Biomedical subjects

M R Bernsen

Publications and source records attributed to M R Bernsen.

18 recordsLinked to original sources

PubMed2005

In vitro imaging of single living human umbilical vein endothelial cells with a clinical 3.0-T MRI scanner.

Iron oxide-labelled, single, living human umbilical vein endothelial cells (HUVECs) were imaged over time in vitro using a clinical 3.0-T magnetic resonance (MR) microscopy system. Labelling efficiency, toxicity, cell viability, proliferation and differentiation were assessed using flow cytometry, magnetic cell sorting and a phenanthroline assay. MR images were compared with normal light and fluorescence microscopy. Efficient uptake of iron oxide into HUVECs was shown, although with higher label uptake dose-dependent cytotoxic effects were observed, affecting cell viability. For MR imaging, a T2* weighted three-dimensional protocol was used with in-plane resolution of 39 x 48 microm2 and 100-microm slices with a scan time of 13 min. MRI could detect living cells in standard culture dishes at single-cell resolution, although label loss was observed that corresponded with the intracellular iron measurements. MR microscopy using iron oxide labels is a promising tool for studying HUVEC migration and cell biology in vitro and in vivo, but possible toxic effects of label uptake and loss of label over time should be taken into account.

Cell Proliferation↗

PubMed2003

On the biological relevance of MHC class II and B7 expression by tumour cells in melanoma metastases.

A large number of studies have indicated that specific immune reactivity plays a crucial role in the control of malignant melanoma. In this context, expression of MHC I, MHC II and B7 molecules by melanoma cells is seen as relevant for the immune response against the tumour. For a better understanding of the biological relevance of MHC II and B7 expression by tumour cells in metastatic melanoma, we studied the expression of these molecules in melanoma metastases in relation to the inflammatory response, regression of the tumour and survival from 27 patients treated with biochemotherapy (30 mg m(-2) Cisplatin and 250 mg m(-2) decarbazine (dimethyl-triazene-imidazole-carboxamide, DTIC) on days 1-3 i.v., and 10(7) IU IFN-alpha 2b 3 days a week s.c., q. 28d). In 19 out of 27 lesions studied, we found expression of MHC II by the tumour cells, while only in one out of 11 tumour biopsies obtained from untreated metastatic melanoma patients, MHC II expression was detected. Expression of B7.1 and B7.2 by tumour cells was found in nine out of 24 and 19 out of 24 lesions, respectively. In all cases where B7.1 expression was found, expression of B7.2 by the tumour cells was also seen. In general, no or only few inflammatory cells positive for B7 were found. Expression of MHC II by tumour cells was positively correlated with the presence of tumour-infiltrating lymphocytes, regression of the lesion, and with time to progression (TTP) and overall survival (OS) of the patient. However, no significant correlation between B7.1 or B7.2 expression and regression of the tumour, TTP or OS was found. In light of other recent findings, these data altogether do support a role as biomarker for MHC II expression by tumour cells; however, its exact immunological pathomechanism(s) remain to be established.

Adult↗

PubMed2003

Improved resolution by mounting of tissue sections for laser microdissection.

BACKGROUND: Laser microbeam microdissection has greatly facilitated the procurement of specific cell populations from tissue sections. However, the fact that a coverslip is not used means that the morphology of the tissue sections is often poor. AIMS: To develop a mounting method that greatly improves the morphological quality of tissue sections for laser microbeam microdissection purposes so that the identification of target cells can be facilitated. METHODS: Fresh frozen tissue and formalin fixed, paraffin wax embedded tissue specimens were used to test the morphological quality of mounted and unmounted tissue. The mounting solution consisted of an adhesive gum and blue ink diluted in water. Interference of the mounting solution with DNA quality was analysed by the polymerase chain reaction using 10-2000 cells isolated by microdissection from mounted and unmounted tissue. RESULTS: The mounting solution greatly improved the morphology of tissue sections for laser microdissection purposes and had no detrimental effects on the isolation and efficiency of amplification of DNA. One disadvantage was that the mounting solution reduced the cutting efficiency of the ultraviolet laser. To minimise this effect, the mounting solution should be diluted as much as possible. Furthermore, the addition of blue ink to the mounting medium restores the cutting efficiency of the laser. CONCLUSIONS: The mounting solution is easy to prepare and apply and can be combined with various staining methods without compromising the quality of the DNA extracted.

Coloring Agents↗

PubMed1999

[Laser-assisted microdissection and molecular analysis at the cellular level].

Developments of molecular biological techniques have allowed the analysis of small tissue samples. In order to obtain optimal results it is essential to work with pure cell populations. The procurement of pure samples has, however, been one of the main limiting factors in biomedical research. Recently developed systems for laser-assisted microdissection now allow the isolation of pure cell populations from tissue sections, even at a single cell level, that are suitable for subsequent molecular analyses. Since morphological features can be directly related to molecular characteristics, studies concerning molecular genetic backgrounds underlying disease can be performed more easily and accurately. By future combined use of laser microdissection and new molecular analysis methods, the applications for laser-micro-dissection will increase further.

Cell Separation↗

PubMed1999

Interleukin 2 (IL-2) therapy: potential advantages of locoregional versus systemic administration.

There has been an apparent discrepancy between the results obtained with IL-2 based immunotherapy in animal tumour models, including veterinary cancer patients, and human cancer patients. We argue that this is due to differences in the therapeutic regimens used to treat human and veterinary cancer patients. The main differences are systemic therapy and surgical removal of the primary tumour in case of human cancer patients, whereas these treatment modalities are not used in IL-2 treated veterinary cancer cases. We have developed a treatment protocol, in which IL-2 is applied locally, that has been successful against transplanted tumours as well as spontaneous tumours of varying origin and immunogenicity. In view of immunobiological considerations we conclude that local treatment of cancer with IL-2 makes more sense than systemic treatment, as usually applied in the clinic, and that surgical removal of tumours may be detrimental to successful IL-2 therapy.

Animals↗

PubMed1999

The VX2 carcinoma in the rabbit auricle as an experimental model for intra-arterial embolization of head and neck squamous cell carcinoma with dextran microspheres.

A head and neck cancer model is developed using the VX2 carcinoma cell line injected s.c. in both ears of New Zealand White (NZW) rabbits. The study is focused on the effects of intraarterial embolization of the carcinomas with a new type of dextran hydrogel microspheres. During the phase of exponential growth the tumour-surface doubling-time was 7.1+/-2.0 days. Standard deviation in growth of the tumours was significantly larger between separate animals than between tumours growing in the left and right auricle of each individual animal (2.0 versus 0.65 days). A fresh cell suspension containing at least 10 x 10(6) vital tumour cells was necessary to yield a tumour-take of 85%. The caudal auricular artery perfuses the caudal half of the external ear and is very suitable for macroscopic cannulation. Histological evaluation shows, that the use of dextran hydrogel microspheres of at least 25 microm in combination with ligation of non-tumour perfusing branches of the central auricular artery yields diffuse embolization of the VX2 carcinoma. This tumour model can be of use in further studies to optimize particle size and dosage for embolization as well as to evaluate the effect of different anti-neoplastic drugs, slowly released by controlled degradation of dextran microspheres.

Animals↗

PubMed1999

Local low-dose IL-2 therapy.

Interleukin-2 (IL-2) is a powerful drug for treating cancer. However, it is only powerful if it is properly applied. That is, IL-2 should be applied at the tumor site, because at the transition of normal and malignant tissue are the tumor infiltrating cells. These should be activated by IL-2. Local application implies that IL-2 can be used in relatively low doses. It is becoming clear that even a single injection of IL-2 can cure cancer. IL-2 can also enhance the therapeutic effects of irradiation and Cisplatin. Locally applied IL-2 therapy is virtually non-toxic.

Animals↗

PubMed1998

Interleukin-2: hope in cases of cisplatin-resistant tumours.

To establish whether or not local low-dose recombinant interleukin-2 (rIL-2) therapy might result in therapeutic benefit for ovarian cancer patients treated with cisplatin, the antitumour effects of rIL-2 and of combined treatment with cisplatin and rIL-2 in a mouse ovarian tumour (MOT) model were studied. In addition, some possible mechanistic aspects underlying the observed antitumour responses were analysed. MOT cells were injected i.p. into syngeneic, immunocompetent, female C3HeB mice. Tumour-bearing mice received i.p. treatment with cisplatin, rIL-2 or both. The MOT tumour appeared to be hardly responsive to treatment with cisplatin only or rIL-2 only. In contrast, combined local treatment with low doses of cisplatin (1 and 5 mg/kg body weight) and rIL-2 (60000 U/day) resulted in an effective antitumour response in MOT-bearing mice. Complete rejection of the i.p. (local) tumour occurred in up to 60% of the cases. In vitro studies showed that cisplatin and rIL-2 do not have cumulative direct toxic effects on MOT cells. Mice cured after combined treatment with cisplatin and rIL-2 were not able to reject a rechallenge with tumour cells, indicating that these mice had not developed immunity to the tumour. Analysis of tumour-associated leucocytes, however, showed that combined treatment with cisplatin and rIL-2 did result in enhanced non-specific cytolytic activity of peritoneal leucocytes. We have thus demonstrated that, in the MOT model, combined local treatment with low doses of cisplatin and of rIL-2 is far more effective than therapy with cisplatin alone. Non-specific cytotoxicity of leucocytes appears to be involved in antitumour responses induced by combined treatment with cisplatin and rIL-2. These results suggest that, in human ovarian carcinoma, much better results may be obtained with the combined treatment of cisplatin and low (non-toxic) doses of rIL-2 than with cisplatin only. This may also apply to cisplatin-resistant ovarian carcinoma.

Animals↗

PubMed1998

Identification of multiple mRNA and DNA sequences from small tissue samples isolated by laser-assisted microdissection.

Molecular analysis of small tissue samples has become increasingly important in biomedical studies. Using a laser dissection microscope and modified nucleic acid isolation protocols, we demonstrate that multiple mRNA as well as DNA sequences can be identified from a single-cell sample. In addition, we show that the specificity of procurement of tissue samples is not compromised by smear contamination resulting from scraping of the microtome knife during sectioning of lesions. The procedures described herein thus allow for efficient RT-PCR or PCR analysis of multiple nucleic acid sequences from small tissue samples obtained by laser-assisted microdissection.

Base Sequence↗

PubMed1997

Local low-dose interleukin-2 induces systemic immunity when combined with radiotherapy of cancer. A pre-clinical study.

Tumor recurrence and outgrowth of metastases limit the therapeutical effect of radiotherapy. We have tested whether these problems can be overcome by supplementing radiotherapy with locoregional interleukin-2 (IL-2) treatment. The SL2 lymphoma and the M8013 mammary carcinoma were used. Mice bearing a 10-day-old s.c. tumor were locally irradiated and were treated daily with IL-2 peritumorally for 5 or 10 days. Low-dose IL-2 therapy improved local response (LR) and increased disease-free survival (DFS) in both tumor models following either single-dose irradiation or fractionated irradiation. For example, 93% of SL2-bearing mice treated with single-dose irradiation and 10 days of IL-2 experienced long-term DFS, compared with 17% for irradiation alone (p < 0.0001). Additionally, treatment of one tumor with irradiation +IL-2 led to anti-tumor effects in a second, untreated tumor in 80% of SL2-bearing mice. LR was increased to 100% and DFS to 70% when the second, non-irradiated tumor was also treated with peritumoral IL-2. We conclude that supplementing local radiotherapy with low doses of IL-2 results in increased local tumor control and regression of distant, non-irradiated tumors. This type of radioimmunotherapy is a promising new approach for the clinic.

Animals↗

PubMed1997

Anti-tumor effects of local irradiation in combination with peritumoral administration of low doses of recombinant interleukin-2 (rIL-2).

The aim of this work was to improve radiotherapy results by immune stimulation. We tested the effects of a combination of radio- and immunotherapy, i.e., local low dose recombinant interleukin-2 (rIL-2) treatment, in two murine tumor models. Syngeneic tumors (SL2 lymphoma or M8013 mammary carcinoma) were induced subcutaneously on one or both flanks of mice. Irradiation was given either as a single dose (20 Gy) or fractionated (25 Gy) in 2 weeks. One or two cycles of rIL-2 were given concurrent with or subsequent to radiotherapy. One cycle of rIL-2 consisted of peritumoral injections administered on 5 consecutive days. Treatment effects were measured in terms of local tumor response and disease-free survival (DFS). The combined treatment modality was significantly better than treatment with either irradiation alone or rIL-2 alone. When tumors were inoculated on both flanks of the mice, combined radioimmunotherapy of one of the tumors also resulted in regression of the contralateral untreated tumor, indicating that a systemic anti-tumor immune reaction was induced. Additional rIL-2 injections did not enhance radiation toxicity. In conclusion supplementing irradiation with locally administered low doses of rIL-2 results in better local anti-tumor responses and DFS rates than either treatment alone without enhanced treatment toxicity. Furthermore, the local treatment induces a systemic anti-tumor reaction, influencing the growth patterns of a second, untreated tumor.

Animals↗

PubMed1997

Priming of the antitumor response promotes efficacy of interleukin-2 therapy.

We have studied the effect of active specific immunization (ASI) on the antitumor response induced by locoregional, low-dose interleukin-2 (IL-2) therapy. On day 0, mice were injected i.p. with viable, syngeneic tumor cells and with irradiated tumor cells (ASI). Low-dose IL-2 treatment was given i.p. for 5 consecutive days. ASI led to extended survival in two out of seven models tested. In these two models, enhanced efficacy was observed when both ASI and IL-2 were administered. In the five models in which ASI had no therapeutic value, ASI + IL-2 treatment was no more effective than IL-2 therapy. In the SL2 lymphoma model, use of ASI prior to IL-2 therapy given as early as days 1-5 led to at least 60% cure, whereas IL-2 therapy without ASI was only effective when administered after day 9. In the P815 mastocytoma model, however, ASI, IL-2, and the combination caused negative (suppressive) effects when administered on days 6-10. IL-2 administered on days 6-10 was therapeutically effective in this model when mice were treated with cyclophosphamide on day 6. In both the SL2 and the P815 tumor models, cured mice were specifically immune. The positive and negative effects observed were not due to the increased number of cells injected (non-specific inflammation) nor to possible antigenic alteration of the ASI cells by irradiation, as ASI with fragmented tumor cells was also effective in inducing synergy. Investigations into the underlying mechanism indicated that CD4+ cells play an important role. In total, the results indicate that ASI may be a good supplement to locoregional IL-2 treatment if care is taken to alleviate immunosuppressive activities.

Animals↗

PubMed1997

Analgesics in mice used in cancer research: reduction of discomfort?

During the last decades, an increase is apparent in the use of analgesics for laboratory animals in situations where this was previously considered unnecessary. Mice with advanced tumours often show clear signs of discomfort which may be a result of chronic pain or a result of general ill-being. The syngeneic murine tumour model most frequently used in our experiments was used to investigate whether this discomfort can be reduced with an analgesic. Twenty DBA/2 mice bearing SL2 lymphoma were given 0.5 mg/kg buprenorphine (Temgesic) in food gel twice daily, 20 tumour-bearing mice were given control food gel at the same times. Indicators of well-being were monitored daily. These included behavioural parameters such as exploration, grooming, and posture; food and water consumption and fur quality. All mice showed a clear increase of discomfort with time: explorative behaviours and grooming decreased, while sitting in hunched posture increased. Food and water consumption and fur quality also decreased. Major significant differences between the buprenorphine treated group and the control group were not apparent. In conclusion, we could not document a positive effect or buprenorphine on discomfort in mice as evaluated by our scoring system. It remains possible that pain itself was not the primary cause of the discomfort in mice bearing these tumours, or that the analgesic effect of buprenorphine was insufficient under these circumstances.

Analgesia↗

PubMed1996

Optimal regimes for local IL-2 tumour therapy.

In this report we present studies on optimal regimes for regional IL-2 therapy, focused on dose, schedule and site of injection. Original data obtained in 2 murine tumour models show that all 3 factors are of importance. Anti-tumour responses were most effective when IL-2 was administered regionally 5 to 10 times, at doses ranging from 7,000 to 33,000 IU/day every day or every other day. This resulted in cure rates of more than 40% in mice bearing ascitic tumour that had also disseminated to liver and lungs. The importance of these data is discussed in the light of previous results of our group. These results illustrate that the doses and schedules used in this study are not effective exclusively in these 2 tumour models but may have a more general applicability.

Animals↗

PubMed1996

The success of locoregional, low-dose recombinant interleukin-2 therapy in tumor-bearing mice is dependent on the time of rIL-2 administration.

The extremely different administration schedules that are used in testing recombinant interleukin-2 (rIL-2) therapies for cancer may account for the extreme variation in efficacy reported in various studies in animal models. A major point may be the variation of the time interval between tumor transplantation and rIL-2 therapy. We hypothesized that administration of rIL-2 before the immune system has mounted a specific cellular reaction against the tumor (associated antigens) might result in lesser efficacies than later rIL-2 administration. This hypothesis was tested in DBA/2 mice bearing a syngeneic SL2 lymphoma. When 7000 IV/day rIL-2 was administered to tumor-bearing mice for 5 consecutive days starting on day 1, 3, 4, 5, or 6 after tumor inoculation, the survival curve of the mice did not significantly differ from that of diluent-treated mice. In contrast, a significant difference was observed when treatment was begun on day 7, 8, 9, 10, or 12 (p < or = 0.004). rIL-2 therapies begun on day 9 or 10 were most effective, curing up to 80% of mice treated, despite there being an enormous burden of disseminated tumor present at that time (1-4% of the total body weight). When rIL-2 was administered for fewer than 5 consecutive days, beginning on day 10, the efficacy of the therapy dropped radically (p < or = 0.055). Involvement of a specific anti-tumor reaction was also tested. All mice that were cured of the tumor as a result of rIL-2 therapy proved to be specifically immune to the SL2 tumor. Furthermore, day 10-14 administration of rIL-2 was completely ineffective in CD4(+)-cell depleted mice (p = 0.0116 vs. rIL-2 therapy in non-depleted mice). Together, this implies that this form of rIL-2 therapy is mediated by tumor-specific T-cells. As a whole, these results indicate that T-cell mediated rIL-2 therapy of cancer in animal models is sensitive to the time when the rIL-2 is administered and to the length of time for which the rIL-2 is given. This should be taken into account when planning new therapy protocols and when analyzing published data.

Animals↗

PubMed1995

Reevaluation of the superiority of polyethylene glycol-modified interleukin-2 over regular recombinant interleukin-2.

Other groups have reported a superior antitumor efficacy of polyethylene glycol-modified interleukin-2 (PEG-IL-2) compared with regular recombinant interleukin-2 (rIL-2). However, detailed comparison of the antitumor efficacies of locally applied PEG-IL-2 and rIL-2 in the well-established DBA/2-SL2 model shows a higher antitumor efficacy of PEG-IL-2 only at doses < 800 micrograms IL-2 protein/kg body weight. At doses > 800 micrograms IL-2 protein/kg body weight, rIL-2 has better therapeutic efficacy. The superiority of rIL-2 at doses > 800 micrograms IL-2 protein/kg body weight is a result of the toxicity of PEG-IL-2 at these doses. With either IL-2 preparation, cure rates of approximately 90% can be obtained at nontoxic doses. We conclude that PEG-IL-2 does not have superior antitumor efficacy to rIL-2. The main advantage of PEG-IL-2 is that for optimal therapeutic efficacy a daily injection schedule is not required as seems to be the case for rIL-2.

Animals↗

PubMed1993

Dualistic effects of cis-diammine-dichloro-platinum on the anti-tumor efficacy of subsequently applied recombinant interleukin-2 therapy: a tumor-dependent phenomenon.

The efficacy with which disseminated SL2 and P815 tumors, in syngeneic DBA/2 mice, can be eradicated with low-dose recombinant interleukin-2 (rIL-2) therapy is about equal. Treatment (i.p.) of DBA/2 mice, injected i.p. with SL2 or P815 cells on day 0, with rIL-2 (Proleukin) on days 10 to 14 results in a cure rate of 50 to 60% in each case. The in vitro sensitivity of SL2 and P815 cells to cis-diammine-dichloro-platinum [II] (cisplatin) is also comparable, although P815 appears to be slightly more sensitive. In vivo, however, therapy with cisplatin is far less effective against P815 than against SL2. In the DBA/2-SL2 model, at all doses tested, combination therapy with cisplatin (administered on day 2) and rIL-2 (administered on days 10-14) resulted in anti-tumor efficacy greater than that of either drug separately. In contrast, in the DBA/2-P815 model, cisplatin decreased the anti-tumor efficacy of subsequently applied rIL-2 therapy. As the only difference between the 2 tumor models is the tumor itself, the success of combination therapy with cisplatin and rIL-2 was dependent on tumor characteristics. We suggest that in these 2 tumor models, neither the sensitivity of these tumors to cisplatin nor their growth and dissemination patterns were responsible for the contrasting results of combination therapy in these models. Instead, tumor-dependent immune-modulating effects of cisplatin may be the cause of these effects. These immune-modulating effects may comprise (a) effects of cisplatin on the tumor cells, resulting in changes in their susceptibility to immune effector cells, or changes in their immunogenicity; (b) activating or suppressive effects of cisplatin on immune effector cells; or (c) a combination of these effects. These effects could then either synergize or antagonize with the immune activating properties of rIL-2.

Animals↗

PubMed1991

Lymphokine-activated killer-cell-mediated killing of WiDr colon carcinoma cells is inhibited by K562 erythroleukemia cells.

Lymphokine activated killer (LAK) activity was induced in human peripheral mononuclear blood cells by human recombinant interleukin-2. Monocytes were required for optimal rapid proliferation of cells with LAK activity. They had no influence on the expression of tumoricidal activity by the LAK cells. The effector cells killed K562 erythroleukemia cells and WiDr colon cells differently, i.e. contact areas with WiDr cells were limited, whereas the contact areas between effector cells and K562 cells were much longer. Using mixtures of hot and cold target cells it was shown that effector cells preferably bind with K562 cells, impeding the binding of WiDr cells. Differences in expression of cytotoxicity of LAK cells against WiDr and K562 cells respectively was also observed after culturing the LAK cells for a relatively longer period. Cytotoxicity against WiDr was maximal at 3-16 days after starting LAK cell generation, whereas cytotoxicity against K562 was kept constantly high for at least 21 days. The addition of biological response modifiers [PHA and anti-CD3 antibody (OKT3)] during the LAK cell induction also had different effects on the expression of LAK activity against WiDr and K562 cells. Whereas PHA, in combination with rIL-2 had no significant influence on the cytotoxicity against WiDr cells, the cytotoxicity against K562 was significantly inhibited. Addition of anti-CD3 antibody diminished the cytotoxicity against WiDr target cells and had no influence on the cytotoxicity against K562 cells.

Carcinoma↗