Informing Antibiotic Treatment Decisions: Evaluating Rapid Molecular Diagnostics To Identify Susceptibility and Resistance to Carbapenems against Acinetobacter spp. in PRIMERS III.

The widespread dissemination of carbapenem-resistant Acinetobacter spp. has created significant therapeutic challenges. At present, rapid molecular diagnostics (RMDs) that can identify this phenotype are not commercially available. Two RMD platforms, PCR combined with electrospray ionization mass spectrometry (PCR/ESI-MS) and molecular beacons (MB), for detecting genes conferring resistance/susceptibility to carbapenems in Acinetobacter spp. were evaluated. An archived collection of 200 clinical Acinetobacter sp. isolates was tested. Predictive values for susceptibility and resistance were estimated as a function of susceptibility prevalence and were based on the absence or presence of beta-lactamase (bla) NDM, VIM, IMP, KPC, and OXA carbapenemase genes (e.g., blaOXA-23, blaOXA-24/40, and blaOXA-58 found in this study) against the reference standard of MIC determinations. According to the interpretation of MICs, 49% (n = 98) of the isolates were carbapenem resistant (as defined by either resistance or intermediate resistance to imipenem). The susceptibility sensitivities (95% confidence interval [CI]) for imipenem were 82% (74%, 89%) and 92% (85%, 97%) for PCR/ESI-MS and MB, respectively. Resistance sensitivities (95% CI) for imipenem were 95% (88%, 98%) and 88% (80%, 94%) for PCR/ESI-MS and MB, respectively. PRIMERS III establishes that RMDs can discriminate between carbapenem resistance and susceptibility in Acinetobacter spp. In the context of a known prevalence of resistance, SPVs and RPVs can inform clinicians regarding the best choice for empiric antimicrobial therapy against this multidrug-resistant pathogen.

Rapid Molecular Diagnostics, Antibiotic Treatment Decisions, and Developing Approaches to Inform Empiric Therapy: PRIMERS I and II.

BACKGROUND: Rapid molecular diagnostic (RMD) platforms may lead to better antibiotic use. Our objective was to develop analytical strategies to enhance the interpretation of RMDs for clinicians. METHODS: We compared the performance characteristics of 4 RMD platforms for detecting resistance against ß-lactams in 72 highly resistant isolates of Escherichia coli and Klebsiella pneumoniae (PRIMERS I). Subsequently, 2 platforms were used in a blinded study in which a heterogeneous collection of 196 isolates of E. coli and K. pneumoniae (PRIMERS II) were examined. We evaluated the genotypic results as predictors of resistance or susceptibility against ß-lactam antibiotics. We designed analytical strategies and graphical representations of platform performance, including discrimination summary plots and susceptibility and resistance predictive values, that are readily interpretable by practitioners to inform decision-making. RESULTS: In PRIMERS I, the 4 RMD platforms detected ß-lactamase (bla) genes and identified susceptibility or resistance in >95% of cases. In PRIMERS II, the 2 platforms identified susceptibility against extended-spectrum cephalosporins and carbapenems in >90% of cases; however, against piperacillin/tazobactam, susceptibility was identified in <80% of cases. Applying the analytical strategies to a population with 15% prevalence of ceftazidime-resistance and 5% imipenem-resistance, RMD platforms predicted susceptibility in >95% of cases, while prediction of resistance was 69%-73% for ceftazidime and 41%-50% for imipenem. CONCLUSIONS: RMD platforms can help inform empiric ß-lactam therapy in cases where bla genes are not detected and the prevalence of resistance is known. Our analysis is a first step in bridging the gap between RMDs and empiric treatment decisions.

Adipocyte derived paracrine mediators of mammary ductal morphogenesis controlled by retinoic acid receptors.

We generated a transgenic (Tg)-mouse model expressing a dominant negative-(DN)-RARα, (RARαG303E) under adipocytes-specific promoter to explore the paracrine role of adipocyte retinoic acid receptors (RARs) in mammary morphogenesis. Transgenic adipocytes had reduced level of RARα, ß and γ, which coincided with a severely underdeveloped pubertal and mature ductal tree with profoundly decreased epithelial cell proliferation. Transplantation experiments of mammary epithelium and of whole mammary glands implicated a fat-pad dependent paracrine mechanism in the stunted phenotype of the epithelial ductal tree. Co-cultures of primary adipocytes, or in vitro differentiated adipocyte cell line, with mammary epithelium showed that when activated, adipocyte-RARs contribute to generation of secreted proliferative and pro-migratory factors. Gene expression microarrays revealed a large number of genes regulated by adipocyte-RARs. Among them, pleiotrophin (PTN) was identified as the paracrine effectors of epithelial cell migration. Its expression was found to be strongly inhibited by DN-RARα, an inhibition relieved by pharmacological doses of all-trans retinoic acid (atRA) in culture and in vivo. Moreover, adipocyte-PTHR, another atRA responsive gene, was found to be an up-stream regulator of PTN. Overall, these results support the existence of a novel paracrine loop controlled by adipocyte-RAR that regulates the mammary ductal tree morphogenesis.

RARα1 control of mammary gland ductal morphogenesis and wnt1-tumorigenesis.

INTRODUCTION: Retinoic acid signaling pathways are disabled in human breast cancer suggesting a controlling role in normal mammary growth that might be lost in tumorigenesis. We tested a single receptor isotype, RARα1, for its role in mouse mammary gland morphogenesis and MMTV-wnt1-induced oncogenesis. METHODS: The role of RARα1 in mammary morphogenesis was tested in RARα1-knockout (KO) mice and in mammary tumorigenesis in bi-genic (RARα1/KO crossed with MMTV-wnt1) mice. We used whole mounts analysis, stem cells/progenitor quantification, mammary gland repopulation, Q-PCR, test of tumor-free survival, tumor fragments and cell transplantation. RESULTS: In 2 genetic backgrounds (129/Bl-6 and FVB) the neo-natal RARα1/KO-mammary epithelial tree was 2-fold larger and the pubertal tree had 2-fold more branch points and 5-fold more mature end buds, a phenotype that was predominantly epithelial cell autonomous. The stem/progenitor compartment of the RARα1/KO mammary, defined as CD24(low)/ALDH(high activity) was increased by a median 1.7 fold, but the mammary stem cell (MaSC)-containing compartment, (CD24(low)/CD29(high)), was larger (~1.5 fold) in the wt-glands, and the mammary repopulating ability of the wt-gland epithelium was ~2-fold greater. In MMTV-wnt1 transgenic glands the progenitor (CD24(low)/ALDH(high activity)) content was 2.6-fold greater than in the wt and was further increased in the RARα1/KO-wnt1 glands. The tumor-free survival of RARα1/KO-wnt1 mice was significantly (p=0.0002, Kaplan Meier) longer, the in vivo growth of RARα1/KO-wnt1 transplanted tumor fragments was significantly (p=0.01) slower and RARα1/KO-wnt1 tumors cell suspension produced tumors after much longer latency. CONCLUSIONS: In vitamin A-replete mice, RARα1 is required to maintain normal mammary morphogenesis, but paradoxically, also efficient tumorigenesis. While its loss increases the density of the mammary epithelial tree and the content of luminal mammary progenitors, it appears to reduce the size of the MaSC-containing compartment, the mammary repopulating activity, and to delay significantly the MMTV-wnt1-mammary tumorigenesis. Whether the delay in tumorigenesis is solely due to a reduction in wnt1 target cells or due to additional mechanisms remains to be determined. These results reveal the intricate nature of the retinoid signaling pathways in mammary development and carcinogenesis and suggest that a better understanding will be needed before retinoids can join the armament of effective anti- breast cancer therapies.

Dominant negative retinoic acid receptor initiates tumor formation in mice.

BACKGROUND: Retinoic acid suppresses cell growth and promotes cell differentiation, and pharmacological retinoic acid receptor (RAR) activation is anti-tumorigenic. This begs the question of whether chronic physiological RAR activation by endogenous retinoids is likewise anti-tumorigenic. RESULTS: To address this question, we generated transgenic mice in which expression of a ligand binding defective dominant negative RARalpha (RARalphaG303E) was under the control of the mouse mammary tumor virus (MMTV) promoter. The transgene was expressed in the lymphoid compartment and in the mammary epithelium. Observation of aging mice revealed that transgenic mice, unlike their wild type littermates, developed B cell lymphomas at high penetrance, with a median latency of 40 weeks. MMTV-RARalphaG303E lymphomas were high grade Pax-5+, surface H+L Ig negative, CD69+ and BCL6- and cytologically and phenotypically resembled human adult high grade (Burkitt's or lymphoblastic) lymphomas. We postulated that mammary tumors might arise after a long latency period as seen in other transgenic models of breast cancer. We tested this idea by transplanting transgenic epithelium into the cleared fat pads of wild type hosts, thus bypassing lymphomagenesis. At 17 months post-transplantation, a metastatic mammary adenocarcinoma developed in one of four transplanted glands whereas no tumors developed in sixteen of sixteen endogenous glands with wild type epithelium. CONCLUSION: These findings suggest that physiological RAR activity may normally suppress B lymphocyte and mammary epithelial cell growth and that global RAR inactivation is sufficient to initiate a stochastic process of tumor development requiring multiple transforming events. Our work makes available to the research community a new animal resource that should prove useful as an experimental model of aggressive sporadic lymphoma in immunologically uncompromised hosts. We anticipate that it may also prove useful as a model of breast cancer.