Comparative genomic analysis reveals a high prevalence of inter-species in vivo transfer of carbapenem-resistance plasmids in patients with haematological malignancies.

OBJECTIVES: Conjugative gene transfer has been considered as one of the driving factors in the transmission and dissemination of multidrug resistance in bacteria. The abundance of antimicrobial resistance genes and bacteria in the gut microbiome may provide the ideal platform for plasmid exchange. Systematic data on in vivo horizontal gene transfer (HGT) and its frequency are scarce. MATERIALS AND METHODS: One hundred and ninety-six carbapenem-resistant gram-negative bacilli (CRGNBs) from 179 patients (158 inpatients and 21 outpatients) between January 2016 and April 2017 were analysed retrospectively. Alignment of plasmid content for 32 isolates from 16 patients with multiple CRGNB species was performed from whole-genome sequencing (WGS) data. RESULTS: Sixteen of the 179 patients (8.9%) were colonized and/or infected with more than one CRGNB species; 11/179 (6.1%) were colonized by multiple carbapenem-resistant Enterobacteriaceae (CREs) and 5/179 (2.8%) by carbapenem-resistant non-fermenters (CRNFs) and CREs. WGS suggested interspecies transfer as the predominant mechanism rather than independent acquisition in 8/10 patients (80%, one non-recoverable isolate) with multiple CREs but not in CRNF-CRE combinations; 30/158 inpatients (20%) had underlying haematological malignancies, and they are more likely to exhibit multiple CRGNB strains (OR 3.0, 95%CI 0.98-8.89, p 0.05) and CRE strains (OR 3.9, 95%CI 1.02-14.58, p 0.04) during hospital stay compared to other patient groups. CONCLUSION: Our data give insight into the occurrence of natural in vivo HGT in a clinical setting. Better understanding of HGT will help optimize containment measures and may guide antibiotic stewardship programmes.

Induction of tumor-specific CTL responses using the C-terminal fragment of Viral protein R as cell penetrating peptide.

The discovery of tumor-associated antigens recognized by T lymphocytes opens the possibility of vaccinating cancer patients with defined antigens. However, one of the major limitation of peptide-based vaccines is the low immunogenicity of antigenic peptides. Interestingly, if these epitopes are directly delivered into the cytoplasm of antigen presenting cells, they can be efficiently presented via the direct MHC class I presentation pathway. To improve antigen entry, one promising approach is the use of cell penetrating peptides (CPPs). However, most studies use a covalent binding of the CPP with the antigen. In the present study, we focused on the C-terminal domain of Vpr which was previously demonstrated to efficiently deliver plasmid DNA into cells. We provide evidence that the peptides Vpr55-91 and Vpr55-82 possess the capacity of delivering proteins and epitopes into cell lines as well as into human primary dendritic cells, without the necessicity for a chemical linkage. Moreover, immunization of HLA-A2 transgenic mice with Vpr55-91 as the sole adjuvant is able to induce antigen-specific cytotoxic T lymphocytes against multiple tumor epitopes.

Cytolethal distending toxin promotes Helicobacter cinaedi-associated typhlocolitis in interleukin-10-deficient mice.

Helicobacter cinaedi colonizes a wide host range, including rodents, and may be an emerging zoonotic agent. Colonization parameters, pathology, serology, and inflammatory responses to wild-type H. cinaedi (WT(Hc)) were evaluated in B6.129P2-IL-10(tm1Cgn) (IL-10(-/-)) mice for 36 weeks postinfection (WPI) and in C57BL/6 (B6) mice for 12 WPI. Because cytolethal distending toxin (CDT) may be a virulence factor, IL-10(-/-) mice were also infected with the cdtB(Hc) and cdtB-N(Hc) isogenic mutants and evaluated for 12 WPI. Consistent with other murine enterohepatic helicobacters, WT(Hc) did not cause typhlocolitis in B6 mice, but mild to severe lesions developed at the cecocolic junction in IL-10(-/-) mice, despite similar colonization levels of WT(Hc) in the cecum and colon of both B6 and IL-10(-/-) mice. WT(Hc) and cdtB mutants also colonized IL-10(-/-) mice to a similar extent, but infection with either cdtB mutant resulted in attenuated typhlocolitis and hyperplasia compared to infection with WT(Hc) (P < 0.03), and only WT(Hc) infection caused dysplasia and intramucosal carcinoma. WT(Hc) and cdtB(Hc) mutant infection of IL-10(-/-) mice elevated mRNA expression of tumor necrosis factor alpha, inducible nitric oxide synthase, and gamma interferon in the cecum, as well as elevated Th1-associated serum immunoglobulin G2a(b) compared to infection of B6 mice (P < 0.05). Although no hepatitis was noted, liver samples were PCR positive at various time points for WT(Hc) or the cdtB(Hc) mutant in approximately 33% of IL-10(-/-) mice and in 10 to 20% of WT(Hc)-infected B6 mice. These results indicate that WT(Hc) can be used to model inflammatory bowel disease in IL-10(-/-) mice and that CDT contributes to the virulence of H. cinaedi.

Prospective assessment of pain and functional status after vertebroplasty for treatment of vertebral compression fractures.

BACKGROUND AND PURPOSE: There has been no prospective evaluation of vertebroplasty using a validated instrument. We describe the pain and functional status of 72 patients before and after vertebroplasty, as prospectively evaluated by the Vertebral Compression Fracture Pain and Functional Disability Questionnaire. METHODS: Of 161 consecutive patients, 72 consented to participate in the study and self-completed the questionnaire prior to undergoing vertebroplasty. Differences in pain and distress before and after vertebroplasty, and between the first and second follow-up intervals, were evaluated. Mean scores for each of 24 activities of daily living (ADLs) were plotted at the baseline and first and second follow-up intervals. RESULTS: The mean (SD) patient age was 74 (10) years; 80% were female. On the 0 (no pain) to 10 (pain as bad as it could be) visual analog pain scale, patients reported significantly more pain, on average, before undergoing percutaneous vertebroplasty (PV) than at the first follow-up interval (mean 5.8 vs 3.5, p<0.001). The reduction in reported pain following vertebroplasty persisted at the second follow-up on both the visual analog and adjectival pain scales. Among the 24 ADLs, between 25% and 69% of patients reported a mean improvement of at least 1 level on the 5-point ADL scale, and between 14% and 55% reported a mean improvement of at least two levels. The majority of the improvement in reported functional status following vertebroplasty was sustained at the second follow-up interval. CONCLUSION: PV resulted in substantial, lasting reduction in pain and improvement in ability to perform ADLs.

Enhanced lentiviral transduction of monocyte-derived dendritic cells in the presence of conditioned medium from dying monocytes.

Lentiviral vectors (LVs) are attractive vehicles for the transduction of human dendritic cells (DCs) in order to mobilize their endogenous antigen presentation pathways. We analyzed here how to improve the efficiency of LV transduction, which we performed at the initial stages of the differentiation of purified monocytes into dendritic cells (Mo-DCs). Using LVs pseudotyped with the vesicular stomatitis virus envelope G glycoprotein (VSV-G), we found that a conditioned medium derived from dying monocytes (MCM) improved by 2- to 10- fold the proportion of transduced Mo-DCs. This enhanced transduction efficiency requires the presence of MCM during the initial stage of LV transduction and does not affect the phenotype and antigen presentation function of terminally differentiated Mo-DCs. Importantly, we found that MCM derived from a human acute monocytic leukemia cell line, THP-1, was equally effective. The MCM activity was heat stable (56 degrees C) and was present in the soluble fraction after high-speed centrifugation. Altogether our results show that a soluble factor present in dying monocyte cultures can replace advantageously facilitating agents such as Polybrene, to achieve high LV transductions levels. This protocol can be performed with autologous monocytes and is therefore applicable in clinical settings.

Global gene expression profiling: a complement to conventional histopathologic analysis of neoplasia.

Transcriptional profiling of entire tumors has yielded considerable insight into the molecular mechanisms of heterogeneous cell populations within different types of neoplasms. The data thus acquired can be further refined by microdissection methods that enable the analyses of subpopulations of neoplastic cells. Separation of the various components of a neoplasm (i.e., stromal cells, inflammatory infiltrates, and blood vessels) has been problematic, primarily because of a paucity of tools for accurate microdissection. The advent of laser capture microdissection combined with powerful tools of linear amplification of RNA and high-throughput microarray-based assays have allowed the transcriptional mapping of intricate and highly complex networks within pure populations of neoplastic cells. With this approach, specific "molecular signatures" can be assigned to tumors of distinct or even similar histomorphology, thereby aiding the desired objective of pattern recognition, tumor classification, and prognostication. This review highlights the potential benefits of global gene expression profiling of tumor cells as a complement to conventional histopathologic analyses.

Characterization of cis-acting sequences involved in canine adenovirus packaging.

The cis-acting packaging domain in adenovirus serotype 5 (Ad5) is a series of redundant, albeit not functionally equivalent, "A-repeats" made up of the consensus sequence 5'-TTTGN(8)CG-3'. A-repeats may bind trans-acting factors that direct packaging of the adenovirus genome into the preformed capsid. To try to understand this basic mechanism, we examined the packaging domain from a nonhuman adenovirus. We delimited the canine adenovirus type 2 (CAV-2) packaging domain to within 156 bp via a conditional mutation based on the Cre/loxP excision. Using an insertion, deletion, and substitution strategy, we generated packaging-defective CAV-2 vectors. Our results demonstrate that, like Ad5, CAV-2 cis-acting packaging sequences are located near the left inverted terminal repeat and are redundant, but not functionally equivalent. However, the bipartite motif found in Ad5 is present only once in CAV-2 and deletion of it caused only a minor variation in the packaging efficiency. We have identified at least four functional cis-acting packaging sequences in CAV-2. The CAV-2 vectors that we generated were not replication-defective in an E1-transcomplementing cell line and as heat stable as the parental vectors that did not contain mutations.

Canine adenovirus type 2 attachment and internalization: coxsackievirus-adenovirus receptor, alternative receptors, and an RGD-independent pathway.

The best-characterized receptors for adenoviruses (Ads) are the coxsackievirus-Ad receptor (CAR) and integrins alpha(v)beta(5) and alpha(v)beta(3), which facilitate entry. The alpha(v) integrins recognize an Arg-Gly-Asp (RGD) motif found in some extracellular matrix proteins and in the penton base in most human Ads. Using a canine adenovirus type 2 (CAV-2) vector, we found that CHO cells that express CAR but not wild-type CHO cells are susceptible to CAV-2 transduction. Cells expressing alpha(M)beta(2) integrins or major histocompatibility complex class I (MHC-I) molecules but which do not express CAR were not transduced. Binding assays showed that CAV-2 attaches to a recombinant soluble form of CAR and that Ad type 5 (Ad5) fiber, penton base, and an anti-CAR antibody partially blocked attachment. Using fluorescently labeled CAV-2 particles, we found that in some cells nonpermissive for transduction, inhibition was at the point of internalization and not attachment. The transduction efficiency of CAV-2, which lacks an RGD motif, surprisingly mimicked that of Ad5 when tested in cells selectively expressing alpha(v)beta(5) and alpha(v)beta(3) integrins. Our results demonstrate that CAV-2 transduction is augmented by CAR and possibly by alpha(v)beta(5), though transduction can be CAR and alpha(v)beta(3/5) independent but is alpha(M)beta(2), MHC-I, and RGD independent, demonstrating a transduction mechanism which is distinct from that of Ad2/5.

Canine adenovirus vectors: an alternative for adenovirus-mediated gene transfer.

Preclinical studies have shown that gene transfer following readministration of viral vectors is often inefficient due to the presence of neutralizing antibodies. Vectors derived from ubiquitous human adenoviruses may have limited clinical use because preexisting humoral and cellular immunity is found in 90% of the population. Furthermore, risks associated with the use of human adenovirus vectors, such as the need to immunosuppress or tolerize patients to a potentially debilitating virus, are avoidable if efficient nonhuman adenovirus vectors are feasible. Plasmids containing recombinant canine adenovirus (CAV) vectors from which the E1 region had been deleted were generated and transfected into a CAV E1-transcomplementing cell line. Vector stocks, with titers greater than or equal to those obtained with human adenovirus vectors, were free of detectable levels of replication-competent CAV and had a low particle-to-transduction unit ratio. CAV vectors were replication defective in all cell lines tested, transduced human-derived cells at an efficiency similar to that of a comparable human adenovirus type 5 vector, and are amenable to in vivo use. Importantly, 49 of 50 serum samples from healthy individuals did not contain detectable levels of neutralizing CAV antibodies.

Amino-acid change in the Epstein-Barr-virus ZEBRA protein in undifferentiated nasopharyngeal carcinomas from Europe and North Africa.

Different Epstein-Barr-virus(EBV) variants were found to be associated with nasopharyngeal carcinoma (NPC). The type-C variant lacks the BamHI site between the BamHI W1* and I* regions and the type-f variant has an extra BamHI site in the BamHI F fragment. The BNLF1 gene (which encodes the LMP1 protein) from a nude-mouse-passaged CAO strain and from NPC biopsies from Taiwanese patients also exhibits variations resulting in structural and functional differences in the protein. The BZLF1 gene encodes the ZEBRA protein which triggers the EBV lytic cycle. A difference has been observed in 8 amino acids in the ZEBRA sequence in B95-8 (Z95) and P3HR1 (ZP3) cell lines. EBV found in NPC biopsies and peripheral-blood cells from Asians was predominantly of the ZP3 type (72%), while 81% of samples from different EBV-associated diseases and peripheral-blood cells from North Africa or Europe were of the Z95 type. We found that an alanine 206 had been replaced by a serine in the Z95 sequence in 72% of the NPC biopsies from European and North African patients. The Zser206 variant is found in a significantly lower percentage (p < 0.001) of other EBV-positive tissues from individuals in the same region (10-33%). In contrast, a 30-bp deletion is observed near the 3' end of the LMP1 gene in the majority of EBV (86%) from NPC and peripheral-blood cells from Asians, whereas a significantly lower percentage (p < 0.001) of NPC biopsies from European and North African patients (56%) have this deletion, as do lymphocytes from control individuals from the same region (36 and 55% respectively).

A recombinant E1-deleted canine adenoviral vector capable of transduction and expression of a transgene in human-derived cells and in vivo.

Human adenovirus (HAV) serotypes 2 and 5 are commonly used as vector backbones for adenovirus-mediated gene transfer. However, HAVs were chosen as a backbone for the vectors for historical reasons and have a number of significant disadvantages when used as a shuttle for gene transfer in humans. As an initial trial to circumvent some of the shortcomings of HAV vectors, we have produced an E1-deleted canine adenovirus type 2 (CAV-2) vector for gene transfer. Initially, we demonstrated that CAV-2 undergoes an abortive viral cycle in a wide range of human-derived cell lines. Second, we assayed human sera containing HAV-5 neutralizing antibodies for their ability to inhibit CAV-2-induced plaques on permissive cells. In the cohort tested, our data demonstrate that the humoral response directed against HAV-5 does not inhibit CAV-2 plaque formation in the majority of cases. Canine cell lines expressing the E1 region of CAV-2 were generated and characterized. A recombinant CAV vector (CAVRSVbetagal) deleted in the E1 region and harboring lacZ was constructed. We show that CAVRSVbetagal is able to transduce and direct expression of the transgene in vitro in a variety of mammalian cells, most notably primary human-derived cells. In addition, gene transfer is demonstrated in vivo using chick embryos.

Patients' opinion of mammography screening services: immediate results versus delayed results due to interpretation by two observers.

OBJECTIVE: The purpose of this study was to evaluate by random questionnaire mailings the preferences of women who have undergone mammography in our region regarding communication of mammography screening results. MATERIALS AND METHODS: Questionnaires were mailed to 400 randomly selected women who were more than 35 years old and who had been treated at our institution for medical or surgical reasons. Questions regarding use of mammography screening at our institution versus services at other locations were included. The questionnaire described two possible mammography services; either a double reading service that would provide delayed reports (DRDR) with the benefit of extra cancers detected, and a service that provides immediate reports given directly to the patient by an on-site radiologist. The presentation of the services was reversed in half the questionnaires to avoid bias. Patients' choices were collected, as were demographic data. The choice of one system over the other was evaluated using the one-sample lest for binomial probability. The chi-square test was used to determine if the order of questions on the survey or the site of patients' screening mammography affected responses. RESULTS: The response rate was 42% (n = 168). Of these, one response informed us of the death of a patient. Of the remaining 167 respondents, 75% (n = 126) preferred the DRDR system, 13% (n = 22) preferred the system providing immediate results (p < .0001), and the other 19 respondents did not select a preference. Of the 167 respondents, 156 answered the question regarding previous screening mammography experience. Of the 105 patients who had undergone screening mammography at our institution, 78% (n = 82) preferred the DRDR system. Of the 51 patients who had undergone mammography elsewhere or who had never undergone mammography, 75% (n = 38) preferred the DRDR system. We found that ordering of presentation of the systems in the questionnaire had no effect on responses. Likewise, whether a respondent had undergone mammography at our institution had no effect on responses (p = 1.0). CONCLUSION: A statistically significant number of women who responded to our questionnaire preferred the DRDR system of reporting screening mammographic results. Educational material about double reading that we included with each patient's questionnaire could account for these results. If the use of a second interpreter is feasible and is done for batch interpretation of screening mammograms, then education of patients about this process may increase acceptance of a delayed mammographic report.