Hepatitis E virus blood donor NAT screening: as much as possible or as much as needed?

BACKGROUND: The cost-benefit question of general screening of blood products for the hepatitis E virus (HEV) is currently being discussed. One central question is the need for individual nucleic acid amplification techniques (NAT) screening (ID-NAT) versus minipool NAT screening (MP-NAT) approaches to identify all relevant viremias in blood donors. Here, the findings of ID-NAT versus MP-NAT in pools of 96 samples were compared. STUDY DESIGN AND METHODS: From November 2017 to January 2018, a total of 10,141 allogenic blood donations from 7650 individual German blood donors were screened for the presence of HEV RNA using MP-NAT (96 samples) (RealStar HEV RT-PCR Kit) compared to ID-NAT (cobas HEV assay) on the fully automated cobas 6800 platform. RESULTS: Parallel screening of MP (n = 122, 96 samples/MP) using both methods detected seven reactive pools. After pool resolution, 8 HEV RNA-positive donations were identified by the in-house detection method, whereas 17 HEV RNA-positive donations were identified by ID-NAT with the cobas HEV assay. This resulted in an incidence of 1:1268 donations (0.079%) for MP-NAT screening and 1:597 donations (0.168%) for ID-NAT screening. CONCLUSIONS: The detection frequency of HEV RNA was approximately 50% higher if ID-NAT was used compared to MP-NAT. However, viral loads of ID-NAT-only samples were below 25 IU/mL and will often not result in transfusion-transmitted HEV (TT-HEV) infection, taking into account the currently known infectious dose of 5.0E + 04 IU inevitably resulting in TT-HEV infection. The clinical relevance and need for identification of these low-level HEV-positive donors still require further investigation.

Randomized supplementation of 4000 IU vitamin D3 daily vs placebo on the prevalence of anemia in advanced heart failure: the EVITA trial.

BACKGROUND: Low 25-hydroxyvitamin D (25OHD) levels (< 75 nmol/l) are inversely associated with anemia prevalence. Since anemia and low 25OHD levels are common in patients with heart failure (HF), we aimed to investigate whether vitamin D supplementation can reduce anemia prevalence in advanced HF. METHODS: EVITA (Effect of Vitamin D on Mortality in Heart Failure) is a randomized, placebo-controlled clinical trial in patients with initial 25OHD levels < 75 nmol/l. Participants received either 4000 IU vitamin D3 daily or a matching placebo for 36 months. A total of 172 patients (vitamin D group: n = 85; placebo group: n = 87) were investigated in this pre-specified secondary data analysis. Hemoglobin (Hb) and other hematological parameters were measured at baseline and study termination. Assessment of between-group differences in anemia prevalence and Hb concentrations was performed at study termination, while adjusting for baseline differences. RESULTS: In the vitamin D and placebo group, baseline proportions of patients with anemia (Hb < 12.0 g/dL in females and < 13.0 g/dL in males) were 17.2% and 10.6%, respectively (P = 0.19). At study termination, the proportion of patients with anemia in the vitamin D and placebo groups was 32.2% and 31.8%, respectively (P > 0.99). There was no between-group difference in change in the Hb concentrations (- 0.04 g/dL [95%CI:-0.53 to 0.45 g/dL]; P = 0.87). Results regarding anemia risk and Hb concentrations were similar in the subgroup of patients with chronic kidney disease (vitamin D group: n = 26; placebo group: n = 23). Moreover, results did not differ substantially when data analysis was restricted to patients with deficient baseline 25OHD levels. CONCLUSIONS: A daily vitamin D supplement of 4000 IU did not reduce anemia prevalence in patients with advanced HF. Data challenge the clinical relevance of vitamin D supplementation to increase Hb levels. TRIAL REGISTRATION: The study was registered at EudraCT (No. 2010-020793-42) and clinicaltrials.gov ( NCT01326650 ).

Establishment of a proficiency panel for an external quality assessment programme for the detection of bacterial contamination in platelet concentrates using rapid and cultural detection methods.

BACKGROUND: Platelet concentrates (PCs) are the main focus regarding the residual risk of transfusion-transmitted bacterial infections. Rapid screening methods for bacterial detection in platelets have been optimized over the last decade, but their external evaluation represents a complicated process. We developed a new type of proficiency panel for bacterial detection in PCs using currently available screening methods (especially rapid methods) suitable for external quality assessment programmes (EQAP). METHODS: PC samples were inoculated with different bacteria at two concentrations (10E+03 CFU/ml, 10E+05 CFU/ml) and stored under temperature-controlled conditions (1-5 days). Bacterial growth was further prevented by the addition of 0-20 µg/ml cotrimoxazole. Samples were analysed prior to and after storage using rapid detection methods (Bactiflow (BF), bacteria-generic NAT) and cultural methods to determine the influence of storage and antibiotic treatment on bacterial counts and the result outcome. A pilot EQAP was performed with four participants. RESULTS: Testing under the evaluated conditions demonstrated that bacterial counts remained constant prior to and after storage. The supplementation of 10 µg/ml cotrimoxazole did not influence bacterial detection using the two rapid detection methods BF and NAT. Furthermore, the detection of bacteria using cultural methods is still possible despite of antibiotic supplementation. The pilot EQAP confirmed these results. A storage time of up to 3 days proved practicable, showing no considerable influence on bacterial count and outcome of test results. CONCLUSION: The established proficiency panel provided PC matrix-conform samples with stabilized bacterial counts which can be analysed in parallel by rapid and cultural detection methods.

Vitamin D metabolites and fibroblast growth factor-23 in patients with left ventricular assist device implants: association with stroke and mortality risk.

PURPOSE: Stroke and mortality risk in patients with left ventricular assist device (LVAD) implants continue to be high. Whether nonclassical cardiovascular risk markers such as vitamin D metabolites and fibroblast growth factor (FGF)-23 contribute to this risk remains to be studied, and this was the objective of our work. METHODS: In 154 LVAD patients (91 HeartWare and 63 HeartMate II implants), we measured circulating 25-hydroxyvitamin D (25OHD), 1,25-dihydroxyvitamin D3 (1,25[OH]2D3), parathyroid hormone (PTH) and FGF-23 shortly before LVAD implantation and investigated their association with stroke and mortality risk during 1-year follow-up. RESULTS: Of the study cohort, 34.4 and 92.2%, respectively, had deficient 25OHD (<25 nmol/l) and 1,25(OH)2D3 (<41 pmol/l) values, whereas 42.6 and 98.7%, respectively, had elevated PTH levels (>6.7 pmol/l) and FGF-23 values above the reference range (100 RU/ml). One-year freedom from stroke was 80.9 %, and 1-year survival was 64.3%. The multivariable-adjusted hazard ratio of stroke was 2.44 (95% CI: 1.09-5.45; P = 0.03) for the subgroup of 25OHD levels <25 nmol/l (reference group: 25OHD levels ≥25 nmol/l). The multivariable-adjusted hazard ratio of 1-year mortality was 2.78 (95% CI: 1.52-5.09; P = 0.001) for patients with 25OHD levels <25 nmol/l compared with patients with 25OHD levels ≥25 nmol/l. PTH, FGF-23 and 1,25(OH)2D3 were not associated with stroke or mortality risk. CONCLUSIONS: In LVAD patients, deficient 25OHD levels are independently associated with high stroke and mortality risk. If confirmed in randomized controlled trials, preoperative correction of deficient vitamin D status could be a promising measure to reduce stroke and mortality risk in LVAD patients.

Systematic Evaluation of Different Nucleic Acid Amplification Assays for Cytomegalovirus Detection: Feasibility of Blood Donor Screening.

Acute primary cytomegalovirus (CMV) infections, which commonly occur asymptomatically among blood donors, represent a significant risk for serious morbidity in immunocompromised patients (a major group of transfusion recipients). We implemented a routine CMV pool screening procedure for plasma for the identification of CMV DNA-positive donors, and we evaluated the sensitivities and performance of different CMV DNA amplification systems. Minipools (MPs) of samples from 18,405 individual donors (54,451 donations) were screened for CMV DNA using the RealStar CMV PCR assay (Altona Diagnostic Technologies), with a minimum detection limit of 11.14 IU/ml. DNA was extracted with a high-volume protocol (4.8 ml, Chemagic Viral 5K kit; PerkinElmer) for blood donor pool screening (MP-nucleic acid testing [NAT]) and with the Nuclisens easyMAG system (0.5 ml; bioMérieux) for individual donation (ID)-NAT. In total, six CMV DNA-positive donors (0.03%) were identified by routine CMV screening, with DNA concentrations ranging from 4.35 × 10(2) to 4.30 × 10(3) IU/ml. Five donors already showed seroconversion and detectable IgA, IgM, and/or IgG antibody titers (IgA(+)/IgM(+)/IgG(-) or IgA(+)/IgM(+)/IgG(+)), and one donor showed no CMV-specific antibodies. Comparison of three commercial assays, i.e., the RealStar CMV PCR kit, the Sentosa SA CMV quantitative PCR kit (Vela Diagnostics), and the CMV R-gene PCR kit (bioMérieux), for MP-NAT and ID-NAT showed comparably good analytical sensitivities, ranging from 10.23 to 11.14 IU/ml (MP-NAT) or from 37.66 to 57.94 IU/ml (ID-NAT). The clinical relevance of transfusion-associated CMV infections requires further investigation, and the evaluated methods present powerful basic tools providing sensitive possibilities for viral testing. The application of CMV MP-NAT facilitated the identification of one donor with a window-phase donation during acute primary CMV infection.

Association between circulating 25-hydroxyvitamin D levels and medication use in patients scheduled for cardiac surgery.

BACKGROUND AND AIM: Low vitamin D status, i.e. circulating 25-hydroxyvitamin D (25OHD) levels <50 nmol/l, is independently associated with increased CVD risk. Medication use may influence 25OHD levels. We therefore investigated the association of circulating 25OHD with medication use in patients scheduled for cardiac surgery. METHODS AND RESULTS: A total of 11,256 patients were included in this cross-sectional study. We compared 25OHD levels of medication users (18 groups of continuously used and 5 groups of intermittently used medications) with levels of non-users. Moreover, we assessed variables (medications, demographic and clinical parameters) that were independently associated with 25OHD levels <50 nmol/l. The prevalence of 25OHD levels <50 nmol/l was 65.7%. The use of statins and immunosuppressive agents was significantly associated with higher 25OHD levels and lower odds ratios of 25OHD levels <50 nmol/l. The use of ACE-inhibitors, catecholamines and antibiotics was associated with lower 25OHD levels and higher odds ratios of 25OHD levels <50 nmol/l. However, only use of antibiotics, immunosuppressive agents and catecholamines showed clinically relevant differences in 25OHD levels, i.e. differences of more than +4 nmol/l or -4 nmol/l, compared with respective non-users. These medications were prescribed either intermittently (antibiotics, catecholamines) and/or infrequently (<2%; immunosuppressive agents, catecholamines) and/or its causal relationship with circulating 25OHD is questionable (antibiotics). Female sex and blood drawing during wintertime were associated with the highest odds ratios of 25OHD levels <50 nmol/l. CONCLUSION: Data indicate that in patients with high cardiovascular risk profile medication use does not substantially contribute to 25OHD levels <50 nmol/l.

Comparison of real-time PCR and antigen assays for detection of hepatitis E virus in blood donors.

Hepatitis E virus (HEV) infection is recognized as an emerging and often undiagnosed disease in industrialized countries, with asymptomatic infections actually occurring in blood donors. Sensitive detection of HEV-RNA is crucial for diagnosis and monitoring of disease progression. We evaluated the analytical sensitivity and performance of three HEV RT-PCR assays (RealStar HEV reverse transcription-PCR [RT-PCR], hepatitis@ceeramTools, and ampliCube HEV RT-PCR) for screening of individuals for HEV infections (ID-nucleic acid amplification technology [ID-NAT]) and for blood donor pool screening (minipool-NAT [MP-NAT]). RNA was extracted using NucliSens easyMAG (ID-NAT) and a high-volume extraction protocol (4.8 ml, chemagic Viral 5K, MP-NAT). Three NAT assays were evaluated for ID-NAT but only two assays for MP-NAT due to inhibition of the ampliCube HEV RT-PCR kit using the corresponding RNA extract. Assays provided good analytical sensitivity, ranging from 37.8 to 180.1 IU/ml (ID-NAT) and from 4.7 to 91.2 IU/ml (MP-NAT). The applicability of HEV antigen (HEV-Ag) screening was compared to that of RT-PCR screening and detection of HEV-IgM antibodies using seroconversion panels of 10 HEV genotype 3-infected individuals. Four individuals revealed a positive HEV-Ag detection result, with corresponding viremias ranging from 1.92 E + 03 to 2.19 E + 05 IU/ml, while the progression of HEV-Ag followed that of HEV viremia. The other six individuals showed no presence of HEV-Ag although the corresponding viremias were also in the range of >1.0 E + 03. Anti-HEV-IgM antibodies were detectable in seven donors; one donor presented parallel positivities of HEV-Ag and anti-HEV IgM. The evaluated NAT methods present powerful tools providing sensitive HEV detection. Application of HEV-Ag or anti-HEV IgM screening is currently inferior for the early detection of HEV infection due to the decreased sensitivity compared to NAT methods.

Development and application of a multilocus sequence typing scheme for Streptococcus gallolyticus subsp. gallolyticus.

Streptococcus gallolyticus subsp. gallolyticus (formerly known as S. bovis biotype I) is a commensal of the gastrointestinal tract in animals and in up to 15% of healthy humans. Furthermore, it is a facultative pathogen that can cause infectious endocarditis, mastitis, and septicemia. The number of infections is increasing, but the transmission routes and zoonotic potential remain unknown. To assess the zoonotic potential and characterize the epidemiological structure of S. gallolyticus subsp. gallolyticus, we established a multilocus sequence typing (MLST) scheme. We amplified and sequenced internal fragments of seven housekeeping genes. The resulting sequences were analyzed with BioNumerics software 6.6 by using the unweighted-pair group method using average linkages algorithm. A total of 101 S. gallolyticus subsp. gallolyticus strains isolated from animals, humans, and environmental samples were analyzed and divided into 50 sequence types. Our first results highlight the importance of this MLST scheme for investigating the epidemiology, transmission patterns, and infection chains of S. gallolyticus subsp. gallolyticus.

Human xylosyltransferase-I - a new marker for myofibroblast differentiation in skin fibrosis.

Skin fibrosis is a severe type of fibrotic disorder emerging in terms of hypertrophic scars or systemic sclerosis. Key event of fibrogenesis is the transition of fibroblasts to matrix-producing myofibroblasts. In the presence of fibrotic triggers, for instance secretion of profibrotic growth factors like transforming growth factor-ß1 (TGF-ß1) or mechanical strain, myofibroblasts persist. Current research focuses on discovering innovative myofibroblast biomarkers which are regulated in fibrotic development and accessible for antifibrotic inhibition. Here, we consider the suitability of xylosyltransferase-I (XT-I) as a myofibroblast biomarker in skin fibrosis. XT-I catalyzes the initial step of glycosaminoglycan biosynthesis. Its increase in enzymatic activity is known to refer only to manifested diseases which are characterized by an abnormal rate of proteoglycan biosynthesis. In this study, treatment of normal human dermal fibroblasts (NHDF) with TGF-ß1 was followed by increased relative XYLT1 mRNA expression. Remarkably, this upregulation was strongly dependent on myofibroblast content, increasing during fibrogenesis. Moreover, XT activity increased time-dependently in response to progressive myofibroblast transformation. XYLT1 expression was inhibited by TGF-ß receptor I (ALK5) inhibitor SB431542. In contrast, XYLT2 expression was only marginally affected by TGF-ß1 as well as ALK5 inhibition. Our results strengthen the significance of XT expression and activity in fibrotic remodeling. Therefore, we propose XT activity, in addition to α-SMA expression, as a new biomarker for myofibroblast differentiation and fibrotic development. Further studies are now needed to evaluate the option to control and inhibit fibrotic remodeling by interfering with XT expression.

Inter-laboratory comparison of different rapid methods for the detection of bacterial contamination in platelet concentrates.

BACKGROUND: Bacterial contamination of platelet concentrates still represents a major risk in transfusion medicine, and a variety of screening methods have been available to improve the safety of PCs. In the present study, the analytical quality of three different rapid screening methods (BactiFlow flow cytometry, Pan Genera Detection Assay, 23S rRNA RT-PCR) was evaluated in an inter-laboratory comparison in three different German blood services. METHODS: Samples were inoculated with different bacteria [Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli (two strains), Klebsiella pneumoniae (two strains), Enterobacter aerogenes (one strain), Serratia marcescens (one strain)] at different counts (4·5 × 10(3) -4·5 × 10(8) CFU/ml) alternating with negative samples in one transfusion facility. Samples were blinded with a random order for each screening method, shipped to partners and analysed immediately after receipt with different rapid screening methods. RESULTS: The inter-laboratory comparison revealed that the BactiFlow assay and 23S rRNA RT-PCR-screening detected all samples correctly (positive: 12/12, negative: 8/8). The Pan Genera Detection Assay test detected only four of the positive samples. Four of the non-detected positive samples were below the assay's detection limit. Another four inoculated samples with comparatively high bacteria counts were detected false negative (E. coli (two strains): 9·87 × 10(5) and 2·10 × 10(7) CFU/ml, respectively, K. pneumoniae: 4·79 × 10(6) CFU/ml, S. aureus: 6·03 × 10(5) CFU/ml). All rapid screening methods revealed no false-positive results. CONCLUSIONS: Both BactiFlow and 23S rRNA RT-PCR demonstrated a high sensitivity to detecting bacterial contamination in PCs. The Pan Genera Detection Assay had some shortcomings regarding sensitivity, especially for the detection of Gram-negative strains.

Detection of bacterial contamination in platelet concentrates by a sensitive flow cytometric assay (BactiFlow): a multicentre validation study.

BACKGROUND: Bacterial contamination of platelet concentrates (PCs) still represents an ongoing risk. As a result of septic complications, particularly observed with older PCs, the shelf life of PCs has been reduced in Germany to 4 days. In this study, bacterial screening of PCs by BactiFlow (BF) flow cytometry was introduced in three German blood services to evaluate the robustness and applicability of the assay. Results were used to discuss the potential for the extension of PC shelf life to 5 days. STUDY DESIGN AND METHODS: A total of 1956 PCs were tested on days 4 or 5+ after PC production using the BF, whereas the BacT/Alert culture system served as reference method. RESULTS: Two PCs were confirmed positive by culture only and were identified as Propionibacterium acnes and Staphylococcus species. Two PCs were confirmed positive for Streptococcus mitis by BF and culture. Additionally, two PCs were culture-positive only in one culture bottle (aerobic: S. mitis and anaerobic: S. hominis). Retrospective analysis of bacterial growth kinetics provide the indication that corresponding bacterial titres were most likely below the BF analytical detection limit (<150 CFU mL(-1) ) and had probably no transfusion relevance. All remaining specimens were tested negative. CONCLUSIONS: Testing of PCs by BF was successfully implemented. The BF proved sufficient as a rapid screening method to improve PC safety. This study further provides data supporting the extension of PC shelf life to 5 days after negative BF testing on day 4.

Vitamin D supplementation of 4000 IU daily and cardiac function in patients with advanced heart failure: The EVITA trial.

BACKGROUND: Data regarding the effects of vitamin D on cardiac function are inconclusive. METHODS: In a post-hoc analysis of the EVITA (Effect of vitamin D on mortality in heart failure) trial, we investigated whether a daily vitamin D3 supplement of 4000 IU for three years affects echocardiography parameters like left ventricular end-diastolic diameter (LVEDD), LV end-systolic diameter (LVESD), and LV ejection fraction (LVEF) in patients with advanced heart failure (HF) and 25­hydroxyvitamin D levels <75 nmol/L. Of 400 patients enrolled, 199 were assigned to vitamin D and 201 to placebo. We assessed time × treatment interaction effects using linear mixed models and analyzed in subgroups vitamin D effects at 12 and 36 months post-randomization using analysis of covariance with adjustments for baseline values. RESULTS: At baseline, values of LVEDD, LVESD, and LVEF were 67.5 ±â€¯10.5 mm, 58.9 ±â€¯12.0 mm, and 30.47 ±â€¯10.2%, respectively. There were no time × treatment interaction effects on LV echocardiographic parameters in the entire study cohort, neither at 12 months nor at 36 months post-randomization (P-values > 0.05). However, in the subgroup of patients aged ≥50 years, vitamin D treatment was associated with an increase in LVEF of 2.73% (95%CI: 0.14 to 5.31%) at 12 months post-randomization (n = 311). The increase was slightly attenuated to 2.60% (95%CI: -2.47 to 7.67%) at 36 months post-randomization (n = 242). CONCLUSION: Our data indicate that vitamin D supplementation does not significantly improve cardiac function in all patients with advanced HF. However, vitamin D probably improves LV function in HF patients aged ≥50 years.

Heart Transplantation in Systemic Sclerosis: New Impulses for Conventional Scleroderma Transplantation Regimen and Scleroderma Diagnostic Monitoring: 2 Case Reports.

BACKGROUND: Although low (but increasing) rates of lung/lung-heart transplantations of scleroderma (systemic sclerosis [SSc]) patients have been reported, exclusive heart transplantation is a rare approach for treatment of heart failure due to SSc. CASES: We report on 2 cases of SSc patients receiving a heart transplantation (HTx) due to severe and progressive right heart failure without pulmonary artery hypertension. One patient received a hepatitis C virus (HCV)-positive donor heart and recovered excellently from viral transmission after administration of a direct-acting antiviral (DAA) regimen. This is the first published case of an SSc patient who underwent HTx using an HCV-positive donor heart. The clinical course of both patients was monitored by different serum SSc biomarkers. Only xylosyltransferase activity proved to be a promising biomarker for disease stage determination and therapeutic monitoring, precisely reflecting fibrotic remodeling and successful organ recovery. CONCLUSIONS: Successful implementation of the 2 cases described here demonstrates that HTx is a safe and effective therapeutic option for defined SSc sub-patient groups despite the progressive character of the underlying disease. In the future, xylosyltransferase activity might be conducive to simplify the identification of patients with low systemic involvement but a strong indication for single heart transplantation. Finally, we demonstrate that treatment of HCV viral transmission from HCV-positive donor to organ recipient using DAA gives us new opportunities to consider HCV-positive donor organs for HTx and might reveal new possibilities to ease the lack of donor organs.

Interference of DOACs in different DRVVT assays for diagnosis of lupus anticoagulants.

OBJECTIVE: Determination of lupus anticoagulants (LA) is an important, but still challenging test in the diagnosis of antiphospholipid syndrome (APS). This is especially the case in patients using one of the direct oral anticoagulants (DOACs). The aim of our study was to examine the influence of these drugs on DRVVT assays from two companies (in each case: screening test, confirming test and calculated ratio) and on aPTT and lupus-sensitive aPTT. METHODS: We used plasma samples from healthy volunteers spiked with the DOACs dabigatran, rivaroxaban and apixaban (0, 10, 30, 50, 100 ng/mL) for testing. Furthermore, samples from patients receiving a DOAC were investigated. The plasma concentrations of the DOACs were determined using ultra-performance liquid chromatography/electrospray ionization-tandem mass spectrometry (UPLC-MS/MS). RESULTS: Depending on type and concentration, all the DOACs resulted in pathological values in the DRVVT screening assays. In samples spiked with apixaban, no influence on the DRVVT normalized ratio of the two assays was observed, but 7 to 15% of samples from patients receiving apixaban displayed pathological values. In contrast, up to 71% of dabigatran-spiked samples showed normalized ratio values above the cut-off, whereas there was no influence in the patients' samples. In both spiked and patient samples containing rivaroxaban, the DRVVT assays were influenced. CONCLUSION: LA diagnostics should, under DOAC therapy, be limited to situations in which time-critical evaluation is warranted. It is crucial to take into account the finding that even samples containing DOAC concentrations below the limit of detection of the drugs may lead to false-positive DRVVT measurements.

Effect of v-rasH oncogene transfection on estrogen-independent tumorigenicity of estrogen-dependent human breast cancer cells.

Spontaneous or therapeutically induced progression of hormone-dependent human breast cancer to a form not amenable to endocrine treatment has been frequently recorded in clinical settings. In an experimental model system, we have changed the estrogen-dependent tumorigenicity of a human breast cancer cell line, MCF-7, to an independent state by stably introducing a model oncogene, v-rasH, into this cell line by means of DNA transfection. We now show that the oncogene-transfected hormone-independent MCF-7 cells may secrete diffusible tumorigenic factors that not only support their own tumor growth in vivo, but are also humorally active in partially triggering the tumor growth of wild type previously nontumorigenic MCF-7 cells, even when the wild type cells are implanted at a distant anatomical site in the same animal. Estrogen-independent tumor formation by MCF-7 cells was also induced in 50% of animals given injection by continuous administration of conditioned media from MCF-7-ras cells. However, the wild type tumors had limited tumor growth. Tumors were verified as adenocarcinomas and by Southern blotting were shown to be derived from the cells injected. In an in vitro coculture assay, a 5- to 7-fold enhancement in anchorage-independent growth of MCF-7 cells was observed in the presence of MCF-7-ras feeder cell layer. These data suggest that v-rasH-induced estrogen-independent tumorigenicity of human breast cancer cells occurs by secretion of mitogens which may function in an endocrine manner.

Transforming growth factor beta 2 (TGF-beta 2) levels in plasma of patients with metastatic breast cancer treated with tamoxifen.

Blood levels of transforming growth factor beta 2 (TGF-beta 2) were measured in 20 patients with metastatic breast cancer before and during treatment with the antiestrogen tamoxifen, and in a control group of 7 patients with primary breast cancer before and during adjuvant tamoxifen treatment. The results of this study reveal typical time patterns for TGF-beta 2 in relation to the clinical outcome. Patients in remission showed a significant increase of TGF-beta 2 in the first 4-6 weeks of therapy, followed by a subsequent decrease. Patients who did not respond showed unchanged or diminished TGF-beta 2 values after start of therapy, followed by a later increase preceding the clinical manifestation of tumor progression. Thus, TGF-beta 2 blood levels after 4 weeks of tamoxifen treatment can be used as an early marker for prediction of response.

Partial antagonism between steroidal and nonsteroidal antiestrogens in human breast cancer cell lines.

Nonsteroidal antiestrogens, such as tamoxifen, are well established in the treatment of breast cancer. The development of new steroidal compounds without partial agonist activity allows deeper insights into the mechanism of antiestrogen action, but thus far, the combined use of steroidal and nonsteroidal antiestrogens has not been studied extensively. We compared the nonsteroidal 4-trans-hydroxytamoxifen (OHT) with the two steroidal antiestrogens, ICI 182780 and RU 58668, in the estrogen receptor-positive human breast cancer cell lines MCF-7 and T47D. The effect of each compound alone or of OHT in combination with one of the steroidal antiestrogens was studied in regard to cell proliferation, expression of estrogen receptors (ERs) and progesterone receptors, and secretion of transforming growth factor beta2 (TGF-beta2). All antiestrogens examined led to enhanced secretion of TGF-beta2, which is correlated with their individual growth-inhibitory potential. OHT partially counteracts the larger growth inhibition of human breast cancer cells exerted by the steroidal antiestrogens ICI 182780 and RU 58668. Also, OHT antagonizes the higher induction of TGF-beta2 seen after treatment of MCF-7 cells with steroidal antiestrogens. The loss of ER and down-regulation of progesterone receptor under treatment with the steroidal antiestrogens is prevented by OHT, whereas the steroidal antiestrogens prevent the ability of hydroxytamoxifen to increase the ER content. These results indicate that TGF-beta2 is a marker of action for both types of compounds, but steroidal and nonsteroidal antiestrogens partially antagonize each other in blocking ER-mediated cellular events. It would appear that no additive or synergistic effect of the two types of antiestrogens can be expected in the treatment of breast cancer.

Transforming growth factor beta production and responsiveness in normal human melanocytes and melanoma cells.

Previous studies have shown that some human melanoma cells express transforming growth factor beta (TGF-beta) mRNA and are growth inhibited by exogenous TGF-beta, suggesting a possible negative autocrine role for this melanoma-derived growth factor. To better understand the role of endogenous TGF-beta in the development of melanoma, we investigated patterns of TGF-beta protein production and responsiveness of human melanoma cells as compared to normal melanocytes. Both cultured melanoma cells and normal melanocytes secreted biologically inactive, latent TGF-beta protein which, upon acid treatment, became biologically active. In melanoma cells, TGF-beta production occurred constitutively, i.e., in the absence of exogenous polypeptide growth factors. By contrast, in melanocytes, TGF-beta production depended on stimulation by exogenous growth factors such as insulin-like growth factor I. Exogenous, bioactive TGF-beta 1 at picomolar concentrations inhibited tritiated thymidine uptake of normal melanocytes, whereas melanoma cells demonstrated various degrees of resistance to TGF-beta-induced inhibition of DNA synthesis. Five of six cell lines were less sensitive than any of the melanocyte lines tested, and one cell line was completely resistant to inhibitory effects of TGF-beta on DNA synthesis. In vivo selection of melanoma cells for metastatic ability in athymic mice produced a variant cell line that was resistant to TGF-beta 1-induced inhibition of DNA synthesis and proliferation. Development of TGF-beta resistance in the variant cell line was not associated with changes in TGF-beta cell surface binding. Stable transfection of melanocytes with a plasmid expressing the Simian Virus 40 large T-antigen rendered these cells resistant to growth inhibition by TGF-beta, suggesting that TGF-beta inhibits melanoma/melanocyte growth via interaction with Simian Virus 40 large T-antigen-responsive transcription elements.