Background: Toxic element exposure and essential trace element consumption may have changed after the Chinese economy transformed to a market-oriented system. The objectives of this study were to measure urinary concentrations of toxic (arsenic, cadmium, lead) and essential trace (selenium, zinc, copper) elements among rural residents in Hainan, China and to examine if variations in economic development are linked to differences in toxic and trace element exposure. Methods: We conducted a questionnaire-based survey and undertook anthropometric measurements of residents aged ≥20 years (n = 599). Urinary samples were collected and analyzed using inductively coupled plasma mass spectrometry. Results: The median (μg/g creatinine) element concentrations were: arsenic, 73.2; cadmium, 1.8; lead, 3.1; selenium, 36.5; zinc, 371; and copper, 11.0. Intra-community variation in element concentrations was explained by age (arsenic, cadmium, zinc and copper), sex (arsenic, cadmium and selenium: higher in females; zinc: higher in males), body mass index (cadmium) and individual involvement in the market economy as indexed by agrochemical use (lead and selenium). The degree of community-level economic development, which was determined by the proportion of people living in better housing among the study communities, was positively associated with cadmium concentration. Conclusions: The degree of community-level economic development was positively associated with urinary cadmium concentration while individual involvement in the market economy was positively associated with lead and selenium.
toxic elements; heavy; trace elements; urine; economic development; China
We report here the complete sequence of novel duck reovirus (N-DRV) strain SD12 isolated from diseased wild mallard ducklings in the Shandong Province of China in 2012. The complete genome consists of 23,420 nucleotide base pairs (bp), including 10 segments ranging from 1,191 bp (S4) to 3,959 bp (L1).
The study was to investigate the effects of oxygen concentration at different levels for culturing pre-compaction embryos on human embryo development competence. A total of 1254 oocytes from 92 patients treated with conventional in vitro fertilization (IVF) were harvested in this study. Oocytes were randomly assigned to the atmospheric (~20%) or low (~5%) oxygen concentration groups on the retrieval day (day 0). Groups were compared with respect to fertilization rates, embryo development, and reproductive outcome. We failed to detect a significant difference on fertilization rate between two groups. However, the low oxygen group yielded more optimal embryos on day 3 when compared with the atmospheric group (72.4% vs. 64.2%). The low oxygen group had a significantly higher blastocyst formation rate than the atmospheric oxygen group (64.5% vs. 52.9%). It is seemly that the optimal blastocyst and frozen blastocyst rates was higher in the low oxygen group, but the data did not reach a statistical significance. Although the use of low oxygen will not affect the clinical outcome in the fresh cleavage-transfer cycles, but it will result in more favorable clinical outcomes in the subsequent warming blastocyst-transfer cycles, with statistically significantly higher clinical pregnancy rate (CPR) and implantation rate (IR) compared with atmospheric oxygen. In conclusion, a low oxygen concentration may significantly improve the developmental potential of pre-compaction embryos, thus resulting in a positive effect on subsequent blastocyst cultivation and optimizing the treatment cycle.
Oxygen concentration; atmospheric oxygen; embryo development; sibling oocytes
Erysipelothrix rhusiopathiae is the causative agent of animal erysipelas and, to a fewer occurrences, human erysipeloid. It is ubiquitous in nature and commensal in diverse species of animals, wild or domestic, from mammals and birds to reptiles and fish. Mechanisms of its virulence and pathogenicity are poorly understood.
Making use of the complete genome sequencing of E. rhusiopathiae strain SY1027 and comparative genome analysis between the three highly pathogenic strains (SY1027, Fujisawa and ATCC19414), the genomic structure and putative functional elements, such as pathogenicity island (PAI)-like regions, potential virulence factors and horizontal transferring genes of the bacteria are identified. Strain SY1027 genome is 1,752,910 base pairs long, just 30 kilobases smaller than strain Fujisawa, with the same GC level of 36.36%. It contains 1,845 open reading frames (ORF) predicted by GLIMMER 3.02, of which 1,775 were annotated by PGAAP, 1,757 (~95.23%) were annotated by NCBI nr blast, 1,209 by COG database and 1,076 by KEGG database. 37 potential virulence factors were annotated in strain SY1027 by VFDB, while 19 (~51.35%) of them are common in the 2 strains, 7 of which are potentially related to antibiotic resistance and highly conserved (~98-100% match identity (ID)) amongst the three strains of E. rhusiopathiae and modestly homologous to other gastrointestinal tract-inhabiting Firmicutes (~40% match ID), e.g. Clostridium spp., Enterococcus spp. Genomic island- and pathogenicity island-like regions were also predicted, in which some showed association with tRNA and potential virulence factors.
Complete genome sequencing of Erysipelothrix rhusiopathiae, the causative agent of animal erysipelas, was performed. Molecular identification of various genomic elements pave the way to the better understanding of mechanisms underlying metabolic capabilities, pathogenicity of swine erysipelas and prospective vaccine targets besides the widely used SpaA antigens.
Erysipelothrix rhusiopathiae; Complete genome assembly; Genome characterization; Erysipelas; Virulence factors
Dehydroepiandrosterone (DHEA) is now widely used as an adjuvant to IVF treatment protocols in poor responders. However, clinical evidence for DHEA on improvement of ovarian response and IVF outcome is still limited, the validity of the results of the earlier studies, especially the varied inclusion criteria, is a subject of debate. Recently, the ESHRE Working Group developed a new definition, the Bologna criteria. The aim of the current study was to investigate the potential effect of DHEA treatment on in vitro fertilization (IVF) outcome of poor ovarian responders that fulfill the Bologna criteria.
This study investigated 386 poor ovarian responders that fulfill the Bologna criteria. Patients underwent IVF-ET treatment with the GnRH antagonist protocol. The study group contained 189 patients, who received 75 mg of DHEA daily (25 mg three times daily) before the IVF cycle. The control group was composed of 197 patients who received infertility treatment, but did not receive DHEA. The IVF outcome parameters in each group were compared.
The study and control groups did not show statistically significant differences in terms of patient demographics characteristics, mean numbers of oocytes retrieved, mature oocytes, fertilization rate, cleavage rate, or embryo availability. While the DHEA group demonstrated significantly higher implantation rates (18.7% vs. 10.1%; P<0.01) and ongoing PRs (26.7% vs. 15.8%; P<0.05) as compared with the control.
DHEA pre-treatment does not significantly increase oocyte yield. However, the ongoing PRs in this subgroup of women are significantly higher after DHEA administration, suggesting that DHEA may increase IVF results by improving oocyte and embryo quality.
West Nile Virus (WNV) is a mosquito-borne flavivirus that has caused ongoing seasonal epidemics in the United States since 1999. It is estimated that ≤1% of WNV-infected patients will develop neuroinvasive disease (West Nile encephalitis and/or myelitis) that can result in debilitating morbidities and long-term sequelae. It is essential to collect longitudinal information about the recovery process and to characterize predicative factors that may assist in therapeutic decision-making in the future.
We report a longitudinal study of the neurological outcomes (as measured by neurological examination, Glascow Coma Scale, and Modified Mini-Mental State Examination) for 55 subjects with WNV neuroinvasive disease (confirmed by positive CSF IgM) assessed on day 7, at discharge, and on days 14, 30, and 90. The neurological outcome measures were coma (presence and degree), global cognitive status, presence of cranial neuropathy, tremors and/or weakness.
At initial clinical presentation 93% presented with a significant neurological deficit (49% with weakness, 35% with tremor, and 16% with cranial neuropathy). The number of patients with a cognitive deficit fell from 25 at initial evaluation to 9 at their last evaluation. Cranial neuropathy was present in 9 at onset and in only 4 patients at study conclusion. Of the 19 patients who had a tremor at enrollment, 11 continued to exhibit a tremor at follow-up. Seven patients died after initial enrollment in the study, with 5 of those having presented in a coma. The factors that predict either severity or long-term recovery of neurological function include age (older individuals were weaker at follow-up examination), gender (males recovered better from coma), and presentation in a coma with cranial nerve deficits (had a poorer recovery particularly with regard to cognition).
This study represents one of the largest clinical investigations providing prospectively-acquired neurological outcomes data among American patients with WNV central nervous system disease. The findings show that the factors that influence prognosis from the initial presentation include age, gender, and specific neurological deficits at onset.
ClinicalTrials.gov identifier: NCT00138463 and NCT00069316.
West Nile virus; Encephalitis; Neurological deficit; 3MS; Coma
Tumor cells are characterized by their high rate of glycolysis and clotrimazole has been shown to disrupt the glycolysis pathway thereby arresting the cells in the G1 cell cycle phase. Herein, we present data to support our hypothesis that clotrimazole arrests tumor cells in a radiosensitizing, late G1 phase. The effects of clotrimazole were studied using the glioblastoma cell line, U-87 MG. Flow cytometry was used to analyze cell cycle redistribution and induction of apoptosis. Immunoblots were probed to characterize a late G1 cell cycle arrest. Nuclear and cytoplasmic fractions were collected to follow the clotrimazole-induced translocation of hexokinase II. Clonogenic assays were designed to determine the radiosensitizing effect by clotrimazole. Our studies have shown a dose-dependent and time-dependent clotrimazole arrest in a late G1 cell cycle phase. Concurrent with the late G1 arrest, we observed an overexpression of p27Kip along with a decreased expression of p21Cip, cyclin-dependent kinase 1, cyclin-dependent kinase 4, and cyclin D. Clotrimazole induced the translocation of mitochondrial-bound hexokinase II to the cytoplasm and the release of cytochrome c into the cytoplasm. Clotrimazole-induced apoptosis was enhanced when combined with radiation. Clotrimazole was shown to sensitize tumor cells to radiation when the cells were irradiated for 18 h post-clotrimazole treatment. The disruption of the glycolysis pathway by clotrimazole leads to cell cycle arrest of U-87 MG cells in the radiosensitizing late G1 phase. The use of clotrimazole as a radiosensitizing agent for cancer treatment is novel and may have broad therapeutic applications.
antitumor; clotrimazole; glioblastoma; hexokinase II; radiosensitizing
This study measured the antioxidant activity of follicular fluid (FF) in infertile patients and assessed its possible correlation between ovarian stimulation and pregnancy outcomes. Samples from 191 infertile patients undergoing in vitro fertilization-embryo transfer (IVF-ET) were determined by α-diphenyl-β-picrylhydrazyl (DPPH) radical scavenging, reducing power, superoxide radical scavenging, β-Carotene bleaching assay, ferrothiocyanate and thiobarbituric acid assays. The comparison between a positive IVF outcome and FF’s antioxidant activity was also studied. The results showed FF had strong antioxidant activity, which equated to common antioxidants Vc and BHT (100 μg/mL). Patients with endometriosis had less efficient antioxidant activity in FF than that of patients with tubal occlusion or polycystic ovary syndrome. In conclusion, this study detected, for the first time, the antioxidant activity of FF from patients undergoing an IVF and the FF exhibited strong antioxidant activity.
Antioxidant activity; follicular fluid; in vitro fertilization; endometriosis
Influenza disease is a global health issue that causes significant morbidity and mortality through seasonal epidemics. Currently, inactivated influenza virus vaccines given intramuscularly or live attenuated influenza virus vaccines administered intranasally are the only approved options for vaccination against influenza virus in humans. We evaluated the efficacy of a synthetic toll-like receptor 4 agonist CRX-601 as an adjuvant for enhancing vaccine-induced protection against influenza infection. Intranasal administration of CRX-601 adjuvant combined with detergent split-influenza antigen (A/Uruguay/716/2007 (H3N2)) generated strong local and systemic immunity against co-administered influenza antigens while exhibiting high efficacy against two heterotypic influenza challenges. Intranasal vaccination with CRX-601 adjuvanted vaccines promoted antigen-specific IgG and IgA antibody responses and the generation of polyfunctional antigen-specific Th17 cells (CD4+IL-17A+TNFα+). Following challenge with influenza virus, vaccinated mice transiently exhibited increased weight loss and morbidity during early stages of disease but eventually controlled infection. This disease exacerbation following influenza infection in vaccinated mice was dependent on both the route of vaccination and the addition of the adjuvant. Neutralization of IL-17A confirmed a detrimental role for this cytokine during influenza infection. The expansion of vaccine-primed Th17 cells during influenza infection was also accompanied by an augmented lung neutrophilic response, which was partially responsible for mediating the increased morbidity. This discovery is of significance in the field of vaccinology, as it highlights the importance of both route of vaccination and adjuvant selection in vaccine development
Influenza virus remains a global health risk causing significant morbidity and mortality each year, with the elderly (>65 years) and the very young particularly prone to severe respiratory disease. Scientists are working to develop highly efficacious vaccines capable of eliciting broad cross-clade protection from influenza infection. Adjuvants as well as the route of immunization are known to modulate the type, quality and breadth of immune responses to vaccines. In this study, we demonstrated intranasal vaccination with influenza antigens, and a novel synthetic TLR4-based adjuvant system provided protection against a lethal heterologous viral challenge. Immunization stimulated mucosal influenza-specific IgA antibody responses together with systemic IgG antibodies. While intranasal immunization stimulated the production of protective antibodies, vaccination via this route also promoted the generation of influenza-specific Th17 CD4+ T cells. These vaccine-induced Th17 cells increased inflammation and morbidity without contributing to viral clearance following challenge. Antibody neutralization of IL-17A during influenza infection significantly reduced the enhanced lung neutrophilic response, which was partially responsible for mediating the increased morbidity. This discovery is of significance in the field of vaccinology, as it demonstrates the importance of both route of immunization and adjuvant selection in vaccine development.
The Risk Model is a validated outcome predictor for patients with head and neck squamous cell carcinoma (Brandwein-Gensler et al. in Am J Surg Pathol 20:167–178, 2005; Am J Surg Pathol 34:676–688, 2010). This model may potentially shift treatment paradigms for patients with low-stage cancers, as current protocols dictate that they might receive only primary surgery. Here we test the hypothesis that the Risk Model has added prognostic value for low-stage oral cavity squamous cell carcinoma (OCSCC) patients. 299 patients with Stage I/II OCSCC were characterized according to the Risk Model (Brandwein-Gensler et al. in Am J Surg Pathol 20:167–178, 2005; Am J Surg Pathol 34:676–688, 2010). Cumulative incidence and competing risk analysis were performed for locoregional recurrence (LRR) and disease-specific survival (DSS). Receiver operating characteristic analyses were performed for worst pattern of invasion (WPOI) and the risk categories. 292 patients were analyzed; 30 T1N0 patients (17 %) and 26 T2N0 patients (23 %) developed LRR. Disease-specific mortality occurred in 9 T1N0 patients (6 %) and 9 T2N0 patients (10 %). On multivariable analysis, the Risk Model was significantly predictive of LRR (p = 0.0012, HR 2.41, 95 % CI 1.42, 4.11) and DSS (p = 0.0005, HR 9.16, 95 % CI 2.65, 31.66) adjusted for potential confounders. WPOI alone was also significantly predictive for LRR adjusted for potential confounders with a cut-point of either WPOI-4 (p = 0.0029, HR 3.63, 95 % CI 1.56, 8.47) or WPOI-5 (p = 0.0008, HR 2.55, 95 % CI 1.48, 4.41) and for DSS (cut point WPOI-5, p = 0.0001, HR 6.34, 95 % CI 2.50, 16.09). Given a WPOI-5, the probability of developing locoregional recurrence is 42 %. Given a high-risk classification for a combination of features other than WPOI-5, the probability of developing locoregional recurrence is 32 %. The Risk Model is the first validated model that is significantly predictive for the important niche group of low-stage OCSCC patients.
Risk Model; Low-stage; Oral cavity; Pattern of invasion; Squamous cell carcinoma
In this study, we assessed the specific role of BRAF(V600E) signaling in modulating the expression of immune regulatory genes in melanoma, in addition to analyzing downstream induction of immune suppression by primary human melanoma tumor-associated fibroblasts (TAFs).
Primary human melanocytes and melanoma cell lines were transduced to express WT or V600E forms of BRAF, followed by gene expression analysis. The BRAF(V600E) inhibitor vemurafenib was used to confirm targets in BRAF(V600E)-positive melanoma cell lines and in tumors from melanoma patients undergoing inhibitor treatment. TAF lines generated from melanoma patient biopsies were tested for their ability to inhibit the function of tumor antigen-specific T-cells, prior to and following treatment with BRAF(V600E)-upregulated immune modulators. Transcriptional analysis of treated TAFs was conducted to identify potential mediators of T-cell suppression.
Expression of BRAF(V600E) induced transcription of IL-1α and IL-1β in melanocytes and melanoma cell lines. Furthermore, vemurafenib reduced the expression of IL-1 protein in melanoma cell lines and most notably in human tumor biopsies from 11 of 12 melanoma patients undergoing inhibitor treatment. Treatment of melanoma-patient-derived TAFs with IL-1α/β significantly enhanced their ability to suppress the proliferation and function of melanoma-specific cytotoxic T cells, and this inhibition was partially attributable to upregulation by IL-1 of COX-2 and the PD-1 ligands PD-L1 and PD-L2 in TAFs.
This study reveals a novel mechanism of immune suppression sensitive to BRAF(V600E) inhibition, and suggests that clinical blockade of IL-1 may benefit patients with BRAF wild-type tumors and potentially synergize with immunotherapeutic interventions.
Melanoma; BRAF(V600E); interleukin-1; tumor-associated fibroblasts (TAFs); cytotoxic T lymphocytes (CTL)
The aim of the present study was to investigate the effects of exercise training on the characteristics of long-term potentiation (LTP) and N-methyl-D aspartate (NMDA) receptor channels in the hippocampal CA3 neurons of rats with cerebral infarction. Wistar rats were randomly allocated into the model without any training and rehabilitation with exercise training. A model of cerebral infarction was established by middle cerebral artery occlusion. Using chronically embedded electrodes combined with an electrophysiological method, the population spike (PS) amplitude and latency, as well as changes in the NMDA single channel current in the hippocampal neurons were determined prior to and following Y-maze discrimination learning 60 times in the two groups. The formation of learning-dependent LTP and synaptic efficacy in the hippocampal CA3 area after exercise training in the rehabilitation group was significantly faster compared with that in the model group without any training (P<0.05). The incubation period of the PS in the CA3 area of the rats in the rehabilitation group was significantly shorter compared with that in the model group. The PS amplitude in the rehabilitation group was significantly higher compared with that in the model group. Furthermore, the opening probability of the NMDA receptor channel in the rehabilitation group was significantly higher compared with that in the model group. In conclusion, exercise training improved the opening conductance level, time and probability of NMDA receptor channels and accelerated the formation of learning-dependent LTP in the contralateral hippocampal CA3 area.
cerebral infarction; exercise training; long-term potentiation; N-methyl-D aspartate receptor channel
Haemophilus parasuis is the etiological agent of Glässer's disease in pigs and 15 standard serovars were identified. The widespread disease causes great economic loss in the swine industry worldwide. Aiming to investigate the differences in genome composition and functions among various strains, a highly virulent strain ZJ0906 of H. parasuis serovar 12 from China was analyzed and compared with serovar 5 SH0165. Strain ZJ0906 genome is 2,324,740 base pairs with 40.06% genomic GC content. It contains 2,484 open reading frames (ORF) predicted by Glimmer 3.02, of which 2,352 (∼94.7%) were annotated by NCBI nr blast, 1,745 by COG database and 1,829 by KEGG database. 109 potential virulence factors were annotated in strain ZJ0906 and 3 of which are potentially related to antibiotic resistance. Strain ZJ0906 genome is ∼55 kilobases longer than SH0165 genome, with an extra 211 predicted ORFs. VFDB, ARDB, and PAIDB blast searches showed that ZJ0906 and SH0165 shared a nearly identical panel of potential virulence factors, drug resistant genes and four PAI-like regions which showed high homology to Enterococcus, Escherichia and Salmonella. Synteny analysis showed that gene rearrangements are frequent between the two strains, which may lead to variations in pathogenicity and cross-protection among serovars. KEGG pathway analyses showed strain ZJ0906 shared similar metabolic pathways to strain SH0165. Molecular identification of these genomic elements and potential virulence factors pave the way to the better understanding of mechanisms underlying metabolic capabilities and pathogenicity of H. parasuis and prospective vaccine targets besides the widely used method of inactivated bacteria.
Methylation-specific fluorescence in situ hybridization (MeFISH) was developed for microscopic visualization of DNA methylation status at specific repeat sequences in individual cells. MeFISH is based on the differential reactivity of 5-methylcytosine and cytosine in target DNA for interstrand complex formation with osmium and bipyridine-containing nucleic acids (ICON). Cell nuclei and chromosomes hybridized with fluorescence-labeled ICON probes for mouse major and minor satellite repeats were treated with osmium for crosslinking. After denaturation, fluorescent signals were retained specifically at satellite repeats in wild-type, but not in DNA methyltransferase triple-knockout (negative control) mouse embryonic stem cells. Moreover, using MeFISH, we successfully detected hypomethylated satellite repeats in cells from patients with immunodeficiency, centromeric instability and facial anomalies syndrome and 5-hydroxymethylated satellite repeats in male germ cells, the latter of which had been considered to be unmethylated based on anti-5-methylcytosine antibody staining. MeFISH will be suitable for a wide range of applications in epigenetics research and medical diagnosis.
Since the first reported cases of ducks infected with a previously unknown flavivirus in eastern China in April 2010, the virus, provisionally designated Duck Tembusu Virus (DTMUV), has spread widely in domestic ducks in China and caused significant economic losses to poultry industry. In this study, we examined in detail structural, antigenic, and evolutionary properties of envelope (E) proteins of six DTMUV isolates spanning 2010–2012, each being isolated from individual farms with different geographical locations where disease outbreaks were documented. Structural analysis showed that E proteins of DTMUV and its closely related flavivirus (Japanese Encephalitis Virus) shared a conserved array of predicted functional domains and motifs. Among the six DTMUV strains, mutations were observed only at thirteen amino acid positions across three separate domains of the E protein. Interestingly, these genetic polymorphisms resulted in no detectable change in viral neutralization properties as demonstrated in a serum neutralization assay. Furthermore, phylogenetic analysis of the nucleotide sequences of the E proteins showed that viruses evolved into two distinct genotypes, termed as DTMUV.I and DTMUV.II, with II emerging as the dominant genotype. New findings described here shall give insights into the antigenicity and evolution of this new pathogen and provide guidance for further functional studies of the E protein for which no effective vaccine has yet been developed.
Despite the fact that both gonadotropin-releasing hormone (GnRH) agonist and antagonist protocol are effective in suppressing the incidence of premature luteinizing hormone (LH) surges through reversibly blocking the secretion of pituitary gonadotropins, the exact impact of these two distinctive protocols on the clinical setting of patients for in vitro fertilization and embryo transfer (IVF-ET) treatment, however, remained controversial. We thus in the present report conducted a retrospective study to compare the impact of GnRH agonist and antagonist protocol on the same patients during controlled ovarian stimulation cycles. A total of 81 patients undergoing 105 agonist and 88 antagonist protocol were analyzed. We failed to detect a significant difference between two protocols for the difference in duration of ovarian stimulation, number of recombinant FSH (Gonal-F) ampoules used, number of oocytes retrieved, serum levels for estradiol (E2) and progestone (P), thickness of endometrium, and the zygote- and blastocyst-development rate. It is seemly that high quality embryo rate was higher in the antagonist protocol, but the data did not reach a statistical significance. Nevertheless, Implantation rate and clinical pregnancy rate were significantly higher in the antagonist protocol (10.64% and 30.26%, respectively) than that of the agonist protocol (5.26% and 15.82%, respectively). Our data also suggest that the GnRH antagonist protocol is likely to have the advantage for improving the outcome of pregnancy in those patients with a history of multiple failures for the IVF-ET treatment.
Gonadotropin-releasing hormone (GnRH); agonist; antagonist; in vitro fertilization; embryo transfer; assisted reproduction; controlled ovarian stimulation cycles
Perhaps one of the most important developments in head and neck oncology of the past decade is the demonstration that patients with human papillomavirus (HPV)-mediated oropharyngeal cancers have significantly improved outcomes, compared to HPV-negative counterpart patients. This has become the basis for clinical trials investigating the impact on “treatment deintensification” for patients with HPV-mediated oropharyngeal cancers. Unfortunately, the significance of HPV in non-oropharyngeal head and neck cancers is much less certain. Our goal is to systematically review the published data regarding the role HPV in carcinomas of the oral cavity, larynx, sinonasal tract and nasopharynx with respect to HPV detection frequency, viral activity, and association with outcome. We also present preliminary data on HPV16/18 transcriptional status in oral cavity carcinomas, as well as salivary gland neoplasia, as determined by nested reverse transcription PCR for HPV E6/E7 RNA. The weighted prevalence (WP) of HPV DNA detection in 4,195 oral cavity cancer patients is 20.2 %, (95 % CI 16.0 %, 25.2 %). HPV16 is the most common type detected. Importantly, no data currently demonstrates a significant association between the presence of HPV DNA and improved outcome. The WP of HPV DNA in 1,712 laryngeal cancer patients is 23.6 %, (95 % CI 18.7 %, 29.3 %). Similarly, no association has yet been demonstrated between HPV DNA status and outcome. The WP of HPV DNA detection in 120 sinonasal cancer patients is 29.6 % (95 % CI 17.8 %, 44.9 %), and in 154 nasopharyngeal carcinoma patients is 31.1 %, (95 % CI 20.3 %, 44.5 %). Recent preliminary data also suggests an association between HPV and certain salivary gland neoplasms. The clinical significance of these findings is unclear. The published data strongly support the need for studies on patients with oral and laryngeal carcinomas that will be powered to find any differences in clinical outcome with respect to HR-HPV and p16 overexpression.
HPV; Squamous carcinoma; Oral cavity; Laryngeal; Larynx; Mucoepidermoid carcinoma; Salivary
Tumor-specific T-cells are frequently induced naturally in melanoma patients and infiltrate tumors. It is enigmatic why these patients fail to experience tumor regression. Given that CD8+ T cells mediate antigen-specific killing of tumor cells, the focus of this study was to identify alterations in the differentiation of CD8+ residing at the tumor site, with emphasis on a population expressing CD57, a marker for terminal differentiation.
We performed flow cytometric analysis of CD8+ tumor-infiltrating lymphocytes (TIL) isolated from 44 resected melanoma metastases using known T-cell differentiation markers. For comparison, PBMC were isolated from matched melanoma patients. We sorted different CD8+ subsets found in TIL and determined their effector functions. In addition, we performed Vβ spectratyping of T-cell receptors to determine lineage relationship between the CD8+ TIL subsets.
The majority of CD8+ TIL were in the early effector-memory stage of differentiation. A significant population consisted of an oligoclonal subset of cells co-expressing early effector-memory markers and end-stage CTL marker, CD57, yet having low to absent perforin expression. These cells could be induced to proliferate, produce a high level of IFN-γ, and differentiate into CD27−CD57+, perforinhigh mature CTL in vitro. Addition of TGF-β1 prevented this further differentiation.
Our studies identified a novel subset of incompletely differentiated CD8+ CTL co-expressing early effector-memory and late CTL markers. This population resembles that found by in patients with uncontrolled chronic viral infections. TGF-β1, frequently produced by melanoma tumors, may be a key cytokine inhibiting the further maturation of this subset.
Melanoma; tumor-infiltrating lymphocyte; CTL; CD8+ effector-memory; CD57
Porcine reproductive and respiratory syndrome virus (PRRSV) is largely responsible for heavy economic losses in the swine industry worldwide because of its high mutation rate and subsequent emergence of virulent strains. However, the immunological and pathological responses of pigs to PRRSV strains with different virulence have not been completely elucidated.
Twenty-four piglets were divided into 4 groups (n = 6 each) and inoculated with highly pathogenic PRRSV isolate BB0907 (HP), low pathogenic PRRSV NT0801 (LP), LP derivative strain NT0801-F70 (LP-der), and DMEM medium (control), respectively. The changes in TLR2, 3, 7, and 8 gene expression and TNF-α, IL-1β, IL-6, IFN-γ, and IL-10 secretion were evaluated using real-time PCR and ELISA at 6, 9, and 15 days post inoculation (d.p.i.). The cytokine levels were evaluated in the supernatants of porcine alveolar macrophages (PAMs) and peripheral blood mononuclear cells (PBMCs) following stimulation with LTA, poly(I:C), CL097, and PRRSV individually.
HP caused more severe clinical signs and pathological lesions in swine than LP and LP-der had almost no virulence compared with LP. The serum levels of IL-1β, IL-6, TNF-α, and IFN-γ were increased in HP-infected piglets, which were greater than in those infected with LP or LP-der. The mRNA levels of TLR3, 7, and 8 were significantly up-regulated in PAMs in HP-infected pigs compared to those in groups LP and LP-der. Furthermore, TNF-α and IL-1β secretion in PAMs from group LP was statistically greater than those from the control group after stimulation with either poly(I:C) or CL097. Meanwhile, TNF-α, IL-1β, and IL-6 levels in CL097-stimulated PBMCs from HP-infected pigs were markedly higher than those from the LP- and LP-der-infected groups.
We found that HP was a stronger inducer of TLR 3, 7, and 8 expression and IL-1β, IL-6, TNF-α, and IFN-γ production compared to LP and LP-der. HP enhanced production of TNF-α, IL-1β, and IL-6 in PBMCs following CL097-stimulation more than LP and LP-der, whereas LP enhanced the secretion of TNF-α and IL-1β in poly(I:C)- and CL097-stimulated PAMs. Our data regarding cellular reactivity to different isolates should be useful in the development of more efficacious vaccines.
PRRSV; Immunogenicity; Pathogenicity; TLRs; Cytokines
Porcine reproductive and respiratory syndrome (PRRS) is characterized by a delayed and defective adaptive immune response. The viral nonstructural protein 1 (NSP1) of the PRRS virus (PRRSV) is able to suppress the type I interferon (IFN) response in vitro. In this study, recombinant adenoviruses (rAds) expressing NSP1 (rAd-NSP1), glycoprotein 5 (GP5) (rAd-GP5), and the NSP1-GP5 fusion protein (rAd-NSP1-GP5) were constructed, and the effect of NSP1 on immune responses was investigated in pigs. Pigs inoculated with rAd-NSP1 or rAd-NSP1-GP5 had significantly lower levels of IFN-γ and higher levels of the immunosuppressive cytokine IL-10 than pigs inoculated with rAd-GP5, wild-type adenovirus, or cell culture medium alone. The antibody response to vaccination against classic swine fever virus (CSFV) was significantly decreased by inoculation of NSP1 7 d after CSFV vaccination in pigs. Thus, NSP1-mediated immune suppression may play an important role in PRRSV pathogenesis.
The purpose is to evaluate sensitivity of basal-like breast cancer to treatment with anti-DR5 alone and in combination with chemotherapy. Cytotoxicity of TRA-8 anti-DR5 alone and in combination with doxorubicin or paclitaxel was examined. The role of a DR5-associated molecule (DDX3) in the regulation of apoptosis by recruitment of cIAP1 to the DR5/DDX3 complex was studied. SUM159 and 2LMP orthotopic xenografts were treated with TRA-8 alone and in combination with Abraxane or doxorubicin, and tumor growth inhibition determined. Diffusion-weighted magnetic resonance imaging was used to monitor early tumor response. The majority (12/15) of basal-like cell lines were very sensitive to TRA-8-induced cytotoxicity (IC50 values of 1.0–49 ng/ml). In contrast, 8/11 luminal or HER2-positive cell lines were resistant (IC50 > 1,000 ng/ml). Enhanced killing of basal-like cell lines was produced by combination treatment with TRA-8 and doxorubicin. Majority of basal cell lines expressed lower levels of DR5-associated DDX3 and cIAP1 than luminal and HER2-positive cell lines. TRA-8 inhibited growth of basal xenografts and produced 20% complete 2LMP tumor regressions. TRA-8 and chemotherapy produced greater 2LMP growth inhibition than either alone. An increase in apparent diffusion coefficient in 2LMP tumors was measured in a week of therapy with TRA-8 and Abraxane. Basal-like cell lines were more sensitive to TRA-8-mediated cytotoxicity than HER2-over-expressing and luminal cell lines, and chemotherapy enhanced cytotoxicity. High sensitivity of basal cells to TRA-8 correlated with low expression of DR5/DDX3/cIAP1 complex. Treatment with TRA-8 and chemotherapy may be an effective therapy for basal-like breast cancer.
Basal-like breast cancer; Anti-DR5 antibody; Chemotherapy
Breast cancer stem cells (BrCSC) are resistant to common therapeutic modalities including chemotherapy, radiation, and hormonal agents. They are thought to contribute to treatment resistance, relapse, and metastases. This study examines the effect of a monoclonal anti-DR5 antibody (TRA-8) and chemotherapy (adriamycin, taxol) on BrCSC populations from basal-like breast cancer cell lines. Doubly enriched BrCSC (CD44+, CD24−, ALDH+) cells were exposed to TRA-8 and control reagents and examined for cytotoxicity, caspase activation, tumorsphere formation and tumorigenicity. Doubly enriched BrCSC populations expressed cell surface DR5 and were sensitive to TRA-8 mediated cytotoxicity with induction of caspase 8 and 3 activation. TRA-8 at sub-nanomolar concentrations inhibited 2LMP and SUM159 BrCSC tumorsphere formation and was more than 50-fold more inhibitory than TRAIL or anti-DR4 at equimolar concentrations. Chemotherapy treatment of 2LMP and SUM159 cell lines resulted in a relative increase of BrCSC, whereas TRA-8 produced a decrease in the percentage of BrCSC. TRA-8 exposure to 2LMP and SUM159 BrCSC preparations produced significant inhibition of tumorigenicity. DR5 maybe a therapeutic target on the surface of basal-like BrCSC which is amenable to agonistic monoclonal anti-DR5 therapy.
Anti-DR5; Tigatuzumab; Basal-like breast cancer; Breast cancer stem cells; Tumor initiating cells; Tumorspheres; Death receptor 5
Encephalomyocarditis virus (EMCV) can infect many host species and cause acute myocarditis and sudden death in preweaned piglets. In this study, an EMCV strain (NJ08) was isolated from newborn pigs with clinical signs on a pig farm in mideastern China. It was identified by indirect immunofluorescence assay and reverse-transcription polymerase chain reaction. Experiments showed that the isolate could cause severe clinical symptoms and pathological changes in mice but no obvious clinical and pathological changes in commercial piglets. Complete genomic sequencing showed that the NJ08 strain was 78.3% to 100% identical with other isolates in regions coding for various proteins. Phylogenetic analysis showed that the NJ08 isolate belonged to subgroup Ia. This study confirmed that an EMCV isolate from pigs could be fatal to mice and provided new epidemiologic data on EMCV in China.
Notch and Hedgehog activate cell-cycle progression of adult and cancer stem cells. Notch is activated by DLL and Jag presents on neighboring cells. We investigated the effects of density of the Notch-activating ligand, Jag-1, and targeting Gli-1, in activation of division of paclitaxel/taxol-resistant, (PTXRes) ovarian cancer cells SKOV3 (SKOV3). We used the specific γ-presenilin inhibitor, DAPT, to identify the specificity of activating signals for Notch-1 and created ‘butterfly-duplex-3548-Gli-1-inhibitory RNA’ (i-Gli-1.RNA) to inhibit cell division. To accurately quantify kinetics of division, the expression of CD44 and CD24 was determined in each gated population of divided cells. CD44High proliferated when activated by Jag-1Low and poorly when activated by Jag-1High. DAPT inhibited proliferation of cells activated by Jag-1Low, and increased proliferation of cells activated by Jag-1High. Only 5–10% of cells activated by Jag-1High and Jag-1Low divided fast, polynomial, and symmetric. i-Gli-1.RNA eliminated more than 50% of the small CD44High/CD24Neg cells in divisions 3 and 4. This effect appeared specific compared with cells transfected with negative control siRNA. i-Gli-1.RNA had no effect on large CD44High/CD24Neg cells, but inhibited the population of CD44High/CD24Low cells. Expansion of CD44High inversely correlated with Jag-1 density on activating autologous tumor and fibrosarcoma cells. Created i-RNAs may decrease the resting CSC pool. Notch and Gli-1 signals play an important role in proliferation/division and survival of cancer stem cells. Targeting Notch-1 through its enhancer Gl-1, should be significant for novel treatments to eliminate taxol-resistant cancer stem cells (CSC). i.Gli-1 RNA should be more effective if used together with Taxol.
drug-resistance; cancer stem cell; Notch; Gli-1; micro-RNA
To evaluate the stability and heterogeneity of cytokine and chemokine profiles in 80 youth with and without HIV-1 infection, we tested plasma samples at repeated visits without antiretroviral therapy. Among nine analytes that were quantified using multiplexing assays, interleukin 10 (IL-10), IL-18, and soluble CD30 persistently showed a positive correlation with HIV-1 viral load (Spearman ρ = 0.40–0.59, p < 0.01 for all). A negative correlation with CD4+ T cell counts (ρ = −0.40 to −0.60, p < 0.01 for all) was also persistent for the three analytes. Analyses restricted to 48 AIDS-free youth (96 visits) yielded similar findings, as did multivariable models in which race, sex, age, body mass index, and time interval between visits were treated as covariates. These relationships reflected two novel features observed for all three analytes. First, their presence in plasma was relatively stable between visits (ρ = 0.50–0.90, p < 0.03), regardless of HIV-1 infection status. Second, pairwise correlation was strong and persistent in HIV-1-seropositive youth (ρ = 0.40–0.59, p < 0.01), but not in HIV-1, seronegatives (p > 0.13). Additional analytes, especially eotaxin/CCL11 and SDF-1β/CXCL12, had no correlation with HIV-1-related outcomes despite their stability between visits. Overall, circulating IL-10, IL-18, and soluble CD30 could partially track unfavorable responses to HIV-1 infection in youth. These markers of persistent immune activation are individually and collectively indicative of HIV-1 pathogenesis.