue of different thyroid disorders. Thyroid-derived lymphocytes are potential TNF-alpha producers and may thus locally influence thyroid function.

Adult↗
PubMed1996

Different cytokine mRNA profiles in Graves' disease, Hashimoto's thyroiditis, and nonautoimmune thyroid disorders determined by quantitative reverse transcriptase polymerase chain reaction (RT-PCR).

Intrathyroidal lymphocytes are a source of cytokines thought to stimulate or maintain the immune process within the thyroid in Graves' disease (GD) and Hashimoto's thyroiditis (HT). Quantitative assessment of the cytokine profile may provide important clues as to the Th1/Th2 balance prevailing in these diseases. We analyzed cytokine mRNA expression levels in thyroid tissue samples from 13 patients with GD, 2 with HT, 5 with nontoxic multinodular goiter (NTG), and 4 with thyroid autonomy (nodular = TAnod and perinodular = TAperi tissue) using multispecific competitor fragments with primer sequences for IL-1 beta, IL-2, IL-4, IL-6, IL-8, IL-10, IFN-gamma, CD25, and CD3 delta-chain mRNA. Patients with GD were subdivided into two groups according to their serum levels of antibodies to thyroperoxidase (anti-TPO; GDhigh > 4000 U/mL, GDlow < or = 200 U/mL). These levels correlated positively with the CD3 delta-chain mRNA levels (r = 0.83) and with the T cell infiltration (r = 0.71) as determined by immunohistochemistry. Patients with GDhigh demonstrated 2- to 4-fold higher IL-4 mRNA levels (as compared to all other investigated groups) and significantly higher IL-10 mRNA levels as compared to HT, GDlow, and TAnod patients. Patients with GDhigh also had significantly higher levels of IFN-gamma, IL-1 beta, IL-8, and CD25 mRNA as compared to GDlow. The highest IFN-gamma, IL-2, and CD25 mRNA levels were found in HT. The lowest mRNA levels of all the investigated groups were detected in TAnod. No significant differences in IL-6 and IL-8 mRNA levels were found between most of the patient groups. In summary, patients with GDhigh showed a shift to a more Th2-driven cytokine pattern. In contrast, the increase mRNA levels of Th1-related cytokines found in HT indicate predominantly T cell-mediated cytotoxic processes.

Adult↗
PubMed1990

Expression of an antigenic polypeptide of the human parvovirus B19.

The DNA fragment of the human parvovirus B19, with 715 nucleotides between nucleotide positions 3141-3856 was expressed in Escherichia coli as a beta-galactosidase fusion protein. The plasmid vector pSS20d used for this purpose permits cleavage of the viral gene product from the beta-galactosidase moiety by collagenase. After purification by p-aminophenyl-beta-D-thiogalactoside-sepharose and superose, a soluble protein with a molecular mass of 28 kDa was isolated. It represents a common part of the viral capsid proteins VP1 and VP2. This bacterially derived parvoviral gene product can be used for detection of anti-B19 antibodies in human sera.

Antibodies, Viral↗
PubMed1990

[A comparison of imaging procedures in the diagnosis of gallbladder and bile duct carcinomas].

Fourteen patients with carcinomas of the gallbladder and eight patients with biliary duct carcinomas were examined by CT, sonography, percutaneous transhepatic cholangiography (PTC), endoscopic retrograde cholangiography (ERC) and angiography. The results were compared retrospectively. Sonography proved suitable as a screening method. For accurate demonstration, this should be supplemented by CT. PTC and ERC demonstrate intraductal extension of biliary carcinomas and of gallbladder carcinomas infiltrating the bile ducts. The value of angiography consists merely in the preoperative demonstration of the vascular anatomy.

Adult↗