As part of the NIH-sponsored Glucosamine/Chondroitin Sulfate Arthritis Intervention Trial (GAIT) our objective here was to examine 1) the pharmacokinetics (PK) of glucosamine (GlcN) and chondroitin sulfate (CS) when taken separately or in combination as a single dose in normal individuals (n=29) and 2) the PK of GlcN and CS when taken as a single dose after 3 months daily dosing with GlcN, CS or GlcN+CS, in patients with symptomatic knee pain (n=28).
The concentration of GlcN in the circulation was determined by established fluorophore-assisted carbohydrate electrophoresis (FACE) methods. The hydrodynamic size and disaccharide composition of CS chains in the circulation and dosage samples was determined by Superose 6 chromatography and FACE.
We show that circulating levels of CS in human plasma are about 20ug/ml. Most significantly, the endogenous concentration and CS disaccharide composition were not detectably altered by ingestion of CS, when the CS was taken alone or in combination with GlcN. On the other hand, the Cmax (single dose study) and AUC values (multiple dose study) for ingested GlcN were significantly reduced by combination dosing with CS, relative to GlcN dosing alone.
We conclude that pain relief perceived following ingestion of CS probably does not depend on simultaneous or prior intake of GlcN. Further, such effects on joint pain, if present, probably do not result from ingested CS reaching the joint space but may result from changes in cellular activities in the gut lining or in the liver, where concentrations of ingested CS, or its breakdown products, could be substantially elevated following oral ingestion. Moreover, since combined dosing of GlcN with CS was found to reduce the plasma levels seen with GlcN dosing alone, any improved pain relief by combination dosing cannot be explained by higher circulating concentrations of GlcN.
Elk3/Net/Sap2 (here referred to as Elk3) is an Ets ternary complex transcriptional repressor known for its involvement in angiogenesis during embryonic development. Although Elk3 is expressed in various tissues, additional roles for the protein outside of vasculature development have yet to be reported. Here, we characterize the early spatiotemporal expression pattern of Elk3 in the avian embryo using whole mount in situ hybridization and quantitative RT-PCR and examine the effects of its loss of function on neural crest development. At early stages, Elk3 is expressed in the head folds, head mesenchyme, intersomitic vessels, and migratory cranial neural crest (NC) cells. Loss of the Elk3 protein results in the retention of Pax7+ precursors in the dorsal neural tube that fail to upregulate neural crest specifier genes, FoxD3, Sox10 and Snail2, resulting in embryos with severe migration defects. The results putatively place Elk3 downstream of neural plate border genes, but upstream of neural crest specifier genes in the neural crest gene regulatory network (NC-GRN), suggesting that it is critical for the progression from progenitor to definitive neural crest cell.
Elk3; Neural crest specification; Progenitors; Pax7
The use of molecular methods to diagnose Clostridium difficile infection (CDI) has improved diagnostic yield compared to conventional methods. However, PCR testing can detect colonization and has introduced several practical challenges pertaining to need for treatment and isolation of cases.
For all new cases detected by real-time PCR, concurrent cytotoxin assay was performed and genetic characterization with MLVA (multi-locus variable number tandem repeat analysis) was done to determine relatedness. We used PCR cycle threshold (Ct) of detection as surrogate marker for bacterial burden in stool.
Overall, 54 cases of CDI were detected during the study period. 42 were concurrently tested by CYT and characterized by MLVA .MLVA analysis revealed marked genetic diversity with no ongoing outbreaks; four cases were due to NAP1 strain. CYT −/PCR + cases had a higher median Ct value of detection compared to CYT+/PCR + cases (28.2 vs 22.5; p = 0.01). Among 25 strains that were genetically related, 9/11 isolates in this dominant cluster were positive by CYT compared to 4/14 in non-dominant clusters (p = 0.02).
CYT−/PCR+ cases contribute to hospital based transmission. However, the risk of transmission of C. difficile from CYT +/PCR+ cases may be higher than those that are CYT−/PCR+.
Nanoemulsions are adjuvants that enhance antigen penetration in the nasal mucosa, increase cellular uptake of antigens by both epithelial dendritic cells, and promote migration of antigen-loaded dendritic cells to regional lymph nodes within a day of vaccine administration. The objective of this study was to determine whether the W805EC nanoemulsion adjuvant enhances immune response not only by direct uptake of antigen by dendritic cells, but also indirectly, by phagocytosis of antigen-primed, apoptotic, epithelial cells. Consistent with this, we show that exposure of both epithelial cells (TC-1s) and dendritic cells (JAWS II or bone marrow derived dendritic cells (BMDCs)) to nanoemulsion exhibited augmented antigen uptake in cell culture. TC-1 cells subsequently underwent G2/M cell cycle arrest and apoptosis, and when co-cultured with JAWS II or BMDCs were rapidly engulfed by the dendritic cells, which responded by up-regulating dendritic cell maturation marker CD86. Altogether these results suggest that the effectiveness of nanoemulsions as adjuvants stems, at least in part, from the engulfment of antigen-loaded epithelial cells, leading to enhanced antigen processing and a strong and balanced mucosal and systemic immune response.
Immunization; antigens; cytokines; antibodies
Genome-wide association studies (GWAS) and candidate gene studies have identified a number of risk loci associated with the smoking-related disease COPD, a disorder that originates in the airway epithelium. Since airway basal cell (BC) stem/progenitor cells exhibit the earliest abnormalities associated with smoking (hyperplasia, squamous metaplasia), we hypothesized that smoker BC have a dysregulated transcriptome, enriched, in part, at known GWAS/candidate gene loci. Massive parallel RNA sequencing was used to compare the transcriptome of BC purified from the airway epithelium of healthy nonsmokers (n = 10) and healthy smokers (n = 7). The chromosomal location of the differentially expressed genes was compared to loci identified by GWAS to confer risk for COPD. Smoker BC have 676 genes differentially expressed compared to nonsmoker BC, dominated by smoking up-regulation. Strikingly, 166 (25%) of these genes are located on chromosome 19, with 13 localized to 19q13.2 (p<10−4 compared to chance), including 4 genes (NFKBIB, LTBP4, EGLN2 and TGFB1) associated with risk for COPD. These observations provide the first direct connection between known genetic risks for smoking-related lung disease and airway BC, the population of lung cells that undergo the earliest changes associated with smoking.
Over 10 million Americans have osteoporosis, and is the predominant cause of fractures in the elderly. Treatment of fractures in the setting of osteoporosis is complicated by a suboptimal bone regenerative response due to a decline in the number of osteoblasts, their function, and survival. Consequently, an osteogenic therapeutic to prevent and treat fractures in patients with osteoporosis is needed. Nel-like molecule-1 (NELL-1), a novel osteoinductive growth factor, has been shown to promote bone regeneration. In this study, we aim to demonstrate the capacity of recombinant NELL-1 to prevent ovariectomy (OVX)-induced osteoporosis in a senile rat model. Ten-month-old female Sprague-Dawley rats underwent either sham surgery or OVX. Subsequently, 50 μL of 600 μg/mL NELL-1 lyophilized onto a 0–50-μm tricalcium phosphate (TCP) carrier was injected into the femoral bone marrow cavity while phosphate-buffered saline (PBS) control was injected into the contralateral femur. Our microcomputed tomography results showed that OVX+PBS/TCP control femurs showed a continuous decrease in the bone volume (BV) and bone mineral density (BMD) from 2 to 8 weeks post-OVX. In contrast, OVX+NELL-1/TCP femurs showed resistance to OVX-induced bone resorption showing BV and BMD levels similar to that of SHAM femurs at 8 weeks post-OVX. Histology showed increased endosteal-woven bone, as well as decreased adipocytes in the bone marrow of NELL-1-treated femurs compared to control. NELL-1-treated femurs also showed increased immunostaining for bone differentiation markers osteopontin and osteocalcin. These findings were validated in vitro, in which addition of NELL-1 in OVX bone marrow stem cells resulted in increased osteogenic differentiation. Thus, NELL-1 effectively enhances in situ osteogenesis in the bone marrow, making it potentially useful in the prevention and treatment of osteoporotic fractures.
On the basis of studies in experimental animals demonstrating that AdVEGF121, an E1−E3− serotype 5 adenovirus coding the 121 isoform of vascular endothelial growth factor (VEGF), could mediate the generation of new blood vessels and reverse coronary ischemia, a clinical study of direct myocardial administration of AdVEGF121 was initiated in patients with late-stage, diffuse coronary artery disease. This study provides long-term (median, 11.8 years) follow-up on these patients. From 1997 to 1999, AdVEGF121 was administered by direct myocardial injection to an area of reversible ischemia in 31 patients with severe coronary disease, either as an adjunct to conventional coronary artery bypass grafting (group A) or as minimally invasive sole (MIS) therapy, using a minithoracotomy (group B). There was no control group; the study participants served as the control subjects. The 5- and 10-year survival was 10 of 15 (67%) and 6 of 15 (40%) for the group A patients, and 11 of 16 (69%) and 5 of 16 (31%) for group B sole therapy patients, respectively. In comparison, maximal medical therapy in comparable groups in the literature have a 3- to 5-year survival rate of 52 to 59%. For the survivors, the angina score for group A was 3.4±0.5 at time 0 and 1.9±1.0 at last follow-up, and for group B it was 3.4±0.6 and 2.0±1.1, respectively. The incidences of malignancy and retinopathy were no greater than that expected for the age-matched general population. We conclude that adenovirus-mediated VEGF direct myocardial administration to patients with severe coronary artery disease is safe, and future larger trials are warranted to assess efficacy.
Rosengart and colleagues report long-term safety data (median, 11.8 years) from a clinical study involving 31 research subjects with late-stage, diffuse coronary artery disease. Research subjects who received direct myocardial administration of an adenovirus encoding the 121 isoform of vascular endothelial growth factor (Ad-VEGF121) did not have an increased incidence of malignancy and retinopathy compared with the age-matched general population.
Nine cancer patients were treated with adoptive cell therapy using autologous anti-MAGE-A3 TCR engineered T cells. Five patients experienced clinical regression of their cancers including two on-going responders. Beginning 1–2 days post-infusion, three patients (#’s 5, 7, and 8) experienced mental status changes, and two patients (5 and 8) lapsed into comas and subsequently died. Magnetic resonance imagining analysis of patients 5 and 8 demonstrated periventricular leukomalacia, and examination of their brains at autopsy revealed necrotizing leukoencephalopathy with extensive white matter defects associated with infiltration of CD3+/CD8+ T cells. Patient 7, developed Parkinson-like symptoms, which resolved over 4 weeks and fully recovered. Immunohistochemical staining of patient and normal brain samples demonstrated rare positively staining neurons with an antibody that recognizes multiple MAGE-A family members. The TCR used in this study recognized epitopes in MAGE-A3/A9/A12. Molecular assays of human brain samples using Q-RT-PCR, Nano string quantitation, and deep-sequencing indicated that MAGE -A12 was expressed in human brain (and possibly MAGE-A1, MAGE-A8, and MAGE-A9). This previously unrecognized expression of MAGE-A12 in human brain was possibly the initiating event of a TCR-mediated inflammatory response that resulted in neuronal cell destruction and raises caution for clinical applications targeting MAGE-A family members with highly active immunotherapies.
Cancer Testes Antigen; Immunotherapy; TCR; Gene Therapy
A growing body of research suggests that rats represent and remember specific earlier events from the past. An important criterion for validating a rodent model of episodic memory is to establish that the content of the representation is about a specific event in the past rather than vague information about remoteness. Recent evidence suggests that rats may also represent events that are anticipated to occur in the future. An important capacity afforded by a representation of the future is the ability to plan for the occurrence of a future event. However, relatively little is known about the content of represented future events and the cognitive mechanisms that may support planning. This article reviews evidence that rats remember specific earlier events from the past, represent events that are anticipated to ccur in the future, and develops criteria for validating a rodent model of future planning. These criteria include representing a specific time in the future, the ability to temporarily disengage from a plan and reactivate the plan at an appropriate time in the future, and flexibility to deploy a plan in novel conditions.
animal model; comparative cognition; episodic memory; incidental encoding; prospective cognition; prospective memory
Basic research toward understanding and treating disc pathology in the spine has utilized numerous animal models, with delivery of small molecules, purified factors, and genes of interest. To date, gene delivery to the rat lumbar spine has only been described utilizing genetically programmed cells in a matrix which has required partial disc excision, and expected limitation of treatment diffusion into the disc.
This study was designed to develop and describe a surgical technique for lumbar spine exposure and disc space preparation, and use of a matrix-free method for gene delivery.
Naïve or genetically programmed isogeneic bone marrow stromal cells were surgically delivered to adolescent male Lewis rat lumbar discs, and utilizing quantitative biochemical and qualitative immunohistological assessments, the implanted cells were detected 3 days post-procedure.
Statistically significant differences were noted for recovery of the β-galactosidase marker gene comparing delivery of naïve or labeled cells (105 cells per disc) from the site of implantation, and between delivery of 105 or 106 labeled cells per disc at the site of implantation and the adjacent vertebral body. Immunohistology confirmed that the β-galactosidase marker was detected in the adjacent vertebra bone in the zone of surgical implantation.
The model requires further testing in larger cohorts and with biologically active genes of interest, but the observations from the pilot experiments are very encouraging that this will be a useful comparative model for basic spine research involving gene or cell delivery, or other locally delivered therapies to the intervertebral disc or adjacent vertebral bodies in rats.
gene delivery; lumbar; disc; matrix-free
Weight monitoring is an important element of HF self-care, yet the most clinically meaningful way to evaluate weight monitoring adherence is uncertain. We conducted this study to evaluate the association of (1) self-reported recall and (2) daily diary-recorded weight monitoring adherence with heart failure-related (HF-related) hospitalization.
We conducted a prospective cohort study among 216 patients within a randomized trial of HF self-care training. All patients had an initial self-care training session followed by 15 calls (median) to reinforce educational material; patients were also given digital scales, instructed to weigh daily, record weights in a diary, and mail diaries back monthly. Weight monitoring adherence was assessed with a self-reported recall question administered at 12 months and dichotomized into at least daily versus less frequent weighing. Diary-recorded weight monitoring was evaluated over 12 months and dichotomized into ≥80% and <80% adherence. HF-related hospitalizations were ascertained through patient report and confirmed through record review.
Over 12 months in 216 patients, we identified 50 HF-related hospitalizations. Patients self-reporting daily or more frequent weight monitoring had an incidence rate ratio of 1.34 (95% CI 0.24-7.32) for HF-related hospitalizations compared to those reporting less frequent weight monitoring. Patients who completed ≥80% of weight diaries had an IRR of 0.37 (95% CI 0.18-0.75) for HF-related hospitalizations compared to patients who completed <80% of weight diaries.
Self-reported recall of weight monitoring adherence was not associated with fewer HF hospitalizations. In contrast, diary-recorded adherence ≥80% of days was associated with fewer HF-related hospitalizations. Incorporating diary-based measures of weight monitoring adherence into HF self-care training programs may help to identify patients at risk for HF-related hospitalizations.
Heart failure; Patient compliance; Monitoring; Physiologic
The low pH of the stomach serves as a barrier to ingested microbes and must be overcome or bypassed when delivering live bacteria for vaccine or probiotic applications. Typically, the impact of stomach acidity on bacterial survival is evaluated in vitro, as there are no small animal models to evaluate these effects in vivo. To better understand the effect of this low pH barrier to live attenuated Salmonella vaccines, which are often very sensitive to low pH, we investigated the value of the histamine mouse model for this application. A low pH gastric compartment was transiently induced in mice by the injection of histamine. This resulted in a gastric compartment of approximately pH 1.5 that was capable of distinguishing between acid-sensitive and acid-resistant microbes. Survival of enteric microbes during gastric transit in this model directly correlated with their in vitro acid resistance. Because many Salmonella enterica serotype Typhi vaccine strains are sensitive to acid, we have been investigating systems to enhance the acid resistance of these bacteria. Using the histamine mouse model, we demonstrate that the in vivo survival of S. Typhi vaccine strains increased approximately 10-fold when they carried a sugar-inducible arginine decarboxylase system. We conclude that this model will be a useful for evaluating live bacterial preparations prior to clinical trials.
During early postnatal life, extensive changes in gene expression occur concomitantly in multiple major organs, indicating the existence of a common core developmental genetic program. This program includes hundreds of growth-promoting genes that are downregulated with age in liver, kidney, lung, and heart, and there is evidence that this component of the program drives the widespread decline in cell proliferation that occurs in juvenile life, as organs approach adult sizes. To investigate epigenetic changes that might orchestrate this program, we performed chromatin immunoprecipitation-promoter tiling array to assess temporal changes in histone H3K4 and H3K27 trimethylation (me3) at promoter regions throughout the genome in kidney and lung, comparing 1- to 4-wk-old mice. We found extensive genome-wide shifts in H3K4me3 and H3K27me3 occurring with age in both kidney and lung. The number of genes with concordant changes in the two organs was far greater than expected by chance. Temporal changes in H3K4me3 showed a strong, positive association with changes in gene expression, assessed by microarray, whereas changes in H3K27me3 showed a negative association. Gene ontology analysis indicated that shifts in specific histone methylation marks were associated with specific developmental functions. Of particular interest, genes with decreases in H3K4me3 with age in both organs were strongly implicated in cell cycle and cell proliferation functions. Taken together, the findings suggest that the common core developmental program of gene expression which occurs in multiple organs during juvenile life is associated with a common core developmental program of histone methylation. In particular, declining H3K4me3 is strongly associated with gene downregulation and occurs in the promoter regions of many growth-regulating genes, suggesting that this change in histone methylation may contribute to the component of the genetic program that drives juvenile body growth deceleration.
To qualitatively explore clinician and pharmacist attitudes toward use of a web-application virtual pharmacist-clinician partnership (VPCP) to assist with co-managed care of illicit drug-using patients prescribed post-exposure prophylaxis (PEP).
Qualitative, descriptive, non-experimental study.
New York City (NYC) from February 2011 to March 2012.
4 pharmacists and 9 clinicians.
Main outcome measure
Prior to VPCP implementation in selected study pharmacies in NYC, we qualitatively explored: 1) the potential impact of the VPCP on pharmacist-clinician communication, and 2) potential barriers to use of the VPCP when co-managing PEP patients among pharmacists and clinicians.
Pharmacists and clinicians were supportive of an interactive web-application that would expand the role of pharmacists to include assistance with PEP access and patient management. Participants noted that the VPCP would facilitate communication between pharmacists and clinicians and have potential to support adherence among patients. Pharmacists and clinicians were concerned about not having time to use the VPCP and security of patient information on the site. Pharmacist and clinician concerns informed final development of the VPCP including creation of a user-friendly interface, linkage to users’ email accounts for timeline notification, and attention to security.
Use of web-based technology to support communication between pharmacists and clinicians was seen as being a potentially feasible method for improving patient care, particularly in the delivery of PEP to drug users and other high risk groups. These findings highlight the need for further study of a technology-supported partnership, particularly for co-management of patients who face challenges with adherence.
The Met allele of the catechol-o-methyltransferase (COMT) Val158Met polymorphism is associated with increased cortical dopamine and risk behaviors including illicit drug use and unprotected sex. Therefore, we examined whether or not the distribution of the Val158Met genotype differed between HIV-infected and HIV-uninfected women. We conducted an Armitage-Cochran Test and logistic regression to compare genotype frequencies between 1,848 HIV-infected and 612 HIV-uninfected women from the Women’s Interagency HIV Study (WIHS). The likelihood of carrying one or two Met alleles was greater in HIV-infected women (61%) compared to HIV-uninfected women (54%), Z = −3.60, p < 0.001. We report the novel finding of an association between the Val158Met genotype and HIV serostatus that may be mediated through the impact of dopamine function on propensity for risk-taking.
Smoking and COPD are associated with decreased mucociliary clearance, and healthy smokers have shorter cilia in the large airway than nonsmokers. We hypothesized that changes in cilia length are consistent throughout the airway, and we further hypothesized that smokers with COPD have shorter cilia than healthy smokers. Because intraflagellar transport (IFT) is the process by which cilia of normal length are produced and maintained, and alterations in IFT lead to short cilia in model organisms, we also hypothesized that smoking induces changes in the expression of IFT-related genes in the airway epithelium of smokers and smokers with COPD. To assess these hypotheses, airway epithelium was obtained via bronchoscopic brushing. Cilia length was assessed by measuring 100 cilia (10 cilia on each of 10 cells) per subject and Affymetrix microarrays were used to evaluate IFT gene expression in nonsmokers and healthy smokers in 2 independent data sets from large and small airway as well as in COPD smokers in a data set from the small airway. In the large and small airway epithelium, cilia were significantly shorter in healthy smokers than nonsmokers, and significantly shorter in COPD smokers than in both healthy smokers and nonsmokers. The gene expression data confirmed that a set of 8 IFT genes were down-regulated in smokers in both data sets; however, no differences were seen in COPD smokers compared to healthy smokers. These results support the concept that loss of cilia length contributes to defective mucociliary clearance in COPD, and that smoking-induced changes in expression of IFT genes may be one mechanism of abnormally short cilia in smokers. Strategies to normalize cilia length may be an important avenue for novel COPD therapies.
Control of parasite transmission is critical for the eradication of malaria. However, most antimalarial drugs are not active against P. falciparum gametocytes, responsible for the spread of malaria. Consequently, patients can remain infectious for weeks after the clearance of asexual parasites and clinical symptoms. Here we report the identification of 27 potent gametocytocidal compounds (IC50 < 1 μM) from screening 5,215 known drugs and compounds. All these compounds were active against three strains of gametocytes with different drug sensitivities and geographical origins, 3D7, HB3 and Dd2. Cheminformatic analysis revealed chemical signatures for P. falciparum sexual and asexual stages indicative of druggability and suggesting potential targets. Torin 2, a top lead compound (IC50 = 8 nM against gametocytes in vitro), completely blocked oocyst formation in a mouse model of transmission. These results provide critical new leads and potential targets to expand the repertoire of malaria transmission-blocking reagents.
The successful USA300 Community-Associated Methicillin-Resistant Staphylococcus aureus (CA-MRSA) lineage predominantly causes skin and soft tissue infections (SSTIs) and is highly associated with carriage of the Arginine Catabolic Mobile Element (ACME). However, the contribution of ACME to USA300 fitness during SSTIs remains incompletely understood. We show that the constitutive ACME-encoded arginine-deiminase system (Arc) allows USA300 to thrive in acidic environments that mimic human skin. Consequently, ACME-Arc drives excessive production of host polyamines, compounds uniquely toxic to S. aureus. To mitigate this, ACME also encodes SpeG, a polyamine-resistance enzyme that is essential for combating excess host polyamines in a murine SSTI model. Inhibiting host polyamine production not only restored ΔspeG persistence within infected wounds but also severely altered the host healing process, implying that polyamines play integral roles in coordinating the wound-healing response. Together, these data underscore the functional modularity of ACME and its contribution to the success of USA300 CA-MRSA.
Severe trauma renders patients susceptible to infection. In sepsis, defective bacterial clearance has been linked to specific deviations in the innate immune response. We hypothesized that innate immune modulations observed during sepsis also contribute to increased bacterial susceptibility after severe trauma. A well-established murine model of burn injury, used to replicate infection following trauma, showed that wound inoculation with P. aeruginosa quickly spreads systemically. The systemic IL-10/IL-12 axis was skewed after burn injury with infection as indicated by a significant elevation in serum IL-10 and polarization of neutrophils into an anti-inflammatory (“N2”; IL-10+ IL-12−) phenotype. Infection with an attenuated P. aeruginosa strain (ΔCyaB) was cleared better than the wildtype strain and was associated with an increased pro-inflammatory neutrophil (“N1”; IL-10−IL-12+) response in burn mice. This suggests that neutrophil polarization influences bacterial clearance after burn injury. Administration of a TLR5 agonist, flagellin, after burn injury restored the neutrophil response towards a N1 phenotype resulting in an increased clearance of wildtype P. aeruginosa after wound inoculation. This study details specific alterations in innate cell populations after burn injury that contribute to increased susceptibility to bacterial infection. In addition, for the first time, it identifies neutrophil polarization as a therapeutic target for the reversal of bacterial susceptibility after injury.
With the ever-increasing global demand for high quality rice in both local production regions and with Western consumers, we have a strong desire to understand better the importance of the different traits that make up the quality of the rice grain and obtain a full picture of rice quality demographics. Rice is by no means a ‘one size fits all’ crop. Regional preferences are not only striking, they drive the market and hence are of major economic importance in any rice breeding / improvement strategy. In this analysis, we have engaged local experts across the world to perform a full assessment of all the major rice quality trait characteristics and importantly, to determine how these are combined in the most preferred varieties for each of their regions. Physical as well as biochemical characteristics have been monitored and this has resulted in the identification of no less than 18 quality trait combinations. This complexity immediately reveals the extent of the specificity of consumer preference. Nevertheless, further assessment of these combinations at the variety level reveals that several groups still comprise varieties which consumers can readily identify as being different. This emphasises the shortcomings in the current tools we have available to assess rice quality and raises the issue of how we might correct for this in the future. Only with additional tools and research will we be able to define directed strategies for rice breeding which are able to combine important agronomic features with the demands of local consumers for specific quality attributes and hence, design new, improved crop varieties which will be awarded success in the global market.
Immune responses are generally impaired in aged mammals. T cells have been extensively studied in this context due to the initial discovery of their reduced proliferative capacity with aging. The decreased responses involve altered signaling events associated with the early steps of T cell activation. The underlying causes of these changes are not fully understood but point to alterations in assembly of the machinery for T cell activation. Here, we have tested the hypothesis that the T cell pool in elderly subjects displayed reduced functional capacities due to altered negative feedback mechanisms that participate in the regulation of the early steps of T cell activation. Such conditions tip the immune balance in favor of altered T cell activation and a related decreased response in aging.
We present evidence that the tyrosine phosphatase SHP-1, a key regulator of T cell signal transduction machinery is, at least in part, responsible for the impaired T cell activation in aging. We used tyrosine-specific mAbs and Western blot analysis to show that a deregulation of the Csk/PAG loop in activated T cells from elderly individuals favored the inactive form of tyrosine-phosphorylated Lck (Y505). Confocal microscopy analysis revealed that the dynamic movements of these regulatory proteins in lipid raft microdomains was altered in T cells of aged individuals. Enzymic assays showed that SHP-1 activity was upregulated in T cells of aged donors, in contrast to young subjects. Pharmacological inhibition of SHP-1 resulted in recovery of TCR/CD28-dependent lymphocyte proliferation and IL-2 production of aged individuals to levels approaching those of young donors. Significant differences in the active (Y394) and inactive (Y505) phosphorylation sites of Lck in response to T cell activation were observed in elderly donors as compared to young subjects, independently of CD45 isoform expression.
Our data suggest that the role of SHP-1 in T cell activation extends to its increased effect in negative feedback in aging. Modulation of SHP-1 activity could be a target to restore altered T cell functions in aging. These observations could have far reaching consequences for improvement of immunosenescence and its clinical consequences such as infections, altered response to vaccination.
Aging; Confocal microscopy; Lck; Lipid rafts; Protein tyrosine kinases; Protein tyrosine phosphatases; SHP-1; Lck; T cells; TCR/CD28 signaling
Helicobacter pylori infection, linked to gastric cancer, is responsible for a large worldwide disease burden. H pylori prevalence and gastric cancer rates are elevated among indigenous Arctic communities, but implementation of prevention strategies is hampered by insufficient information. Some communities in northern Canada have advocated for H pylori prevention research. As a first step, community-driven research was undertaken to describe the H pylori-associated disease burden in concerned communities.
Participants in this cross-sectional study completed a clinical interview and gastroscopy with gastric biopsies taken for histopathological examination in February 2008.
Study procedures were carried out at the health centre in Aklavik, Northwest Territories, Canada (population ∼600).
All residents of Aklavik were invited to complete a clinical interview and gastroscopy; 194 (58% female participants; 91% Aboriginal; age range 10–80 years) completed gastroscopy and had gastric biopsies taken.
Primary and secondary outcome measures
This analysis estimates the prevalence of gastric abnormalities detected by endoscopy and histopathology, and associations of demographic and clinical variables with H pylori prevalence.
Among 194 participants with evaluable gastric biopsies, 66% were H pylori-positive on histology. Among H pylori-positive participants, prevalence was 94% for acute gastritis, 100% for chronic gastritis, 21% for gastric atrophy and 11% for intestinal metaplasia of the gastric mucosa, while chronic inflammation severity was mild in 9%, moderate in 47% and severe in 43%. In a multivariable model, H pylori prevalence was inversely associated with previous gastroscopy, previous H pylori therapy and aspirin use, and was positively associated with alcohol consumption.
In this population, H pylori-associated gastric histopathology shows a pattern compatible with elevated risk of gastric cancer. These findings demonstrate that local concern about health risks from H pylori is warranted and provide an example of how epidemiological research can address health priorities identified by communities.
To characterize patient-derived xenografts (PDXs) for functional studies,
we made whole-genome comparisons with originating breast cancers representative
of the major intrinsic subtypes. Structural and copy number aberrations were
found to be retained with high fidelity. However, at the single-nucleotide
level, variable numbers of PDX-specific somatic events were documented, although
they were only rarely functionally significant. Variant allele frequencies were
often preserved in the PDXs, demonstrating that clonal representation can be
transplantable. Estrogen-receptor-positive PDXs were associated with
ESR1 ligand-binding-domain mutations, gene amplification,
or an ESR1/YAP1 translocation. These events produced different
endocrine-therapy-response phenotypes in human, cell line, and PDX
endocrine-response studies. Hence, deeply sequenced PDX models are an important
resource for the search for genome-forward treatment options and capture
endocrine-drug-resistance etiologies that are not observed in standard cell
lines. The originating tumor genome provides a benchmark for assessing genetic
drift and clonal representation after transplantation.
The importance and advantages of three-dimensional (3D) cell cultures have been well-recognized. Tumor cells cultured in a 3D culture system as multicellular tumor spheroids (MTS) can bridge the gap between in vitro and in vivo anticancer drug evaluations. An in vitro 3D tumor model capable of providing close predictions of in vivo drug efficacy will enhance our understanding, design, and development of better drug delivery systems. Here, we developed an in vitro 3D tumor model by adapting the hydrogel template strategy to culture uniformly sized spheroids in a hydrogel scaffold containing microwells. The in vitro 3D tumor model was to closely simulate an in vivo solid tumor and its microenvironment for evaluation of anticancer drug delivery systems. MTS cultured in the hydrogel scaffold are used to examine the effect of culture conditions on the drug responses. Free MTS released from the scaffold are transferred to a microfluidic channel to simulate a dynamic in vivo microenvironment. The in vitro 3D tumor model that mimics biologically relevant parameters of in vivo microenvironments such as cell-cell and cell-ECM interactions, and a dynamic environment would be a valuable device to examine efficiency of anticancer drug and targeting specificity. These models have potential to provide in vivo correlated information to improve and optimize drug delivery systems for an effective chemotherapy.
3D tumor model; multicellular tumor spheroids; hydrogel; microfluidic channel; drug delivery systems
ALS is a devastating neurodegenerative disease whose causes are still poorly understood. To identify additional genetic risk factors, here we assess the role of de novo mutations in ALS by sequencing the exomes of 47 ALS patients and both of their unaffected parents (n=141 exomes). We found that amino acid-altering de novo mutations are enriched in genes encoding chromatin regulators, including the neuronal chromatin remodeling complex component SS18L1/CREST. CREST mutations inhibit activity-dependent neurite outgrowth in primary neurons, and CREST associates with the ALS protein FUS. These findings expand our understanding of the ALS genetic landscape and provide a resource for future studies into the pathogenic mechanisms contributing to sporadic ALS.