Reference Susceptibility Testing and Genomic Surveillance of Clostridioides difficile, United States, 2012-17.

BACKGROUND: Antimicrobial susceptibility testing (AST) is not routinely performed for Clostridioides difficile and data evaluating minimum inhibitory concentrations (MICs) are limited. We performed AST and whole genome sequencing (WGS) for 593 C. difficile isolates collected between 2012 and 2017 through the Centers for Disease Control and Prevention's Emerging Infections Program. METHODS: MICs to 6 antimicrobial agents (ceftriaxone, clindamycin, meropenem, metronidazole, moxifloxacin, and vancomycin) were determined using the reference agar dilution method according to Clinical and Laboratory Standards Institute guidelines. Whole genome sequencing was performed on all isolates to detect the presence of genes or mutations previously associated with resistance. RESULTS: Among all isolates, 98.5% displayed a vancomycin MIC ≤2 µg/mL and 97.3% displayed a metronidazole MIC ≤2 µg/mL. Ribotype 027 (RT027) isolates displayed higher vancomycin MICs (MIC50: 2 µg/mL; MIC90: 2 µg/mL) than non-RT027 isolates (MIC50: 0.5 µg/mL; MIC90: 1 µg/mL) (P < .01). No vanA/B genes were detected. RT027 isolates also showed higher MICs to clindamycin and moxifloxacin and were more likely to harbor associated resistance genes or mutations. CONCLUSIONS: Elevated MICs to antibiotics used for treatment of C. difficile infection were rare, and there was no increase in MICs over time. The lack of vanA/B genes or mutations consistently associated with elevated vancomycin MICs suggests there are multifactorial mechanisms of resistance. Ongoing surveillance of C. difficile using reference AST and WGS to monitor MIC trends and the presence of antibiotic resistance mechanisms is essential.

Trends in Incidence of Methicillin-resistant Staphylococcus aureus Bloodstream Infections Differ by Strain Type and Healthcare Exposure, United States, 2005-2013.

BACKGROUND: Previous reports suggested that US methicillin-resistant Staphylococcus aureus (MRSA) strain epidemiology has changed since the rise of USA300 MRSA. We describe invasive MRSA trends by strain type. METHODS: Data came from 5 Centers for Disease Control and Prevention Emerging Infections Program sites conducting population-based surveillance and collecting isolates for invasive MRSA (ie, from normally sterile body sites), 2005-2013. MRSA bloodstream infection (BSI) incidence per 100 000 population was stratified by strain type and epidemiologic classification of healthcare exposures. Invasive USA100 vs USA300 case characteristics from 2013 were compared through logistic regression. RESULTS: From 2005 to 2013, USA100 incidence decreased most notably for hospital-onset (6.1 vs 0.9/100 000 persons, P < .0001) and healthcare-associated, community-onset (10.7 vs 4.9/100 000 persons, P < .0001) BSIs. USA300 incidence for hospital-onset BSIs also decreased (1.5 vs 0.6/100 000 persons, P < .0001). However, USA300 incidence did not significantly change for healthcare-associated, community-onset (3.9 vs 3.3/100 000 persons, P = .05) or community-associated BSIs (2.5 vs 2.4/100 000 persons, P = .19). Invasive MRSA was less likely to be USA300 in patients who were older (adjusted odds ratio [aOR], 0.97 per year [95% confidence interval {CI}, .96-.98]), previously hospitalized (aOR, 0.36 [95% CI, .24-.54]), or had central lines (aOR, 0.44 [95% CI, .27-.74]), and associated with USA300 in people who inject drugs (aOR, 4.58 [95% CI, 1.16-17.95]). CONCLUSIONS: Most of the decline in MRSA BSIs was from decreases in USA100 BSI incidence. Prevention of USA300 MRSA BSIs in the community will be needed to further reduce burden from MRSA BSIs.

Carbapenem-Resistant Pseudomonas aeruginosa at US Emerging Infections Program Sites, 2015.

Pseudomonas aeruginosa is intrinsically resistant to many antimicrobial drugs, making carbapenems crucial in clinical management. During July-October 2015 in the United States, we piloted laboratory-based surveillance for carbapenem-resistant P. aeruginosa (CRPA) at sentinel facilities in Georgia, New Mexico, Oregon, and Tennessee, and population-based surveillance in Monroe County, NY. An incident case was the first P. aeruginosa isolate resistant to antipseudomonal carbapenems from a patient in a 30-day period from any source except the nares, rectum or perirectal area, or feces. We found 294 incident cases among 274 patients. Cases were most commonly identified from respiratory sites (120/294; 40.8%) and urine (111/294; 37.8%); most (223/280; 79.6%) occurred in patients with healthcare facility inpatient stays in the prior year. Genes encoding carbapenemases were identified in 3 (2.3%) of 129 isolates tested. The burden of CRPA was high at facilities under surveillance, but carbapenemase-producing CRPA were rare.

Overexpression of cytosolic, plasma membrane bound and extracellular heat shock protein 70 (Hsp70) in primary glioblastomas.

A unique feature in several non-CNS-tumors is the overexpression of heat shock protein 70 (Hsp70, HSPA1A) in the cytosol, but also its unusual plasma membrane expression and release. Although in gliomas, cytosolic Hsp70 levels are not associated with histological grading, the role of membrane bound and released Hsp70 is still completely unknown. Membrane bound as well as cytosolic Hsp70 can be detected in viable tumor cells with the monoclonal antibody (mAb) cmHsp70.1. Herein, we analysed membrane bound Hsp70 levels in primary and secondary gliomas of different grades and on isolated glioma subpopulations (endothelial cells, CD133-positive cells, primary cultures) by immunohistochemistry and flow cytometry using cmHsp70.1 mAb. Extracellular Hsp70 was determined by a commercial Hsp70 sandwich ELISA (R&D) in plasma samples of glioblastoma patients and healthy volunteers. We found an overexpression of Hsp70 in primary glioblastomas compared to low-grade, anaplastic, or secondary gliomas as determined by immunohistochemistry. Especially in flow cytometry, a strong plasma membrane Hsp70 expression was only observed in primary but not secondary glioblastomas. Within the heterogeneous tumor mass, CD133-positive tumor-initiating and primary glioblastoma cells showed a high membrane Hsp70 expression density, whereas endothelial cells, isolated from glioblastoma tissues only showed a weak staining pattern. Also in plasma samples, secreted Hsp70 protein was significantly increased in patients harbouring primary glioblastomas compared to those with secondary and low grade glioblastomas. Taken together, we show for the first time that cytosolic, membrane bound and extracellular Hsp70 is uniquely overexpressed in primary glioblastomas.

Anticalins directed against the fibronectin extra domain B as diagnostic tracers for glioblastomas.

The standard of care for diagnosis and therapy monitoring of gliomas is magnetic resonance imaging (MRI), which however, provides only an indirect and incomplete representation of the tumor mass, offers limited information for patient stratification according to WHO-grades and may insufficiently indicate tumor relapse after antiangiogenic therapy. Anticalins are alternative binding proteins obtained via combinatorial protein design from the human lipocalin scaffold that offer novel diagnostic reagents for histology and imaging applications. Here, the Anticalins N7A, N7E and N9B, which possess exquisite specificity and affinity for oncofetal fibronectin carrying the extra domain B (ED-B), a well-known proangiogenic extracellular matrix protein, were applied for immunohistochemical studies. When investigating ED-B expression in biopsies from 41 patients with confirmed gliomas of WHO grades I to IV, or in non-neoplastic brain samples, we found that Anticalins specifically detect ED-B in primary glioblastoma multiforme (GBM; WHO IV) but not in tumors of lower histopathological grade or in tumor-free brain. In primary GBM samples, ED-B specific Anticalins locate to fibronectin-rich perivascular areas that are associated with angiogenesis. Anticalins specifically detect ED-B both in fixed tumor specimen and on vital cells, as evidenced by cytofluorometry. Beyond that, we labeled an Anticalin with the γ-emitter (123) I and demonstrated specific binding to GBM-tissue samples using in vitro autoradiography. Overall, our data indicate that ED-B specific Anticalins are useful tools for the diagnosis of primary GBM and related angiogenic sites, presenting them as promising tracers for molecular tumor imaging.

Staphylococcus aureus Colonization and Strain Type at Various Body Sites among Patients with a Closed Abscess and Uninfected Controls at U.S. Emergency Departments.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is a prevalent cause of skin and soft tissue infections (SSTI), but the association between CA-MRSA colonization and infection remains uncertain. We studied the carriage frequency at several body sites and the diversity of S. aureus strains from patients with and without SSTI. Specimens from the nares, throat, rectum, and groin of case subjects with a closed skin abscess (i.e., without drainage) and matched control subjects without a skin infection (n = 147 each) presenting to 10 U.S. emergency departments were cultured using broth enrichment; wound specimens were cultured from abscess cases. Methicillin resistance testing and spa typing were performed for all S. aureus isolates. S. aureus was found in 85/147 (57.8%) of abscesses; 49 isolates were MRSA, and 36 were methicillin-susceptible S. aureus (MSSA). MRSA colonization was more common among cases (59/147; 40.1%) than among controls (27/147; 18.4%) overall (P < 0.001) and at each body site; no differences were observed for MSSA. S. aureus-infected subjects were usually (75/85) colonized with the infecting strain; among MRSA-infected subjects, this was most common in the groin. The CC8 lineage accounted for most of both infecting and colonizing isolates, although more than 16 distinct strains were identified. Nearly all MRSA infections were inferred to be USA300. There was more diversity among colonizing than infecting isolates and among those isolated from controls versus cases. CC8 S. aureus is a common colonizer of persons with and without skin infections. Detection of S. aureus colonization, and especially MRSA, may be enhanced by extranasal site culture.

Vancomycin-Resistant Staphylococcus aureus - Delaware, 2015.

Vancomycin-resistant Staphylococcus aureus (VRSA) is a rare, multidrug-resistant bacterium of public health concern that emerged in the United States in 2002. VRSA (S. aureus with vancomycin minimum inhibitory concentration [MIC] ≥16 µg/mL) arises when vancomycin resistance genes (e.g., the vanA operon, which codes for enzymes that result in modification or elimination of the vancomycin binding site) from vancomycin-resistant enterococci (VRE) are transferred to S. aureus (1). To date, all VRSA strains have arisen from methicillin-resistant S. aureus (MRSA). The fourteenth VRSA isolate (VRSA 14) identified in the United States was reported to CDC in February 2015.

Report of the 13th vancomycin-resistant Staphylococcus aureus isolate from the United States.

Vancomycin-resistant Staphylococcus aureus (VRSA), an important multidrug-resistant organism of public health concern, has been infrequently identified in the United States since 2002. All previous VRSA isolates belonged to clonal complex 5, a lineage associated primarily with health care. This report describes the most recent (13th) U.S. VRSA isolate, the first to be community associated.

Comparison of carbapenem-susceptible and carbapenem-resistant Enterobacterales at nine sites in the USA, 2013-2016: a resource for antimicrobial resistance investigators.

Carbapenem-resistant Enterobacterales (CRE) are an urgent public health threat. Genomic sequencing is an important tool for investigating CRE. Through the Division of Healthcare Quality Promotion Sentinel Surveillance system, we collected CRE and carbapenem-susceptible Enterobacterales (CSE) from nine clinical laboratories in the USA from 2013 to 2016 and analysed both phenotypic and genomic sequencing data for 680 isolates. We describe the molecular epidemiology and antimicrobial susceptibility testing (AST) data of this collection of isolates. We also performed a phenotype-genotype correlation for the carbapenems and evaluated the presence of virulence genes in Klebsiella pneumoniae complex isolates. These AST and genomic sequencing data can be used to compare and contrast CRE and CSE at these sites and serve as a resource for the antimicrobial resistance research community.

Carbapenem-Resistant enterobacterales in individuals with and without health care risk factors -Emerging infections program, United States, 2012-2015.

BACKGROUND: Carbapenem-resistant Enterobacterales (CRE) are usually healthcare-associated but are also emerging in the community. METHODS: Active, population-based surveillance was conducted to identify case-patients with cultures positive for Enterobacterales not susceptible to a carbapenem (excluding ertapenem) and resistant to all third-generation cephalosporins tested at 8 US sites from January 2012 to December 2015. Medical records were used to classify cases as health care-associated, or as community-associated (CA) if a patient had no known health care risk factors and a culture was collected <3 days after hospital admission. Enterobacterales isolates from selected cases were submitted to CDC for whole genome sequencing. RESULTS: We identified 1499 CRE cases in 1194 case-patients; 149 cases (10%) in 139 case-patients were CA. The incidence of CRE cases per 100,000 population was 2.96 (95% CI: 2.81, 3.11) overall and 0.29 (95% CI: 0.25, 0.35) for CA-CRE. Most CA-CRE cases were in White persons (73%), females (84%) and identified from urine cultures (98%). Among the 12 sequenced CA-CRE isolates, 5 (42%) harbored a carbapenemase gene. CONCLUSIONS: Ten percent of CRE cases were CA; some isolates from CA-CRE cases harbored carbapenemase genes. Continued CRE surveillance in the community is critical to monitor emergence outside of traditional health care settings.

Prevalence of methicillin-resistant staphylococcus aureus as an etiology of community-acquired pneumonia.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is a common cause of skin infections. Recent case series describe severe community-acquired pneumonia (CAP) caused by MRSA, but the prevalence and risk factors are unknown. METHODS: We prospectively enrolled adults hospitalized with CAP from 12 university-affiliated emergency departments during the winter-spring of 2006 and 2007. Clinical information and culture results were collected, and factors associated with MRSA were assessed. RESULTS: Of 627 patients, 595 (95%) had respiratory (50%) and/or blood cultures (92%) performed. A pathogen was identified in 102 (17%); MRSA was identified in 14 (2.4%; range by site, 0%-5%) patients and in 5% of patients admitted to the intensive care unit. Two (14%) MRSA pneumonia patients died. All 9 MRSA isolates tested were pulsed-field type USA300. Features significantly associated with isolation of MRSA (as compared with any other or no pathogen) included patient history of MRSA; nursing home admission in the previous year; close contact in the previous month with someone with a skin infection; multiple infiltrates or cavities on chest radiograph; and comatose state, intubation, receipt of pressors, or death in the emergency department. CONCLUSIONS: Methicillin-resistant Staphylococcus aureus remains an uncommon cause of CAP. Detection of MRSA was associated with more severe clinical presentation.

Molecular Epidemiology of Carbapenem-Resistant Acinetobacter baumannii in the United States, 2013-2017.

Healthcare-associated carbapenem-resistant Acinetobacter baumannii (CRAB) infections are a serious threat associated with global epidemic clones and a variety of carbapenemase gene classes. In this study, we describe the molecular epidemiology, including whole-genome sequencing analysis and antimicrobial susceptibility profiles of 92 selected, nonredundant CRAB collected through public health efforts in the United States from 2013 to 2017. Among the 92 isolates, the Oxford (OX) multilocus sequence typing scheme identified 30 sequence types (STs); the majority of isolates (n = 59, 64%) represented STs belonging to the international clonal complex 92 (CC92OX). Among these, ST208OX (n = 21) and ST281OX (n = 20) were the most common. All isolates carried an OXA-type carbapenemase gene, comprising 20 alleles. Ninety isolates (98%) encoded an intrinsic OXA-51-like enzyme; 67 (73%) harbored an additional acquired blaOXA gene, most commonly blaOXA-23 (n = 45; 49%). Compared with isolates harboring only intrinsic oxacillinase genes, acquired blaOXA gene presence was associated with higher prevalence of resistance and a higher median minimum inhibitory concentration to the carbapenem imipenem (64 µg/mL vs. 8 µg/mL), and antibiotics from other drug classes, including penicillin, aminoglycosides, cephalosporins, and polymyxins. These data illustrate the wide distribution of CC92OX and high prevalence of acquired blaOXA carbapenemase genes among CRAB in the United States.

Integrative analysis of therapy resistance and transcriptomic profiling data in glioblastoma cells identifies sensitization vulnerabilities for combined modality radiochemotherapy.

BACKGROUND: Inherent resistance to radio/chemotherapy is one of the major reasons for early recurrence, treatment failure, and dismal prognosis of glioblastoma. Thus, the identification of resistance driving regulators as prognostic and/or predictive markers as well as potential vulnerabilities for combined modality treatment approaches is of pivotal importance. METHODS: We performed an integrative analysis of treatment resistance and DNA damage response regulator expression in a panel of human glioblastoma cell lines. mRNA expression levels of 38 DNA damage response regulators were analyzed by qRT-PCR. Inherent resistance to radiotherapy (single-shot and fractionated mode) and/or temozolomide treatment was assessed by clonogenic survival assays. Resistance scores were extracted by dimensionality reduction and subjected to correlation analyses with the mRNA expression data. Top-hit candidates with positive correlation coefficients were validated by pharmacological inhibition in clonogenic survival assays and DNA repair analyses via residual γH2AX/53BP1-foci staining. RESULTS: Inherent resistance to single-shot and similarly also to fractionated radiotherapy showed strong positive correlations with mRNA expression levels of known vulnerabilities of GBM, including PARP1, NBN, and BLM, as well as ATR and LIG4-two so far underestimated targets. Inhibition of ATR by AZD-6738 resulted in robust and dose-dependent radiosensitization of glioblastoma cells, whereas LIG4 inhibition by L189 had no noticeable impact. Resistance against temozolomide showed strong positive correlation with mRNA expression levels of MGMT as to be expected. Interestingly, it also correlated with mRNA expression levels of ATM, suggesting a potential role of ATM in the context of temozolomide resistance in glioblastoma cells. ATM inhibition exhibited slight sensitization effects towards temozolomide treatment in MGMT low expressing glioblastoma cells, thus encouraging further characterization. CONCLUSIONS: Here, we describe a systematic approach integrating clonogenic survival data with mRNA expression data of DNA damage response regulators in human glioblastoma cell lines to identify markers of inherent therapy resistance and potential vulnerabilities for targeted sensitization. Our results provide proof-of-concept for the feasibility of this approach, including its limitations. We consider this strategy to be adaptable to other cancer entities as well as other molecular data qualities, and its upscaling potential in terms of model systems and observational data levels deserves further investigation.

Molecular Characterization of Carbapenem-Resistant Enterobacterales Collected in the United States.

Carbapenem-resistant Enterobacterales (CRE) are a growing public health concern due to resistance to multiple antibiotics and potential to cause health care-associated infections with high mortality. Carbapenemase-producing CRE are of particular concern given that carbapenemase-encoding genes often are located on mobile genetic elements that may spread between different organisms and species. In this study, we performed phenotypic and genotypic characterization of CRE collected at eight U.S. sites participating in active population- and laboratory-based surveillance of carbapenem-resistant organisms. Among 421 CRE tested, the majority were isolated from urine (n = 349, 83%). Klebsiella pneumoniae was the most common organism (n = 265, 63%), followed by Enterobacter cloacae complex (n = 77, 18%) and Escherichia coli (n = 50, 12%). Of 419 isolates analyzed by whole genome sequencing, 307 (73%) harbored a carbapenemase gene; variants of blaKPC predominated (n = 299, 97%). The occurrence of carbapenemase-producing K. pneumoniae, E. cloacae complex, and E. coli varied by region; the predominant sequence type within each genus was ST258, ST171, and ST131, respectively. None of the carbapenemase-producing CRE isolates displayed resistance to all antimicrobials tested; susceptibility to amikacin and tigecycline was generally retained.

Comparison of Staphylococcus aureus from skin and soft-tissue infections in US emergency department patients, 2004 and 2008.

BACKGROUND: In the past decade, new methicillin-resistant Staphylococcus aureus (MRSA) strains have emerged as a predominant cause of community-associated skin and soft-tissue infections (SSTIs). Little information exists regarding trends in MRSA prevalence and molecular characteristics or regarding antimicrobial susceptibility profiles of S. aureus isolates. METHODS: We enrolled adults with acute, purulent SSTIs presenting to a US network of 12 emergency departments during August 2008. Cultures and clinical information were collected. S. aureus isolates were characterized by antimicrobial susceptibility testing, pulsed-field gel electrophoresis, and toxin genes detection. The prevalence of S. aureus and MRSA and isolate genetic characteristics and susceptibilities were compared with those from a similar study conducted in August 2004. RESULTS: The prevalence of MRSA was 59% among all SSTIs during both study periods; however, the prevalence by site varied less in 2008 (38%-84%), compared with 2004 (15%-74%). Pulsed-field type USA300 continued to account for almost all MRSA isolates (98%). Susceptibility to trimethoprim-sulfamethoxazole, clindamycin, and tetracycline among MRSA isolates remained greater than 90% in 2008. A higher proportion of MRSA infections were treated with an agent to which the infecting isolate was susceptible in vitro in 2008 (97%), compared with 2004 (57%). CONCLUSIONS: Similar to 2004, MRSA remained the most common identifiable cause of purulent SSTIs among patients presenting to a network of US emergency departments in 2008. The infecting MRSA isolates continued to be predominantly pulsed-field type USA300 and susceptible to recommended non-ß-lactam oral agents. Clinician prescribing practices have shifted from MRSA-inactive to MRSA-active empirical antimicrobial regimens.

Inhibition of HSP90 as a Strategy to Radiosensitize Glioblastoma: Targeting the DNA Damage Response and Beyond.

Radiotherapy is an essential component of multi-modality treatment of glioblastoma (GBM). However, treatment failure and recurrence are frequent and give rise to the dismal prognosis of this aggressive type of primary brain tumor. A high level of inherent treatment resistance is considered to be the major underlying reason, stemming from constantly activated DNA damage response (DDR) mechanisms as a consequence of oncogene overexpression, persistent replicative stress, and other so far unknown reasons. The molecular chaperone heat shock protein 90 (HSP90) plays an important role in the establishment and maintenance of treatment resistance, since it crucially assists the folding and stabilization of various DDR regulators. Accordingly, inhibition of HSP90 represents a multi-target strategy to interfere with DDR function and to sensitize cancer cells to radiotherapy. Using NW457, a pochoxime-based HSP90 inhibitor with favorable brain pharmacokinetic profile, we show here that HSP90 inhibition at low concentrations with per se limited cytotoxicity leads to downregulation of various DNA damage response factors on the protein level, distinct transcriptomic alterations, impaired DNA damage repair, and reduced clonogenic survival in response to ionizing irradiation in glioblastoma cells in vitro. In vivo, HSP90 inhibition by NW457 improved the therapeutic outcome of fractionated CBCT-based irradiation in an orthotopic, syngeneic GBM mouse model, both in terms of tumor progression and survival. Nevertheless, in view of the promising in vitro results the in vivo efficacy was not as strong as expected, although apart from the radiosensitizing effects HSP90 inhibition also reduced irradiation-induced GBM cell migration and tumor invasiveness. Hence, our findings identify the combination of HSP90 inhibition and radiotherapy in principle as a promising strategy for GBM treatment whose performance needs to be further optimized by improved inhibitor substances, better formulations and/or administration routes, and fine-tuned treatment sequences.

Sentinel Surveillance Reveals Emerging Daptomycin-Resistant ST736 Enterococcus faecium and Multiple Mechanisms of Linezolid Resistance in Enterococci in the United States.

Enterococcus faecalis and faecium with resistance to daptomycin and/or linezolid are emerging globally. We present the genomic characterization of daptomycin- and linezolid-resistant E. faecalis and E. faecium surveillance isolates from the United States, 2013-2016. Daptomycin resistance was low among E. faecalis (2/364, 0.5%) and E. faecium (17/344, 5%). The majority (71%, 12/17) of daptomycin-resistant E. faecium isolates belonged to the emerging ST736 clone and contained mutations in liaFSR and cls previously associated with resistance. However, 1/2 E. faecalis and 3/17 E. faecium did not contain these mutations previously associated with daptomycin resistance. Linezolid resistance was rare among E. faecalis (1/364, 0.3%) and E. faecium (2/344, 0.6%). These two E. faecium isolates, one of which was also resistant to daptomycin and vancomycin, contained the 23S rRNA nucleotide mutation (G2576T) associated with linezolid resistance. Long-read sequencing revealed the linezolid-resistant E. faecalis isolate contained chromosomal- and plasmid-encoded copies of optrA. The chromosomal optrA was located on the recently described Tn6674 multiresistance transposon. The second copy of optrA was encoded on an ∼65 kb mosaic plasmid, with component regions sharing high sequence identity to optrA-encoding multiresistance plasmids of animal origin. The optrA-encoding plasmid contained open reading frames predicted to encode proteins associated with a pheromone-responsive plasmid transfer system, and filter mating experiments confirmed the plasmid was conjugative. Continued surveillance of enterococci is necessary to assess the prevalence and trends of daptomycin and linezolid resistance in the United States, characterize resistance mechanisms and how they transfer, and monitor for emerging sequence types associated with resistance.

Contrast-enhanced, conebeam CT-based, fractionated radiotherapy and follow-up monitoring of orthotopic mouse glioblastoma: a proof-of-concept study.

BACKGROUND: Despite aggressive treatment regimens comprising surgery and radiochemotherapy, glioblastoma (GBM) remains a cancer entity with very poor prognosis. The development of novel, combined modality approaches necessitates adequate preclinical model systems and therapy regimens that closely reflect the clinical situation. So far, image-guided, fractionated radiotherapy of orthotopic GBM models represents a major limitation in this regard. METHODS: GL261 mouse GBM cells were inoculated into the right hemispheres of C57BL/6 mice. Tumor growth was monitored by contrast-enhanced conebeam CT (CBCT) scans. When reaching an average volume of approximately 7 mm3, GBM tumors were irradiated with daily fractions of 2 Gy up to a cumulative dose of 20 Gy in different beam collimation settings. For treatment planning and tumor volume follow-up, contrast-enhanced CBCT scans were performed twice per week. Daily repositioning of animals was achieved by alignment of bony structures in native CBCT scans. When showing neurological symptoms, mice were sacrificed by cardiac perfusion. Brains, livers, and kidneys were processed into histologic sections. Potential toxic effects of contrast agent administration were assessed by measurement of liver enzyme and creatinine serum levels and by histologic examination. RESULTS: Tumors were successfully visualized by contrast-enhanced CBCT scans with a detection limit of approximately 2 mm3, and treatment planning could be performed. For daily repositioning of the animals, alignment of bony structures in native CT scans was well feasible. Fractionated irradiation caused a significant delay in tumor growth translating into significantly prolonged survival in clear dependence of the beam collimation setting and margin size. Brain sections revealed tumors of similar appearance and volume on the day of euthanasia. Importantly, the repeated contrast agent injections were well tolerated, as liver enzyme and creatinine serum levels were only subclinically elevated, and liver and kidney sections displayed normal histomorphology. CONCLUSIONS: Contrast-enhanced, CT-based, fractionated radiation of orthotopic mouse GBM represents a versatile preclinical technique for the development and evaluation of multimodal radiotherapeutic approaches in combination with novel therapeutic agents in order to accelerate translation into clinical testing.

Tolerance induced via TLR2 and TLR4 in human dendritic cells: role of IRAK-1.

BACKGROUND: While dendritic cells (DCs) can induce tolerance in T cells, little is known about tolerance induction in DCs themselves. We have analysed tolerance induced in human in-vitro generated DCs by repeated stimulation with ligands for TLR4 and TLR2. RESULTS: DCs stimulated with the TLR4 ligand LPS did show a rapid and pronounced expression of TNF mRNA and protein. When DCs were pre-cultured for 2 days with 5 ng LPS/ml then the subsequent response to stimulation with a high dose of LPS (500 ng/ml) was strongly reduced for both TNF mRNA and protein. At the promoter level there was a reduced transactivation by the -1173 bp TNF promoter and by a construct with a tetrameric NF-kappaB motif. Within the signalling cascade leading to NF-kappaB activation we found an ablation of the IRAK-1 adaptor protein in LPS-tolerant DCs. Pre-culture of DCs with the TLR2 ligand Pam3Cys also led to tolerance with respect to TNF gene expression and IRAK-1 protein was ablated in such tolerant cells as well, while IRAK-4 protein levels were unchanged. CONCLUSION: These data show that TLR-ligands can render DCs tolerant with respect to TNF gene expression by a mechanism that likely involves blockade of signal transduction at the level of IRAK-1.