Targeting the ABC transporter ABCB5 sensitizes glioblastoma to temozolomide-induced apoptosis through a cell-cycle checkpoint regulation mechanism.

Glioblastoma multiforme (GBM) is a malignant brain tumor with a poor prognosis resulting from tumor resistance to anticancer therapy and a high recurrence rate. Compelling evidence suggests that this is driven by subpopulations of cancer stem cells (CSCs) with tumor-initiating potential. ABC subfamily B member 5 (ABCB5) has been identified as a molecular marker for distinct subsets of chemoresistant tumor-initiating cell populations in diverse human malignancies. In the current study, we examined the potential role of ABCB5 in growth and chemoresistance of GBM. We found that ABCB5 is expressed in primary GBM tumors, in which its expression was significantly correlated with the CSC marker protein CD133 and with overall poor survival. Moreover, ABCB5 was also expressed by CD133-positive CSCs in the established human U-87 MG, LN-18, and LN-229 GBM cell lines. Antibody- or shRNA-mediated functional ABCB5 blockade inhibited proliferation and survival of GBM cells and sensitized them to temozolomide (TMZ)-induced apoptosis in vitro Likewise, in in vivo human GBM xenograft experiments with immunodeficient mice, mAb treatment inhibited growth of mutant TP53, WT PTEN LN-229 tumors, and sensitized LN-229 tumors to TMZ therapy. Mechanistically, we demonstrate that ABCB5 blockade inhibits TMZ-induced G2/M arrest and augments TMZ-mediated cell death. Our results identify ABCB5 as a GBM chemoresistance marker and point to the potential utility of targeting ABCB5 to improve current GBM therapies.

ATP-binding cassette member B5 (ABCB5) promotes tumor cell invasiveness in human colorectal cancer.

ABC member B5 (ABCB5) mediates multidrug resistance (MDR) in diverse malignancies and confers clinically relevant 5-fluorouracil resistance to CD133-expressing cancer stem cells in human colorectal cancer (CRC). Because of its recently identified roles in normal stem cell maintenance, we hypothesized that ABCB5 might also serve MDR-independent functions in CRC. Here, in a prospective clinical study of 142 CRC patients, we found that ABCB5 mRNA transcripts previously reported not to be significantly expressed in healthy peripheral blood mononuclear cells are significantly enriched in patient peripheral blood specimens compared with non-CRC controls and correlate with CRC disease progression. In human-to-mouse CRC tumor xenotransplantation models that exhibited circulating tumor mRNA, we observed that cancer-specific ABCB5 knockdown significantly reduced detection of these transcripts, suggesting that the knockdown inhibited tumor invasiveness. Mechanistically, this effect was associated with inhibition of expression and downstream signaling of AXL receptor tyrosine kinase (AXL), a proinvasive molecule herein shown to be produced by ABCB5-positive CRC cells. Importantly, rescue of AXL expression in ABCB5-knockdown CRC tumor cells restored tumor-specific transcript detection in the peripheral blood of xenograft recipients, indicating that ABCB5 regulates CRC invasiveness, at least in part, by enhancing AXL signaling. Our results implicate ABCB5 as a critical determinant of CRC invasiveness and suggest that ABCB5 blockade might represent a strategy in CRC therapy, even independently of ABCB5's function as an MDR mediator.

Evaluation of apramycin against spectinomycin-resistant and -susceptible strains of Neisseria gonorrhoeae.

BACKGROUND: The emergence of Neisseria gonorrhoeae resistant to all currently available antimicrobial therapies poses a dire public health threat. New antimicrobial agents with activity against N. gonorrhoeae are urgently needed. Apramycin is an aminocyclitol aminoglycoside with broad-spectrum in vitro activity against MDR Gram-negative pathogens and Staphylococcus aureus. However, its activity against N. gonorrhoeae has not been described. OBJECTIVES: The activity spectrum of apramycin against a collection of MDR N. gonorrhoeae was assessed. Isolates tested included those susceptible and resistant to the structurally distinct aminocyclitol, spectinomycin. RESULTS: The modal MICs for apramycin and spectinomycin were 16 mg/L and 32 mg/L, respectively. The epidemiological cut-off (ECOFF) for apramycin was 64 mg/L. No strains among 77 tested had an MIC above this ECOFF, suggesting very low levels of acquired apramycin resistance. In time-kill analysis, apramycin demonstrated rapid bactericidal activity comparable to that of spectinomycin. CONCLUSIONS: Apramycin has broad-spectrum, rapidly bactericidal activity against N. gonorrhoeae. Future pharmacokinetic and pharmacodynamic studies will be needed to determine whether apramycin and/or apramycin derivatives hold promise as new therapeutics for N. gonorrhoeae infection.

Activity of Fosfomycin against Extended-Spectrum-ß-Lactamase-Producing Uropathogens in Patients in the Community and Hospitalized Patients.

Few oral antibiotics exist for the empirical treatment of extended-spectrum ß-lactamase (ESBL) urinary tract infections (UTI). In this study, we sought to determine the activity of fosfomycin against ESBL-producing uropathogens from patients at 3 Veterans Affairs (VA) facilities between 2010 and 2013. Among the ESBL uropathogens, 19.9% were fosfomycin resistant. Klebsiella species were more likely than Escherichia coli to be resistant (46% versus 4%; P < 0.001). Fosfomycin remains active against a majority of the ESBL uropathogens, although resistance among Klebsiella spp. was higher than that in previous reports.

Molecular epidemiology of methicillin-resistant Staphylococcus aureus isolates from patients newly identified as nasal carriers.

We aimed to determine whether additional molecular and microbiological evaluations of methicillin-resistant Staphylococcus aureus (MRSA) isolated from patients newly identified as nasal carriers were useful for control strategies and whether longitudinal testing during the same or repeat hospitalization changed MRSA status. Nasal swabs from patients positive by Xpert MRSA PCR and not known to be colonized in the previous year were cultured for S. aureus. Isolates were tested for resistance to a variety of antibiotics, including high-level mupirocin resistance (HLMR) and low-level mupirocin resistance (LLMR) and the presence of genes mecA and mupA and those for Panton-Valentine leukocidin (PVL), USA300, and USA400. Repeat nasal screens during the 6-month study were tested for continued presence of MRSA. Among 130 patients, cultures revealed MRSA in 85 (65.4%), methicillin-susceptible S. aureus in 19 (14.6%), and no growth in 26 (20%). MRSA isolates were USA300 positive in 13/85 (15.3%) and LLMR in 8/85 (9.4%) patients. No isolates were HLMR or mupA positive. mecA dropout was detected in 9/130 (6.9%) patients. The rate of subsequent MRSA infections in USA300-positive versus -negative patients was not different. MRSA nasal status remained concordant in 69/70 (98.6%) patients who had follow-up testing. The findings do not support expanding MRSA surveillance to include routine detection of genes for USA300, PVL, or mupA, all of which were either of low frequency or not significantly associated with MRSA infection risk in our population of newly identified nasal carriers. Repeat nasal screening for MRSA during the same or subsequent hospitalizations over 6 months could also be deferred, reducing costs associated with screening.

Identification, structure, and characterization of an exopolysaccharide produced by Histophilus somni during biofilm formation.

BACKGROUND: Histophilus somni, a gram-negative coccobacillus, is an obligate inhabitant of bovine and ovine mucosal surfaces, and an opportunistic pathogen responsible for respiratory disease and other systemic infections in cattle and sheep. Capsules are important virulence factors for many pathogenic bacteria, but a capsule has not been identified on H. somni. However, H. somni does form a biofilm in vitro and in vivo, and the biofilm matrix of most bacteria consists of a polysaccharide. RESULTS: Following incubation of H. somni under growth-restricting stress conditions, such as during anaerobiosis, stationary phase, or in hypertonic salt, a polysaccharide could be isolated from washed cells or culture supernatant. The polysaccharide was present in large amounts in broth culture sediment after H. somni was grown under low oxygen tension for 4-5 days (conditions favorable to biofilm formation), but not from planktonic cells during log phase growth. Immuno-transmission electron microscopy showed that the polysaccharide was not closely associated with the cell surface, and was of heterogeneous high molecular size by gel electrophoresis, indicating it was an exopolysaccharide (EPS). The EPS was a branched mannose polymer containing some galactose, as determined by structural analysis. The mannose-specific Moringa M lectin and antibodies to the EPS bound to the biofilm matrix, demonstrating that the EPS was a component of the biofilm. The addition of N-acetylneuraminic acid to the growth medium resulted in sialylation of the EPS, and increased biofilm formation. Real-time quantitative reverse transcription-polymerase chain reaction analyses indicated that genes previously identified in a putative polysaccharide locus were upregulated when the bacteria were grown under conditions favorable to a biofilm, compared to planktonic cells. CONCLUSIONS: H. somni is capable of producing a branching, mannose-galactose EPS polymer under growth conditions favorable to the biofilm phase of growth, and the EPS is a component of the biofilm matrix. The EPS can be sialylated in strains with sialyltransferase activity, resulting in enhanced density of the biofilm, and suggesting that EPS and biofilm formation may be important to persistence in the bovine host. The EPS may be critical to virulence if the biofilm state is required for H. somni to persist in systemic sites.

Evaluation of the fully automated AIX1000 rapid plasma reagin system compared to a manual plasma reagin testing method for the diagnosis of syphilis.

The analytical performance of the FDA-cleared AIX1000 automated RPR testing platform was evaluated in comparison to manual RPR card testing. Eight hundred thirty-three patient serum samples were analyzed, 87 samples were positive by the AIX1000, 108 were positive by the manual test method; overall agreement between methods was 96.5% (κ = 0.83). Cases were further classified by clinical and laboratory-based confirmation of disease, to which reactivity rates were compared, yielding sensitivities of 96.4% and 100%, and specificities of 99.2% and 96.8% for the automated and manual RPR methods, respectively. The difference in specificity between methods was statistically significant (P < 0.001). Twenty-five of 29 samples with discordant results were reactive by manual testing (titers of 1:1 or 1:2); 21 of 25 patients with negative AIX100 results were identified to have histories of remote, treated syphilis. Overall, the AIX1000 platform demonstrated excellent agreement with the manual RPR method; discrepancies occurred with specimens at the threshold of reactivity.

Cohorting based on nasal methicillin-resistant Staphylococcus aureus status: an opportunity to share more than a room.

BACKGROUND: Hospital roommates are cohorted with similarly colonized patients to decrease methicillin-resistant Staphylococcus aureus (MRSA) transmission risk. However, little is known about differences in S aureus nasal and extranasal carriage between hospital roommates who are in MRSA or non-MRSA designated rooms. METHODS: Patients sharing hospital rooms were cultured for S aureus in the nose, throat, and other body sites. Differences in S aureus methicillin and mupirocin susceptibility and USA300 type were evaluated. RESULTS: Eighty-two patients comprising 48 roommate pairs were studied. Among 6 roommate pairs in MRSA rooms, 3 (50%) had differences in carriage based on having methicillin-susceptible S aureus at an extranasal body site. In non-MRSA rooms, 19 (45%) roommate pairs had differences in S aureus carriage. Extranasal colonization was significantly associated with discordance between roommates, P < .001. Antibiotic exposure, ward type, and the duration of room sharing were not associated with discordance. CONCLUSION: Patients have almost a 50% chance of having differences in S aureus colonization compared with their hospital roommate, even in MRSA-designated rooms. Cohorting by MRSA status at the time of admission may not be as effective a control strategy as horizontal measures that do not rely on known colonization with S aureus or other pathogens.

Isolation and mutagenesis of a capsule-like complex (CLC) from Francisella tularensis, and contribution of the CLC to F. tularensis virulence in mice.

BACKGROUND: Francisella tularensis is a category-A select agent and is responsible for tularemia in humans and animals. The surface components of F. tularensis that contribute to virulence are not well characterized. An electron-dense capsule has been postulated to be present around F. tularensis based primarily on electron microscopy, but this specific antigen has not been isolated or characterized. METHODS AND FINDINGS: A capsule-like complex (CLC) was effectively extracted from the cell surface of an F. tularensis live vaccine strain (LVS) lacking O-antigen with 0.5% phenol after 10 passages in defined medium broth and growth on defined medium agar for 5 days at 32°C in 7% CO2. The large molecular size CLC was extracted by enzyme digestion, ethanol precipitation, and ultracentrifugation, and consisted of glucose, galactose, mannose, and Proteinase K-resistant protein. Quantitative reverse transcriptase PCR showed that expression of genes in a putative polysaccharide locus in the LVS genome (FTL_1432 through FTL_1421) was upregulated when CLC expression was enhanced. Open reading frames FTL_1423 and FLT_1422, which have homology to genes encoding for glycosyl transferases, were deleted by allelic exchange, and the resulting mutant after passage in broth (LVSΔ1423/1422_P10) lacked most or all of the CLC, as determined by electron microscopy, and CLC isolation and analysis. Complementation of LVSΔ1423/1422 and subsequent passage in broth restored CLC expression. LVSΔ1423/1422_P10 was attenuated in BALB/c mice inoculated intranasally (IN) and intraperitoneally with greater than 80 times and 270 times the LVS LD50, respectively. Following immunization, mice challenged IN with over 700 times the LD50 of LVS remained healthy and asymptomatic. CONCLUSIONS: Our results indicated that the CLC may be a glycoprotein, FTL_1422 and -FTL_1423 were involved in CLC biosynthesis, the CLC contributed to the virulence of F. tularensis LVS, and a CLC-deficient mutant of LVS can protect mice against challenge with the parent strain.

Bronchus-associated lymphoid tissue (BALT) and survival in a vaccine mouse model of tularemia.

BACKGROUND: Francisella tularensis causes severe pulmonary disease, and nasal vaccination could be the ideal measure to effectively prevent it. Nevertheless, the efficacy of this type of vaccine is influenced by the lack of an effective mucosal adjuvant. METHODOLOGY/PRINCIPAL FINDINGS: Mice were immunized via the nasal route with lipopolysaccharide isolated from F. tularensis and neisserial recombinant PorB as an adjuvant candidate. Then, mice were challenged via the same route with the F. tularensis attenuated live vaccine strain (LVS). Mouse survival and analysis of a number of immune parameters were conducted following intranasal challenge. Vaccination induced a systemic antibody response and 70% of mice were protected from challenge as showed by their improved survival and weight regain. Lungs from mice recovering from infection presented prominent lymphoid aggregates in peribronchial and perivascular areas, consistent with the location of bronchus-associated lymphoid tissue (BALT). BALT areas contained proliferating B and T cells, germinal centers, T cell infiltrates, dendritic cells (DCs). We also observed local production of antibody generating cells and homeostatic chemokines in BALT areas. CONCLUSIONS: These data indicate that PorB might be an optimal adjuvant candidate for improving the protective effect of F. tularensis antigens. The presence of BALT induced after intranasal challenge in vaccinated mice might play a role in regulation of local immunity and long-term protection, but more work is needed to elucidate mechanisms that lead to its formation.