A new test for long-term spatial memory using an operant chamber in mice

As part of ongoing efforts to develop fully automated and standardized behavioral tasks to probe cognitive and mnemonic capabilities of mice, we have constructed a new rectangular operant chamber. The chamber contains numerous nose poke holes, distributed over three of its inner walls that are identifiable by their spatial locations. Using this apparatus, we have developed a 'spatial' memory task using a successive reversal discrimination paradigm. Mice learn to discriminate, by trial and error, the position of a single valid hole during a Presentation session wherein they obtained a maximum of 20 reinforcements or 15 min time elapsed. Following a delay interval, they were resubmitted to the same task (Test) using the same reinforced hole. Results indicated that C57BL/6 mice exhibited a significant improvement during the Test, the magnitude of the improvement (memory savings) being dependent on the length of retention intervals ranging from 5 min to 24h. In addition, discrimination performance was sensitive to scopolamine in a dose dependent manner. The simplicity in task set up and the minimal labor and space requirements make this task suitable for high throughput behavioral characterization of genetically modified mice.     

Combined measurement of procalcitonin and soluble TREM-1 in the diagnosis of nosocomial sepsis

This prospective, non-interventional study was conducted in a medical adult intensive care unit to determine the usefulness of procalcitonin (PCT) and soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) determinations in the diagnosis of nosocomial sepsis. Serum PCT and bronchoalveolar lavage fluid sTREM-1 concentrations were measured in 50 critically ill patients suffering from nosocomial sepsis. Ventilator-associated pneumonia (VAP) was diagnosed in 31 patients and extrapulmonary sepsis in 19. Increase serum PCT concentration (>0.15 ng/ml) was found in 44 (88%) patients and was higher in those suffering from a non-pulmonary sepsis. The concomitant BAL sTREM-1 determination correctly classified pulmonary (VAP) versus non-pulmonary origin in 41 out of 44 cases (93%). Even when PCT concentration remained low, sTREM-1 assessment allowed for the detection of the sepsis (VAP) in 50% of cases. Both PCT and sTREM-1 concentrations were low in only 3 patients (6%) in whom sepsis could have been missed if only diagnosed by the measurement of these 2 biomarkers. We therefore concluded that the combined measurement of serum PCT and BAL sTREM-1 concentrations could be of interest in detecting the presence of a nosocomial sepsis and in discriminating VAP versus extrapulmonary infection.     

Cellular cofactors affecting hepatitis C virus infection and replication

Recently identified hepatitis C virus (HCV) isolates that are infectious in cell culture provide a genetic system to evaluate the significance of virus-host interactions for HCV replication. We have completed a systematic RNAi screen wherein siRNAs were designed that target 62 host genes encoding proteins that physically interact with HCV RNA or proteins or belong to cellular pathways thought to modulate HCV infection. This includes 10 host proteins that we identify in this study to bind HCV NS5A. siRNAs that target 26 of these host genes alter infectious HCV production >3-fold. Included in this set of 26 were siRNAs that target Dicer, a principal component of the RNAi silencing pathway. Contrary to the hypothesis that RNAi is an antiviral pathway in mammals, as has been reported for subgenomic HCV replicons, siRNAs that target Dicer inhibited HCV replication. Furthermore, siRNAs that target several other components of the RNAi pathway also inhibit HCV replication. MicroRNA profiling of human liver, human hepatoma Huh-7.5 cells, and Huh-7.5 cells that harbor replicating HCV demonstrated that miR-122 is the predominant microRNA in each environment. miR-122 has been previously implicated in positively regulating the replication of HCV genotype 1 replicons. We find that 2'-O-methyl antisense oligonucleotide depletion of miR-122 also inhibits HCV genotype 2a replication and infectious virus production. Our data define 26 host genes that modulate HCV infection and indicate that the requirement for functional RNAi for HCV replication is dominant over any antiviral activity this pathway may exert against HCV.     

Changes in adult olfactory bulb neurogenesis in mice expressing the A30P mutant form of alpha-synuclein

In familial and sporadic forms of Parkinson's disease (PD), alpha-synuclein pathology is present in the brain stem nuclei and olfactory bulb (OB) long before Lewy bodies are detected in the substantia nigra. The OB is an active region of adult neurogenesis, where newly generated neurons physiologically integrate. While accumulation of wild-type alpha-synuclein is one of the pathogenic hallmarks of non-genetic forms of PD, the A30P alpha-synuclein mutation results in an earlier disease onset and a severe clinical phenotype. Here, we study the regulation of adult neurogenesis in the subventricular zone (SVZ)/OB system in a tetracycline-suppressive (tet-off) transgenic model of synucleinopathies, expressing human mutant A30P alpha-synuclein under the control of the calcium/calmodulin-dependent protein kinase II alpha (CaMK) promoter. In A30P transgenic mice alpha-synuclein was abundant at the site of integration in the glomerular cell layer of the OB. Without changes in proliferation in the SVZ, significantly fewer newly generated neurons were observed in the OB granule cell and glomerular layers of A30P transgenic mice than in controls, most probably due to increased cell death. By tetracycline-dependent abrogation of A30P alpha-synuclein expression, OB neurogenesis and programmed cell death was restored to control levels. Our results indicate that, using A30P conditional (tet-off) mice, A30P alpha-synuclein has a negative impact on olfactory neurogenesis and suppression of A30P alpha-synuclein enhances survival of newly generated neurons. This finding suggests that interfering with alpha-synuclein pathology can rescue newly generated neurons, possibly leading to new targets for therapeutic interventions in synucleinopathies.     

Vaccination with human anti-trastuzumab anti-idiotype scFv reverses HER2 immunological tolerance and induces tumor immunity in MMTV.f.huHER2(Fo5) mice

Introduction

Novel adjuvant therapies are needed to prevent metastatic relapses in HER2-expressing breast cancer. Here, we tested whether trastuzumab-selected single-chain Fv (scFv) could be used to develop an anti-idiotype-based vaccine to inhibit growth of HER2-positive tumor cells in vitro and in vivo through induction of long-lasting HER-specific immunity.

Methods

BALB/c mice were immunized with anti-trastuzumab anti-idiotype (anti-Id) scFv (scFv40 and scFv69), which mimic human HER2. Their sera were assessed for the presence of HER2-specific Ab1'' antibodies and for their ability to reduce viability of SK-OV-3 cells, a HER2-positive cancer cell line, in nude mice. MMTV.f.huHER2(Fo5) transgenic mice were immunized with scFv40 and scFv69 and, then, growth inhibition of spontaneous HER2-positive mammary tumors, humoral response, antibody isotype as well as splenocyte secretion of IL2 and IFN-γ were evaluated.

Results

Adoptively-transferred sera from BALB/c mice immunized with scFv40 and scFv69 contain anti-HER2 Ab1'' antibodies that can efficiently inhibit growth of SK-OV-3 cell tumors in nude mice. Similarly, prophylactic vaccination with anti-Id scFv69 fully protects virgin or primiparous FVB-MMTV.f.huHER2(Fo5) females from developing spontaneous mammary tumors. Moreover, such vaccination elicits an anti-HER2 Ab1'' immune response together with a scFv69-specific Th1 response with IL2 and IFN-γ cytokine secretion.

Conclusions

Anti-trastuzumab anti-Id scFv69, used as a therapeutic or prophylactic vaccine, protects mice from developing HER2-positive mammary tumors by inducing both anti-HER2 Ab1'' antibody production and an anti-HER2 Th2-dependent immune response. These results suggest that scFv69 could be used as an anti-Id-based vaccine for adjuvant therapy of patients with HER2-positive tumors to reverse immunological tolerance to HER2.