Serious Stem Cell Donation Events and Recipient Adverse Reactions Related to Severe Acute Respiratory Syndrome Coronavirus 2: Review of Reports to the World Marrow Donor Association.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has deeply impacted hematopoietic stem cell (HSC) donation and transplantation. Numerous changes in practice have been introduced, and monitoring the impact of these changes on donations and transplantations is of vital importance. As part of a global response to this pandemic, the World Marrow Donor Association (WMDA) asked that its member registries and cord blood banks submit SARS-CoV-2-related adverse events to the WMDA-operated Serious Product Events and Adverse Reactions (SPEAR) database. Here we review SARS-CoV-2-related SPEAR events that occurred in 2020. The WMDA SPEAR Committee reviewed reports submitted via an online tool. The Committee reviewed each report following the European Union definitions of a serious adverse event or reaction and determined the imputability and its impact. Reports submitted in 2020 were included in this analysis. A TOTAL OF: 74 such reports were received, and events were classified as donor-related (n = 41; 55.4%), recipient-related (n = 3; 4.1%), technical issues (n = 31; 41.8%), or transport-related issues (n = 4; 5.4%). Five cases appeared in multiple categories. The most frequently reported adverse events were of cells being unused. Many of these cases were caused by the uncoupling of the donation and transplantation consequent on the cryopreservation of products, as well as technical issues related to cell viability. Experience in some registries suggests that these issues have become less frequent as transplantation centers have become used to the changes in practice. Lessons learned include the importance of confirming recipient eligibility before the start of donor mobilization or collection and of minimizing the time between cell collection and transplantation. Transplantation centers should familiarize themselves with the expected cell losses when peripheral blood stem cell and bone marrow products are cryopreserved and should have validated viability assays in place for quality assurance. Reassuringly, there were no reports of donors becoming severely unwell because of G-CSF or transmission of SARS-CoV-2 to recipients and only 1 report of complete failure of transport of a donation.

Validation of a rapid potency assay for cord blood stem cells using phospho flow cytometry: The IL-3-pSTAT5 assay.

INTRODUCTION: Public cord blood banks (CBBs) are required to measure cord blood units (CBUs) potency before their release, allowing for the identification of units that may be unsuitable for haematopoietic transplantation. We have developed a rapid flow cytometry assay based on the measurement of STAT-5 phosphorylation of CD34+ stem cells in response to IL-3 stimulation. METHOD: To adapt the assay from a research setting to its implementation within our CBB regulated operations, we proceded with a full method validation and a correlation comparison of the IL-3-pSTAT5 assay results with the colony-forming unit assay (CFU) results. A total of 60 CBUs cryopreserved in vials were analysed by flow cytometry to determine the sensitivity, specificity, intra-assay precision, robustness, reproducibility, and inter-laboratory agreement of the assay. The CFU assay was also done on the same samples for comparison purposes. RESULTS: The assay threshold was established at 50% CD34+CD45+pSTAT5+, which provides a 100% sensitivity and a 98.3% specificity. An average intra-assay CV of 7.3% was determined. All results met our qualitative results acceptance criteria regarding the inter-user and inter-laboratory agreements, IL-3 stimulation time, post-thaw incubation delay and staining time. The IL-3-pSTAT5 assay results correlated well with the total CFU determined using the CFU assay (r2  = 0.82, n = 56). CONCLUSION: This study shows that our rapid flow cytometry assay can be successfully validated and that the potency data obtained display good sensitivity, specificity and robustness. These results demonstrate the feasibility of implementing this assay within CBB operations, as a validated potency assay.

Multicenter evaluation of the IL-3-pSTAT5 assay to assess the potency of cryopreserved stem cells from cord blood units: The BEST Collaborative study.

BACKGROUND: The IL-3-pSTAT5 assay, a new, rapid, and standardized flow-cytometry-based assay may compensate for several limitations of the colony-forming unit (CFU) assay typically used for stem cell potency assessments of cord blood units (CBU). We performed an inter-laboratory evaluation of the performance of this new assay. STUDY DESIGN AND METHODS: This Biomedical Excellence for Safer Transfusion (BEST) Collaborative multicenter, international study included 15 participants from public cord blood banks (CBBs), CBB-supporting research laboratories, and stem cell laboratories. To perform the IL-3-pSTAT5 assay, participating centers received reagents, instructions, and 10 blind CBU samples, including eight normal samples and two samples exposed to a transient warming event. We measured inter-laboratory agreement qualitatively (proportion of correctly classified samples) and quantitatively (coefficient of variation [CV], correlation coefficients, receiver operating characteristics (ROC) curve, and intraclass correlation coefficient [ICC]). RESULTS: The qualitative agreement was 97.3% (i.e., 107/110; Fleiss' kappa = 0.835). The average CV on a per-sample basis was 11.57% among all samples, 8.99% among normal samples, and on a per-center basis was 9.42% among normal samples. In a correlation matrix that compared results across centers, the mean Pearson's correlation coefficient was 0.88 (standard deviation = 0.04). The ICC was 0.83 (95% confidence interval = 0.68-0.95). The area under the curve (AUC) from the ROC curve was 0.9974. DISCUSSION: Excellent qualitative and quantitative agreement was exhibited across laboratories. The IL-3-pSTAT5 assay may therefore be implemented in flow cytometry laboratories to rapidly and reliably provide standardized measures of stem cell potency in CBUs.

Rapid potency assessment of autologous peripheral blood stem cells by intracellular flow cytometry: the PBSC-IL-3-pSTAT5 assay.

BACKGROUND AIMS: The current gold standard for stem cell product potency assessment, the colony-forming unit (CFU) assay, delivers results that are difficult to standardize and requires a substantial amount of time (up to 14 days) for cellular growth. Recently, the authors developed a rapid (<24 h) flow cytometry assay based on the measurement of intracellular phosphorylated STAT5 (pSTAT5) in CD34+ cord blood stem and progenitor cells in response to IL-3 stimulation. The present work presents a novel adaptation of the protocol for use with autologous peripheral blood stem cells (PBSCs) and a performance comparison with the CFU assay. METHODS: The flow cytometry intracellular staining assay was optimized for PBSCs, and patient samples were analyzed using the PBSC-IL-3-pSTAT5 and CFU assays. Warming events were also simulated to emulate impaired potency products. RESULTS: Optimization led to minor protocol adjustments, such as removal of the red blood cell lysis step, the addition of a formaldehyde fixation step and an increase in anticoagulant concentration. The PBSC-IL-3-pSTAT5 assay discriminated between normal and impaired samples and identified 100% (18 of 18) of the impaired samples, thus showing better specificity than the CFU assay. CONCLUSIONS: The updated IL-3-pSTAT5 potency assay has several important advantages, such as accelerating the release of autologous stem cell products and enabling the detection of potentially impaired products. The assay could also be used to rapidly assess the potency of any cryopreserved allogeneic stem cell product, such as those processed during the coronavirus disease 2019 pandemic.

Cryopreserved hematopoietic stem/progenitor cells stability program-development, current status and recommendations: A brief report from the AABB-ISCT joint working group cellular therapy product stability project team.

BACKGROUND: The AABB-ISCT Joint Working Group Stability Project Team (SPT) was assigned to roadmap a path toward standardization of cryopreserved hematopoietic stem/progenitor cell (HSPC) stability programs. HSPC stability encompasses a broad scope of conditions including non-frozen ("fresh") and cryopreserved cell products, and varying methods for storage, thaw, and administration. This report assessed current practices and focused solely on cryopreserved HSPC cell therapy products to establish preliminary recommendations for a stability program roadmap. METHODS: A survey was prepared by the SPT and distributed to ISCT and AABB members. Survey results were summarized and recommendations were outlined based on the responses from the survey. This report highlights current practices for cryopreserved HSPC stability programs, including additional considerations and recommendations. RESULTS AND DISCUSSION: Eighty-two (82) centers worldwide participated in the survey. Survey results indicate variability across programs. HSPC stability depends on multiple factors within the processing facility (e.g., cryopreservation techniques, reagents used, and storage temperature) and independent variables (e.g., donor-related factors and starting material variability). While retention of hematopoietic engraftment potential is the primary goal for cryopreserved HSPC stability, engraftment results should not be used as the sole metric for stability programs. Based on the survey results, the SPT provides recommendations for consideration. CONCLUSIONS: The SPT recommendations for best practices are not intended to replace existing standards. The survey results emphasize the need for the community to optimize best practices and consider initiating collaborative projects to improve the standardization of cryopreserved HSPC stability programs for cell therapy products.

Cryopreserved hematopoietic stem/progenitor cells stability program-development, current status and recommendations: A brief report from the AABB-ISCT joint working group cellular therapy product stability project team.

BACKGROUND: The AABB-ISCT Joint Working Group Stability Project Team (SPT) was assigned to roadmap a path toward standardization of cryopreserved hematopoietic stem/progenitor cell (HSPC) stability programs. HSPC stability encompasses a broad scope of conditions including non-frozen ("fresh") and cryopreserved cell products, and varying methods for storage, thaw, and administration. This report assessed current practices and focused solely on cryopreserved HSPC cell therapy products to establish preliminary recommendations for a stability program roadmap. METHODS: A survey was prepared by the SPT and distributed to ISCT and AABB members. Survey results were summarized and recommendations were outlined based on the responses from the survey. This report highlights current practices for cryopreserved HSPC stability programs, including additional considerations and recommendations. RESULTS AND DISCUSSION: Eighty-two (82) centers worldwide participated in the survey. Survey results indicate variability across programs. HSPC stability depends on multiple factors within the processing facility (e.g., cryopreservation techniques, reagents used, and storage temperature) and independent variables (e.g., donor-related factors and starting material variability). While retention of hematopoietic engraftment potential is the primary goal for cryopreserved HSPC stability, engraftment results should not be used as the sole metric for stability programs. Based on the survey results, the SPT provides recommendations for consideration. CONCLUSIONS: The SPT recommendations for best practices are not intended to replace existing standards. The survey results emphasize the need for the community to optimize best practices and consider initiating collaborative projects to improve the standardization of cryopreserved HSPC stability programs for cell therapy products.

Preparation and growth factor characterization of cord blood-derived plasma, serum, growth factor-rich plasma and induced serum.

BACKGROUND: In recent years, there has been a significant decline in the demand for cord blood units (CBUs). This trend has led to cord blood banks (CBBs) exploring complementary uses of CBUs in order to exploit the full potential of this unique, valuable, and readily available product. The aim of this study was to establish standardized protocols for the preparation of four cord blood (CB) derivatives: plasma (CB-P), serum (CB-S), induced-serum (CB-IS) and growth factor-rich plasma (GFRP); to measure their respective growth-factor content and their potential as cell culture supplements, and finally, to identify the characteristics of each derivative beyond their growth-factor composition, in the context of optimizing CBBs resources. STUDY DESIGN AND METHOD: To this end, CB plasma and serum were prepared and their concentrations for ten growth factors (TGF-ß1, TGF-ß2, TGF-ß3, EGF, HGF, PDGF-BB, VEGF-A, VEGF-D, IGF-I, IGF-II) were determined using a multiplex bead array, and compared to adult plasma and serum. Protocols for the preparation of CB-IS and GFRP with reverse anticoagulation using CaCl2·2H2O were also established, and all four derivatives were compared for their EGF and TGF-ß1 content by enzyme-linked immunosorbent assay (ELISA), and also for their cell culture supplement capacity. RESULTS: CB plasma was shown to be richer than adult plasma for TGF-ß2 and TGF-ß3, with a lower concentration of IGF-I and IGF-II. CB serum was shown to be richer than adult serum for TGF-ß2, TGF-ß3, EGF, HGF, PDGF-BB, and VEGF-A and D, and poorer in IGF-I and II. The measure of EGF and TGF-ß1 concentrations has shown that CB serum is the most concentrated (1874 pg/ml and 41 094 pg/ml) of the CB derivatives, followed by induced-serum (405 pg/ml and 16 735 pg/ml), growth factor-rich plasma (131 and 7947 pg/ml) and plasma (8 and 2845 pg/ml). All four CB derivatives were shown to be superior to FBS in sustaining cell growth at low doses. CONCLUSION: Our study shows that four CB derivatives can be easily prepared and pooled to provide significant volume of products that vary in their growth factor composition. A cord blood bank interested in introducing such manufacturing will need to evaluate the financial and processing characteristics of each derivative. The use of standardized manufacturing protocols such as the ones we suggest could help research initiatives exploring the potential therapeutic uses of such rich and high-quality starting material.

Multi-laboratory assay for harmonization of enumeration of viable CD34+ and CD45+ cells in frozen cord blood units.

BACKGROUND AIMS: In 2016, specifications for both pre-cryopreserved and post-thawed cord blood were defined in the sixth edition of NetCord Foundation for the Accreditation of Cellular Therapy (FACT) Standards for Cord Blood Banks. However, for several experts, harmonization regarding flow cytometry analysis performed on post-thawed samples is still a concern. A multicenter study led by Héma-Québec aimed to provide scientific data to support the cord blood accreditation bodies such as NetCord FACT in the revision of standards. METHODS: Twelve cord blood units were processed for plasma and red cell reduction following standard operating procedures. Cord blood unit aliquots were shipped to eight participating centers under cryogenic conditions for analysis before and after standardization of protocol. Repeatability of stem cell count, measured pre- and post-intervention with the centers, was estimated using multilevel linear regression models with a heterogeneous compound symmetry correlation structure among repeated measures. RESULTS: Excellent inter-center repeatability was reported by each participant regarding the viable CD34+ cells concentration, and a successful improvement effect of protocol standardization was also observed. However, we observed that better control over the critical parameters of the protocol did not have a significant effect on improving homogeneity in the enumeration of CD45+ cells. CONCLUSIONS: The current practice in cord blood selection should now also consider relying on post-thaw CD34+ concentration, providing that all cord blood banks or outsourcing laboratories in charge of the analysis of post-thaw CB samples take into account the consensual recommendations provided in this work and adhere to a good-quality management system.

An objective flow cytometry method to rapidly determine cord blood potency in cryopreserved units.

BACKGROUND: Cord blood banks have to determine the regenerative potential of cord blood units (CBUs) on a representative sample of the cryopreserved product before release to the transplant center. Potency can be measured by using a colony-forming unit (CFU) method, which delays the release of CBU by 7 to 14 days. To accelerate CBU qualification, we have developed a rapid method to assess the response of CD34 cells to interleukin (IL)-3. Flow cytometry was used to measure IL-3-induced STAT5 phosphorylation within CD34-cells. This IL-3 test was compared to the CFU method, as well as the aldehyde dehydrogenase (ALDH) enzyme-based assay. STUDY DESIGN AND METHODS: Ten cryopreserved CBUs were analyzed for their contents in CD34 and CD45 viable cells, total CFUs, ADLHbright cells, and IL-3-responsive CD34+ cells. Extreme and mild warming event scenarios were simulated on CBUs and used as poor-quality samples. Segments, tubes, and bags from five CBUs were compared for their potency using IL-3 and CFU methods. RESULTS: The IL-3 test was accurate in identifying the samples handled following standard operating procedures and those subjected to extreme warming events. Based on these results, a threshold of 55% of IL-3-responsive CD34 cells was established to identify good-quality samples. The IL-3 test was also the most sensitive to detect samples subjected to milder warming events. CONCLUSIONS: Our new method for determining CBU functionality is rapid, unbiased, and robust. The IL-3 test described herein fulfills the requirements for validation, and we intend to implement this method in our cord blood bank facility.

Optimization of cord blood unit sterility testing: impact of dilution, analysis delay, and inhibitory substances.

BACKGROUND: Different methods are used by cord blood banks to prepare samples for sterility testing. Suboptimal methods can result in the release of contaminated products. In our organization, samples are prepared by diluting the final product in RPMI-1640 medium. In this work, we have compared our method with different approaches to verify whether optimization should be sought. STUDY DESIGN AND METHODS: Cord blood units (n = 6 units per bacterial strain) characterized to contain inhibitory substances or not were inoculated (10 colony-forming units/mL) with Streptococcus agalactiae, Staphylococcus epidermidis, Klebsiella pneumoniae, Escherichia coli, or Bacteroides fragilis. After plasma and red blood cell removal, stem cell concentrates were diluted in RPMI-1640, thioglycollate, or the unit's plasma. These products, as well as final product, plasma, and red blood cell fractions, were held from 0 to 72 hours at 20 to 24°C before inoculation in culture bottles and detection using the BacT/ALERT 3D system. RESULTS: Dilution of cell concentrates in RPMI-1640 allowed bacterial detection in 93.3% of noninhibitory cord blood samples after a 24-hour storage period. Thioglycollate medium better promoted bacterial growth in inhibitory cord blood samples that were held for 72 hours before testing (66.7%) compared with RPMI-1640 (45.0%). Less than 33% of all spiked plasma samples were detected by the BacT/ALERT 3D system. CONCLUSION: Diluting cord blood samples in culture medium containing bacterial growth promoting substances is a suitable option for sterility testing, whereas the use of plasma should be proscribed, because it might lead to false-negative results. Because inhibitory substances affect bacterial growth, inoculation of culture bottles should be done rapidly after sample preparation.

An alternative to dextran for the thawing of cord blood units.

BACKGROUND: Preparation of umbilical cord blood units (CBUs) for infusion requires a step of dilution or washing to reduce the toxicity of the dimethyl sulfoxide present in the freezing solution. However, the worldwide shortage of clinical-grade dextran 40, the most widely used cord blood dilution or washing solution, prompted us to search for an alternative solution. We elected to evaluate the performance of alternative solutions that could be used as potential replacements for the dextran 40-based solution. STUDY DESIGN AND METHODS: Frozen CBUs were rapidly thawed and immediately diluted 1:4 in each of 10 dilution solution variants of dextran 40, Plasma-lyte, or Hespan. We compared these alternatives by assessing viability, CD34, CD45, and colony-forming units recovery postthaw. RESULTS: A significantly lower CD34 and CD45 recovery was observed in all solutions without human serum albumin (HSA). All solutions with 5% HSA gave recovery, viability, and potency figures similar to the dextran 40/HSA solution. CONCLUSION: Based on our results and considering that Plasma-lyte A (PA)/HSA is already used for the thawing of CD34 from mobilized blood, we conclude that PA/HSA could be a safe and efficient solution for the replacement of dextran 40-based dilution solutions.

Cost-effectiveness analysis of antiviral treatment in the management of seasonal influenza A: point-of-care rapid test versus clinical judgment.

BACKGROUND: A point-of-care rapid test (POCRT) may help early and targeted use of antiviral drugs for the management of influenza A infection. OBJECTIVE: (i) To determine whether antiviral treatment based on a POCRT for influenza A is cost-effective and, (ii) to determine the thresholds of key test parameters (sensitivity, specificity and cost) at which a POCRT based-strategy appears to be cost effective. METHODS: An hybrid « susceptible, infected, recovered (SIR) ¼ compartmental transmission and Markov decision analytic model was used to simulate the cost-effectiveness of antiviral treatment based on a POCRT for influenza A in the social perspective. Data input parameters used were retrieved from peer-review published studies and government databases. The outcome considered was the incremental cost per life-year saved for one seasonal influenza season. RESULTS: In the base-case analysis, the antiviral treatment based on POCRT saves 2 lives/100,000 person-years and costs $7600 less than the empirical antiviral treatment based on clinical judgment alone, which demonstrates that the POCRT-based strategy is dominant. In one and two way-sensitivity analyses, results were sensitive to the POCRT accuracy and cost, to the vaccination coverage as well as to the prevalence of influenza A. In probabilistic sensitivity analyses, the POCRT strategy is cost-effective in 66% of cases, for a commonly accepted threshold of $50,000 per life-year saved. CONCLUSION: The influenza antiviral treatment based on POCRT could be cost-effective in specific conditions of performance, price and disease prevalence.

Antimicrobial activity in cord blood units: occurrence and levels of antibiotics.

BACKGROUND: Antibiotic prophylaxis treatment at delivery is highly recommended for reducing the risk of infection for mothers positive for group B streptococcus. It is therefore expected that some cord blood (CB) products will contain residual antibiotics. This study aimed to determine the incidence and level of ß-lactam antibiotics in CB products. STUDY DESIGN AND METHODS: The antimicrobial activity of 60 CB plasma by-products was evaluated using disk diffusion assays on 10 bacteria species. Plasma samples showing antimicrobial activity were either treated with ß-lactamase enzyme to inhibit ß-lactam antibiotics or heated to 56°C for 30 minutes to inhibit complement proteins. ß-Lactam antibiotic concentrations were determined by comparison with a standard curve obtained with known concentrations of antibiotics. RESULTS: Antimicrobial activity against mostly Gram-positive microorganisms was observed in 33% of CB units. The ß-lactamase enzyme abolished the antimicrobial activity in the majority of these CB products. Up to 5 µg/mL penicillin and 14 µg/mL ampicillin were measured in these products. CONCLUSION: Approximately one-third of CB products contain significant amounts of plasma with residual antibiotics, which can affect the survival and growth of bacterial contaminants when performing the sterility test and potentially lead to false-negative results. Additional work is required to better understand whether residual antibiotics in CB affect penicillin-allergic patients.

Dabigatran versus warfarin under standard or pharmacogenetic-guided management for the prevention of stroke and systemic thromboembolism in patients with atrial fibrillation: a cost/utility analysis using an analytic decision model.

BACKGROUND: Atrial fibrillation (AF) is the most common form of heart arrhythmia and a leading cause of stroke and systemic embolism. Chronic anticoagulation is recommended for preventing those complications. Our study aimed to compare the cost/utility (CU) of three main anticoagulation options: 1) standard warfarin dosing (SD-W) 2) warfarin dosage under the guidance of CYP2C9 and VKORC1 genotyping (GT-W) and 3) dabigatran 150 mg twice a day. METHODS: A Markov state transition model was built to simulate the expected C/U of dabigatran, SD-W and GT-W anticoagulation therapy for the prevention of stroke and systemic thromboembolism in patients with atrial fibrillation over a period of 5 years under the perspective of the public health care system. Model inputs were derived from extensive literature search and government's data bases. Outcomes considered were the number of total major events (thromboembolic and hemorrhagic events), total costs in Canadian dollars (1CAD$ = 1$US), total quality-adjusted life years (QALYs), costs/QALYs and incremental costs/QALYs gained (ICUR). RESULTS: Raw base case results show that SD-W has the lowest C/U ratio. However, the dabigatran option might be considered as an alternative, as its cost per additional QALY gained compared to SD-W is CAD $ 4 765, i.e. less than 50 000, the ICUR threshold generally accepted to adopt an intervention. At the same threshold, GT-W doesn't appear to be an alternative to SD-W. Our results were robust to one-way and multi-way sensitivity analyses. CONCLUSION: SD-W has the lowest C/U ratio among the 3 options. However, dabigatran might be considered as an alternative. GT-W is not C/U and should not currently be recommended for the routine anticoagulotherapy management of AF patients.

Chemical synthesis and evaluation of 17α-alkylated derivatives of estradiol as inhibitors of steroid sulfatase.

Steroid sulfatase (STS) controls the levels of 3-hydroxysteroids available from circulating steroid sulfates in several normal and malignant tissues. This and the known involvement of active estrogens and androgens in diseases such as breast and prostate cancers thus make STS an interesting therapeutic target. Here we describe the chemical synthesis and characterization of an extended series of 17α-derivatives of estradiol (E2) using different strategies. A variant of the samarium-Barbier reaction with stoichiometric samarium metal and catalytic Kagan reagent formation was used for introducing low reactive benzyl substrates in position 17 of estrone (E1) whereas heterocyclic substrates were metalated and reacted with either the carbonyl or the 17-oxirane of E1. In vitro evaluation of the inhibitory potency of the new compounds against STS identified new inhibitors and allowed a more complete structure-activity relationship study of this family of 17α-derivatives of E2.

Binary and ternary crystal structure analyses of a novel inhibitor with 17beta-HSD type 1: a lead compound for breast cancer therapy.

Oestradiol is a well-characterized sex hormone that stimulates breast cancer and other oestrogen-related diseases. 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1) catalyses the last step in the synthesis of oestradiol and androstenediol in breast tumour tissue. The enzyme's high expression and activity after simultaneous blockade of oestrogen receptors and inhibition of aromatase in the tumour shows the necessity for its inhibition as a requirement for breast cancer therapy. In the present paper, we report structures of the binary and ternary complexes of 17beta-HSD1 with a new inhibitor E2B {3-[3',17'beta-dihydroxyestra-1',3',5'(10')-trien-16'beta-methyl]benzamide}, and the enzyme inhibition by the later. The IC50 value for E2B was determined to be 42 nM in T47D cells. Multiple interactions between E2B and the enzyme include hydrogen bonds and hydrophobic interactions, as well as pi-pi interactions. A kinetic study demonstrated that E2B inhibits the enzyme's reduction forming oestradiol from oestrone, with a Ki of 0.9+/-0.15 nM. Such strong inhibition is in agreement with its extensive interaction with the enzyme, suggesting its potential as a lead compound for breast cancer therapy. In fact, this possibility is enhanced by its capacity for cell penetration similar to natural steroids. Such inhibitors that block oestrogen synthesis to suppress the sulfatase pathway producing oestradiol can be used in adjuvant therapies with oestrogen receptor blockade, opening a new orientation of breast cancer treatment.

Aerobic biodegradation of N-nitrosodimethylamine by the propanotroph Rhodococcus ruber ENV425.

The propanotroph Rhodococcus ruber ENV425 was observed to rapidly biodegrade N-nitrosodimethylamine (NDMA) after growth on propane, tryptic soy broth, or glucose. The key degradation intermediates were methylamine, nitric oxide, nitrite, nitrate, and formate. Small quantities of formaldehyde and dimethylamine were also detected. A denitrosation reaction, initiated by hydrogen atom abstraction from one of the two methyl groups, is hypothesized to result in the formation of n-methylformaldimine and nitric oxide, the former of which decomposes in water to methylamine and formaldehyde and the latter of which is then oxidized further to nitrite and then nitrate. Although the strain mineralized more than 60% of the carbon in [(14)C]NDMA to (14)CO(2), growth of strain ENV425 on NDMA as a sole carbon and energy source could not be confirmed. The bacterium was capable of utilizing NDMA, as well as the degradation intermediates methylamine and nitrate, as sources of nitrogen during growth on propane. In addition, ENV425 reduced environmentally relevant microgram/liter concentrations of NDMA to <2 ng/liter in batch cultures, suggesting that the bacterium may have applications for groundwater remediation.