Clinical outcomes linked to expression of gene subsets for protein hormones and their cognate receptors from LCM-procured breast carcinoma cells

Purpose

Certain peptide hormones and/or their cognate receptors influencing normal cellular pathways also have been detected in breast cancers. The hypothesis is that gene subsets of these regulatory molecules predict risk of breast carcinoma recurrence in patients with primary disease.

Methods

Gene expression levels of 61 hormones and 81 receptors were determined by microarray with LCM-procured carcinoma cells of 247 de-identified biopsies. Univariable and multivariable Cox regressions were determined using expression levels of each hormone/receptor gene, individually or as a pair.

Results

Molecular signatures for ER+/PR+, ER?/PR?, and ER? carcinoma cells deciphered by LASSO were externally validated at HRs (CI) of 2.8 (1.84–4.4), 1.53 (1.01–2.3), and 1.72 (1.15–2.56), respectively. Using LCM-procured breast carcinoma cells, a 16-gene molecular signature was derived for ER+/PR+ biopsies, whereas a 10-gene signature was deciphered for ER?/PR? cancers. Four genes, POMC, CALCR, AVPR1A, and GH1, of this 10-gene signature were identified in a 6-gene molecular signature for ER? specimens.

Conclusions

Applying these signatures, Kaplan–Meier plots definitively identified a cohort of patients with either ER?/PR? or ER? carcinomas that exhibited low risk of recurrence. In contrast, the ER+/PR+ signature identified a cohort of patients with high risk of breast cancer recurrence. Each of the three molecular signatures predicted clinical outcomes of breast cancer patients with greater accuracy than observed with either single-gene analysis or by ER/PR protein content alone. Collectively, our results suggest that gene expression profiles of breast carcinomas with suspected poor prognosis (ER?/PR?) have identified a subset of patients with decreased risk of recurrence.

     

Liver transplantation for hepatocellular carcinoma: analysis of factors predicting outcome in 1074 patients in OPTN Region 5

Previous studies on loco‐regional therapy (LRT) and alpha‐fetoprotein (AFP) in predicting outcome after liver transplant (LT) for hepatocellular carcinoma (HCC) have shown inconsistent results. We analyzed the OPTN database in Region 5 from January 2004 to January 2009 and performed univariate and multivariate analysis of 11 pre‐transplant recipient and donor variables in 1074 patients with HCC meeting Milan criteria to detect association with post‐LT tumor recurrence or mortality. Mean waitlist time was 438 d. The 1‐ and 5‐yr post‐LT survival was 91.1% and 71.1%, respectively. In multivariate analysis, AFP before LT was the only predictor of HCC recurrence. The association between AFP and HCC recurrence was observed only in the subgroup receiving LRT but not in the subgroup without LRT. Predictors of mortality in multivariate analysis were HCC recurrence, Donor Risk Index, last AFP before LT, and MELD score. AFP before LT was the strongest predictor of post‐transplant HCC recurrence or death in multivariate analysis. In conclusion, in Region 5 with prolonged waitlist time, high AFP was the only pre‐transplant variable predicting post‐transplant tumor recurrence and mortality for HCC meeting Milan criteria. Our results also supported the importance of the effects of LRT on AFP in predicting prognosis.     

Burkholderia bacteria infectiously induce the proto-farming symbiosis of Dictyostelium amoebae and food bacteria

Symbiotic associations can allow an organism to acquire novel traits by accessing the genetic repertoire of its partner. In the Dictyostelium discoideum farming symbiosis, certain amoebas (termed “farmers”) stably associate with bacterial partners. Farmers can suffer a reproductive cost but also gain beneficial capabilities, such as carriage of bacterial food (proto-farming) and defense against competitors. Farming status previously has been attributed to amoeba genotype, but the role of bacterial partners in its induction has not been examined. Here, we explore the role of bacterial associates in the initiation, maintenance, and phenotypic effects of the farming symbiosis. We demonstrate that two clades of farmer-associated Burkholderia isolates colonize D. discoideum nonfarmers and infectiously endow them with farmer-like characteristics, indicating that Burkholderia symbionts are a major driver of the farming phenomenon. Under food-rich conditions, Burkholderia-colonized amoebas produce fewer spores than uncolonized counterparts, with the severity of this reduction being dependent on the Burkholderia colonizer. However, the induction of food carriage by Burkholderia colonization may be considered a conditionally adaptive trait because it can confer an advantage to the amoeba host when grown in food-limiting conditions. We observed Burkholderia inside and outside colonized D. discoideum spores after fruiting body formation; this observation, together with the ability of Burkholderia to colonize new amoebas, suggests a mixed mode of symbiont transmission. These results change our understanding of the D. discoideum farming symbiosis by establishing that the bacterial partner, Burkholderia, is an important causative agent of the farming phenomenon.Symbiotic interactions are ubiquitous in nature and can play a central role in the evolutionary trajectory of organisms. For instance, symbiosis can drive rapid lateral procurement of novel traits as interacting organisms gain access to the genetic capabilities of their partner (1, 2). The evolutionary power of symbiosis is apparent in the many major life forms that owe their very existence to past and present symbiotic partnerships (3, 4). A famous example is the emergence of eukaryotes through their ancestor’s acquisition of bacteria that subsequently evolved into organelles indispensable for energy generation (5). Although many classic examples of symbiosis are conspicuously mutualistic, the characteristics of other symbiotic associations can be complex, dynamic, and less definable. In some cases, symbionts have good or bad effects on their host that vary depending on genotypic and environmental details (6, 7). The length and transmission mode of symbiosis also can have strong effects on the selection and evolution of partner traits. Although older symbiotic associations are often obligate and stable, recent associations can be transient and protean (8). Additionally, vertical transmission may favor mutualistic interactions, whereas horizontal transmission can allow the emergence and spread of more pathogenic characteristics (9–11). However, as a whole, the fates of symbioses are often the result of a delicate balance between mutualism and pathogenesis, requiring pathogenic characteristics at the least to facilitate infection and beneficial properties to promote maintenance (12, 13). Indeed, many cases of mutualistic associations are thought to have evolved from ancient parasitic infections (14, 15). Examining nascent, malleable, or less definable forms of symbiosis may provide insight into the mechanisms that promote or corrode this balance and their subsequent evolutionary consequences.Amoeba–bacteria interactions make a promising system for gaining insight into diverse and dynamic symbiotic relationships. Amoebas interact with bacteria in multiple ways. Most apparently, they are predators of bacteria. However, other amoeba–bacteria interactions are less favorable for the amoebas. Some bacteria can evade amoeba phagocytosis and thereby diminish amoeba predatory prowess and food acquisition (16). Still worse, amoebas can fall victim to bacterial processes or exploitation, with some bacteria producing products detrimental to amoeba fitness or surviving phagocytosis to invade and multiply within amoeba cells (17, 18). There also are stable symbiotic interactions between amoebas and bacteria in which the origins, mechanisms, and impacts on both species are less defined. For instance, several bacterial endosymbionts inhabit amoebas, incurring variable and not always obvious consequences to the amoeba host (19–22). In addition, certain isolates of the soil-dwelling amoeba Dictyostelium discoideum persistently associate with bacteria, an association that has beneficial or detrimental outcomes depending on the environmental conditions (23–25). Amoebas can serve as environmental reservoirs for bacterial pathogens (26, 27) or as training facilities for the adaptation of bacteria to evade eukaryotic phagocytosis or to survive intracellularly after phagocytosis (17, 28). Although, for the eukaryotic host, some of these interactions may have decidedly unfortunate outcomes, such as the emergence of bacterial pathogenesis through intracellular adaptation, others may illuminate important evolutionary advances, such as the transition of bacterial endosymbionts into organelles that provide novel functionality. Thus, the diversity and persistence of amoeba–bacteria interactions may have several ecological and health-related consequences.The stable association between bacteria and some wild isolates of D. discoideum supplies a particularly interesting study system for elucidating symbiotic causes and consequences. D. discoideum is a soil-dwelling amoeba that transitions between solitary and social life stages (29). In the solitary stage, D. discoideum cells consume bacteria through phagocytosis and divide by binary fission. When food is exhausted, amoebas coaggregate, ultimately forming a multicellular structure called a “fruiting body.” As the fruiting body develops, ∼20% of the cells sacrifice themselves to form an erect stalk. The remaining cells ascend the stalk, forming a globular sorus at the top where they differentiate into sturdy spores (29). In the sorus, spores are positioned for dispersal into more favorable environments, presumably through contact and transport upon animals (30). Multiple wild isolates of the amoeba D. discoideum, but not all of them, have been found to be stably associated with several bacteria species (24). These amoebas persistently carry both edible and inedible bacteria with them through the social stage and are termed “farmers” because of their ability to reseed new environments with a food source (23, 24). Interestingly, farmer isolates produce fewer spores under optimal conditions than their non–bacteria-carrying counterparts; however, this cost is countered by farmers’ advantage in being able to replenish their food supply when dispersing to food-poor environments (24). Additionally, some of the bacterial isolates that farmers carry produce compounds detrimental to nonfarmer clones but not to their host farmer, giving the host farmer a competitive advantage in a mixed population (23, 25).The ease of D. discoideum manipulation coupled with the variability inherent in the farming symbiosis provide a promising platform for addressing questions concerning symbiosis between microbes and eukaryotes. For instance, are mutualistic associations and their resulting phenotypes driven by the host, by the bacteria, or by a very specific interaction between the two? How much coevolution is required to reach a beneficial outcome? In aphids and other insects, defensive symbionts can sweep through populations and be horizontally transferred to new hosts (31, 32). Even Buchnera, a vertically transferred obligate symbiont of aphids, still confers its benefits to new hosts after experimental infection (33). Taken together, this horizontal transfer suggests that little coevolution may be needed for the formation of novel symbiosis. However, in some plant–rhizobia or –mycorrhizal associations, the symbiont phenotype can vary substantially among hosts, and the outcome of the association is determined by the interaction of host and symbiont-derived factors (34–38). Similarly, a synergistic interplay between host and symbiont components mediates the initiation and persistence of the bobtail squid–Vibrio fischeri symbiosis (39–41). For the D. discoideum farming symbiosis, it previously had been assumed that farmers were genetically distinct from nonfarmers, suggesting the role of a host-specific factor in establishing the symbiosis (24). This study aims to characterize more thoroughly the partner dynamics within the D. discoideum farming symbiosis by specifically analyzing the role of bacterial associates in farming phenotypes.To determine the role bacterial partners play in farming, we first examined the diversity of bacterial passengers associated with our present collection of stable farmer clones. We confirmed that several different bacterial species can be isolated from farmer D. discoideum; however isolates belonging to the Burkholderia genus were ubiquitous among our tested farmers. These Burkholderia isolates fail to support amoeba growth when provided as the only food source and therefore are considered inedible. Because of their prevalence in our farmer clones and because Burkholderia species form symbiotic relationships with diverse organisms, we hypothesized that these Burkholderia isolates could be crucial for the symbiotic relationship with D. discoideum that results in the farming phenomenon (42). Therefore we asked whether nonfarmer D. discoideum could be colonized by farmer-associated Burkholderia isolates and whether colonization could induce secondary bacterial carriage. We established that the Burkholderia associated with D. discoideum fall into two distinct phylogenetic clades. We found that the tested Burkholderia isolates from each clade robustly colonized nonfarmer D. discoideum sori, with this colonization persisting through multiple rounds of D. discoideum spore dispersal, germination, and vegetative growth. Like their farmer counterparts, nonfarmers colonized with Burkholderia can carry bacterial food, allowing them to reseed new territories with food bacteria following spore dispersal. Inversely, removal of Burkholderia from wild farmers by antibiotic treatment results in the loss of detectable bacterial food carrying. These findings suggest that Burkholderia colonization drives secondary bacterial carriage (farming) in D. discoideum. In total, these results suggest that specific Burkholderia isolates stably colonize D. discoideum and induce a novel adaptive trait of ecological relevance, the carriage of bacterial food. Our initial evidence suggests that the consequences of Burkholderia carriage may differ according to symbiont and host genotypes. For instance, some Burkholderia isolates impose a higher cost to their hosts, and the extent of this cost appears to be more severe for newer hosts than for the original host. We also observe Burkholderia (and occasionally, our laboratory food bacterium, Klebsiella pneumoniae) inside colonized D. discoideum spores after fruiting body formation. This observation, together with Burkholderia’s ability to be horizontally transmitted to new hosts and to associate stably with old hosts, suggests a mixed mode of Burkholderia transmission. The less severe fitness costs exerted by Burkholderia colonization in the original hosts suggests that long-term vertical transmission may lead to compensatory host adaptation.     

Compromised GCF total antioxidant capacity in periodontitis: cause or effect?

BACKGROUND: Oxidative stress is implicated in the pathogenesis of periodontitis. The total antioxidant capacity (TAOC) of gingival crevicular fluid volume (GCF) and plasma appears compromised in periodontitis, but it is unclear whether this predisposes to, or results from the inflammatory process. AIM: To investigate longitudinal changes in GCF and plasma TAOC following reductions in periodontal inflammation with successful non-surgical therapy. MATERIALS AND METHODS: Two longitudinal studies were run in series on non-smokers with chronic periodontitis (CP). Study-1 (n=17) assessed index sites with mild disease; Study-2 (n=18) investigated deep sites. GCF sampling and clinical measures were performed at baseline and 3 months post-therapy. Plasma and GCF TAOC was determined by enhanced chemiluminescence and 32 age/sex-matched periodontally healthy controls were used. RESULTS: Therapy improved clinical outcomes consistent with the literature. There were no differences in plasma TAOC between periodontitis patients (507+/-92 microMTeq) and controls (520+/-100 microMTeq; p=0.57) at baseline, but GCF TAOC was lower (p<0.0001) in CP patients (680+/-371 microMTeq) than controls (1129+/-722 microMTeq). Successful periodontal therapy did not alter plasma TAOC (p=0.56), but GCF TAOC increased (by 449+/-722 microMTeq, p<0.001) to control subject levels (p=0.47) CONCLUSIONS: Local total antioxidant capacity in CP appears to reflect increased oxygen radical activity during periodontal inflammation and can be restored to control subject levels by successful non-surgical therapy.     

The impact of circulation in a heart–lung machine on function and survival characteristics of red blood cells

Extracorporeal circulation is accompanied by changes in red blood cell morphology and structural integrity that affect cell function and survival, and thereby may contribute to the various side effects of heart–lung machine-assisted surgery. Our main objectives were to determine the effect of circulation of red blood cells in a stand-alone extracorporeal circuit on several parameters that are known to be affected by, as well as contribute to red blood cell aging. As a source of RBCs, we employed blood bank storage units of different ages. In order to assess the relevance of our in vitro observations for the characterization of extracorporal circulation technology, we compared these changes in those of patients undergoing extracorporeal circulation-assisted cardiac surgery. Our results show that circulation in a heart–lung machine is accompanied by changes in red blood cell volume, an increase in osmotic fragility, changes in deformability and aggregation behavior, and alterations in the exposure of phosphatidylserine and in microvesicle generation. RBCs from 1-week-old concentrates showed the highest similarities with the in vivo situation. These changes in key characteristics of the red blood cell aging process likely increase the susceptibility of red blood cells to the various mechanical, osmotic, and immunological stress conditions encountered during and after surgery in the patient’s circulation, and thereby contribute to the side effects of surgery. Thus, aging-related parameters in red blood cell structure and function provide a foundation for the validation and improvement of extracorporeal circulation technology.