Molecular subtyping of Vibrio cholerae O1 and O139 by pulsed-field gel electrophoresis in Hong Kong: correlation with epidemiological events from 1994 to 2002

Two hundred twenty isolates of Vibrio cholerae O1 and O139 collected from 1994 to 2002 in Hong Kong were analyzed by pulsed-field gel electrophoresis (PFGE). Chromosomal DNAs from all V. cholerae isolates in agarose plugs were digested with the restriction enzyme NotI, resulting in 20 to 27 bands. Sixty distinctive PFGE patterns in the range of 10 to 300 kb were noted among 213 isolates typeable by PFGE. By comparing the common PFGE patterns obtained from four well-defined outbreaks of V. cholerae O1 and O139 with those obtained from other, epidemiologically unrelated isolates during the study period, indistinguishable and similar PFGE patterns were identified, indicating their close relatedness, in agreement with the results of epidemiological investigations. Heterogeneous PFGE patterns (with four to six banding differences), however, were identified among strains that were imported from other parts of Asia, including Indonesia, India, and Pakistan. Correlations with epidemiological information further support the usefulness of PFGE as an epidemiological tool in laboratory investigations of suspected outbreaks. Standardization of PFGE methodology will allow international comparison of fingerprint patterns and will form the basis of a laboratory network for tracking V. cholerae.     

Chronic intracerebroventricular exposure to β-amyloid(1–40) impairs object recognition but does not affect spontaneous locomotor activity or sensorimotor gating in the rat

This study examined the cognitive effects of chronic in vivo exposure to beta-amyloid(1-40) via the intracerebroventricular route on two distinct paradigms. The first test evaluated a form of early attentional control referred to as sensorimotor gating in which an antecedent weak prepulse stimulus modulates the reactivity to a subsequent startle-eliciting stimulus. The second test utilized the spontaneous preference for a novel object over that of a familiar one in rats as a measure of object recognition memory. We found that beta-amyloid exposure leads to a severe deficit in the object memory test but spares sensorimotor gating. Moreover, unlike the water maze deficit induced by beta-amyloid (Nag et al., in preparation), the deficit on object recognition was resistant to amelioration by systemic physostigmine treatment at a dose of 0.06 mg/kg per day intraperitoneally. The present results add to previous reports that beta-amyloid exposure can lead to deficits on hippocampal lesion sensitive tasks, suggesting that dysfunction of the rhinal cortices in addition to that of the septohippocampal system is implicated in beta-amyloid-induced behavioral impairments. It therefore lends support to the hypothesis that beta-amyloid exposure can lead to severe impairment across multiple memory systems.     

Development of a generic real-time PCR assay for simultaneous detection of proviral DNA of simian Betaretrovirus serotypes 1, 2, 3, 4 and 5 and secondary uniplex assays for specific serotype identification

Simian betaretroviruses (formerly Type D retroviruses; SRV) are a group of closely related retroviruses for which the natural host species are Asian monkeys of the genus Macaca. Five serotypes have been identified by classical neutralization assays and three additional untyped variants have been reported (SRV_Tsukuba, SRV-6, SRV-7). These viruses may be significant pathogens in macaque colonies, causing a broad spectrum of clinical disease secondary to viral-induced immune suppression. Undetected SRV infections in research macaques also represent a potential confounding variable in research protocols and a concern for human caretakers. Intensive testing efforts have been implemented to identify infected animals in established colonies. A real-time quantitative generic multiplex PCR assay was developed that is capable of simultaneous detection of proviral DNA of SRV serotypes 1, 2, 3, 4 and 5. This assay incorporates amplification of the oncostatin M (OSM) gene for confirmation of amplifiable DNA and allows quantitation of the number of proviral copies per cell analyzed in each multiplex reaction. Detection of multiple serotypes by PCR increases the efficiency and cost-effectiveness of SRV screening programs. A panel of SRV serotype-specific uniplex real-time PCR assays for discrimination among the five recognized serotypes is also described.     

PCR-based detection of Bacillus anthracis in formalin-fixed tissue from a patient receiving ciprofloxacin

We demonstrate that Bacillus anthracis may be detected from a formalin-fixed, paraffin-embedded biopsy specimen, even after the patient has received antibiotic treatment. Although traditional PCR methods may not be sufficiently sensitive for anthrax detection in such patients, cycle numbers can be increased or PCR can be repeated by using an aliquot from a previous PCR as the template.     

Ricolinostat (ACY‐1215) induced inhibition of aggresome formation accelerates carfilzomib‐induced multiple myeloma cell death

Proteasome inhibition induces the accumulation of aggregated misfolded/ubiquitinated proteins in the aggresome; conversely, histone deacetylase 6 (HDAC6) inhibition blocks aggresome formation. Although this rationale has been the basis of proteasome inhibitor (PI) and HDAC6 inhibitor combination studies, the role of disruption of aggresome formation by HDAC6 inhibition has not yet been studied in multiple myeloma (MM). The present study aimed to evaluate the impact of carfilzomib (CFZ) in combination with a selective HDAC6 inhibitor (ricolinostat) in MM cells with respect to the aggresome‐proteolysis pathway. We observed that combination treatment of CFZ with ricolinostat triggered synergistic anti‐MM effects, even in bortezomib‐resistant cells. Immunofluorescent staining showed that CFZ increased the accumulation of ubiquitinated proteins and protein aggregates in the cytoplasm, as well as the engulfment of aggregated ubiquitinated proteins by autophagosomes, which was blocked by ricolinostat. Electron microscopy imaging showed increased autophagy triggered by CFZ, which was inhibited by the addition of ACY‐1215. Finally, an in vivo mouse xenograft study confirmed a decrease in tumour volume, associated with apoptosis, following treatment with CFZ in combination with ricolinostat. Our results suggest that ricolinostat inhibits aggresome formation, caused by CFZ‐induced inhibition of the proteasome pathway, resulting in enhanced apoptosis in MM cells.     

Cooperation of the proapoptotic receptor agonist rhApo2L/TRAIL with the CD20 antibody rituximab against non-Hodgkin lymphoma xenografts

Recombinant human rhApo2L/TRAIL selectively stimulates apoptosis in various cancer cells through its receptors DR4 and DR5, and is currently in clinical trials. Preclinical studies have established antitumor activity of rhApo2L/TRAIL in models of epithelial cancers; however, efficacy in non-Hodgkin lymphoma (NHL) models is not well studied. Of 7 NHL cell lines tested in vitro, rhApo2L/TRAIL stimulated apoptosis in BJAB, Ramos RA1, and DoHH-2 cells. Rituximab, a CD20 antibody used to treat certain types of NHL, augmented rhApo2L/TRAIL-induced caspase activation in Ramos RA1 and DoHH2 but not BJAB or SC-1 cells, through modulation of intrinsic rather than extrinsic apoptosis signaling. In vivo, rhApo2L/TRAIL and rituximab cooperated to attenuate or reverse growth of tumor xenografts of all 4 of these cell lines. Depletion of natural killer (NK) cells or serum complement substantially reduced combined efficacy against Ramos RA1 tumors, suggesting involvement of antibody-dependent cell- and complement-mediated cytotoxicity. Both agents exhibited greater activity against disseminated than subcutaneous BJAB xenografts, and worked together to inhibit or abolish disseminated tumors and increase survival. Moreover, rhApo2L/TRAIL helped circumvent acquired rituximab resistance of a Ramos variant. These findings provide a strong rationale for clinical investigation of rhApo2L/TRAIL in combination with rituximab as a novel strategy for NHL therapy.     

Pilot study on combination of azacitidine and low-dose cytarabine for patients with refractory anemia with excess blast

This study analyzed the outcomes of the combination of azacitidine and low-dose cytarabine in patients newly diagnosed with refractory anemia with excess blast (RAEB). Patients were treated with azacitidine 75 mg/m² for 7 days subcutaneously and cytarabine 20 mg/m² intravenously for 7 days every 28 days. The assigned regimen was repeated for two cycles, then the patients treated with azacytidine alone until progression or allogeneic stem cell transplantation (allo-SCT). Eighteen patients with 5 RAEB-1 and 13 RAEB-2 were enrolled in the current study. After two cycles of the combination therapy, responses were achieved in nine patients (50.0%): four complete response (CR) (22.2%), one partial response (5.6%), two marrow-CR (11.1%), and two hematologic improvement (11.1%). Four patients (22.2%) progressed to acute leukemia during two cycles of the combination therapy. The 1-year overall survival (OS) was 87.5% for the early response group (responses at two cycles) and 0% for the late response group (responses at four cycles, p = 0.042). Plus, the median survival time was 476 days (range, 37–718 days) for the early response group and 221 days (range, 193–249 days) for the late response group. The 1-year OS was 100% for the patients who underwent allo-SCT and 73.4% for those without allo-SCT. In summary, the combination therapy showed promising response rate when compared to treatment with azacitidine alone. However, it was limited in terms of preventing leukemic transformation. Allo-SCT would seem to be the only available treatment that can alter disease progression.