Glioblastoma (GBM) is the most common primary brain cancer in adults and is virtually incurable. Recent studies have shown that CMV is present in the majority of GBMs. To evaluate if the CMV antigens pp65 and IE1, which are expressed in GBMs, can be targeted with CMV-specific T cells, we measured the frequency of T cells targeting pp65 and IE1 in the peripheral blood of a cohort of 11 sequentially-diagnosed CMV-seropositive GBM patients, and evaluated whether it was feasible to expand autologous CMV-specific T cells for future clinical studies. All 11 CMV-seropositive GBM patients had T cells specific for pp65 and IE1 in their peripheral blood assessed by IFNγ ELIspot assay. However, the precursor frequency of pp65-specific T cells was decreased in comparison to healthy donors (p=0.001). We successfully reactivated and expanded CMV-specific T cells from 6 out of 6 GBM patients using antigen presenting cells transduced with an adenoviral vector encoding pp65 and IE1. CMV-specific T-cell lines contained CD4-positive as well as CD8-positive T cells, recognized pp65- and IE1-positive targets and killed CMV-infected autologous GBM cells. Infusion of such CMV-specific T-cell lines may extend the benefits of T-cell therapy to patients with CMV-positive GBMs.
Glioblastoma; GBM; CMV; T cells; immunotherapy; pp65; IE1
Epstein-Barr virus (EBV) infection leads to Hodgkin’s disease (HD) in some immunocompetent hosts. The malignant Reed-Sternberg cells of HD only express a limited array of subdominant EBV antigens to evade preexisting immune responses to EBV. The EBV-encoded latent membrane proteins (LMP1 and LMP2), which are expressed by HD and various EBV-associated malignancies, have been proposed as a potential target for CTL-based therapy. However, the precursor frequency for LMP-specific CTL is generally low in healthy EBV-infected hosts, and immunotherapy based on these antigens is often compromised by the poor immunogenicity and the oncogenic potential. In the present study, we report that transitively expressing an inhibitor of A20, a key negative regulator of inflammatory signaling pathways, together with the LMP antigens (truncated LMP1 and full-length LMP2) greatly enhances maturation and cytokine production of human (h) monocyte-derived dendritic cells (DCs). As a consequence, LMP1/2-expressed, A20-silenced hDCs have an enhanced potency to prime LMP-specific T cell response. When the in vitro primed T cells are adoptively transferred into tumor-xenografted, severe combined immunodeficient (SCID) mice, some of the xenografted tumors approach complete regression. Thus, the study may provide an available resource of LMP-specific T cells for T cell immunotherapy.
A20; dendritic cells; cytotoxic T lymphocyte; Epstein-Barr virus; latent membrane proteins
An optimized protocol was developed for the simultaneous detection and differentiation of Haemophilus parasuis, Streptococcus suis, and Mycoplasma hyorhinis in formalin-fixed, paraffin-embedded (FFPE) tissues with multiplex nested polymerase chain reaction (PCR). This method also determines the prevalence of these bacteria in pigs with polyserositis. DNA extraction with a combination of a commercial reagent and proteinase K resulted in more frequent detection of the pathogens than DNA extraction with proteinase K alone. Among FFPE tissue samples from 312 cases of polyserositis in which at least 1 bacterial species was detected, multiplex nested PCR detected H. parasuis in 239 (77%), S. suis in 124 (40%), and M. hyorhinis in 40 (13%). The disease was caused by a single pathogen in 224 (72%) of the cases and multiple pathogens in 88 (28%). Among the pigs positive for H. parasuis, S. suis, and M. hyorhinis by multiplex nested PCR, the pathogen was isolated from only 11%, 35%, and 28%, respectively. Therefore, the PCR protocol developed in this study is a useful diagnostic method when samples are negative after isolation methods and even for samples in which only 1 pathogen was isolated.
Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Here we describe red, single-wavelength GECIs, “RCaMPs,” engineered from circular permutation of the thermostable red fluorescent protein mRuby. High-resolution crystal structures of mRuby, the red sensor RCaMP, and the recently published red GECI R-GECO1 give insight into the chromophore environments of the Ca2+-bound state of the sensors and the engineered protein domain interfaces of the different indicators. We characterized the biophysical properties and performance of RCaMP sensors in vitro and in vivo in Caenorhabditis elegans, Drosophila larvae, and larval zebrafish. Further, we demonstrate 2-color calcium imaging both within the same cell (registering mitochondrial and somatic [Ca2+]) and between two populations of cells: neurons and astrocytes. Finally, we perform integrated optogenetics experiments, wherein neural activation via channelrhodopsin-2 (ChR2) or a red-shifted variant, and activity imaging via RCaMP or GCaMP, are conducted simultaneously, with the ChR2/RCaMP pair providing independently addressable spectral channels. Using this paradigm, we measure calcium responses of naturalistic and ChR2-evoked muscle contractions in vivo in crawling C. elegans. We systematically compare the RCaMP sensors to R-GECO1, in terms of action potential-evoked fluorescence increases in neurons, photobleaching, and photoswitching. R-GECO1 displays higher Ca2+ affinity and larger dynamic range than RCaMP, but exhibits significant photoactivation with blue and green light, suggesting that integrated channelrhodopsin-based optogenetics using R-GECO1 may be subject to artifact. Finally, we create and test blue, cyan, and yellow variants engineered from GCaMP by rational design. This engineered set of chromatic variants facilitates new experiments in functional imaging and optogenetics.
calcium imaging; genetically encoded calcium indicator; multi-color imaging; protein engineering; optogenetics
Thermacetogenium phaeum is a thermophilic strictly anaerobic bacterium oxidizing acetate to CO2 in syntrophic association with a methanogenic partner. It can also grow in pure culture, e.g., by fermentation of methanol to acetate. The key enzymes of homoacetate fermentation (Wood-Ljungdahl pathway) are used both in acetate oxidation and acetate formation. The obvious reversibility of this pathway in this organism is of specific interest since syntrophic acetate oxidation operates close to the energetic limitations of microbial life.
The genome of Th. phaeum is organized on a single circular chromosome and has a total size of 2,939,057 bp. It comprises 3.215 open reading frames of which 75% could be assigned to a gene function. The G+C content is 53.88 mol%. Many CRISPR sequences were found, indicating heavy phage attack in the past. A complete gene set for a phage was found in the genome, and indications of phage action could also be observed in culture. The genome contained all genes required for CO2 reduction through the Wood-Ljungdahl pathway, including two formyl tetrahydrofolate ligases, three carbon monoxide dehydrogenases, one formate hydrogenlyase complex, three further formate dehydrogenases, and three further hydrogenases. The bacterium contains a menaquinone MQ-7. No indications of cytochromes or Rnf complexes could be found in the genome.
The information obtained from the genome sequence indicates that Th. phaeum differs basically from the three homoacetogenic bacteria sequenced so far, i.e., the sodium ion-dependent Acetobacterium woodii, the ethanol-producing Clostridium ljungdahlii, and the cytochrome-containing Moorella thermoacetica. The specific enzyme outfit of Th. phaeum obviously allows ATP formation both in acetate formation and acetate oxidation.
To examine the process of transition from pediatric to adult healthcare services from the perspectives of young adults with chronic disease and pediatric and adult healthcare providers.
A qualitative approach using focus group interviews was performed to investigate transition experiences. Novel innovation games were also utilized to generate data. Content and narrative analyses of interview transcripts were performed.
Six focus groups were conducted with 10 young adults with chronic disease and 24 healthcare providers. Content analysis yielded 3 content domains: transition experiences in the context of relationships between patients, parents, and healthcare providers; differences between pediatric and adult-oriented medicine and how these differences inhibit or facilitate transition; and identifying transition services that should be provided to young patients with chronic disease.
This study demonstrates the need for gradual transfer of disease management from parent to child and better communication between adult and pediatric services during the transition process. Pediatric medicine and adult medicine represent different subcultures; acknowledging these differences may improve cooperation during transition from pediatric to adult providers. Young adult patients with chronic disease embrace the use of technology for specific interventions to improve the transition experience.
Tibial tubercle fractures often occur in athletic adolescents close to skeletal maturity. These fractures can present with marked displacement of the apophysis, intra-articular extension, and associated soft tissue injuries, such as tibial meniscal ligament tears. Here, we present our surgical technique which focuses on recreating the meniscal–articular relationship (using suture anchors) in severely displaced fractures.
We retrospectively reviewed all tibial tuberosity fractures treated with this technique over the last 2.5 years. Fractures with a minimum of a 12-month follow-up post-fixation were identified. Clinical records and radiographs were reviewed. Data included patient age, gender, involved side, injury classification (modified Ogden), mechanism of injury, treatment, return to activity, and complications.
Six patients met the inclusion criteria. Mean age at time of surgery was 14.9 (range 13.2–16.8) years. All patients were male and the mean follow-up period was 14 (12–26) months. Range of motion was started at 4 weeks post-operatively in a hinged knee brace, and return to sports occurred at an average of 3.75 months postoperatively (range 3–5 months). No evidence of growth disturbance of the proximal tibia or recurvatum at final follow-up was evident.
We speculate that patients who sustain a tibial tubercle avulsion fracture types III or V will likely have intra-articular pathology, specifically capsular avulsion or coronary ligament disruption. By utilizing suture anchors, our technique emphasizes renewing the anatomic articular environment to ensure better long-term results and maintaining these active individuals in sports.
Tibial tuberosity fracture; Open reduction tibial tubercle; Pediatric avulsion injury; Intra-articular involvement
To determine the relationship between opioid consumption and cognitive impairment following hip fracture repair.
Prospective study of consecutive patients.
Johns Hopkins Bayview Medical Center; Baltimore, Maryland.
Two hundred thirty-six patients ≥65 years old undergoing hip fracture repair.
Elderly patients without preoperative delirium who underwent hip fracture repair between April 2005 and July 2009 were followed for pain, opioid consumption, and postoperative delirium. Patients were tested for delirium with the confusion assessment method preoperatively and mid-morning on postoperative day 2. Pain was assessed by the nursing staff with a numeric 0–10 verbal scale. Opioid analgesia was provided in response to pain at rest to achieve scores of ≤3. Opioid consumption was analyzed with respect to the occurrence of incident postoperative delirium, presence of dementia, and other demographic variables.
Of the 236 patients, 66 (28%) had dementia with 213 (90%) receiving opioids postoperatively; including 55 (83%) demented patients and 158 (93%) non-demented patients. There was no association between the use of any postoperative opioid and incident delirium (P=0.615) in both demented (p=0.333) and non-demented patients (P=0.398). Dementia, but not postoperative delirium, was associated with less opioid use (P<0.001 for dementia; P=0.120 for delirium; P=0.038, for their interaction; Wald χ2 =142.8 with 7 d.o.f.). Furthermore, opioid dose (P≥0.591) on postoperative days 1 and 2 was not predictive of incident delirium. Dementia (P<0.001) and intensive care unit admission (P=0.006), not opioid consumption, were the most important predictors of incident postoperative delirium.
Concern for postoperative delirium should not prevent the use of opioid analgesic therapy sufficient to achieve a generally accepted level of comfort in patients with or without preexisting cognitive impairment.
delirium; hip fracture; opioids; surgery (complications)
Some drugs are known for their fetal nephrotoxicity and should be avoided during pregnancy. We report on a pregnant woman suffering from breast cancer who received a weekly neoadjuvant trastuzumab (Herceptin®) therapy from 15 weeks of gestation onward, in addition to a 3-weekly carboplatin/docetaxel chemotherapy. Fetal renal insufficiency with anhydramnios and missing visualization of the fetal bladder developed at 21 weeks. After discontinuation of trastuzumab and repeated instillation of amniotic fluid, the amount of amniotic fluid remained stable after 24 weeks of gestation. After caesarean section at 34 weeks because of fetal growth restriction, the renal function of the neonate was normal postnatally. In accordance with the current literature, our case shows a reversible adverse effect of trastuzumab on the fetal renal function and confirms the current recommendation that trastuzumab in pregnancy should be avoided. In pregnancies exposed to trastuzumab, treatment should be discontinued and the fetus should be closely monitored, with particular attention to the amniotic fluid and the fetal bladder volume, as these reflect fetal renal function.
Fetus; Renal insufficiency; Trastuzumab; Breast cancer; Pregnancy
Based on a multi-factorial model of delirium, we compared the types and magnitude of pre- and intra-operative predisposing factors for incident delirium in a stratified sample of acute hip fracture repair patients with and without pre-operative dementia.
DESIGN and SETTING
A prospective cohort study based in an academic medical center.
425 non-delirious, acute hip fracture patients (mean age: 80.2 +/− 6.8; female: 73.2%; “probable dementia”: 33.1%) admitted to the multi-disciplinary hip fracture repair service.
Each participant was assessed for delirium by a research nurse based on the Confusion Assessment Method (CAM) before study enrollment and from the second postoperative day until hospital discharge.
The incidence of delirium was higher in the Probable Dementia Group than in the No Dementia Group (54% vs. 26%; p≤ 0.001). In the No Dementia group (n = 284), age (OR: 1.07; 95% CI: 1.02-1.13), male gender (OR: 2.81; 95% CI: 1.40-5.64), BMI (OR: 0.92; 95% CI: 0.86-0.99), number of medical comorbidities (OR: 1.15; 95% CI: 1.01-1.32), and duration of surgery longer than two hours (OR: 2.53; 95% CI: 1.20-4.88) were independently associated with a post-operative delirium. In the Probable Dementia group, only the lag time from emergency room to operation room was significantly associated (OR: 2.83; 95% CI: 1.24-2.25) with delirium.
Pre-operative determination of dementia status is important for risk stratification for incident delirium after acute hip fracture repair surgery because types and magnitude of predisposing risk factors for post-operative delirium substantially differ based on their pre-operative dementia status.
dementia; delirium; hip fracture; surgery; risk factor
Identification of discrete states is a common task when studying biological systems on microscopic scales. Here, we present a novel step detection algorithm that is ideally suited to locate steplike features separating adjacent plateaus, even if they are smooth and hidden by noise. It can be adjusted to detect very low or narrow steps that cannot be recognized by conventional methods. We demonstrate the applicability of the technique on various experimental data and show strong evidence of sub-10-pN steps in atomic force spectroscopy measurements performed with living lymphocytes.
Since, most pathogens enter through the mucosa it is important to develop vaccines that induce mucosal immunity. To this end, we generated a novel adenovirus (Ad) vaccine that displays the sigma1 protein from reovirus to target junctional adhesion molecule 1 (JAM1) and sialic acid.
Replication-defective Ad5 vectors were modified by replacement of the Ad fiber protein with Sigma 1 (T3Dσ1) protein of reovirus T3D in previous work. Ad5 and Ad5-Sigma1 were compared in mouse models for gene delivery and vaccination to monitor cytokine, antibody, and T cell responses. The viruses were also tested for the ability to transduce and mature dendritic cells.
Ad5-Sigma1 was 40-fold less efficient at gene delivery in vivo, yet was capable of inducing equal or greater cellular immune responses and systemic IFN-γ levels than Ad5 after intranasal administration. Despite weaker gross transduction, intranasal administration of Ad5-Sigma1 produced more GFP-positive MHCII cells in the draining lymph nodes, less GFP+/MHCII+ cells in the lungs and mediated modestly better maturation of dendritic cells in vitro. These data suggest that targeting gene-based vaccination via the Sigma1 protein may enhance the T cell immune response perhaps by skewing immune responses to encoded antigens.
Falling is a common and morbid condition among elderly persons. Effective strategies to prevent falls have been identified but are underutilized.
Using a nonrandomized design, we compared rates of injuries from falls in a region of Connecticut where clinicians had been exposed to interventions to change clinical practice (intervention region) and in a region where clinicians had not been exposed to such interventions (usual-care region). The interventions encouraged primary care clinicians and staff members involved in home care, outpatient rehabilitation, and senior centers to adopt effective risk assessments and strategies for the prevention of falls (e.g., medication reduction and balance and gait training). The outcomes were rates of serious fall-related injuries (hip and other fractures, head injuries, and joint dislocations) and fall-related use of medical services per 1000 person-years among persons who were 70 years of age or older. The interventions occurred from 2001 to 2004, and the evaluations took place from 2004 to 2006.
Before the interventions, the adjusted rates of serious fall-related injuries (per 1000 person-years) were 31.2 in the usual-care region and 31.9 in the intervention region. During the evaluation period, the adjusted rates were 31.4 and 28.6, respectively (adjusted rate ratio, 0.91; 95% Bayesian credibility interval, 0.88 to 0.94). Between the preintervention period and the evaluation period, the rate of fall-related use of medical services increased from 68.1 to 83.3 per 1000 person-years in the usual-care region and from 70.7 to 74.2 in the intervention region (adjusted rate ratio, 0.89; 95% credibility interval, 0.86 to 0.92). The percentages of clinicians who received intervention visits ranged from 62% (131 of 212 primary care offices) to 100% (26 of 26 home care agencies).
Dissemination of evidence about fall prevention, coupled with interventions to change clinical practice, may reduce fall-related injuries in elderly persons.
Despite strong efforts to improve clinical outcome of ovarian cancer patients by conventional and targeted immuno-based therapies, the prognosis of advanced ovarian cancer is still poor. Natural killer (NK) cells mediate antibody-dependent cellular cytotoxicity (ADCC), release immunostimulatory cytokines and thus function as potent anti-tumour effector cells. However, tumour cells developed mechanisms to escape from an effective immune response. So highly immunogenic substances, like the 38 kDa-preparation of M. tuberculosis, PstS-1, are explored for their potential to enhance cancer-targeted immune responses. In this study we examined the modulation of different NK cell functions by accessory monocytes and PstS-1. We focussed on NK cell activation as well as natural and antibody-dependent cellular cytotoxicity directed against epidermal-growth-factor-receptor (EGFR)-positive ovarian cancer cell lines.
Activation, cytokine release and cytotoxicity of NK cells stimulated by monocytes and PstS-1 were determined by FACS-analysis, ELISA, Bioplex assay and quantitative polymerase-chain reaction (qPCR). Transwell assays were used to discriminate cell-cell contact-dependent from contact-independent mechanisms. Five ovarian cancer cell lines (A2780, IGROV-1, OVCAR-3, OVCAR-4 and SKOV-3) with different EGFR-expression were used as target cells for natural and antibody-dependent cellular cytotoxicity assays. Cetuximab (anti-EGFR-antibody) was used for ADCC studies.
Our data show that monocytes effectively enhance activation as well natural and antibody-dependent cytolytic activity of NK cells. PstS-1 directly stimulated monocytes and further activated monocyte-NK-co-cultures. However, PstS-1 did not directly influence purified NK cells and did also not affect natural and antibody-dependent cellular cytotoxicity directed against EGFR-positive ovarian cancer cells, even in presence of monocytes. Direct cell-cell contact between NK cells and monocytes was required for NK activation, while released cytokines seemed to play a minor role.
Our data suggest that monocytes enhance natural and antibody-dependent cytotoxic activity of NK cells in a cell-cell contact dependent manner. The TLR-agonist PstS-1 provides additional monocyte activation and induces NK activation markers, while NK cytotoxicity remains unaffected. We conclude that monocytes provide accessory function for ADCC exerted by NK during antibody-based cancer immunotherapy directed against EGFR-positive ovarian cancer cells.
NK cell; PstS-1; Ovarian cancer; BCG; Immunotherapy; Cetuximab
The C. elegans nervous system is particularly well suited for optogenetic analyses of circuit function: Essentially all connections have been mapped, and light can be directed at the neuron of interest in the freely moving, transparent animals, while behavior is observed. Thus, different nodes of a neuronal network can be probed for their role in controlling a particular behavior, using different optogenetic tools for photo-activation or –inhibition, which respond to different colors of light. As neurons may act in concert or in opposing ways to affect a behavior, one would further like to excite these neurons concomitantly, yet independent of each other. In addition to the blue-light activated Channelrhodopsin-2 (ChR2), spectrally red-shifted ChR variants have been explored recently. Here, we establish the green-light activated ChR chimera C1V1 (from Chlamydomonas and Volvox ChR1′s) for use in C. elegans. We surveyed a number of red-shifted ChRs, and found that C1V1-ET/ET (E122T; E162T) works most reliable in C. elegans, with 540–580 nm excitation, which leaves ChR2 silent. However, as C1V1-ET/ET is very light sensitive, it still becomes activated when ChR2 is stimulated, even at 400 nm. Thus, we generated a highly efficient blue ChR2, the H134R; T159C double mutant (ChR2-HR/TC). Both proteins can be used in the same animal, in different neurons, to independently control each cell type with light, enabling a further level of complexity in circuit analyses.
The increasing emergence of multidrug-resistant Staphylococcus aureus is a problem of global importance. Here, we report the genome of S. aureus VC40, which is resistant to the last-resort antibiotics vancomycin and daptomycin. Its genome sequence will allow insights into the mechanisms that convey full resistance to these compounds.
Besides inducing apoptosis, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) activates NF-κB. The apoptosis signaling pathway of TRAIL is well characterized involving TRAIL receptors, Fas-associated protein with death domain (FADD) and caspase-8. In contrast, the molecular mechanism of TRAIL signaling to NF-κB remains controversial. Here, we characterized the receptor–proximal mediators of NF-κB activation by TRAIL. Deletion of the DD of TRAIL receptors 1 and 2 revealed that it is essential in NF-κB signaling. Because FADD interacts with the TRAIL receptor DD, FADD was tested. RNAi-mediated knockdown of FADD or FADD deficiency in JURKAT T-cell leukemia cells decreased or disabled NF-κB signaling by TRAIL. In contrast, TRAIL-induced activation of NF-κB was maintained upon loss of receptor interacting protein 1 (RIP1) or knockdown of FLICE-like inhibitory protein (FLIP). Exogenous expression of FADD rescued TRAIL-induced NF-κB signaling. Loss-of-function mutations of FADD within the RHDLL motif of the death effector domain, which is required for TRAIL-induced apoptosis, abrogated FADD's ability to recruit caspase-8 and mediate NF-κB activation. Accordingly, deficiency of caspase-8 inhibited TRAIL-induced activation of NF-κB, which was rescued by wild-type caspase-8, but not by a catalytically inactive caspase-8 mutant. These data establish the mechanism of TRAIL-induced NF-κB activation involving the TRAIL receptor DD, FADD and caspase-8, but not RIP1 or FLIP. Our results show that signaling of TRAIL-induced apoptosis and NF-κB bifurcates downstream of caspase-8.
NF-κB; death receptor; signaling; DISC; p65
Highly infectious diseases (HIDs) are defined as being transmissible from person to person, causing life-threatening illnesses and presenting a serious public health hazard. The sampling, handling and transport of specimens from patients with HIDs present specific bio-safety concerns.
The European Network for HID project aimed to record, in a cross-sectional study, the infection control capabilities of referral centers for HIDs across Europe and assesses the level of achievement to previously published guidelines. In this paper, we report the current diagnostic capabilities and bio-safety measures applied to diagnostic procedures in these referral centers. Overall, 48 isolation facilities in 16 European countries were evaluated. Although 81% of these referral centers are located near a biosafety level 3 laboratory, 11% and 31% of them still performed their microbiological and routine diagnostic analyses, respectively, without bio-safety measures.
The discrepancies among the referral centers surveyed between the level of practices and the European Network of Infectious Diseases (EUNID) recommendations have multiple reasons of which the interest of the individuals in charge and the investment they put in preparedness to emerging outbreaks. Despite the fact that the less prepared centers can improve by just updating their practice and policies any support to help them to achieve an acceptable level of biosecurity is welcome.
Diagnostic techniques and procedures; European Union; Communicable diseases; Infection control; Patient isolation; Critical pathway
Streptococcus suis is an important swine pathogen and an emerging zoonotic agent of septicemia and meningitis. Knowledge on host immune responses towards S. suis, and strategies used by this pathogen for subversion of these responses is scarce. The objective of this study was to identify the immune receptors involved in S. suis recognition by dendritic cells (DCs). Production of cytokines and expression of co-stimulatory molecules by DCs were shown to strongly rely on MyD88-dependent signaling pathways, suggesting that DCs recognize S. suis and become activated mostly through Toll-like receptor (TLR) signaling. Supporting this fact, TLR2−/− DCs were severely impaired in the release of several cytokines and the surface expression of CD86 and MHC-II. The release of IL-12p70 and CXC10, and the expression of CD40 were found to depend on signaling by both TLR2 and TLR9. The release of IL-23 and CXCL1 were partially dependent on NOD2. Finally, despite the fact that MyD88 signaling was crucial for DC activation and maturation, MyD88-dependent pathways were not implicated in S. suis internalization by DCs. This first study on receptors involved in DC activation by S. suis suggests a major involvement of MyD88 signaling pathways, mainly (but not exclusively) through TLR2. A multimodal recognition involving a combination of different receptors seems essential for DC effective response to S. suis.
Optogenetic approaches using light-activated proteins like Channelrhodopsin-2 (ChR2) enable investigating the function of populations of neurons in live Caenorhabditis elegans (and other) animals, as ChR2 expression can be targeted to these cells using specific promoters. Sub-populations of these neurons, or even single cells, can be further addressed by restricting the illumination to the cell of interest. However, this is technically demanding, particularly in free moving animals. Thus, it would be helpful if expression of ChR2 could be restricted to single neurons or neuron pairs, as even wide-field illumination would photostimulate only this particular cell. To this end we adopted the use of Cre or FLP recombinases and conditional ChR2 expression at the intersection of two promoter expression domains, i.e. in the cell of interest only. Success of this method depends on precise knowledge of the individual promoters' expression patterns and on relative expression levels of recombinase and ChR2. A bicistronic expression cassette with GFP helps to identify the correct expression pattern. Here we show specific expression in the AVA reverse command neurons and the aversive polymodal sensory ASH neurons. This approach shall enable to generate strains for optogenetic manipulation of each of the 302 C. elegans neurons. This may eventually allow to model the C. elegans nervous system in its entirety, based on functional data for each neuron.
Streptococcus suis type 2 is a major swine pathogen and a zoonotic agent, causing meningitis in both swine and humans. S. suis infects the host through the respiratory route, reaches the bloodstream, and persists until breaching into the central nervous system. The capsular polysaccharide (CPS) of S. suis type 2 is considered a key virulence factor of the bacteria. Though CPS allows S. suis to adhere to the membrane of cells of the immune system, it provides protection against phagocytosis. In fact, nonencapsulated mutants are easily internalized and killed by macrophages and dendritic cells. The objective of this work was to study the molecular mechanisms by which the CPS of S. suis prevents phagocytosis. By using latex beads covalently linked with purified CPS, it was shown that CPS itself was sufficient to inhibit entry of both latex beads and bystander fluorescent beads into macrophages. Upon contact with macrophages, encapsulated S. suis was shown to destabilize lipid microdomains at the cell surface, to block nitric oxide (NO) production during infection, and to prevent lactosylceramide accumulation at the phagocytic cup during infection. In contrast, the nonencapsulated mutant was easily internalized via lipid rafts, in a filipin-sensitive manner, leading to lactosylceramide recruitment and strong NO production. This is the first report to identify a role for CPS in lipid microdomain stability and to recognize an interaction between S. suis and lactosylceramide in phagocytes.
Essentially any behavior in simple and complex animals depends on neuronal network function. Currently, the best-defined system to study neuronal circuits is the nematode Caenorhabditis elegans, as the connectivity of its 302 neurons is exactly known. Individual neurons can be activated by photostimulation of Channelrhodopsin-2 (ChR2) using blue light, allowing to directly probe the importance of a particular neuron for the respective behavioral output of the network under study. In analogy, other excitable cells can be inhibited by expressing Halorhodopsin from Natronomonas pharaonis (NpHR) and subsequent illumination with yellow light. However, inhibiting C. elegans neurons using NpHR is difficult. Recently, proton pumps from various sources were established as valuable alternative hyperpolarizers. Here we show that archaerhodopsin-3 (Arch) from Halorubrum sodomense and a proton pump from the fungus Leptosphaeria maculans (Mac) can be utilized to effectively inhibit excitable cells in C. elegans. Arch is the most powerful hyperpolarizer when illuminated with yellow or green light while the action spectrum of Mac is more blue-shifted, as analyzed by light-evoked behaviors and electrophysiology. This allows these tools to be combined in various ways with ChR2 to analyze different subsets of neurons within a circuit. We exemplify this by means of the polymodal aversive sensory ASH neurons, and the downstream command interneurons to which ASH neurons signal to trigger a reversal followed by a directional turn. Photostimulating ASH and subsequently inhibiting command interneurons using two-color illumination of different body segments, allows investigating temporal aspects of signaling downstream of ASH.
Afatinib (BIBW 2992) is an ErbB-family blocker that irreversibly inhibits signaling from all relevant ErbB-family dimers. Afatinib has demonstrated preclinical activity in human epidermal growth factor receptor HER2 (ErbB2)-positive and triple-negative xenograft models of breast cancer, and clinical activity in phase I studies. This was a multicenter phase II study enrolling patients with HER2-negative metastatic breast cancer progressing following no more than three lines of chemotherapy. No prior epidermal growth factor receptor-targeted therapy was allowed. Patients received 50-mg afatinib once daily until disease progression. Tumor assessment was performed at every other 28-day treatment course. The primary endpoint was clinical benefit (CB) for ≥4 treatment courses in triple-negative (Cohort A) metastatic breast cancer (TNBC) and objective responses measured by Response Evaluation Criteria in Solid Tumors in patients with HER2-negative, estrogen receptor-positive, and/or progesterone receptor-positive breast cancer (Cohort B). Fifty patients received treatment, including 29 patients in Cohort A and 21 patients in Cohort B. No objective responses were observed in either cohort. Median progression-free survival was 7.4 and 7.7 weeks in Cohorts A and B, respectively. Three patients with TNBC had stable disease for ≥4 treatment courses, one of them for 12 courses (median 26.3 weeks; range 18.9–47.9 weeks). The most frequently observed afatinib-associated adverse events (AEs) were gastrointestinal and skin-related side effects, which were manageable by symptomatic treatment and dose reductions. Afatinib pharmacokinetics were comparable to those observed in previously reported phase I trials. In conclusion, afatinib had limited activity in HER2-negative breast cancer. AEs were generally manageable and mainly affected the skin and the gastrointestinal tract.
Electronic supplementary material
The online version of this article (doi:10.1007/s10549-012-2126-1) contains supplementary material, which is available to authorized users.
Afatinib; Metastatic breast cancer; Triple-negative breast cancer; HER2-negative breast cancer; EGFR TKI
The Streptococcus suis 103 gene product is an immunogenic and protective lipoprotein that is a component of an ATP-binding cassette transporter implicated in zinc uptake. Belonging to the same transcriptional unit and downstream of the 103 gene is a gene that encodes a homologue of the pneumococcal histidine triad (Pht) protein Pht309. In an intraperitoneal mouse model the virulence of a mutant lacking the 103 gene was more than 50 times lower than that of the wild-type (WT) parent strain, S. suis serotype 2 strain P1/7. In addition, the immunogenicity of this mutant was dramatically decreased. In striking contrast, the virulence and immunogenicity of a P1/7 mutant lacking the Pht309 gene were similar to those of the parent strain. These results demonstrate that the 103 lipoprotein is strongly involved in S. suis virulence and support the hypothesis that this lipoprotein might be an excellent candidate for vaccines aiming to achieve broad protection against streptococci.