Peter Kulmburg

Pro-Inflammatory and T Cell Inhibitory Cytokines Are Secreted at High Levels in Tumor Cell Cultures of Human Renal Cell Carcinoma

Objectives: The objectives of this study were to assess cytokine secretion in human renal cell carcinoma (RCC) and to identify cytokines contributing to the immunomodulatory effect of tumor cells. Methods: Cytokine secretion in the supernatant of primary tumor cell cultures (PTCC) and corresponding cell lines (CL) was assayed using ELISA. Tumor cells were characterized by morphology, immunocytochemistry, and flow-cytometric analysis. Tumor-cell-induced T cell activation was determined by coculture of γδ and αβ T cell clones with tumor CL. Results: We assessed the cytokine secretion of tumor cells from 27 PTCC and their corresponding CL (3/27) of RCC. We found that RCC predominantly produced both pro-inflammatory and T-cell-inhibitory cytokines, such as IL-8, IL-6, GM-CSF, TNF-α, IL-10 and TGF-β1. CL were adapted to serum-free medium which may prove as a useful tool in future studies of cytokine secretion in RCC. In addition, we used γδ and αβ T cell clones to assess the immunomodulatory effect of tumor cells from RCC and found that predominantly γδ T cells were activated by RCC. Conclusions: Our data suggest that RCC produce large amounts of both pro-inflammatory and T-cell-inhibitory cytokines that potentially could influence the immune response of the host, especially tumor-specific cytotoxic T cells.

Induction of tumor-specific cytotoxic T lymphocytes by immunization with autologous tumor cells and interleukin-2 gene transfected fibroblasts.

Abstract In a phase I trial designed to study a vaccine composed of autologous tumor cells and interleukin-2 gene transfected fibroblasts we analyzed lymphocytes infiltrating the vaccination site (VIL) in two melanoma patients. Functional studies demonstrated that numbers of MHC class I restricted cytotoxic T cells directed against the autologous tumor had increased at the immunization site in both cases. Analysis of the variability of T cell receptors (TCR) in the VIL of one patient revealed that the cytotoxic T lymphocytes consisted of a predominant population of TCRBV21S3+ T cells. Enrichment of this subpopulation to more than 99% by specific anti-TCRBV21S3 monoclonal antibody linked immunomagnetic beads and sequencing of the TCR-β chain disclosed exactly the same V-D-J junctional sequence in all eight TCRBV21 transcripts from these VIL. The identical sequence was also detected in all eight TCRBV21 transcripts from the patient’s tumor-infiltrating lymphocytes, indicating that the same CTL clone had infiltrated the tumor, circulated in the peripheral blood, and was amplified at the vaccination site. The TCRBV21S3+ T cells were also found to display an MHC class I restricted cytotoxic activity specifically directed against the autologous tumor cells. At the beginning of treatment these cells were undetectable at the vaccination site and delayed-type hypersensitivity testing was negative, contrasting with the positive results after therapy. Thus it is likely that vaccination with autologous tumor cells plus interleukin-2 gene transfected allogeneic fibroblasts had induced not only local accumulation but also an increase in the frequency of circulating tumor specific CTL.

Comparison of Cytogenetics, Cytokine Secretion, and Oncogene Expression in Primary Cultures of Renal Carcinoma Cells

We compared the cytogenetic pattern of 20 different primary tumor cell cultures (PTCC) of renal cell carcinoma (RCC) to their cytokine secretion and oncogene expression. High secretion of IL-6 (gene locus on chromosome 7p21-p14) was correlated with the gain of an additional chromosome 7. Structural changes involving chromosome 5q22, the site of the GM-CSF gene, were matched with the high secretion of GM-CSF in PTCC. No such association was found for beta 2-microglobulin, TGF-beta 1, TNF-alpha, IL-8, and oncogenes, such as c-fos, c-myc, and pan-ras. Our approach may be useful in simultaneously analyzing several factors contributing to tumor progression and may contribute to understanding the multistep development of RCC.

Processing of Tumor Tissues for Vaccination with Autologous Tumor Cells

Vaccination with gene-transfected tumor cells has recently been proposed as a new strategy in the immunotherapy of cancer. Since autologous tumor cells provide an optimal antigen profile, the possibility of generating single cell suspensions from renal cell carcinoma (RCC), malignant melanoma (MM), colon carcinoma (CC), and non-small-cell lung cancer (NSCLC) biopsies was investigated. One hundred and seventy-four tumor biopsies were processed by mechanic and enzymatic dissociation, yielding 1-2 x 10(6) cells/g tumor (median), irrespective of tumor type. Primary tumor cell cultures (PTCC) of > or = 10(7) cells were established from 29 of 86 (34%) RCC, 14 of 38 (37%) MM, 11 of 23 (48%) NSCLC and 4 of 27 (15%) CC specimens. The amount of non-tumor cells, as assessed by morphology and immunocytology, was generally low (< 30%) in RCC (35 of 41) and MM (11 of 17), while it exceeded 60% in 8 of 11 PTCC from NSCLC and 3 of 11 CC. A high tumor cell yield was obtained in biopsies with a high degree of vascularization and in the virtual absence of necrosis. Thus, PTCC > or = 10(7) cells were obtained in 73% of MM with a high degree of vascularization and in 22% of MM with a low degree of vascularization (p < 0.007). Long-term tumor cell cultures exceeding 20 passages were established in 24 of 86 (18%) RCC, 7 of 38 (18%) MM and 3 of 27 (11%) CC, while successful implantation in nude mice was achieved in 8 of 20 RCC and 5 of 10 MM. Thus, under the conditions described, > or = 10(7) primary tumor cells of high purity could be generated from about one third of RCC and MM biopsies, while the success rate increased to > 50 and > 70%, respectively, in samples with a high degree of vascularization generated by an optimized biopsy technique excluding necrotic parts.

Serum Cytokine Levels in Cancer Patients Treated with Different Schedules of Ultra-Low-Dose Interleukin-2

Interleukin-2 (IL-2) induces secondary cytokines in vivo that may mediate antitumor effects as well as toxicity. The course and quantity of this in vivo reaction may depend on scheduling of IL-2 due to changes in responsiveness of the respective producer cells. This was evaluated in a phase-Ib study with ultra-low-dose IL-2 at 0.9 and 4.5 MIU/m2 administered once daily subcutaneously either once weekly (4 doses, stratum I), three times a week every other day (9 doses, stratum II), or five times a week every other week (10 doses, stratum III). Twenty-eight patients with advanced cancer were randomly assigned to the six treatment groups. Serum levels of IL-2, secondary cytokines, and soluble receptors were significantly increased after a single dose of 0.9 MIU/m2 s.c. demonstrating systemic efficacy. Baseline levels and native responsiveness were recovered after a l-week treatment-free interval in stratum I patients with the exception of sCD8 that was still increased although readily inducible at that time. Stratum II patients exhibited a prolonged and possibly continuous elevation of all serum parameters studied. Values did not increase beyond the 2nd week of therapy and even decreased with respect to sCD8 and neopterin. A sequential mode of administration (stratum III) may obviate some of these adaptive mechanisms as evidenced from a progressive increase of neopterin and sCD8 levels after the second treatment cycle, although induction of sTNFRI was saturable under these conditions. Thus, scheduling of IL-2 profoundly affects in vivo responses as evidenced from cytokine and soluble receptor serum levels. These data may be important for the design of IL-2 studies with respect to the induction of specific biological endpoints.

Curriculum in Urology: Phimosis and Circumcision in Children

ly around the glans, but they are blocked in the midline on the ventrum by the incompletely developed urethra. This preputial fold rolls over the base of the glans and leaves a groove between the coronal sulcus and the primitive prepuce. At the same time, an ingrowth of epithelium with thick cells, known as glanular lamella, actively proliferates in the groove in order to form a sheet between the preputial fold and the glans. This active epithelial proliferation in the groove carries the ridge of the preputial fold distally with the help of the mesenchymal growth within the prepuce [1]. The preputial and urethral folds fuse on the ventrum of the glans as the frenulum. The urethra within the glans penis develops by canalization of the urethral plate and the urethral orifice reaches its final destination at the site of the former epithelial tag. Formation of the frenulum seems to be required for completion of glandular urethral development [2]. After the 4th month of gestation, the single epithelial The prepuce, also called the foreskin, is the double-faced covering of the glans of the flaccid penis. Its inner surface is mucosa and outer surface is penile skin. The junction of these two surfaces is known as the preputial ring. Disorders of the prepuce, such as balanitis and phimosis, receive only cursory attention even in urological textbooks and their exact etiopathogenesis still remains unclear. Knowing the development of the prepuce is important to better understand its disorders and surgery.

NTS influence on transgene expression from mono- and bicistronic plasmid vectors after lipofection in a human fibroblast cell line

Abstract We evaluated the survival, transgene production, and copy numbers of integrated plasmid units per host genome after lipofection with mono- and bicistronic plasmid vectors in different cell lines and under various conditions. The addition of an integration enhancing murine sequence nontranscribed spacer (NTS) to the plasmids increased transfection efficiency, survival, and transgene expression. However, in human fibroblast cells this sequence had only marginal effects on overall plasmid copy number in bulk cultures. Clones producing the highest amounts of the transgene contained only one or two copies of plasmid per genome, independent of cell type and plasmid design.

Tumor Immunotherapy by IL-2 and IL-2 Gene Transfected Cells

Tumortherapy is based on a variety of treatment modalities. Directly operating cytoreductive approaches are designed to work either locally like surgery or radiotherapy, or systemically like cytotoxic drugs. Other ways of treatment are going to exploit the interaction of tumor and host. They are designed to block the host supply of hormones and growth factors the tumor relies on, to inhibit angiogenesis, or to recruit the intrinsic host defense system. The latter is translated into practice by immunotherapy that has made considerable progress recently. Aside from using monoclonal antibodies against tumor associated antigens (TAA) to mediate tumor cytotoxic effects, various approaches to recruit cytotoxic lymphocytes have been worked out.