Repurposing proteasome inhibitors for improved treatment of triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is associated with poor prognosis and limited treatment options due to the lack of important receptors (estrogen receptor [ER], progesterone receptor [PR], and human epidermal growth factor receptor 2 [HER2]) used for targeted therapy. However, high-throughput in vitro drug screening of cell lines is a powerful tool for identifying effective drugs for a disease. Here, we determine the intrinsic chemosensitivity of TNBC cell lines to proteasome inhibitors (PIs), thereby identifying potentially potent 2-drug combinations for TNBC. Eight TNBC cell lines (BT-549, CAL-148, HCC1806, HCC38, HCC70, MDA-MB-436, MDA-MB-453, and MDA-MB-468) and two controls (MCF-10A and MCF-7) were first exposed to 18 drugs (11 PIs and 7 clinically relevant chemotherapeutic agents) as monotherapy, followed by prediction of potent 2-drug combinations using the IDACombo pipeline. The synergistic effects of the 2-drug combinations were evaluated with SynergyFinder in four TNBC cell lines (CAL-148, HCC1806, HCC38, and MDA-MB-468) and three controls (BT-474, MCF-7, and T47D) in vitro, followed by further evaluation of tumor regression in zebrafish tumor models established using HCC1806 and MCF-7 cells. Monotherapy identified nine effective drugs (bortezomib, carfilzomib, cisplatin, delanzomib, docetaxel, epoxomicin, MLN-2238, MLN-9708, and nedaplatin) across all cell lines. PIs (e.g., bortezomib, delanzomib, and epoxomicin) were highly potent drugs in TNBC cells, of which bortezomib and delanzomib inhibited the chymotrypsin-like activity of the 20 S proteasome by 100% at 10 µM. Moreover, several potent 2-drug combinations (e.g., bortezomib+nedaplatin and epoxomicin+epirubicin) that killed virtually 100% of cells were also identified. Although HCC1806- and MCF-7-derived xenografts treated with bortezomib+nedaplatin and carboplatin+paclitaxel were smaller, HCC1806 cells frequently metastasized to the trunk region. Taken together, we show that PIs used in combination with platinum agents or topoisomerase inhibitors exhibit increased efficiency with almost 100% inhibition in TNBC cell lines, indicating that PIs are therefore promising compounds to use as combination therapy for TNBC.

Cancer stem cells are prevalent in the basal-like 2 and mesenchymal triple-negative breast cancer subtypes in vitro.

Background: Triple-negative breast cancer (TNBC) is an aggressive subtype with the most unfavorable clinical outcomes, in part due to tumor heterogeneity, treatment resistance, and tumor relapse. The TNBC subtypes [basal-like 1 (BL1), basal-like 2 (BL2), mesenchymal (M), and luminal androgen receptor (LAR)] are biologically and clinically distinct entities that respond differently to local and systemic therapies. Therefore, we need to have a better understanding of cancer stemness relating to drug-resistant populations in the TNBC subtypes. Methods: Breast cancer stem cell (BCSC) distribution was investigated using an integrated flow cytometry approach with the ALDEFLUOR™ assay (ALDH) and CD24/CD44 antibodies. In total, 27 commercially available cell lines derived from normal and malignant mammary tissue were characterized into differentiated tumor cells and/or BCSC subpopulations (ALDH-CD44+CD24-/low enriched mesenchymal-like BCSCs, ALDH+non-CD44+CD24-/low enriched epithelial-like BCSCs, and highly purified ALDH+CD44+CD24-/low BCSCs). Results: BCSCs were not only enriched in estrogen receptor (ER) negative (mean, 49.6% versus 6.9% in ER+) and TNBC cell lines (51.3% versus 2.1% in Luminal A), but certain BCSC subpopulations (e.g., enriched mesenchymal-like BCSCs) were also significantly more common in the M (64.0% versus 6.2% in BL1; 64.0% versus 0% in LAR) and BL2 (77.4% versus 6.2% in BL1; 77.4% versus 0% in LAR; 77.4% versus 10.4% in TNBC UNS) TNBC subtypes. In contrast, ALDH status alone was not indicative of ER status or BC subtype. Conclusion: Taken together, these findings demonstrate the enrichment of potentially treatment-resistant BCSC subpopulations in the M and BL2 triple-negative breast cancer subtypes.

Optimization of Cell Viability Assays for Drug Sensitivity Screens.

During the preclinical stages of the drug discovery process, cell viability assays are fundamental tools for studying the phenotypic properties and overall health of cells following in vitro drug sensitivity screens. Therefore, it is important to optimize your viability assay of choice to obtain reproducible and replicable results, as well as use relevant drug response metrics (e.g., IC50, AUC, GR50, and GRmax) to identify candidate drugs for further evaluation in vivo. Herein, we used the resazurin reduction assay which is a quick, cost-effective, simple-to-use, and sensitive method for examining the phenotypic properties of cells. Using the MCF7 breast cancer cell line, we provide a detailed step-by-step protocol for optimizing drug sensitivity screens using the resazurin assay.

APR-246 Enhances Colorectal Cancer Sensitivity to Radiotherapy.

p53 mutation is common and highly related to radiotherapy resistance in rectal cancer. APR-246, as a small molecule, can restore the tumor-suppressor function to mutant p53. As there is currently no existing study on combining APR-246 with radiation in rectal cancer, our objective was to investigate whether APR-246 could enhance the sensitivity of colorectal cancer cells, regardless of their p53 status, to radiation treatment. The combination treatment had synergistic effects on HCT116p53-R248W/- (p53Mut) cells, followed by HCT116p53+/+ [wild-type p53 (p53WT)] cells, and exhibited an additive effect on HCT116p53-/- (p53Null) cells through inhibiting proliferation, enhancing reactive oxygen species, and apoptosis. The results were confirmed in zebrafish xenografts. Mechanistically, p53Mut and p53WT cells shared more activated pathways and differentially expressed genes following the combination treatment, compared with p53Null cells, although the combination treatment regulated individual pathways in the different cell lines. APR-246 mediated radiosensitization effects through p53-dependent and -independent ways. The results may provide evidence for a clinical trial of the combination in patients with rectal cancer.

Pan-cancer analysis of genomic and transcriptomic data reveals the prognostic relevance of human proteasome genes in different cancer types.

BACKGROUND: The human proteasome gene family (PSM) consists of 49 genes that play a crucial role in cancer proteostasis. However, little is known about the effect of PSM gene expression and genetic alterations on clinical outcome in different cancer forms. METHODS: Here, we performed a comprehensive pan-cancer analysis of genetic alterations in PSM genes and the subsequent prognostic value of PSM expression using data from The Cancer Genome Atlas (TCGA) containing over 10,000 samples representing up to 33 different cancer types. External validation was performed using a breast cancer cohort and KM plotter with four cancer types. RESULTS: The PSM genetic alteration frequency was high in certain cancer types (e.g. 67%; esophageal adenocarcinoma), with DNA amplification being most common. Compared with normal tissue, most PSM genes were predominantly overexpressed in cancer. Survival analysis also established a relationship with PSM gene expression and adverse clinical outcome, where PSMA1 and PSMD11 expression were linked to more unfavorable prognosis in ≥ 30% of cancer types for both overall survival (OS) and relapse-free interval (PFI). Interestingly, PSMB5 gene expression was associated with OS (36%) and PFI (27%), and OS for PSMD2 (42%), especially when overexpressed. CONCLUSION: These findings indicate that several PSM genes may potentially be prognostic biomarkers and novel therapeutic targets for different cancer forms.

Pan-cancer analysis identifies BIRC5 as a prognostic biomarker.

BACKGROUND: The BIRC5 gene encodes for the Survivin protein, which is a member of the inhibitor of apoptosis family. Survivin is found in humans during fetal development, but generally not in adult cells thereafter. Previous studies have shown that Survivin is abundant in most cancer cells, thereby making it a promising target for anti-cancer drugs and a potential prognostic tool. METHODS: To assess genetic alterations and mutations in the BIRC5 gene as well as BIRC5 co-expression with other genes, genomic and transcriptomic data were downloaded via cBioPortal for approximately 9000 samples from The Cancer Genome Atlas (TCGA) representing 33 different cancer types and 11 pan-cancer organ systems, and validated using the ICGC Data Portal and COSMIC. TCGA BIRC5 RNA sequencing data from 33 different cancer types and matching normal tissue samples for 16 cancer types were downloaded from Broad GDAC Firehose and validated using breast cancer microarray data from our previous work and data sets from the GENT2 web-based tool. Survival data were analyzed with multivariable Cox proportional hazards regression analysis and validated using KM plotter for breast-, ovarian-, lung- and gastric cancer. RESULTS: Although genetic alterations in BIRC5 were not common in cancer, BIRC5 expression was significantly higher in cancer tissue compared to normal tissue in the 16 different cancer types. For 14/33 cancer types, higher BIRC5 expression was linked to worse overall survival (OS, 4/14 after adjusting for both age and tumor grade and 10/14 after adjusting only for age). Interestingly, higher BIRC5 expression was associated with better OS in lung squamous cell carcinoma and ovarian serous cystadenocarcinoma. Higher BIRC5 expression was also linked to shorter progressive-free interval (PFI) for 14/33 cancer types (4/14 after adjusting for both age and tumor grade and 10/14 after adjusting only for age). External validation showed that high BIRC5 expression was significantly associated with worse OS for breast-, lung-, and gastric cancer. CONCLUSIONS: Our findings suggest that BIRC5 overexpression is associated with the initiation and progression of several cancer types, and thereby a promising prognostic biomarker.

Integrative genomics approach identifies molecular features associated with early-stage ovarian carcinoma histotypes.

Ovarian cancer comprises multiple subtypes (clear-cell (CCC), endometrioid (EC), high-grade serous (HGSC), low-grade serous (LGSC), and mucinous carcinomas (MC)) with differing molecular and clinical behavior. However, robust histotype-specific biomarkers for clinical use have yet to be identified. Here, we utilized a multi-omics approach to identify novel histotype-specific genetic markers associated with ovarian carcinoma histotypes (CCC, EC, HGSC, and MC) using DNA methylation, DNA copy number alteration and RNA sequencing data for 96 primary invasive early-stage (stage I and II) ovarian carcinomas. More specifically, the DNA methylation analysis revealed hypermethylation for CCC in comparison with the other histotypes. Moreover, copy number imbalances and novel chromothripsis-like rearrangements (n = 64) were identified in ovarian carcinoma, with the highest number of chromothripsis-like patterns in HGSC. For the 1000 most variable transcripts, underexpression was most prominent for all histotypes in comparison with normal ovarian samples. Overall, the integrative approach identified 46 putative oncogenes (overexpressed, hypomethylated and DNA gain) and three putative tumor suppressor genes (underexpressed, hypermethylated and DNA loss) when comparing the different histotypes. In conclusion, the current study provides novel insights into molecular features associated with early-stage ovarian carcinoma that may improve patient stratification and subclassification of the histotypes.

Optimization of cell viability assays to improve replicability and reproducibility of cancer drug sensitivity screens.

Cancer drug development has been riddled with high attrition rates, in part, due to poor reproducibility of preclinical models for drug discovery. Poor experimental design and lack of scientific transparency may cause experimental biases that in turn affect data quality, robustness and reproducibility. Here, we pinpoint sources of experimental variability in conventional 2D cell-based cancer drug screens to determine the effect of confounders on cell viability for MCF7 and HCC38 breast cancer cell lines treated with platinum agents (cisplatin and carboplatin) and a proteasome inhibitor (bortezomib). Variance component analysis demonstrated that variations in cell viability were primarily associated with the choice of pharmaceutical drug and cell line, and less likely to be due to the type of growth medium or assay incubation time. Furthermore, careful consideration should be given to different methods of storing diluted pharmaceutical drugs and use of DMSO controls due to the potential risk of evaporation and the subsequent effect on dose-response curves. Optimization of experimental parameters not only improved data quality substantially but also resulted in reproducible results for bortezomib- and cisplatin-treated HCC38, MCF7, MCF-10A, and MDA-MB-436 cells. Taken together, these findings indicate that replicability (the same analyst re-performs the same experiment multiple times) and reproducibility (different analysts perform the same experiment using different experimental conditions) for cell-based drug screens can be improved by identifying potential confounders and subsequent optimization of experimental parameters for each cell line.

Evaluation of ninety-six periprosthetic hip joint infections seen within five consecutive years.

Periprosthetic hip joint infections (PHJI) are severe complications. In 2003 Zimmerli published a well-noted treatment algorithm for PHJI. The aim of this study is to evaluate outcome, analyze the applied treatment regimen and compare it to the proposed algorithm. We evaluated the outcome of 96 PHJI treated at our institution between 2008 and 2012 and analysed adherence to the algorithm and outcome in coherence with the algorithm. The operations performed were irrigation and debridement with exchange of mobile parts (45%), two-stage exchange (36%), one-stage exchange (12%) and permanent explantation (7%). 47% were acute infections, 53% were chronic. Staphylococcus aureus was the most common pathogen. The overall success rate was 88%. In 12% of the cases the chosen operation didn't follow the algorithm. Of these only 10% was successfully treated with the primary operation. We find that the algorithm proposed by Zimmerli is a useful tool and easy to translate into clinical practice. When followed it yields a high success rate.

Lipopolysaccharide-induced fever depends on prostaglandin E2 production specifically in brain endothelial cells.

Immune-induced prostaglandin E2 (PGE2) synthesis is critical for fever and other centrally elicited disease symptoms. The production of PGE2 depends on cyclooxygenase-2 and microsomal prostaglandin E synthase-1 (mPGES-1), but the identity of the cells involved has been a matter of controversy. We generated mice expressing mPGES-1 either in cells of hematopoietic or nonhematopoietic origin. Mice lacking mPGES-1 in hematopoietic cells displayed an intact febrile response to lipopolysaccharide, associated with elevated levels of PGE2 in the cerebrospinal fluid. In contrast, mice that expressed mPGES-1 only in hematopoietic cells, although displaying elevated PGE2 levels in plasma but not in the cerebrospinal fluid, showed no febrile response to lipopolysaccharide, thus pointing to the critical role of brain-derived PGE2 for fever. Immunohistochemical stainings showed that induced cyclooxygenase-2 expression in the brain exclusively occurred in endothelial cells, and quantitative PCR analysis on brain cells isolated by flow cytometry demonstrated that mPGES-1 is induced in endothelial cells and not in vascular wall macrophages. Similar analysis on liver cells showed induced expression in macrophages and not in endothelial cells, pointing at the distinct role for brain endothelial cells in PGE2 synthesis. These results identify the brain endothelial cells as the PGE2-producing cells critical for immune-induced fever.

Onset time for pharmacologic premedication with clonidine as a nasal aerosol: a double-blind, placebo-controlled, randomized trial.

OBJECTIVES/AIM: To investigate whether nasal aerosol clonidine can reduce the onset time of preoperative sedation. BACKGROUND: Premedication is common in the pediatric population, but the optimal agent and administration route is still a matter of debate. Clonidine has many beneficial effects in the perioperative period. Clonidine nasal drops produce a similar sedative effect as after oral administration but do not reduce the onset time. Nasal aerosol administration of drugs is generally more effective than drops and an option to decrease the onset time of clonidine. METHODS: Pediatric ASA status 1 and 2 patients were randomized to receive placebo (P), clonidine 3-4 µg kg(-1) (C4), or clonidine 7-8 µg kg(-1) (C7) as a nasal aerosol. Acceptance of administration, pre- and postoperative sedation, and adverse events were assessed. RESULTS: A total of 60 patients were enrolled with a median age of 3.5 years (range 0.7-6.9) and median weight of 14.8 kg (range 10-25). In the C7 group, 55% of the children were found adequately sedated at 30 min as compared to 32% in the C4 group (P = 0.1202). At 45 min, adequate sedation was seen in 65% of the patients in both C4 and C7 groups, which were both found to be significantly higher compared with the placebo control group (14%) (P-values = 0.0027 and 0.0013, respectively). The postoperative sedation profile did not differ between the three study groups. CONCLUSIONS: Clonidine administered as nasal aerosol (3-8 µg kg(-1)) was not found to achieve adequate preoperative sedation within 30 min of administration. Despite its sedative properties, no prolongation of postoperative sedation was noted compared with placebo.

Oral bioavailability of clonidine in children.

BACKGROUND: Oral clonidine is used as premedication in children. The bioavailability of clonidine given orally in adults is 75-100% but is unknown in children. METHODS: Children (3-10 years) undergoing adenotonsillectomy were administered oral clonidine 4 mcg·kg(-1) mixed with apple fruit drink as premedication. Intravenous plasma was assayed for clonidine concentration at 5, 15, 30, 45 min and 1, 2, 4, 6, 12, 18 h after administration. Clonidine plasma concentrations were determined by liquid chromatography-mass spectroscopy, and pharmacokinetic parameters were calculated using nonlinear effects mixed-effects models. Current data were pooled with published time-concentration profiles from children (n = 49) administered intravenous clonidine to determine oral bioavailability. RESULTS: There were eight children studied (age 3-10 years, weight 10.5-36 kg). A two-compartment model with first-order absorption and elimination was used to describe time-concentration profiles. Population parameter estimates (CV%; 95% CI), standardized to a 70-kg person, were absorption half-life (Tabs), 0.45 (85.1; 0.221-0.884) h, absorption lag time (Tlag), 0.148 (91.2; 0.002-0.316) h, Clearance (CL) 17.9 (30.3; 16-20.3) l·h(-1) per 70 kg, between compartment clearance (Q) 121 (44.3; 80.1-165) l·h(-1) per 70 kg, central volume (V1) 81.2 (71.5; 60.7-105) l·70 kg(-1), peripheral volume of distribution (V2) 113 (33.9; 91-131) l·70 kg(-1). The oral bioavailability was 55.4% (CV 6.4%; 95% CI 0.469, 0.654). CONCLUSIONS: Clonidine administered with an apple fruit drink displays a variable and relatively slow absorption after oral administration (T(max) 1.04 h, C(max) 0.77 mcg·l(-1)). The oral bioavailability was 55.4%, which is less than reported in adults. Consequently, higher oral doses of clonidine (per kg) are required when this formulation is used to achieve concentrations similar to those reported in adults.

Hypoxia mediates low cell-cycle activity and increases the proportion of long-term-reconstituting hematopoietic stem cells during in vitro culture.

OBJECTIVE: Recent evidence suggests that hematopoietic stem cells (HSCs) in the bone marrow (BM) are located in areas where the environment is hypoxic. Although previous studies have demonstrated positive effects by hypoxia, its role in HSC maintenance has not been fully elucidated, neither has the molecular mechanisms been delineated. Here, we have investigated the consequence of in vitro incubation of HSCs in hypoxia prior to transplantation and analyzed the role of hypoxia-inducible factor (HIF)-1alpha. MATERIALS AND METHODS: HSC and progenitor populations isolated from mouse BM were cultured in 20% or 1% O(2), and analyzed for effects on cell cycle, expression of cyclin-dependent kinase inhibitors genes, and reconstituting ability to lethally irradiated mice. The involvement of HIF-1alpha was studied using methods of protein stabilization and gene silencing. RESULTS: When long-term FLT3(-)CD34(-) Lin(-)Sca-1(+)c-Kit(+) (LSK) cells were cultured in hypoxia, cell numbers were significantly reduced in comparison to normoxia. This was due to a decrease in proliferation and more cells accumulating in G(0). Moreover, the proportion of HSCs with long-term engraftment potential was increased. Whereas expression of the cyclin-dependent kinase inhibitor genes p21(cip1), p27(Kip1), and p57(Kip2) increased in LSK cells by hypoxia, only p21(cip1) was upregulated in FLT3(-)CD34(-)LSK cells. We could demonstrate that expression of p27(Kip1) and p57(Kip2) was dependent of HIF-1alpha. Surprisingly, overexpression of constitutively active HIF-1alpha or treatment with the HIF stabilizer agent FG-4497 led to a reduction in HSC reconstituting ability. CONCLUSIONS: Our results imply that hypoxia, in part via HIF-1alpha, maintains HSCs by decreasing proliferation and favoring quiescence.

The place of premedication in pediatric practice.

Behind the multiple arguments for and against the use of premedication, sedative drugs in children is a noble principle that of minimizing psychological trauma related to anesthesia and surgery. However, several confounding factors make it very difficult to reach didactic evidence-based conclusions. One of the key confounding issues is that the nature of expectations and responses for both parent and child vary greatly in different environments around the world. Studies applicable to one culture and to one hospital system (albeit multicultural) may not apply elsewhere. Moreover, the study of hospital-related distress begins at the start of the patient's journey and ends long after hospital discharge; it cannot be focused completely on just the moment of anesthetic induction. Taking an example from actual practice experience, the trauma caused by the actual giving of a premedication to a child who absolutely does not want it and may struggle may not be recorded in a study but could form a significant component of overall effect and later psychological pathology. Clearly, attitudes by health professionals and parents to the practice of routine pediatric premedication, vary considerably, often provoking strong opinions. In this pro-con article we highlight two very different approaches to premedication. It is hoped that this helps the reader to critically re-evaluate a practice, which was universal historically and now in many centers is more selective.

Clonidine disposition in children; a population analysis.

BACKGROUND: There are few data describing clonidine population pharmacokinetics in children (0-15 years) despite common use. Current pediatric data, described in terms of elimination half-life or C(max) and T(max), poorly explain variability in drug responses among individuals representative of those in whom the drug will be used clinically. METHODS: Published data from four studies investigating clonidine PK after intravenous (i.v.), rectal and epidural administration (n = 42) were combined with an open-label study undertaken to examine the pharmacokinetics of i.v. clonidine 1-2 microg x kg(-1) bolus in children after cardiac surgery (n = 41). A population pharmacokinetic analysis of clonidine time-concentration profiles (380 observations) was undertaken using nonlinear mixed effects modeling. Estimates were standardized to a 70-kg adult using allometric size models. RESULTS: Children had a mean age of 4 (sd 3.6 years, range 1 week-14 years) years and weight 17.8 (sd 12.6, range 2.8-60) kg. A two compartment disposition model with first-order elimination was superior to a one compartment model. Population parameter estimates (between subject variability) were clearance (CL) 14.6 (CV 35.1%) l x h(-1 )70 kg(-1), central volume of distribution (V1) 62.5 (71.1%) l 70 kg(-1), intercompartment clearance (Q) 157 (77.3%) l x h(-1) 70 kg(-1) and peripheral volume of distribution (V2) 119 (22.9%) l 70 kg(-1). Clearance at birth was 3.8 l x h(-1) 70 kg(-1) and matured with a half-time of 25.7 weeks to reach 82% adult rate by 1 year of age. The volumes of distribution, but not clearance, were increased after cardiac surgery (V1 123%, V2 126%). There was a lag time of 2.3 (CV 73.2%) min before absorption began in the rectum. The absorption half-life from the epidural space was slower than that from the rectum (0.98 CV 24.5% h vs 0.26 CV 32.3% h). The relative bioavailability of epidural and rectal clonidine was unity (F = 1). CONCLUSIONS: Clearance in neonates is approximately one-third that described in adults, consistent with immature elimination pathways. Maintenance dosing, which is a function of clearance, should be reduced in neonates and infants when using a target concentration approach.

Combined metabolic gas analyser and dGPS analysis of performance in cross-country skiing.

The purpose of this study was to provide a more detailed analysis of performance in cross-country skiing by combining findings from a differential global positioning system (dGPS), metabolic gas measurements, speed in different sections of a ski-course and treadmill threshold data. Ten male skiers participated in a freestyle skiing field test (5.6 km), which was performed with dGPS and metabolic gas measurements. A treadmill running threshold test was also performed and the following parameters were derived: anaerobic threshold, threshold of decompensated metabolic acidosis, respiratory exchange ratio = 1, onset of blood lactate accumulation and peak oxygen uptake (VO2peak). The combined dGPS and metabolic gas measurements made detailed analysis of performance possible. The strongest correlations between the treadmill data and final skiing field test time were for VO2peak (l x min(-1)), respiratory exchange ratio = 1 (l x min(-1)) and onset of blood lactate accumulation (l x min(-1)) (r = -0.644 to - 0.750). However, all treadmill test data displayed stronger associations with speed in different stretches of the course than with final time, which stresses the value of a detailed analysis of performance in cross-country skiing. Mean oxygen uptake (VO2) in a particular stretch in relation to speed in the same stretch displayed its strongest correlation coefficients in most stretches when VO2 was presented in units litres per minute, rather than when VO2 was normalized to body mass (ml x kg(-1) x min(-1) and ml x min(-1) x kg(-2/3)). This suggests that heavy cross-country skiers have an advantage over their lighter counterparts. In one steep uphill stretch, however, VO2 (ml x min(-1) x kg(-2/3)) displayed the strongest association with speed, suggesting that in steep uphill sections light skiers could have an advantage over heavier skiers.

Advenella incenata gen. nov., sp. nov., a novel member of the Alcaligenaceae, isolated from various clinical samples.

A polyphasic taxonomic study of 14 isolates recovered from various human and veterinary clinical samples was performed. Phenotypically these isolates shared several characteristics with members of the Alcaligenaceae and related genera. Random amplified polymorphic DNA fingerprinting and whole-cell protein analysis suggested the presence of multiple genomic groups, which was confirmed by DNA-DNA hybridization experiments. 16S rRNA gene sequence analysis indicated that these isolates were related to the genera Pelistega, Taylorella, Oligella, Pigmentiphaga, Alcaligenes, Kerstersia, Achromobacter and Bordetella and belonged to the family Alcaligenaceae. Based on the results of the present study the organisms were classified in a novel genus, Advenella gen. nov. This genus comprises one named species, Advenella incenata sp. nov. (type strain LMG 22250T=CCUG 45225T) and five currently unnamed genomic species. The DNA G+C content of members of the novel genus Advenella is between 54.0 and 57.7 mol%.

Inhibition of lipopolysaccharide-mediated human monocyte activation, in vitro, by alpha1-antitrypsin.

alpha1-Antitrypsin (AAT) is a major circulating and tissue inhibitor of serine proteinases. As such AAT is thought to play an important role in limiting host tissue injury at sites of inflammation. There is now increasing evidence, however, that AAT may exhibit biological activity independent of its protease inhibitor function. In this study we compared the effects of native (inhibitory) and modified (non-inhibitory), e.g., polymerised and oxidised forms of AAT on LPS-induced human monocyte activation, in vitro. We found that native AAT inhibited LPS-stimulated synthesis and release of TNFalpha and IL-1beta mRNA and protein, respectively, but enhanced the release of the anti-inflammatory cytokine, IL-10. Similarly, polymerised and oxidised forms of AAT inhibited LPS-stimulated IL-1beta and TNFalpha. The effects of AATs were observed whether added prior to or following removal of LPS, suggesting that sequestration of agonist was unlikely to explain their biological effects. Furthermore, studies with neutralising antibodies indicated that generation of IL-10 was unlikely to be the mechanism responsible for the inhibitory effects of AATs. Thus, our data demonstrate for the first time that AAT exhibits anti-inflammatory activity in vitro that is unrelated to inhibition of serine proteases.