Thilo Schlott

Anaphylatoxin C5a Induces Monocyte Recruitment and Differentiation into Dendritic Cells by TNF-α and Prostaglandin E2-Dependent Mechanisms

Although monocytes can be directed to develop into dendritic cells (DC) in vitro, the molecular mechanisms that induce their transformation in vivo are largely unknown. In the present study we employed an in vivo SCID mouse model to investigate the impact of two proinflammatory chemotaxins, the anaphylatoxin C5a and the chemokine macrophage inflammatory protein-1α (CCL3), on the differentiation of human monocytes and immature DC generated from monocytes in the presence of GM-CSF and IL-4. Both C5a and macrophage inflammatory protein-1α recruited human monocytes and immature DC into the peritoneal cavity of SCID mice, but only C5a induced their differentiation into phenotypically mature DC by 48 h after injection. Macrophages derived from monocytes by in vitro culture were resistant to C5a-mediated transformation in vivo. The effect of C5a was indirect, since C5a-stimulated TNF-α and PGE2 were found to be obligatory as well as sufficient to induce differentiation of monocytes. In contrast to monocytes, in vitro generated immature DC required TNF-α, but not PGE2, for their C5a-mediated maturation in vivo. C5a-transformed monocytes represented an inflammatory type of DC, as they constitutively secreted high amounts of TNF-α, but also retained the capacity to release the Th1 cytokine IL-12 p70 upon stimulation with CD40 ligand. In summary, we identified for the first time a cascade of inflammatory signals that can induce the transformation of monocytes into DC in vivo. This novel function emphasizes the important immunoregulatory role of C5a at the interface of innate and adaptive immunity.

POINT MUTATIONS AND NUCLEOTIDE INSERTIONS IN THE MDM2 ZINC FINGER STRUCTURE OF HUMAN TUMOURS

This study investigates the human oncoprotein MDM2, which interferes with regulation of cell division and apoptosis. Fifteen mixed–type follicular non‐Hodgkins lymphomas, ten leukaemias, two hepatocellular carcinomas, one osteosarcoma, and ten normal cell lines (fibroblasts, osteoblasts, mesothelium, peripheral lymphocytes) were tested for MDM2 expression and MDM2 gene mutation by reverse transcriptase‐polymerase chain reaction (RT‐PCR), immunocytochemistry, and nucleotide sequence analysis. Two follicular lymphomas, three leukaemias, both hepatocellular carcinomas, and the osteosarcoma sample showed transcription of the activated MDM2 gene. These samples lacked amplified MDM2 genes and carried mis‐sense, non‐sense and frame‐shift mutations in a zinc finger region of MDM2, altering the amino acid sequence or causing premature termination of transcription. The mis‐sense mutations were found in tumour cells that showed significant accumulation of MDM2 and lack of nuclear p53. Non‐sense mutations and frame‐shift mutations were found in tumours lacking MDM2 proteins. The mutations may affect the biological properties of MDM2 proteins.

Molecular and immunological characterization of the p83/100 protein of various Borrelia burgdorferi sensu lato strains

The complete coding regions of the chromosomally encoded p83/100 protein of four Borrelia garinii strains and one Borrelia burgdorferi sensu stricto strain have been amplified by the polymerase chain reaction (PCR), cloned and sequenced. From alignment studies with the deduced amino acid sequences presented here, and five other published p83/100 sequences, the most heterologous region of the p83/100 molecule was identified to be located between amino acid position 390–540. To study the structure of this heterogeneous region, and internal fragment of the p83/100 genes from 11 additional B. burgdorferi sensu lato strains was amplified by PCR. The PCR products were analyzed by DNA sequencing and restriction enzyme analysis. These internal p83/100 fragments varied in size and sequence. Cluster analysis of internal p83/100 fragments, as well as restriction enzyme analysis, revealed three major groups in accordance with grouping into the three species causing Lyme disease. Strains within the same species (six B. burgdorferi sensu stricto and six B. afzelii strains) showed similar p83/100 partial structures. Nevertheless, nine B. garinii strains showed more sequence variations and could be further divided into two major subgroups. One group is represented by OspA serotype 4 strains, the other more heterogeneous group is represented by OspA serotypes 3, 5, 6 and 7 strains. Phenotypic analysis with four p83/100-specific monoclonal antibodies revealed four distinct reactivity patterns. Antibody L100 1B4 recognized a common epitope of B. burgdorferi sensu stricto and B. afzelii. Antibodies L100 17D3 and L100 18B4 were reactive with an epitope shared by strains of all three species. The broadest reactivity was shown by L100 18B4 which, in constrast to L100 17D3, additionally recognized the relapsing fever borreliae B. turicatae and B. hermsii. L100 8B8 detected a subgroup of the B. burgdorferi sensu stricto strains. Since comparison of the p83/100 molecule with sequences from protein databases showed similarities with characteristics of eukaryotic cell structures, the p83/100 might mimic these structures and may, therefore, be involved in the immune escape mechanism of the pathogenic agent of Lyme disease.

Downregulation of the Retinoblastoma Gene Expression in the Progression of Malignant Melanoma

The retinoblastoma gene is a cell cycle regulator preventing cells from entering into S-phase. An altered expression of the retinoblastoma gene has been reported in the majority of human malignancies. The main aim of this study was to investigate retinoblastoma gene expression in the full spectrum of melanoma progression from naevus to melanoma metastases by applying immunohistochemistry and RT-PCR. All naevi with and without dysplasia showed high expression of the retinoblastoma gene. In primary melanomas, Rb-positive cells were found in 82 out of 106. Loss of expression correlated with an increase in Clark level and shorter survival rates. An independent prognostic role of the retinoblastoma gene was confirmed by Cox multivariate analyses (p < 0.01). In melanoma metastases, retinoblastoma gene expression (at the RNA level) was found in 18 out of 26 melanoma lymphatic metastases, and in 2 out of 5 liver metastases. Our results indicate a downregulation of the retinoblastoma gene in the progression of melanocytic tumours.

Ex vivo generation of human anti-melanoma autologous cytolytic T cells by dendritic cell/melanoma cell hybridomas

Abstract Due to their central role in controlling immunity, dendritic cells are logical targets for priming naive cytotoxic T lymphocytes against tumour cells. In a strictly autologous system, we fused dendritic cells with melanoma cells, both of which were derived from patients with metastatic malignant melanoma. Hybridomas were positive for major histocompatibility complex (MHC) class II, CD40, CD54, CD83, CD86, and the pro-inflammatory cytokine interleukin-12. Autologous T lymphocytes were co-incubated with hybridomas. After 6 days, in-vitro-primed T lymphocytes revealed a strong proliferation activity and released Th-1-associated, but not Th-2-associated, cytokines. Furthermore they showed effective anti-melanoma activity, resulting in death of 70  ±  9% of autologous melanoma cells. After depletion of CD4+ cells from the mixed population of primed T lymphocytes, the remaining CD8+ cells were able to kill 63  ±  8% of autologous melanoma cells. Following depletion of CD8+ cells, however, the cytotoxic capacity of the remaining T lymphocytes caused death in only 32  ±  6% of autologous melanoma cells. Blocking of MHC class I, but not class II, molecules on hybridomas impaired T cell proliferation, secretion of Th-1-associated cytokines, as well as the cytotoxic activity of primed T cells. These findings strongly suggest that hybridomas deliver melanoma-associated antigens via MHC class I molecules to T lymphocytes, resulting in the generation of CD8+ cytotoxic T lymphocytes with effective anti-melanoma activity in vitro. The data may serve as a basis for the use of hybridomas in the immunotherapy of malignant melanoma in vivo.

Analysis of adenomatous polyposis coli gene expression, APC locus-microsatellite instability and APC promoter methylation in the progression of melanocytic tumours

Adenomatous polyposis coli gene (APC) defects have been demonstrated for the first time in familial adenomatous polyposis. Recent reports indicate that the APC gene is an intermediary between cell adhesion molecules and the cytoskeleton and that it may function as a gatekeeper of colonic epithelial proliferation. The objective of this study was to analyse APCs presence in lentigos, primary melanomas and melanoma metastases. By immunohistochemistry, APC was demonstrated in all lentigos, in 75 out of 88 primary melanomas and in 16 out of 28 melanoma lymphatic metastases. The percentage of immunolabelled tumour cells (APC index) in lentigos ranged between 5 and 69%, in primary melanomas between 0 and 98% and in melanoma metastases between 0 and 52%. Statistically significant differences between lentigos and primary melanomas and between lentigos and metastases in APC expression were found. In a multivariate analysis, APC showed an independent prognostic impact. Analysis of microsatellite instability in the APC locus was performed on 29 melanomas. Microsatellite instability was found in 5/29 melanomas and loss of heterozygosity in 1/29 melanomas. Promoter methylation of APC was found in 6/10 APC-negative primary melanomas and in 9/10 APC-negative melanoma lymphatic metastases investigated. We conclude about important role of APC alterations for melanoma progression.

Specific Autologous Anti-Melanoma T Cell Response in vitro Using Monocyte-Derived Dendritic Cells

Dendritic cells (DC) are antigen-presenting cells initiating primary and secondary immune responses. Since malignant tumors are able to escape immunologic control, DC might be prime candidates to activate the immune system against tumor cells. In an autologous system, a dynamic interaction among monocyte-derived DC (MoDC), T lymphocytes, and tumor cells obtained from melanoma patients could be noted. MoDC were generated from blood monocytes in the presence of GM-CSF, IL-4, and IFN-gamma. T cells were isolated either from peripheral blood or from lymph nodes. Melanoma cells were harvested from surgically removed tumor metastases. They were then gamma-irradiated and co-cultured with autologous MoDC and T lymphocytes. After 5 days, the lymphocytes showed a high proliferative activity and the majority of them were CD8-positive. In five cases tested, they revealed a high cytotoxic activity resulting in apoptosis of tumor cells. These findings suggest that MoDC are capable of initiating an effective specific anti-tumor response in a strictly autologous mixed lymphocyte tumor culture (MLTC), even though tumor-specific antigens had not been individually defined. Therefore (I) whole melanoma cells can serve as a source of antigen, (II) monocyte-derived dendritic cells may process and present melanoma-specific antigens resulting in a strong lymphocyte proliferation, (III) the majority of responding T lymphocytes are CD8-positive, and (IV) an acquired cytotoxic response eventually leads to apoptosis of the melanoma cells. The reaction demonstrated here permits to in vitro and quantitatively monitoring the effect of T cell directed immunotherapies such as the adoptive immunotherapy of tumors.

The In Situ Polymerase Chain Reaction for Detection of Chlamydia trachomatis

The in situ polymerase chain reaction (PCR) is a technique that has important applications in the diagnosis of viral and bacterial diseases. This study investigated an in situ PCR assay established to detect the presence of Chlamydia trachomatis in endocervical swabs. In addition, histological sections of endocervical squamous cell carcinoma were analyzed because previous studies had revealed a significant association with C. trachomatis. A total of 20 cervical neoplasms (squamous cell carcinoma in situ; n = 10; invasive squamous cell carcinoma; n = 10) and endocervical smears taken from five patients with and without inflammatory changes were analyzed by conventional PCR. Chlamydial DNA was found in 10 histological samples (six carcinomas in situ, four invasive carcinomas) and in one endocervical swab from a patient with known C. trachomatis infection. Positive specimens were used for establishing an in situ PCR assay (IS-PCR). After IS-PCR, these samples showed dense cytoplasmic staining of endocervical cells (smears) and non-neoplastic epithelial cells (cervical neoplasms). The other tumor samples and smears did not demonstrate positive PCR reaction. The results indicate that in situ PCR is an effective technique for localizing C. trachomatis in target cells because IS-PCR detection of chlamydial DNA correlated with histological and cytological features.

Reverse transcriptase polymerase chain reaction for detecting Ewing's sarcoma in archival fine needle aspiration biopsies

OBJECTIVE Previous studies on pediatric soft tissue sarcomas have demonstrated a chromosomal 11;22 (q24;q12) translocation in Ewings sarcoma (ES) and peripheral primitive neuroectodermal tumors that appears to be a unique oncogenic marker. To investigate the usefulness of reverse transcriptase polymerase chain reaction (RT-PCR) as an ancillary method for cytodiagnosis, we tested archival aspirates derived from ES patients to establish whether any beta-actin RNA expression or tumor-specific EWS/FLI-1 gene translocation had occurred. STUDY DESIGN Sixteen skeletal specimens aspirated 10-15 years earlier from patients with cytologic and histologic diagnoses of ES were prepared for PCR. The amplification products were sequenced. RESULTS Amplifiable RNA was detected in smears from 12 patients by beta-actin RT-PCR. Seven aspirates from beta-actin-positive patients showed ES-specific genomic translocation (58%). Sequence analysis of the resulting PCR fragments revealed EWS/FLI-1 fusion transcriptions of varying length. CONCLUSION When RNA was retrievable, RT-PCR applied to routinely stained aspirate smears was a highly specific method in differential diagnosis of ES.

Down-regulation of Ku 70 and Ku 80 mRNA expression in transitional cell carcinomas of the urinary bladder related to tumor progression.

DNA-dependent protein kinase (DNA-PK) containing the regulatory subunits Ku 70 and Ku 80 plays a prominent role in the repair of double-stranded DNA breaks by a nonhomologous end-joining pathway maintaining genomic stability. In an attempt to elucidate the significance of the DNA-PK complex for human urothelial carcinogenesis, the expression of Ku 70 and Ku 80 was studied in 71 transitional cell carcinomas (TCC) of the urinary bladder of various grades and stages, and in relation to lifestyle and occupational bladder cancer risk factors. To analyse the mRNA expression of Ku 70 and Ku 80, real-time quantitative reverse transcription-polymerase chain reaction was used and the protein expression assessed by immunohistochemistry. Advanced high-grade, high-stage TCC expressed the mRNA of Ku 70 and Ku 80 at a lower level than superficial low-grade, low-stage carcinomas, suggesting down-regulation of the Ku system to be associated with progression of bladder cancer from a low to a high malignant potential. The protein expression of Ku 70 and Ku 80 was closely related and decreased consistently with increasing grades and stages, paralleling the expression of the mRNA. Among hazardous environmental bladder cancer risk factors, heavy consumption of coffee was associated with a twofold decreased Ku 70 and Ku 80 mRNA expression, whereas tobacco smoke did not substantially affect the activity of the Ku system, except for a trend towards a dose-response relationship in the expression of Ku 70 mRNA. There is some evidence that exposure to polycyclic hydrocarbons, paints and lacquer, and stone dust may modify the expression of Ku 70 mRNA. Although the underlying molecular genetic pathways are not yet clearly understood, our data indicate that down-regulation of the Ku system promotes progression of urothelial carcinogenesis to a more malignant and aggressive clinical behavior, presumably as a result of an impaired capacity for DNA repair.