Genetic variants in the genes of the sex steroid hormone metabolism and depressive symptoms during and after pregnancy.

PURPOSE: The aim of this study was to conduct an association analysis of depressive symptoms and polymorphisms in the ESR1, PGR, CYP19A1, and COMT genes in pregnant and postpartum women. METHODS: The Franconian Maternal Health Evaluation Study (FRAMES) recruited healthy pregnant women prospectively for assessment of maternal and fetal health. The German version of the 10-item Edinburgh Postnatal Depression Scale (EPDS) was completed at three time points in this prospective cohort study. Visit 1 was at study entry in the third trimester of pregnancy, visit 2 was shortly after birth, and visit 3 was 6-8 months after birth. Germline DNA and depression measurements from 361 pregnant women were available for analysis. Six single nucleotide polymorphisms (SNPs) in the above-mentioned genes were genotyped. After reconstruction of haplotypes for PGR (rs1042838 and rs10895068) and CYP19A1 (rs10046 and rs4646), a multifactorial linear mixed model was applied to the data to describe the association between haplotypes and depression values. The single SNPs for ESR1 (rs488133) and COMT (rs4680) were analyzed separately using linear mixed models analogously. RESULTS: The mean antepartum EPDS measurement was 5.1, the mean postpartal measurement after 48-72 h was 3.5, and the mean value 6-8 months postpartum was 4.2. The SNPs in PGR were reconstructed into three haplotypes. The most common haplotype was GG, with 63.43% of patients carrying two copies and 33.52% carrying one copy. For haplotype GA, the group of carriers of two copies (0.28%) was combined with the carriers of one copy (9.70%). Haplotype reconstruction using CYP19A1 SNPs resulted in three haplotypes. The most common haplotype was TC, with 25.48% of patients carrying two copies and 51.52% one copy. None of the haplotype blocks and neither of the two single SNPs showed any significant associations with EPDS values. CONCLUSIONS: The candidate haplotypes analyzed in PGR and CYP19A1 and single SNPs in ESR1 and COMT did not show any association with depression scores as assessed by EPDS in this cohort of healthy unselected pregnant women.

Evaluation of a systematic digitized training program on the effectivity of subgingival instrumentation with curettes and sonic scalers in vitro.

OBJECTIVES: Whereas the key role of subgingival instrumentation in periodontal therapy is well known, the influence of operators' experience/training with different devices on treatment results is yet uncertain. Therefore, we assessed untrained undergraduate students, working on manikins, as to how effectively they learn to use curettes (GRA) and sonic scalers (AIR); hypothesizing that AIR will result in higher relative cleaning efficacy (RCE) than GRA. MATERIAL AND METHODS: Before baseline evaluation (T0), 30 operators (9 males, 21 females) received a 2-h theoretical lesson for both instruments, followed by a 12-week period with a weekly digitized training program for 45 min. During three sessions (T1-T3), the operators had to instrument six equivalent test teeth with GRA and AIR. At T0-T3, treatment time, proportion of removed simulated biofilm (RCE-b), and hard deposits (RCE-d) were measured. RESULTS: At T0, RCE-b was in mean(SD) 64.18(25.74) % for GRA, 62.25(26.69) % for AIR; (p = 0.172) and RCE-d 85.48(12.32) %/ 65.71(15.27) % (p < 0.001). At T3, operators reached highest RCE-b in both groups (GRA/AIR 71.54(23.90) %/71.75(23.05)%; p = 0.864); RCE-d GRA/AIR: 84.68(16.84) %/77.85(13.98) %; p < 0.001). Both groups achieved shorter treatment times after training. At T3, using curettes was faster (GRA/AIR 16.67(3.31) min/19.80(4.52) min; p < 0.001). CONCLUSIONS: After systematic digitized training, untrained operators were able to clean 70% of the root surfaces with curettes and sonic scalers. CLINICAL RELEVANCE: It can be concluded that a systematic digitized and interactive training program in manikin heads is helpful in the training of root surface debridement.

Influence of motivation and a new digitized training program on undergraduate dental students during preclinical scaling training.

BACKGROUND: The current study evaluated whether a new digitized scaling training program (DTP: n = 30; supervisor-student-ratio 1:10) improves the performance of undergraduate dental student during a preclinical course in regard to two different instruments [sonic scalers (AIR) and Gracey curettes (GRA)] compared to a conventional training program (CTP: n = 19; supervisor-student-ratio 1:4). METHODS: All the participants received a two-hour lecture on both instruments, followed by a 12-week period with a weekly training program lasting 45 min (10 sessions); one group was supported by DTP. At the end of the training phase, all the participants performed the subgingival scaling of six equivalent test teeth using GRA and AIR. Treatment time, proportion of removed simulated biofilm (relative cleaning efficacy, RCE-b) and hard deposits (RCE-d) were recorded. By using a pseudonymized questionnaire with a 5-point Likert scale, self-assessment of scaling effort, handling, root surface roughness/destruction and effectiveness were evaluated. In addition, personal data such as age, gender, handedness, regularity of playing computer games/consoles and previous dental/technical or medical education were elevated and correlated with cleaning efficacy. RESULTS: The DTP participants showed higher effectiveness in RCE-b compared to those who used the CTP with GRA (71.54% vs. 67.23%, p = 0.004) and AIR (71.75% vs. 62.63%, p ≤ 0.001), and the DTP students were faster with both instruments (p ≤ 0.001). For RCE-d, there was no significant difference between the DTP and CTP groups (GRA p = 0.471; AIR p = 0.158), whereas DTP showed better RCE-d results with GRA versus AIR (84.68% vs. 77.85%, p < 0.001). According to the questionnaire, no significant differences were detected between the training groups in terms of self-assessment, handling, treatment time, root surface roughness/destruction or effectiveness of the instruments. The CTP group favored AIR compared to GRA regarding the fatigue effect. The CTP and playing computer games/consoles regularly was correlated with lower RCE-b, whereas previous education in medicine/dentistry was correlated with higher RCE-b values. CONCLUSIONS: Within the limitations of the study, the DTP with a reduced supervision effort compared to the CTP resulted in higher effectiveness and lower instrumentation time for removing simulated biofilms.

Long-term periodontitis treatment costs according to the 2018 classification of periodontal diseases.

OBJECTIVES: To assess if long-term treatment costs in periodontitis patients differ between stage III vs. IV and grade B vs. C according to the 2018 classification of periodontal diseases. METHODS: A cohort of 231 periodontitis patients (followed over a mean of 18.4 years) was evaluated. Costs for active periodontal therapy (APT, including scaling and root planning, open flap debridement, root resections) and supportive periodontal therapy (SPT, including also restorative, endodontic, prosthetic and surgical treatment) were estimated from a mixed payer-perspective in Germany (in Euro 2020). Multi-dimensional staging and grading was applied. The impact of stage, grade, sex and age on total and annual costs was assessed. RESULTS: Mean (SD) total and annual treatment costs were 7154 (2554) Euro and 437 (222) Euro. Costs were generated during SPT (92 %) and by periodontal treatment (88 %) and decreased significantly with longer follow-up (p < 0.001). Total costs were 7120 (2692) Euro in stage III (n = 154) vs. 7221 (2271) Euro in stage IV (n = 77; p > 0.05), and 6256 (1605) Euro in grade B (n = 35) vs. 7314 (2660) Euro in grade C (n = 196; p < 0.001). Annual costs were 426 (219) Euro vs. 459 (228) Euro for stage III vs. stage IV (p > 0.05) and 308 (163) Euro vs. 460 (224) Euro for grade B vs. grade C (p < 0.001). Multivariable modelling found grade, but not stage, sex and age significantly associated with costs. CONCLUSIONS: Within the limitations of this study, and in patients with severe periodontitis who were systematically treated long-term, grading, but not staging was associated with costs. CLINICAL SIGNIFICANCE: Treatment costs were higher in patients with more progressive periodontitis and were found to decrease during follow-up. Dentists need to consider costs during treatment planning and communication with patients.

Long-term treatment costs of chronic periodontitis patients in Germany.

AIM: We assessed the long-term costs for treating chronic periodontitis (CP) patients. METHODS: A cohort of compliant CP patients was retrospectively evaluated. Costs for active periodontal therapy (APT, including scaling and root planning, open flap debridement, root resections) and supportive periodontal therapy (SPT including periodontal, restorative, endodontic, prosthetic and surgical treatments) were estimated from a mixed payer perspective in Germany. The impact of tooth- and patient-level factors on annual costs was assessed using mixed-modelling. RESULTS: Two hundred and seventy-three patients (mean ± SD age: 49.6 ± 8.8 years), with 24.3 ± 4.5 teeth, were included. Mean follow-up was 18.7 ± 5.7 years. Total treatment costs per patient and per tooth were 6,146 ± 2,236 and 222 ± 98 Euro, respectively. Costs were generated mainly by periodontal therapy and during SPT. Annualized patient- and tooth-level costs were 348 ± 159 and 12.4 ± 5.7 Euro, respectively. Annual costs increased significantly in older patients, regular attenders, those with fewer teeth (<24) at baseline and teeth with higher probing pocket depths or mobility 3 at baseline. CONCLUSIONS: Annual costs for treating CP patients were lower than those found for aggressive periodontitis patients. Regular attendance and having more severe periodontitis came with higher costs per year.

Genetic risk factors for ovarian cancer and their role for endometriosis risk.

OBJECTIVE: Several genetic variants have been validated as risk factors for ovarian cancer. Endometriosis has also been described as a risk factor for ovarian cancer. Identifying genetic risk factors that are common to the two diseases might help improve our understanding of the molecular pathogenesis potentially linking the two conditions. METHODS: In a hospital-based case-control analysis, 12 single nucleotide polymorphisms (SNPs), validated by the Ovarian Cancer Association Consortium (OCAC) and the Collaborative Oncological Gene-environment Study (COGS) project, were genotyped using TaqMan® OpenArray™ analysis. The cases consisted of patients with endometriosis, and the controls were healthy individuals without endometriosis. A total of 385 cases and 484 controls were analyzed. Odds ratios and P values were obtained using simple logistic regression models, as well as from multiple logistic regression models with adjustment for clinical predictors. RESULTS: rs11651755 in HNF1B was found to be associated with endometriosis in this case-control study. The OR was 0.66 (95% CI, 0.51 to 0.84) and the P value after correction for multiple testing was 0.01. None of the other genotypes was associated with a risk for endometriosis. CONCLUSIONS: As rs11651755 in HNF1B modified both the ovarian cancer risk and also the risk for endometriosis, HNF1B may be causally involved in the pathogenetic pathway leading from endometriosis to ovarian cancer.

Meconium indicators of maternal alcohol abuse during pregnancy and association with patient characteristics.

AIM: Identification of women with moderate alcohol abuse during pregnancy is difficult. We correlated self-reported alcohol consumption during pregnancy and patient characteristics with objective alcohol indicators measured in fetal meconium. METHODS: A total of 557 women singleton births and available psychological tests, obstetric data and meconium samples were included in statistical analysis. Alcohol metabolites (fatty acid ethyl esters (FAEEs) and ethyl glucuronide (EtG)), were determined from meconium and correlated with patient characteristics. RESULTS: We found that 21.2% of the 557 participants admitted low-to-moderate alcohol consumption during pregnancy. Of the parameters analyzed from meconium, only EtG showed an association with alcohol history (P < 0.01). This association was inverse in cases with EtG value above 120 ng/g. These values indicate women with most severe alcohol consumption, who obviously denied having consumed alcohol during pregnancy. No other associations between socioeconomic or psychological characteristics and the drinking status (via meconium alcohol metabolites) could be found. CONCLUSION: Women who drink higher doses of ethanol during pregnancy, according to metabolite measures in meconium, might be less likely to admit alcohol consumption. No profile of socioeconomic or psychological characteristics of those women positively tested via meconium could be established.

Genetic variants in the genes of the stress hormone signalling pathway and depressive symptoms during and after pregnancy.

PURPOSE: The aim of this study was to investigate whether single nucleotide polymorphisms (SNPs) in genes of the stress hormone signaling pathway, specifically FKBP5, NR3C1, and CRHR1, are associated with depressive symptoms during and after pregnancy. METHODS: The Franconian Maternal Health Evaluation Study (FRAMES) recruited healthy pregnant women prospectively for the assessment of maternal and fetal health including the assessment of depressiveness. The German version of the 10-item Edinburgh Postnatal Depression Scale (EPDS) was completed at three time points in this prospective cohort study. Visit 1 was at study entry in the third trimester of the pregnancy, visit 2 was shortly after birth, and visit 3 was 6-8 months after birth. Germline DNA was collected from 361 pregnant women. Nine SNPs in the above mentioned genes were genotyped. After construction of haplotypes for each gene, a multifactorial linear mixed model was performed to analyse the depression values over time. RESULTS: EPDS values were within expected ranges and comparable to previously published studies. Neither did the depression scores differ for comparisons among haplotypes at fixed time points nor did the change over time differ among haplotypes for the examined genes. No haplotype showed significant associations with depressive symptoms severity during pregnancy or the postpartum period. CONCLUSION: The analysed candidate haplotypes in FKBP5, NR3C1, and CRHR1 did not show an association with depression scores as assessed by EPDS in this cohort of healthy unselected pregnant women.

Socioeconomic status and depression during and after pregnancy in the Franconian Maternal Health Evaluation Studies (FRAMES).

PURPOSE: Depression during and after pregnancy can have a negative impact on women's quality of life and on the development of the newborn child. Interventions have been shown to have a positive influence on both mothers and children. Predictive factors for depressive symptoms might possibly be able to identify groups that are at high risk. The aim of this study was to investigate the value of socioeconomic factors in predicting depressive symptoms during and after pregnancy. METHODS: Depressiveness was measured using the German version of the 10-item Edinburgh Postnatal Depression Scale (EPDS) at three time-points, in a prospective cohort study (n = 1,100). Visit 1 (Q1) was at study entry in the third trimester of the pregnancy, visit 2 (Q2) was shortly after birth, and visit 3 (Q3) was 6-8 months after birth. Depression scores were associated with socioeconomic factors and time in linear mixed models. RESULTS: Parity status, education status, monthly income, residential property status, and partnership status, as well as interactions between them, were found to be predictive factors for EPDS scores. The strongest factor influencing depressive symptoms was partnership status. Women who did not have an intact partnership had EPDS scores that were on average four points higher than in women with a partner at all three study visits (P < 0.000001). CONCLUSIONS: Socioeconomic factors define subgroups that have different depression scores during and after pregnancy. Partnership status appears to be one of the most important influencing factors and could be useful for identifying women who should be offered an intervention to prevent possible negative effects on the mother or child.

Influence of levamisole and other angiogenesis inhibitors on angiogenesis and endothelial cell morphology in vitro.

Angiogenesis, the formation of new blood vessels from existing vessels is required for many physiological processes and for growth of solid tumors. Initiated by hypoxia, angiogenesis involves binding of angiogenic factors to endothelial cell (EC) receptors and activation of cellular signaling, differentiation, migration, proliferation, interconnection and canalization of ECs, remodeling of the extracellular matrix and stabilization of newly formed vessels. Experimentally, these processes can be studied by several in vitro and in vivo assays focusing on different steps in the process. In vitro, ECs form networks of capillary-like tubes when propagated for three days in coculture with fibroblasts. The tube formation is dependent on vascular endothelial growth factor (VEGF) and omission of VEGF from the culture medium results in the formation of clusters of undifferentiated ECs. Addition of angiogenesis inhibitors to the coculture system disrupts endothelial network formation and influences EC morphology in two distinct ways. Treatment with antibodies to VEGF, soluble VEGF receptor, the VEGF receptor tyrosine kinase inhibitor SU5614, protein tyrosine phosphatase inhibitor (PTPI) IV or levamisole results in the formation of EC clusters of variable size. This cluster morphology is a result of inhibited EC differentiation and levamisole can be inferred to influence and block VEGF signaling. Treatment with platelet factor 4, thrombospondin, rapamycin, suramin, TNP-470, salubrinal, PTPI I, PTPI II, clodronate, NSC87877 or non-steriodal anti-inflammatory drugs (NSAIDs) results in the formation of short cords of ECs, which suggests that these inhibitors have an influence on later steps in the angiogenic process, such as EC proliferation and migration. A humanized antibody to VEGF is one of a few angiogenesis inhibitors used clinically for treatment of cancer. Levamisole is approved for clinical treatment of cancer and is interesting with respect to anti-angiogenic activity in vivo since it inhibits ECs in vitro with a morphology resembling that obtained with antibodies to VEGF.

Shared decision-making in metastatic breast cancer: discrepancy between the expected prolongation of life and treatment efficacy between patients and physicians, and influencing factors.

Treatment decisions in oncology are based on a balance between the efficacy of therapy and its side effects. Patients with metastases and patients with a limited prognosis are a particular challenge, since communication about the disease situation and the expected therapeutic benefit is difficult not only for patients, but also for physicians. The aim of this study was therefore to compare the benefits expected of therapy by patients and physicians. Questionnaires were sent to 9,000 breast cancer patients and to 6,938 physicians. The questionnaires described 10 cases of breast cancer in the metastatic setting. The patients and physicians were asked to state the treatment benefit they would require to decide for the therapy options chemotherapy, endocrine therapy, antibody therapy, radiotherapy, and bisphosphonates. Additionally, the participants provided data on patient and physician characteristics. Expected treatment benefits were compared between patients and physicians, and influencing factors that modified the expected benefit were identified. Patients expected much greater benefits from the therapies offered than the physicians. For all treatment modalities, about 50 % or more of patients expected more than a 12-month increase in overall survival from all therapies. Among the doctors, this proportion ranged from 7 to 30 %. Among patients, previous experience of side effects and having young children in the family were the strongest influencing factors. Among the doctors, age and level of education had a strong influence on the expected prognostic improvement to indicate a therapy option. As expectations of treatment differ greatly between patients and doctors, a structured approach to solving this conflict is required. There appear to be some indicators that might help address the problem, such as the physicians' level of training and experience and the patients' specific social circumstances.

Delivery mode and the course of pre- and postpartum depression.

PURPOSE: To compare depressiveness scores, both during and after pregnancy, with the delivery mode (DM). METHODS: In a longitudinal, prospective study, standardized questionnaires for the Edinburgh Postnatal Depression Scale were presented to 1,100 women and used to assess the presence and severity of depressiveness at three time points: prenatal, from the 30th gestational week (Q1); 48-72 h postnatal (Q2); and 6-8 months postnatal (Q3). The patients were divided into four groups relative to DM: spontaneous delivery, primary cesarean section (CS), secondary CS, and assisted vaginal delivery. The final number of participating women with both delivery mode and depression information for all three time points was 753. RESULTS: There was a significant difference of the mean EPDS values between the spontaneous delivery and primary CS groups (P=0.04) at Q1 (5.1 vs. 6.3). None of the other comparisons was significant. Significant differences relative to DM were seen at Q2 (P<0.0001), but there were no significant differences between the patient groups at Q3 (P=0.54). CONCLUSIONS: DM only showed coherence with the extent of depression briefly during the peripartal period. A relationship was found between depressiveness during pregnancy and DM, with higher depressiveness scores in the group of patients undergoing primary CS. This should be taken into account when patients requesting an elective cesarean section are being counseled.

Genetic variants in the tryptophan hydroxylase 2 gene (TPH2) and depression during and after pregnancy.

BACKGROUND: A number of studies indicate that altered serotonergic transmission may be a risk factor for depression in the peripartum period. The aim of this study was to investigate whether genetic polymorphisms in the TPH2 gene, the gene product of which is the rate-limiting enzyme in the biosynthesis of serotonin in the central nervous system, are associated with depressive symptoms in pregnancy and the postpartum period. METHODS: In a cohort of 361 Caucasians, the severity of depression was assessed prospectively during pregnancy (third trimester) and the postpartum period (2-3 days and 6-8 months) using the Edinburgh Postnatal Depression Scale (EPDS). Tagging single nucleotide polymorphisms (SNPs) in TPH2 and SNPs that are known to be of functional relevance were genotyped. For each haplotype block or SNP, a multifactorial linear mixed model was performed to analyse the EPDS values over time. RESULTS: The haplotype block in the promoter region of TPH2 showed significant associations with depression values during pregnancy and 6-8 months afterwards. Additionally, a haplotype block in intron 8 had an influence on depression values during pregnancy, but not after birth. There was a significant interaction between time and haplotypes and the severity of depression. The effect of TPH2 haplotypes on EPDS values was strongest during pregnancy and 6 months after birth, with a low depression rating in the first few days after delivery for all women. CONCLUSIONS: In this cohort, TPH2 haplotypes known to be of functional relevance were found to be associated with different EPDS values during and after pregnancy. These haplotypes were associated with depressive symptoms both before and after delivery and were thus not specific for postpartum-onset depression. This underlines the relevance of these functional polymorphisms for depression in general and the importance of longitudinal assessments in research on postpartum depression.

Epidemiology of chronic wound patients and relation to serum levels of mannan-binding lectin.

The aim of this study was to describe the epidemiology of chronic wounds in a large cohort of patients from a tertiary hospital out-patient clinic, and examine the significance of serum mannan-binding lectin for the occurrence and clinical presentation of such wounds. The study comprised 489 consecutive patients with chronic foot and leg ulcers. A clinical classification of wound- aetiology was performed, and mannan-binding lectin was measured in the sera of patients and healthy controls. The patients presented with 639 wounds altogether; diabetic foot ulcers (309), venous leg ulcers (188), arterial ulcers (109), and vasculitis (33). The mannan-binding lectin levels of patients with venous leg ulcer, alone or in combination with other types of wounds, differed significantly from the control group, and the frequency of values < 100 ng/ml was significantly higher. In diabetic and arterial ulcer patients the frequency of values >or= 3000 ng/ml was significantly higher than that of the control group. This suggests a role for the innate immunity in the pathology of venous leg ulcers, and indicates different roles for mannan-binding lectin in the development of ulcers with different aetiologies; it further suggests that mannan-binding lectin substitution should be tested in a controlled clinical trial.

Influence of angiogenesis inhibitors on endothelial cell morphology in vitro.

Human umbilical vein endothelial cells (HUVEC) propagated in co-culture with fibroblasts form capillary-like networks of tubes. Here we characterize the morphology and ultrastructure of HUVEC in such co-cultures and investigate the influence of different angiogenesis inhibitors on endothelial cell morphology. Addition of angiogenesis inhibitors to the co-culture disrupted endothelial network formation and influenced endothelial cell morphology in two distinct ways. Instead of characteristic capillary-like networks, the endothelial cell morphology appeared as either short cords or compact cell clusters of variable size. Electron microscopy (EM) showed that in co-culture untreated HUVEC formed capillary-like tubes with lumina and retained important ultrastructural and physiological properties of endothelial cells in functional vessels as they contained both Weibel-Palade bodies and transport vesicles. Immuno-EM showed that the endothelial cell marker CD 31 stained endothelial membranes at cell-cell contacts, and at the luminal and abluminal side of the capillary-like tubes, although most abundantly at the luminal membranes. No ultrastructural signs of apoptosis were seen in HUVEC in inhibitor-treated co-cultures. Our results demonstrate that treatment with levamisole or anti-VEGF inhibits endothelial cell differentiation into tubes or instead induces formation of compact endothelial cell clusters. Treatment with platelet factor 4, suramin and TNP-470 results in formation of short endothelial cell cords. We discuss the implications of these findings.

Levamisole inhibits angiogenesis in vitro and tumor growth in vivo.

The synthetic anthelmintic compound Levamisole has previously been used in cancer treatment as an adjuvant in combination with 5-fluorouracil. Its mode of action remains unclear, but an immune-stimulatory effect has been suggested. Here, we show that Levamisole inhibits angiogenesis in vitro and tumor growth in vivo. In vitro, Levamisole specifically inhibits proliferation and differentiation of endothelial cells propagated in co-culture with fibroblasts. In vivo, Levamisole inhibits the growth in nude mice of a transplanted human tumor. The use of nude mice as tumor hosts permits the discrimination between the angiogenesis inhibitory effect of Levamisole and its assumed immune-stimulatory effect. Our findings support a possible therapeutic effect of angiogenesis inhibitors in the treatment of cancer and call for further investigations of the mechanism(s) underlying the anti-angiogenic effect of Levamisole.

A quantitative ELISA-based co-culture angiogenesis and cell proliferation assay.

Since solid tumours and metastases depend on adequate blood supply, much research is focused on inhibition of angiogenesis. Unfortunately, most known angiogenesis inhibitors have serious side effects when used as therapeutic agents in man. It is therefore important to develop methods to identify well-tolerated and efficient angiogenesis inhibitors. As a method for identification of new angiogenesis inhibitors we have further developed the procedure described by Bishop et al. (Angiogenesis 1999;3:335-44) to a quantitative ELISA-based fibroblast and endothelial cell co-culture angiogenesis assay. In each well of a 96-microwell plate, human umbilical vein endothelial cells (HUVEC) are seeded onto normal human dermal fibroblasts (NHDF) and propagated in co-culture for 72 h with or without a potential angiogenesis inhibitor. The effect on total cell proliferation is evaluated by quantitative immunochemical measurement of DNA, and on endothelial tube formation by quantification of CD 31, von Willebrand factor, and collagen IV. After ELISA reading, the morphology of the tubular structures formed by HUVEC is visualised with BCIP/NBT, permitting a quantitative result and a qualitative evaluation of cell morphology from the same well. We have used the assay to demonstrate the effect of well-known angiogenesis inhibitors on HUVEC tube formation.

Immune selection in murine tumors. Ph.d thesis.

It must be assumed that all tumor cells produce proteins which do not belong to a normal cell. These are called tumor-associated or tumor-specific antigens. In the classic immune surveillance theory it is believed that the cellular immune defense (the T-cell system) continuously discovers and eliminates newly arisen tumor cells which express such tumor-specific antigens. Since then it has been shown that one of the preconditions for the T-cell system to be able to recognize antigens is that they are presented by MHC class I histocompatibility antigens. There is a continual processing and presentation of all intracellular proteins in a cell. Thus, a tumor cell which produces an abnormal protein will also present this and thereby expose itself to being killed by cytotoxic T cells. The antigens are presented in the form of short peptides (8-9 aminoacids), which arise as a result of controlled degradation of the original proteins. The peptides thus formed are transported by specialised molecules in the so-called endogenous antigen processing and presentation pathway, and are eventually bound to and presented by MHC class I molecules. It has been shown that many tumors express less MHC class I on their surface compared to the normal tissue from which they have arisen, and also that patients with reduced immune function have an increased incidence of certain forms of cancer. It is therefore widely believed that a low MHC class I level contributes to the ability of tumor cells to avoid the T-cell-mediated immune defense. The aim of the present research project was to confirm the existence of a T-cell-mediated immune selection in primary tumors. Another of its goals was to elucidate the extent to which tumor cells with low MHC class I expression showed poor ability to present antigen, and whether the reason for this could be found in one or more of the molecular systems which participate in antigen processing and presentation. By using the chemical carcinogen 3-methylcholanthrene a total of 144 tumors were induced in immunologically normal and T-cell defective mice, respectively. It was assumed that tumors induced in normal mice would be immune selected, whilst this would not be the case for tumors from T-cell defective mice. This enabled us to work with a tumor-material where the two populations only differed in that the one part had undergone selection by a T-cell system and the other had not. Tumor induction time turned out to be shorter in immune defective than in normal mice, and the tumor frequency was higher, which might be due to the fact that in normal mice tumor growth was inhibited and in certain cases stopped by the T cells. On transplantation of the uncloned cell lines which were established from the primary tumors to immunologically normal congenic recipients, we were able to show that most of the tumors which originated from mice with a functional T-cell system, and which must therefore be assumed to have undergone selection in the primary tumor host, were not immunogenic and were therefore accepted. On the other hand, most tumors which originated from T-cell-defective mice were rejected as a sign that immunogenic tumor cells, assumed to have expressed tumor antigen, had not been eliminated in the primary tumor host. Still, we found that the ability of tumor cells to induce an immune response on transplantation was not reflected in their MHC class I expression. Both tumor lines from immunodeficient and normal mice had highly varying MHC class I levels, and contrary to expectations the highest levels were seen in tumor lines from immunologically normal mice. At the same time we found that the expression levels for the three different MHC class I molecules were the same in the individual tumor lines, which might indicate that the three genes are syn-regulated. The MHC class I mRNA content in tumors from normal mice was generally concordant with the surface level of MHC protein. Among the tumor lines from immunodeficient mice, on the other hand, we found several where there was no such agreement, which was taken to indicate that tumor cells with deviant MHC class I gene transcription had not been eliminated, in contrast to in the immunocompetent tumor hosts. The ability of tumor cells to present antigen was investigated by infecting cells with virus and thereafter assessing their ability to function as target cells for virus-specific T cells in a cytotoxic test. Their ability to do this varied considerably, but showed a correlation with their MHC class I expression. Among the transplanted tumor lines that were not able to present viral antigen, the majority were accepted, while most of the tumor lines which were rejected on transplantation possessed the ability to present virus. Closer analysis of the composition of proteasomes, heat shock protein content and TAP molecule function, which are all involved in the antigen processing system, did not immediately reveal any defects. Treatment with interferon gamma, which is known to upregulate the transcription of MHC class I and a number of other proteins which are involved in antigen presentation, showed that by far the majority of the tumor lines were able to respond normally. This was also true for the tumor lines which had deviant MHC class I gene transcription and the cells which showed poor ability to present viral antigen. We did find, however, three cell lines which did not respond to interferon gamma, and they all had defective interferon gamma-signaling, not because they did not express the interferon-receptor on the surface, but possibly on account of their lacking phosphorylation of an intracellular signal molecule, Stat1.