text:
Vaccination With GM-K562 Cells in Patients With Advanced Myelodysplastic Syndrome (MDS) or Acute Myeloid Leukemia (AML) After Allogeneic Hematopoetic Stem Cell Transplantation
This study is ongoing, but not recruiting participants.
Dana-Farber Cancer Institute
ClinicalTrials.gov Identifier:
First Posted: December 17, 2008
Last Update Posted: November 15, 2017
Brigham and Women's Hospital
National Institutes of Health (NIH)
Information provided by (Responsible Party):
Vincent T. Ho, MD, Dana-Farber Cancer Institute
- Full Text View
- Tabular View
- No Study Results Posted
- Disclaimer
- How to Read a Study Record
Detailed Description:
The purpose of this research study is to determine if the GM-K562/leukemia cell vaccine can be safely given soon after allogeneic marrow or blood stem cell transplant. The GM-K562/leukemia cell vaccine is composed of a cultured cell line that has been genetically modified to secrete GM-CSF, a naturally occuring substance in the body that stimulates the immune system. The vaccine is a mixture of the GM-K562 cells (radiated to prevent them from growing in the participants body) with the participant's previously frozen and killed leukemia cells. By mixing the GM-K562 with the leukemia cells, we would like to study whether this vaccine combination will stimulate the participant's new immune system to recognize and fight against their MDS/AML cancer cells.
|Acute Myeloid Leukemia Chronic Myelomonocytic Leukemia Myelodysplastic Syndrome-Refractory Anemia With Excess Blasts
|Biological: GM-K562/leukemia cell vaccine
|Phase 1
|Study Type:
|Study Design:
|Intervention Model: Single Group Assignment
Masking: None (Open Label)
Primary Purpose: Treatment
|Official Title:
|Vaccination With Lethally Irradiated Autologous Myeloblasts With Granulocyte Macrophage-colony Stimulating Factor Secreting K562 Cells (GM-K562) in Patients With Advanced MDS or AML After Allogeneic Hematopoietic Stem Cell Transplantation
Resource links provided by NLM:
Genetics Home Reference related topics: core binding factor acute myeloid leukemia cytogenetically normal acute myeloid leukemia familial acute myeloid leukemia with mutated CEBPA
MedlinePlus related topics: Leukemia
Genetic and Rare Diseases Information Center resources: Myeloid Leukemia Acute Myeloid Leukemia Acute Non Lymphoblastic Leukemia Myelodysplastic Syndromes Chronic Myelomonocytic Leukemia Myelodysplastic/myeloproliferative DiseaseU.S. FDA Resources
Further study details as provided by Vincent T. Ho, MD, Dana-Farber Cancer Institute:
Primary Outcome Measures:
- To assess the safety of vaccination, as measured by vaccine related reactions and incidence of grade III-IV acute GVHD. [ Time Frame: 2 years ]
Secondary Outcome Measures:
- To assess the efficacy of vaccination with GM-K562/leukemia cell vaccine following allogeneic stem cell transplantation in this patient population. [ Time Frame: 2 years ]
- To characterize the biologic responses and leukemia specific immune responses after vaccination with GM-K562/leukemia cell vaccine following allogeneic stem cell transplant. [ Time Frame: 2 years ]
- To determine duration of disease response, disease free and overall survival [ Time Frame: 2 years ]
|Study Start Date:
|November 2008
|Estimated Study Completion Date:
|December 2026
|Primary Completion Date:
|May 2011 (Final data collection date for primary outcome measure)
|Assigned Interventions
Experimental: GM-K562/leukemia cell vaccine
Biological/Vaccine: GM-K562/leukemia cell vaccine Cultured cell line genetically changed to secrete GM-CSF mixed with irradiated leukemia cells obtained from the participant. A total of 6 vaccine will be given. Vaccines 1-3 will be given once a week. Vaccines 4-6 will be given every other week.
Biological: GM-K562/leukemia cell vaccine
Cultured cell line genetically changed to secrete GM-CSF mixed with irradiated leukemia cells obtained from the participant. A total of 6 vaccine will be given. Vaccines 1-3 will be given once a week. Vaccines 4-6 will be given every other week.
Detailed Description:
- Participants will be given the GM-K562/Leukemia call vaccine as in injection under the skin a total of six times. The first 3 vaccines will be given weekly and vaccines 4 through 6 will be given every other week. Therefore, it is expected that the vaccines will be completed over a period of 9 weeks.
- During the 9 week vaccination period, participants will have physical exams to monitor for any side effects or graft-versus-host disease (GVHD). Bone marrow biopsies will be performed a the time of enrollment for this study, 4 weeks after completion of 6 GM-K562/Leukemia cell vaccines, and 1 year after the participants transplant.
- As a way of testing whether the GM-K562/Leukemia cell vaccine is triggering any immune response to the participants leukemia, we will be injecting a small amount of leukemia cells (after they are killed with radiation) under the participants skin to see if the body will generate a reaction to the leukemia cells. This test is called a leukemia cell delayed hypersensitivity test (DTH). This test will be performed three times during the study, on the weeks of the 1st vaccine, 5th vaccine and 4 weeks after the 6th vaccine.
- There are a total of 5 skin biopsies required as part of this study. Biopsies will be taken from the vaccination sites 2-3 days after the first and fifth vaccine. Similar biopsies will be taken from the DTH sites after the 1st vaccination, 5th vaccination and 4-6 weeks after the 6th vaccination.
Information from the National Library of Medicine
Choosing to participate in a study is an important personal decision. Talk with your doctor and family members or friends about deciding to join a study. To learn more about this study, you or your doctor may contact the study research staff using the contacts provided below. For general information, Learn About Clinical Studies.
|Ages Eligible for Study:
|18 Years and older (Adult, Senior)
|Sexes Eligible for Study:
|Accepts Healthy Volunteers:
Inclusion Criteria:
- Patients who have received an allogeneic bone marrow or peripheral blood stem cell transplant for AML, meeting one of the following: 1) AML arising from MDS or MDP 2)AML CR1 associated with high risk cytogenetics 3) AML transplanted in induction failure or relapse 4) AML transplanted in second remission or beyond 5) AML in patient 60 years or older
- Patients who have received an allogeneic bone marrow or peripheral blood stem cell transplant for MDS-RAEB or CMML
- 18 years of age or older
- Donor is a related or unrelated donor who is at least 9/10 matched at HLA-A, B, C, DRB1, and DQB1 by antigen level typing at class 1 and allele level typing at class II
- Recipients of myeloablative or reduced intensity conditioning transplants are eligible
- Patient must have sufficient autologous tumor cells banked at DFCI (on companion tissue banking protocol) for vaccine generation prior to transplantation
- No active GVHD requiring systemic corticosteroid therapy
- No conditions requiring systemic corticosteroid therapy greater than or equal to 20mg methylprednisolone or equivalent
- No uncontrolled infection
- Adequate hematopoietic engraftment with ANC >500 off growth factor support, and platelet >10k without transfusion
- No non-hematologic toxicity of CTC Grade 3 or greater
- ECOG Performance Status 0-2
Exclusion Criteria:
- Recipients of cord blood transplant
- Patients with uncontrolled CNS disease
- Patients with relapsed/persistent disease after transplant who are expected to require rapid withdrawal of immune suppression, cytoreductive therapy, or have a life expectancy of < 3 months
- Concurrent participation in other transplant clinical trials where GVHD and/or disease relapse are primary endpoints
- Patients deemed medically or psychologically unfit by treating physician or study investigator
Contacts and Locations
Information from the National Library of Medicine
To learn more about this study, you or your doctor may contact the study research staff using the contact information provided by the sponsor.
To learn more about this study, you or your doctor may contact the study research staff using the contact information provided by the sponsor.
Please refer to this study by its ClinicalTrials.gov identifier (NCT number): NCT00809250
Please refer to this study by its ClinicalTrials.gov identifier (NCT number): NCT00809250
|United States, Massachusetts
|Dana-Farber Cancer Institute
|Boston, Massachusetts, United States, 02115
Sponsors and Collaborators
Dana-Farber Cancer Institute
Brigham and Women's Hospital
National Institutes of Health (NIH)
|Principal Investigator:
|Vincent Ho, MD
|Dana-Farber Cancer Institute
More Information
Keywords provided by Vincent T. Ho, MD, Dana-Farber Cancer Institute:
Additional relevant MeSH terms:
|Responsible Party:
|Vincent T. Ho, MD, Principal Investigator, Dana-Farber Cancer Institute
|ClinicalTrials.gov Identifier:
|NCT00809250 History of Changes
|Other Study ID Numbers:
|First Submitted:
|December 15, 2008
|First Posted:
|December 17, 2008
|Last Update Posted:
|November 15, 2017
|Last Verified:
|November 2017
Keywords provided by Vincent T. Ho, MD, Dana-Farber Cancer Institute:
Additional relevant MeSH terms:
Leukemia, Myeloid
Leukemia, Myeloid, Acute
Myelodysplastic Syndromes
Leukemia, Myelomonocytic, Chronic
Anemia, Refractory
Anemia, Refractory, with Excess of Blasts
Pathologic Processes
Neoplasms by Histologic Type
Bone Marrow Diseases
Hematologic Diseases
Precancerous Conditions
Myelodysplastic-Myeloproliferative Diseases
Immunologic Factors
Physiological Effects of Drugs


text:
Services for Brandon Lee Sanchez, 4, of Lubbock will be at 2 p.m. Friday in Sanders Memorial Chapel with Albert Gutierrez officiating.
Burial will be in City of Lubbock Cemetery under the direction of Sanders Funeral Home.
He died Tuesday, Jan. 25, 2000, at Covenant Medical Center.
He was born May 18, 1995, in Lubbock. He was a pre-kindergarten student at Martin Head Start Center. He collected Pokemon cards and loved to play video games.
Survivors include his parents, Ted and Betsy Sanchez of Lubbock; two brothers, Alexander and Sean, both of Lubbock; maternal grandmother, Janie Rivera of Lubbock; maternal grandfather, Modesto Hinojosa of Lubbock; paternal grandmother, Rita P. Sotelo of Lubbock; paternal grandfather, Teodoro Sanchez of Lubbock; and maternal great-grandmother, Eloisa Hinojosa of Lubbock.
© 2017. All Rights Reserved. | Contact Us

subset: commoncrawl


text:
Get your party dress on
Published 5:26 pm, Monday, November 19, 2012
From sophisticated elegant lace to glitzy show stopping styles, holiday dressing this season is loaded with everything from simplicity to sequins.
Silhouettes, too, are varied from slinky and body hugging to gathered and roomy. There's no holding back unless the dress you covet is backless. And there's plenty of that, too.
Cocktail dresses and gowns for the holidays are dramatic and bold, romantic and defiant. Fabrics are luxurious, shiny and sexy in jersey and silk, velvet and lace.
With the countdown on for holiday festivities, it soon will be time to make an epic entrance in a hot look during the coolest time of the season.
Lifestyle Channel
Now Playing:
- Getting your goat yoga in the Texas Hill Country
- Severe Winter Storms Target the Midwest and East — Just in Time for ThanksgivingTaLTIME
- Flamingos of Lake Bogoria, KenyaTaLTIME
- Canada's Top 5 CitiesTaLTIME
- TSA PreCheck Members Upset as Wait Times Get LongerBuzz60
- Lantern Festivals of ThailandTaLTIME
- President Trump's Thanksgiving Travels Are Costing Tax Payers A Pretty PennyBuzz60
- Southwest Airlines Pilot Arrested for Having a Loaded Gun in His Carry-onTaLTIME
- Everglades National Park, FloridaTaLTIME
- This Wes Anderson-themed Airbnb Is the Quirky Rental of Your DreamsTaLTIME
Party on.


text:
This product is no longer available.
You can try these similar gifts instead.
Durango Straw Hat
New York Hat
Great for both yard work or riding the range, this hat is a classic in cowboy style but lightweight for summer or hot climates. Chin strap keeps it from blowing away at the beach or on the boat.


text:
BOSTON (CBS) – She thought cancer would be the fight of her life. Now, a local mom is battling to save her daughter who also has the disease.
Doctors diagnosed Kezia Fitzgerald shortly after she gave birth, but as she started making strides she noticed drastic changes in her little girl and it wasn't long before both were fighting to stay alive.
In January Kezia was diagnosed with Hodgkin's lymphoma, "it was definitely a shock, of course at first we were scared."
She called the day she got the news D-day on her blog "New Mom….. New Cancer."
WBZ-TV's Diana Perez reports
Just eight months before that day Kezia and Mike had Saoirse, a healthy baby girl who immediately stole their hearts.
Kezia's new cancer required chemotherapy. It slowed her down a bit but she was determined to beat it, "There's no other options you can just crawl into a hole."
But in May the Fitzgeralds were dealt a second devastating blow. After weeks of mysterious symptoms and fruitless doctors visits Saoirse was diagnosed with Neuroblastoma, a common cancer in infants.
When asked what that day was like Mike broke down, "I just couldn't talk. I just kept crying. We all just sat there, holding each other."
For three months, mother and daughter bonded over the debilitating disease, "it does make it a little bit easier to keep on top of side effects because I know what she feels like."
There have been victories, Kezia's cancer is in remission but Saoirse's fight continues. She needs surgery to remove tumors and in January will undergo stem cell transplant.
But like mom her strength in the face of struggle is unwavering, "She is very hyper and very antsy. She loves learning; she's not letting it get her down."
Saoirse's doctors say she is responding well to her cancer medicine. They've also told mom and dad that the two cancers are entirely unrelated and were not passed on from mom to daughter.
The Fitzgerald's have started a fund to help pay their mounting medical bills and living expenses. Both parents are no longer working.
For more information or to make a donation, you can follow the Fitzgeralds on Twitter @fitzcancerfund and on Facebook.


text:
Published online Feb 7, 2008. doi: 10.3748/wjg.14.701
Revised: September 30, 2007
Published online: February 7, 2008
AIM: To elucidate the influence of quasispecies on virological response and disease severity in patients with chronic hepatitis C.
METHODS: Forty seven patients with hepatitis C [32 with chronic active hepatitis (CAH), 9 with cirrhosis, and 6 with hepatocellular carcinoma (HCC)] were screened for the presence of quasispecies by single stranded conformational polymorphism (SSCP) analysis in the hypervariable region (HVR) and non-structural 5B (NS5B) viral genes of hepatitis C virus. The 41 patients excluding those with HCC were on therapy and followed up for a year with the determination of virological response and disease severity. Virus isolated from twenty three randomly selected patients (11 non-responders and 12 showing a sustained virological response) was sequenced for the assessment of mutations.
RESULTS: The occurrence of quasispecies was proportionately higher in patients with HCC and cirrhosis than in those with CAH, revealing a significant correlation between the molecular evolution of quasispecies and the severity of disease in patients with hepatitis C. The occurrence of complex quasispecies has a significant association (P < 0.05) with the non-responders, and leads to persistence of infection. Significant differences (P < 0.05) in viral load (log10 IU/mL) were observed among patients infected with complex quasispecies (CQS), those infected with simple quasispecies (SQS) and those with no quasispecies (NQS), after 12 wk (CQS-5.2 ± 2.3, SQS-3.2 ± 1.9, NQS-2.8 ± 2.4) and 24 wk (CQS-3.9 ± 2.2, SQS-3.0 ± 2.2, NQS-2.1 ± 2.3) in the HVR region. However, a statistically significant difference (P < 0.05) was observed between the viral loads of patients infected with CQS and those infected with NQS in NS5B viral gene after 24 wk (CQS-3.9 ± 2.2, SQS-3.0 ± 2.2, and NQS-2.1 ± 2.3) and 48 wk (CQS-3.1 ± 2.7, SQS-2.3 ± 2.4, NQS-2.0 ± 2.3) of therapy. Disease severity was significantly associated with viral load during therapy. The strains isolated from non-responders showed close pairing on phylogeny based on the NS5B gene, but dissimilar HVR regions. This revealed the possibility of the selection of resistant strains during the evolution of quasispecies in NS5B.
CONCLUSION: Viral quasispecies may be an important predictor of virological responses to combination therapy in patients with chronic hepatitis C. Complex quasispecies and resistant strains may lead to high viral loads during therapy, with a concerted effect on disease severity.
Citation: Kumar D, Malik A, Asim M, Chakravarti A, Das RH, Kar P. Influence of quasispecies on virological responses and disease severity in patients with chronic hepatitis C. World J Gastroenterol 2008; 14(5): 701-708
Hepatitis C is a noncytopathic hepatotrophic virus having a single stranded, positive sense linear -9.5 kb RNA genome. Hepatitis C virus infection is the leading cause of acute, chronic, liver cirrhosis and hepatocellular carcinoma. Chronic infection due to hepatitis C virus (HCV) occurs in 170-200 million people worldwide[12]. In India, 15%-20% of all patients with chronic liver disease and 5%-10% of hepatocellular carcinoma patients are infected with hepatitis C[3]. The HCV continues to persist, unless the patients are put on interferon therapy. Treatment with interferon-based therapy and error-prone replication of the genome via RNA-dependent RNA polymerase (RDRP) accelerates the evolution of HCV variants during the course of infection[2]. The origination of highly heterogeneous population in hepatitis C infected patients closely related to HCV genome because of interferon therapy, immune pressure and replication strategy called quasispecies[4].
The hypervariable region 1 (HVR-1) is located at the 5'-end of the E2 envelope glycoprotein, and variations occur in this region in association with persistence of viral infection[5]. Significant mutations in HVR-1 arise due to the presence of a specific anti-E2 humoral immune response. It is emerging that genetic heterogeneity extends throughout the entire HCV genome, and that this allows HCV infection to persist. Anti-HCV-specific T cell responses efficiently clear viruses during the acute phase of infection[6]. HVR quasispecies may have important biological consequences for the persistence of hepatitis C viral infection and the severity of liver disease[57].
HCV quasispecies populations result from the error rate (misincorporation frequencies averaging from about 10-4 to 10-5 per base) and the absence of proof-reading by RDRP. Most mutant particles cannot replicate, but the fittest infectious particles are selected on the basis of their replication capacities and selective pressure exerted by their interaction with host cell proteins and by the immune response[8]. Adaptive mutations in NS4B, NS5A or NS5B strongly enhance replication, but are incompatible to each other, whereas adaptive mutations in NS3 tend to be weak and cooperatively enhance replication when combined with strongly adaptive mutations[89]. The adaptive mutations in NS3 and NS5B map to surface residues distant from the enzyme active site, suggesting that these changes are likely to affect the interaction between non-structural protein and cellular factors[8].
Hepatitis C viral quasispecies can be detected by cloning of reverse transcription - polymerase chain reaction products, but sequencing of 15-20 independent clones is expensive and laborious, and so is not practical for a large number of clinical samples. For large numbers of clinical samples, faster methodologies like single-strand conformational polymorphism (SSCP), heteroduplex analysis (HDA) and gel shift assay (GSA) have been developed based on the differential migration of amplified DNA in acrylamide gel electrophoresis. In the present study, we used the established and reliable method of SSCP to detect quasispecies[10–13]. The mobility of mutated DNA fragments is affected by particular changes in the single-strand DNA structure; therefore, every variant migrates in a different way.
In controlled trials on the effect of interferon and ribavirin combination therapy, the independent predictive influence of virological parameters on patients with sustained virological response (SVR) raised questions about the intrinsic properties of HCV strains, like genotype and quasispecies and their effect on sensitivity and resistance to combination therapy[14–16]. The influence of viral quasispecies with variations in the hypervariable region (HVR) and NS5A ISDR (interferon-sensitivity determining region) genes on the effectiveness of interferon has been explored world wide[1417–20]. These studies paid special attention to the importance of quasispecies as independent predictor of SVR. The cause and mechanism of HCV resistance to interferon needs to be well understood and several viral proteins may influence the therapy outcome. Gupta R et al reported HCV mutations in the 5' untranslated region, ISDR and PePHD region, and their influence on therapy responses in a South-Indian population. However, the influence of quasispecies in the HVR and NS5B regions on the persistence of infection and disease severity in chronic hepatitis C patients needs to be explored.
This study enrolled 47 patients with chronic liver disease [32 with chronic active hepatitis (CAH), 9 with cirrhosis and 6 with hepatocellular carcinoma (HCC)] verified by histological examination, Child-Pugh score, and FNAC (fine needle aspiration cytology) wherever applicable, who attended medical outpatient departments (OPDs) and wards of Lok Nayak Hospital, New Delhi, India between 2003 to 2005, on the basis of pre designed performa, with all relevant information about possible risk factors, symptoms, clinical examination, detailed biochemical investigation and serology. They had a mean age of 48.4 ± 14.5 years, a male:female ratio of 25:22 and an alanine aminotransferase (ALT) value (mean ± SD) of 79.6 ± 44.6 (26-296) IU/L. Out of 47 patients, 34 (24 with CAH, 6 with cirrhosis, and 4 with HCC) were infected with virus strain of genotype 3, 11 (7 with CAH, 2 with cirrhosis, and 2 with HCC) with virus of genotype 1 and 2 (1 with CAH and 1 with cirrhosis) with virus of mixed genotype (1 + 3). The most probable risk factor for transmission was transfusion of infected blood and contaminated blood products, in 63.8% (30/47) of the patients. Out of 47 patients, 41 (32 with CAH and 9 with cirrhosis) were on combination therapy, 30 (73.17%) attained sustained virological responses and 11 (26.83%) were non-responders. However, the 6 HCC patients were not on therapy. The 41 chronic hepatitis C (CH-C) patients (32 with chronic active hepatitis and 9 with cirrhosis) were on medication (interferon-α2b 3 MU + 1000 mg ribavarin daily), having ALT ≥ 60 IU/L and HAI score ≥ 3 at baseline. The severity of liver disease was determined by histopathological grading according to the modified Knodell Score for the CAH patients and by Child-Pugh score for the cirrhosis patients[21–23]. These 41 patients were put on 48 wk of therapy or follow up. The viral load was estimated after 0 d, 4, 8, 12, 24 and 48 wk of the therapy. Patients without any detectable HCV viremia (≤ log10 1 IU/mL) after stopping the therapy, and even after 6 mo of therapy withdrawal, were defined as responders, and, if these patients were found to show positivity 6 mo after withdrawal of therapy, they were defined as relapsers. However, the patients with detectable HCV viremia after the end of wk 48 of therapy were defined as non-responders. Liver biopsies of CAH patients and Child-Pugh scoring in cirrhosis patients were performed at baseline, and after 24 and 48 wk of therapy. The SSCP analysis of the HVR and NS5B regions was performed in all sera samples before the initiation of treatment and after 24 wk of therapy. Genotypes were also studied by RFLP for genotypic classification of these patients (data not shown). Patients with > 4 SSCP bands were considered to have complex quasispecies (CQS) of HCV. However, the patients with 3 or 4 bands in the SSCP gel were considered to have simple quasispecies (SQS) and those with 2 bands were considered to have no quasispecies (NQS). The patients were grouped based on the number of bands in the SSCP gel (Figure 1) to study the influence of quasispecies on virological responses and disease severity. RNA extracted from sera samples (11 non-responders and 12 responders) was amplified by RT-PCR using primers specific for the HVR and NS5B genes, and the products were sequenced (Macrogen Inc, Korea) on a commercial basis at three different time (0 d, 24 wk and 48 wk) intervals during therapy in non-responders and two times (0 d and last sample before the clearance of HCV infection) in responders. Sequencing results at baseline were submitted to the GenBank database and also used for phylogeny. A phylogenetic tree was drawn to analyze the distance matrix between the SVR and non-responder (NR) patients on the basis of sequences derived from the HVR and NS5B genes, using the Bio Edit program for sequence analysis.
Biochemical investigations, including routine and specific liver function tests, were carried out in all cases. Serological tests were performed using commercially available ELISA kits, according to the manufacturer's instructions. Sera samples of all 47 patients were screened for HBsAg using a 3rd generation ELISA kit from Biokit, Spain; IgG HBc and HBeAg were screened using an ELISA kit from RADIM SpA, Italy; and anti-HCV antibody using a 3rd generation ELISA kit (General Biological Corporation, Taiwan). Exclusively anti-HCV-positive patients' sera were used for the detection of HCV RNA.
Total RNA was isolated using Tri Reagent (Sigma-Aldrich) according to the manufacturer's instructions and amplified by reverse transcription-PCR. For reverse transcription, 5 &mgr;L of extracted template RNA was added to 20 &mgr;L of reaction master mix containing reaction buffer, dNTPs, RNAsin (10 U), M-MuLV reverse transcriptase (40 units) and outer antisense primer, and incubated at 42°C for one hour for cDNA synthesis. First-round PCR amplification was carried out using outer sense and antisense primers, 2 &mgr;L of cDNA template, reaction buffer containing magnesium, dNTPs, and Taq DNA polymerase (1 unit), in a 25-&mgr;L reaction, by denaturing at 94°C for 5 min and 35 cycles with 60 s each of denaturation at 94°C, annealing at 55°C and extension at 72°C, in a DNA thermal cycler (M.J. Research, USA). After the last cycle, the extension was prolonged for 8 min at 72°C. Second-round PCR was performed using 2 &mgr;L of the first PCR product as template, which was amplified with the second PCR mix containing inner primers (Table 1). The reagent composition and temperature profile were the same as those for the first PCR. Both positive and negative controls were run in parallel with the test samples. The expected PCR products for HVR and NS5B were 403 and 293 bp, respectively; these were detected in ethidium bromide-stained 30 g/L Nusieve agarose gels in a UV Transilluminator.
|Region/Nucleotide position
|Primer set
|Sequence (5'-3')
|Amplicon size (bp)
Briefly, single-strand conformational polymorphism (SSCP) was performed by radiolabelling the PCR products for an additional 10 cycles where dCTP was replaced with (α-p32) dCTP (specific activity, 4000 CI/nmol) in the PCR mix. One &mgr;L of radiolabelled PCR product was diluted with 9 volumes (10 times) of denaturing solution (950 g/L formamide, 20 mmol/L EDTA pH 8.0, 0.5 g/L xylene cyanol and 0.5 g/L bromophenol blue), heat-denatured for 5 min at 95°C, and chilled on ice for 3 min; 3 &mgr;L of this diluted radiolabel product was subjected to non-denaturing electrophoresis in a 60 g/L polyacrylamide gel containing 100 mL/L glycerol. Gels were run in 1 × TBE for 16 h at 250 V in a DNA sequencing apparatus, at 17 ± 1°C, dried on a gel drier, and exposed to X-ray film with an intensifying screen at -20°C. Electrophoretic mobility shifts of single-strand DNA bands were analyzed following development of the X-ray film.
Total RNA was used for real time quantification. The RealArt HCV PCR Reagents constitute a ready-to use system for PCR amplification and the detection of HCV RNA on the Light Cycler instrument (Rotor Gene 3000, Corbett Research Inc.). The RealArt HCV PCR master mixture contains reagents and enzymes for the amplification of a 240-bp region of the HCV genome with the parallel detection (A.Ch 1-FAM) and quantification of the amplified PCR products in comparison to five standard samples diluted 10-fold, which were used to generate the standard curve.
Statistical analysis was performed using the Student's t-test, ANOVA, Chi-square, and Fischer's exact test, wherever applicable, using the software EPISTAT and SPSS. Statistical analysis was performed by comparisons of two groups descriptively. Patients were classified as being infected with complex quasispecies (CQS), simple quasispecies (SQS) and no quasispecies (NQS) on the basis of their SSCP pattern, representing the level of HCV quasispecies variations. The results are expressed as means ± SD. P < 0.05 was considered significant.
Patients were screened for the presence of quasispecies by SSCP analysis, on the basis of differential migration in PAGE denaturing gels. The appearance of more than 2 bands in gels is associated with the presence of quasispecies. Patients with more than 4 bands in SSCP gels were infected with complex quasispecies (CQS). Complex quasispecies were detected both in the NS5B and HVR regions of HCV, but were relatively more abundant in the HVR (n = 19) than the NS5B (n = 9) region. Among the 32 patients with chronic active hepatitis, 18 (56.3%) showed presence of quasispecies (CQS-7, SQS-11, NQS-14) when the HVR gene was used for the analysis, while 9 (28.2%) showed presence of quasispecies (CQS-4, SQS-5, NQS-23) when the NS5B gene was used for the analysis. All 9 cirrhosis patients showed presence of quasispecies (CQS-6, SQS-3) when the HVR gene was used for the analysis, while only 6 (66.7%) showed presence of quasispecies (CQS-2, SQS-4, NQS-3) when the NS5B gene was used (Figure 2). It was interesting to note that all 6 HCC patients were positive for complex quasispecies when the HVR was used for the analysis, while 5 out of the 6 (83.3%) showed presence of quasispecies (CQS-3, SQS-2, NQS-1) on the basis of the NS5B gene (Figure 2). These results showed the influence of the occurrence of HVR quasispecies on the disease severity in hepatitis C infected patients as all cirrhosis and HCC patients showed evolution of quasispecies in this region. However, NS5B quasispecies also showed their influence on disease severity. No difference was observed in the SSCP band patterns of the patients after 24 wk of therapy, confirming the accuracy of the PCR reaction and SSCP profiles. The genotypic classification of quasispecies was not feasible from this data due to sample size. However, the occurrence of quasispecies in both the genotypes (genotypes 1 and 3) was observed.
Among the 41 patients on therapy, 30 had sustained virological responses, whereas 11 were non-responders. HVR quasispecies were detected in 27 out of 41 (65.9%) patients (CQS-13, SQS-14, NQS-14), while NS5B quasispecies were detected in 15 out of 41 (36.6%) (CQS-6, SQS-9, NQS-26) (Figure 3). Out of the 11 non-responders, 10 were infected with complex quasispecies (> 4 bands in the SSCP) and 1 was infected with simple quasispecies, based on analysis of the HVR gene; when the NS5B gene was analyzed, among the 11 non-responders, 5 were infected with complex quasispecies, 4 with simple quasispecies and 2 with no quasispecies. Among the 30 SVR patients, 16 (53.3%) were infected with HVR quasispecies (CQS-3, SQS-13, NQS-14), whereas 6 (20.0%) were infected with NS5B quasispecies (CQS-1, SQS-5, NQS-24) (Figure 3). These results suggest that the presence of complex quasispecies with variations in the HVR gene has an impact on virological response, as 90.9% (10/11) of patients infected with complex quasispecies with variations in this region did not respond to combination therapy, whereas only 10.0% (3/30) of patients infected with complex quasispecies were responders.
Out of 41 patients on therapy, HVR quasispecies were seen in 27 patients. No statistical difference was observed in viral load of patients with CQS, SQS and NQS (mean ± SD log10 IU/mL) from baseline (CQS-6.9 ± 0.29, SQS-6.8 ± 0.26, and NQS-6.9 ± 0.22) to 8 wk (CQS-4.3 ± 1.05, SQS-4.5 ± 1.2, NQS-4.1 ± 1.1) of combination therapy with minimum standard error. A statistically significant difference in the viral load (P < 0.05; CQS vs SQS, NQS) was observed after 12 wk (CQS-5.2 ± 2.3, SQS-3.2 ± 1.9, NQS-2.8 ± 2.4) of therapy in patients with HVR region quasispecies of HCV. A statistically significant difference in viral load (P < 0.05) with high standard error was observed in comparisons of CQS with SQS and NQS after 24 wk (CQS-5.7 ± 2.6, SQS-2.6 ± 2.1, NQS-2.0 ± 2.33) and 48 wk (CQS-6.1 ± 2.1, SQS-2.4 ± 2.3, NQS-1.9 ± 2.5) of therapy (Figure 4A). No significant difference (P > 0.05) was observed in the mean viral load (log10 IU/mL) from baseline (CQS-6.9 ± 0.29, SQS-6.8 ± 0.3, NQS-6.94 ± 0.22) to 12 wk (CQS-3.5 ± 2.7, SQS-3.3 ± 2.5, NQS-3 ± 2.4) in comparisons of patients with different levels quasispecies (CQS, SQS and NQS) in NS5B region. However, the standard error was almost negligible at baseline, but an increased standard error in viral load was observed after 12 wk of therapy. A statistically significant difference (P < 0.05) with high standard error was observed between the mean viral loads of patients infected with CQS and those infected with NQS after 24 wk (CQS-3.9 ± 2.2, SQS-3.0 ± 2.2, and NQS-2.1 ± 2.3) and 48 wk (CQS-3.1 ± 2.7, SQS-2.3 ± 2.4, NQS-2.0 ± 2.3) of therapy (Figure 4B). A continuous decline in mean viral load was observed in patients without complex quasispecies infection, suggestive of an influence of the presence of quasispecies on viral kinetics and therapeutic outcome during combination therapy.
The histological activity index (HAI) score is an important factor for the assessment of disease severity in CAH patients. It was interesting to note that mean HAI scoring (mean ± SD) was comparable among groups based on the degree of quasispecies diversity in the HVR at baseline (CQS-6.64 ± 2.9, SQS-6.72 ± 1.2, NQS-6.9 ± 0.22). Patients grouped on the basis of genetic variability in the NS5B gene showed a statistically significant difference (CQS vs NQS) in the mean HAI scores at baseline (CQS-7.6 ± 3.3, SQS-6.7 ± 2.2, NQS-5.86 ± 2.08). An improvement in the severity of disease was observed in 80.0% (24/30) of the SVR (sustained virological response) patients. Bridging of fibrosis was observed in all NR (non-responders) and 13.3% (4/30) of the SVR patients. No statistical significant difference in the results of liver function tests was observed among patients with different degrees of HVR and NS5B quasispecies, at baseline and on follow-up every 4 wk until 48 wk.
Amino acid variation in the NS5B gene plays a major role in the replication of the virus and may be important for the virological response during combination therapy. It was interesting to note that the number of mutations was higher in genotype 1 as compared to genotype 3 in the HVR region, suggesting an influence of genotype on the rate of mutations in the selected region of HCV and on the evolution of quasispecies. Amino acid variations in the HVR and NS5B regions of different genotypes among patients (n = 23, Genotype 1 = 5, Genotype 3 = 18) during different phases of therapy are suggestive of the occurrence of mutations during the course of therapy. The sequences of virus strains during the course of therapy showed 2-5 amino acid substitutions and synonymous mutations at the nucleotide level in both HVR and NS5B regions, compared with the sequences of virus strains before therapy (data not shown as no specific mutation was observed to be responsible for the persistence of infection during therapy). Phylogenetic analysis of sequenced virus isolated from patient samples showed different distance patterns between responders and non-responders in HVR and NS5B regions, as shown in Figures 5 and 6. The non-responders were on a different branch, suggestive of no close pairing between these non-responder strains of genotype 3 in the HVR region. However, closing pairing was observed between non-responder strains of genotype 3 in the NS5B region: 5 non responder patients were on the same branch and 3 patients were on the other branch. However, all four non-responder were on the same branch in patients of genotype 1. Strains appearing on the same branch (close pairing) during phylogenetic tree analysis possess significant homology in their nucleotide sequences. These results suggest that either the selection of resistant strains or the development of quasispecies may be the cause of unresponsiveness to combination therapy in HCV patients; however, these mechanisms require further analysis.
Diversifications in the HCV genome are the results of viral RNA polymerase infidelity, immune-driven selection and exertion of therapy pressure or a combination of these processes. PCR-SSCP analysis is a well established technique to study genetic variability, including the presence of mutations and quasispecies[10–13]. Millions of viruses are produced via replication and eliminated each day due to exertion of immune pressure and interferon therapy[16]. Ribavirin does not have any effect on the viremia level of HCV-infected patients, but it reduces the ALT level, increases the likelihood of sustained virological response and reduces the probability of relapse[16].
The HVR and NS5B genes were selected in the present study because the persistence of infection may be associated with positive selection of HVR quasispecies by the host immune system and because of the major role of NS5B in the replication of hepatitis C. Factors like age, sex and duration of infection may be possible causes of the occurrence of quasispecies; in this study, we studied virological responses in patients infected with quasispecies before the initiation of therapy and looked for alterations in the SSCP patterns during therapy. Several conflicting reports about a possible correlation of therapy response and disease severity with the presence of quasispecies have been published[152024–27]. The genes encoding HVR protein exhibit a high degree of variability, giving rise to differing phenotypic traits, including alterations in receptor-binding affinity and escape from immune recognition[28]. Substitutions in the NS5B gene of HCV may correlate with the virological response to interferon-based therapy[29–31].
The occurrence of quasispecies was proportionately higher in patients with HCC and cirrhosis than in CAH patients, revealing a significant correlation between the molecular evolution of quasispecies and the severity of disease in hepatitis C patients in this study. The genetic diversity at baseline can be predictive of distinct virological behavior during the course of combination therapy. Among the 13 patients infected with complex quasispecies with variations in the HVR gene, 10 were non-responders at baseline. This correlates significantly with the persistent of infection as the response rate against interferon may be due to two mutually compatible approaches; the infecting virus might have become naturally resistant to interferon or the virus could have evolved into quasispecies. The non-responders had normal signal transduction pathways required for the intracellular activity of interferon[32]. The recognition of the HVR epitope (envelope) by immune cells and the exertion of interferon-based therapy play a major role in the evolution of quasispecies.
Several studies have shown an association of epidemiological factors and treatment regime with the presence of quasispecies[13141933]. Complex quasispecies with variations in the HVR and NS5B genes of HCV were significantly associated with virological response during wk 48 of therapy. Virus quasispecies evolution within patients increases the pathogenicity of HCV. The resulting viral quasispecies are heterogeneous and sensitive to selection pressures. Complex variants escape from immune pressure and the antiviral spectrum of the combination therapy[34]. No mutant strains have the capacity for replication, but only selective mutants can replicate, which may be responsible for persistence of infection. The circulating viremia in blood and liver cells is significantly associated with disease prognosis[35]. There was no statistically significance difference in the HAI scores among patients with different degrees of HVR quasispecies at baseline. However, there was a significant difference in the HAI scores at baseline between patients infected with different genetic variants (CQS vs NQS) of the NS5B gene. Selective adaptive mutations in the resistant strains occurring during replication influence the RDRP activity and lead to increases in viremia level and HAI scoring[7–9]. These results suggest the significance of evolution of quasispecies on virological responses and disease severity.
The amino acid variability in HVR and NS5B was evaluated from cDNA-derived sequences. The occurrence of HVR quasispecies influences the therapeutic outcome; because no strains isolated from non-responder patients showed close pairing, it is possible that some other factors may be responsible for the persistence of infection. However, the possibility of resistant strains can not be denied. The NS5B sequences of non-responder strains were on the same branch, showing the possibility of resistant strain infection in these patients. However, adaptive mutations in NS5B lead to persistence of infection and a severe course of liver disease in patients. These findings reveal that the occurrence of HVR quasispecies, including the presence of resistant strains, is a major factor influencing the therapeutic outcome and disease severity in chronic hepatitis C patients. Genetic variability was not uniformly distributed throughout the HCV genome.
In conclusion, viral quasispecies heterogeneity can be an important marker to predict the response of combination therapy in patients with chronic hepatitis C. Quasispecies complexity on the basis of SSCP pattern could be helpful in differentiation of SVR and NR patients. Complex quasispecies heterogeneity causes high HCV viremia, which was significantly correlated with the severity of liver disease and prognosis during the course of combination therapy. However, there was no significant alteration in SSCP profile during therapy. The occurrence of resistant strains may be another factor contributing to the persistence of HCV infection. Quasispecies in both HVR and NS5B genes may have a major impact on treatment and be a good predictor of virological response. Complex quasispecies are resistant to combination therapy and influence disease prognosis during therapy. Further evaluation of viral quasispecies complexity in the HVR and NS5B genes, as well as other HCV genes (whole genome), is required for a better understanding of the influence of quasispecies on the persistence of infection and disease prognosis during the course of therapy in HCV-infected patients.
The hepatitis C virus (HCV) continues to persist unless patients are put on interferon therapy. Treatment with interferon-based therapy and error prone replication of the viral genome via RNA-dependent RNA polymerase (RDRP) accelerates the evolution of HCV variants during the course of infection. The highly heterogeneous population in HCV-infected patients, consisting of quasispecies, developed because of interferon therapy, immune pressure and replication strategy. Quasispecies are one of the major virological factors other than genotypes that can influence the response to therapy in chronic hepatitis C patients; their role in this needs to be evaluated.
HCV quasispecies can be detected by cloning of reverse transcription-polymerase chain reaction products; however, sequencing of 15-20 independent clones is expensive and laborious, and not practical for a large number of clinical samples. For large numbers of clinical samples, faster methodologies like single-strand conformational polymorphism (SSCP), heteroduplex analysis (HDA) and gel shift assay (GSA) have been developed based on the differential migration of amplified DNA in acrylamide gel electrophoresis. In the present study, we used the established and reliable method of SSCP for the detection of quasispecies with variations in the HVR and NS5B genes. The patients were grouped on the basis of the number of bands in the SSCP gel. Patients with > 4 SSCP bands were considered to be infected with complex quasispecies (CQS) of HCV. Patients with 3 or 4 bands were considered to be infected with simple quasispecies (SQS); and those with 2 bands were considered to be infected with no quasispecies (NQS). Our interest was to evaluate whether or not the occurrence of quasispecies at baseline influences the virological response and disease severity in chronic hepatitis C patients.
The significance of disease severity on the occurrence of HVR quasispecies in serum of hepatitis C infected patients was observed as all of the cirrhosis and HCC patients showed evolution of quasispecies in this region. However, a major role of NS5B quasispecies was also observed with regard to disease severity. No difference was observed in the SSCP patterns of patients after 24 wk of therapy, confirming the validity of the PCR reaction and SSCP profiles. These results suggest an effect of complex quasispecies with variations in the HVR gene on virological response, as 90.9% (10/11) of patients infected with complex quasispecies with variations in this region did not respond to combination therapy, whereas only 10.0% (3/30) patients infected with complex quasispecies showed SVR. A continuous decline of mean viral load was observed in the patients without complex quasispecies viral infection, suggestive of an influence of quasispecies on viral kinetics and therapeutic outcome during combination therapy.
The study suggests that quasispecies complexity in both HVR and NS5B genes had a major impact on treatment and is a good predictor of virological responses. However, the existence of quasispecies based on other HCV genes should be further evaluated for a better understanding of the influence of quasispecies on virological responses and disease severity in patients with chronic hepatitis C.
Chronic active hepatitis (CAH): anorexia, weight loss, jaundice, ascites, edema present singly or in combination, persistently or intermittently and biochemical features (SGOT, SGPT more than twice normal and globulin 1.5 times normal) of hepatocellular disease for more than 6 months duration. Liver biopsy is necessary for the assessment of disease severity. A diagnosis of cirrhosis of the liver was considered on the basis of the following criteria: ascites, splenomegaly, shrunken liver, endoscopy showing esophageal varices and biochemical features of hepatocellular failure characterized by low serum albumin level (< 2.5 gm). Hepatocellular Carcinoma (HCC): patients were evaluated on the basis of history, clinical examination, liver function tests and serological tests for hepatitis B & C, serum alpha-feto protein levels and liver imaging [ultrasound sonography (US), computed tomography (CT), magnetic resonance imaging (MRI)], and final diagnosis of HCC was made on FNAC (fine needle aspiration cytology). Quasispecies: The origination of a highly heterogeneous population in hepatitis C-infected patients as a result of interferon therapy, immune pressure and replication strategies.
In this study the authors assessed whether the occurrence of HCV quasispecies influence responses to antiviral therapy and the severity of chronic hepatitis C. Quasispecies were assessed on the basis of SSCP patterns. The results are interesting and suggest that complex quasispecies are a major factor in non-response to combination therapy. The SSCP pattern remains unaltered during the course of therapy. The concept of this study is interesting; evaluation of other HCV genes should be performed in future work.
|Kim WR. The burden of hepatitis C in the United States. Hepatology. 2002;36:S30-S34.
|Watanabe K, Yoshioka K, Yano M, Ishigami M, Ukai K, Ito H, Miyata F, Mizutani T, Goto H. Mutations in the nonstructural region 5B of hepatitis C virus genotype 1b: their relation to viral load, response to interferon, and the nonstructural region 5A. J Med Virol. 2005;75:504-512.
|Panigrahi AK, Panda SK, Dixit RK, Rao KV, Acharya SK, Dasarathy S, Nanu A. Magnitude of hepatitis C virus infection in India: prevalence in healthy blood donors, acute and chronic liver diseases. J Med Virol. 1997;51:167-174.
|Durante Mangoni E, Forton DM, Ruggiero G, Karayiannis P. Hepatitis C virus E2 and NS5A region variability during sequential treatment with two interferon-alpha preparations. J Med Virol. 2003;70:62-73.
|Martell M, Esteban JI, Quer J, Genesca J, Weiner A, Esteban R, Guardia J, Gomez J. Hepatitis C virus (HCV) circulates as a population of different but closely related genomes: quasispecies nature of HCV genome distribution. J Virol. 1992;66:3225-3229.
|Farci P, Strazzera R, Alter HJ, Farci S, Degioannis D, Coiana A, Peddis G, Usai F, Serra G, Chessa L. Early changes in hepatitis C viral quasispecies during interferon therapy predict the therapeutic outcome. Proc Natl Acad Sci USA. 2002;99:3081-3086.
|Feld JJ, Hoofnagle JH. Mechanism of action of interferon and ribavirin in treatment of hepatitis C. Nature. 2005;436:967-972.
|Bartenschlager R, Frese M, Pietschmann T. Novel insights into hepatitis C virus replication and persistence. Adv Virus Res. 2004;63:71-180.
|Bowen DG, Walker CM. Adaptive immune responses in acute and chronic hepatitis C virus infection. Nature. 2005;436:946-952.
|Gomez J, Martell M, Quer J, Cabot B, Esteban JI. Hepatitis C viral quasispecies. J Viral Hepat. 1999;6:3-16.
|Laskus T, Wilkinson J, Gallegos-Orozco JF, Radkowski M, Adair DM, Nowicki M, Operskalski E, Buskell Z, Seeff LB, Vargas H. Analysis of hepatitis C virus quasispecies transmission and evolution in patients infected through blood transfusion. Gastroenterology. 2004;127:764-776.
|Takeda T, Tatsumi N, Nakayama Y, Yasuda T, Nishiguchi S, Seki S. Mode of HCV infection examined by polymorphism of hypervariable region-1 in cases of acute hepatitis C after accidental exposure to blood-borne pathogens. J Med Virol. 2005;75:35-41.
|Chen S, Wang YM. Multigene tracking of quasispecies in viral persistence and clearance of hepatitis C virus. World J Gastroenterol. 2005;11:2874-2884.
|Vuillermoz I, Khattab E, Sablon E, Ottevaere I, Durantel D, Vieux C, Trepo C, Zoulim F. Genetic variability of hepatitis C virus in chronically infected patients with viral breakthrough during interferon-ribavirin therapy. J Med Virol. 2004;74:41-53.
|Tao Q, Wei L, Chang J, Wang H, Sun Y. Relationship between interferon therapy and variability in nonstructural gene 5b of hepatitis C virus. J Gastroenterol. 1998;33:684-693.
|Davis GL, Esteban-Mur R, Rustgi V, Hoefs J, Gordon SC, Trepo C, Shiffman ML, Zeuzem S, Craxi A, Ling MH. Interferon alfa-2b alone or in combination with ribavirin for the treatment of relapse of chronic hepatitis C. International Hepatitis Interventional Therapy Group. N Engl J Med. 1998;339:1493-1499.
|Gerotto M, Sullivan DG, Polyak SJ, Chemello L, Cavalletto L, Pontisso P, Alberti A, Gretch DR. Effect of retreatment with interferon alone or interferon plus ribavirin on hepatitis C virus quasispecies diversification in nonresponder patients with chronic hepatitis C. J Virol. 1999;73:7241-7247.
|Fan X, Di Bisceglie AM. Genetic complexity and serum reactivity of HVR1 quasispecies of hepatitis C virus in patients with cirrhosis. Am J Gastroenterol. 2002;97:1489-1495.
|Chambers TJ, Fan X, Droll DA, Hembrador E, Slater T, Nickells MW, Dustin LB, Dibisceglie AM. Quasispecies heterogeneity within the E1/E2 region as a pretreatment variable during pegylated interferon therapy of chronic hepatitis C virus infection. J Virol. 2005;79:3071-3083.
|Gupta R, Subramani M, Khaja MN, Madhavi C, Roy S, Habibullah CM, Das S. Analysis of mutations within the 5' untranslated region, interferon sensitivity region, and PePHD region as a function of response to interferon therapy in hepatitis C virus-infected patients in India. J Clin Microbiol. 2006;44:709-715.
|Knodell RG, Ishak KG, Black WC, Chen TS, Craig R, Kaplowitz N, Kiernan TW, Wollman J. Formulation and application of a numerical scoring system for assessing histological activity in asymptomatic chronic active hepatitis. Hepatology. 1981;1:431-435.
|Child CG, Turcotte JG. Surgery and portal hypertension. Major Probl Clin Surg. 1964;1:1-85.
|Pugh RN, Murray-Lyon IM, Dawson JL, Pietroni MC, Williams R. Transection of the oesophagus for bleeding oesophageal varices. Br J Surg. 1973;60:646-649.
|Thelu MA, Baud M, Leroy V, Seigneurin JM, Zarski JP. Dynamics of viral quasispecies during interferon therapy in non responder chronic hepatitis C patients. J Clin Virol. 2001;22:125-131.
|Farci P, Shimoda A, Coiana A, Diaz G, Peddis G, Melpolder JC, Strazzera A, Chien DY, Munoz SJ, Balestrieri A. The outcome of acute hepatitis C predicted by the evolution of the viral quasispecies. Science. 2000;288:339-344.
|Zeuzem S. Heterogeneous virologic response rates to interferon-based therapy in patients with chronic hepatitis C: who responds less well? Ann Intern Med. 2004;140:370-381.
|Abbate I, Lo Iacono O, Di Stefano R, Cappiello G, Girardi E, Longo R, Ferraro D, Antonucci G, Di Marco V, Solmone M. HVR-1 quasispecies modifications occur early and are correlated to initial but not sustained response in HCV-infected patients treated with pegylated- or standard-interferon and ribavirin. J Hepatol. 2004;40:831-836.
|Brown RJ, Juttla VS, Tarr AW, Finnis R, Irving WL, Hemsley S, Flower DR, Borrow P, Ball JK. Evolutionary dynamics of hepatitis C virus envelope genes during chronic infection. J Gen Virol. 2005;86:1931-1942.
|Nelson DR, Gray AH, Kolberg JA, Joh J, Urdea MS, Mizokami M, Davis GL, Lau JY. Variations of hepatitis C virus NS5B sequence (nucleotides 8261-8566) do not correlate with response to interferon-alpha therapy. J Viral Hepat. 1995;2:285-292.
|Duffy M, Salemi M, Sheehy N, Vandamme AM, Hegarty J, Curry M, Nolan N, Kelleher D, McKiernan S, Hall WW. Comparative rates of nucleotide sequence variation in the hypervariable region of E1/E2 and the NS5b region of hepatitis C virus in patients with a spectrum of liver disease resulting from a common source of infection. Virology. 2002;301:354-364.
|Hamano K, Sakamoto N, Enomoto N, Izumi N, Asahina Y, Kurosaki M, Ueda E, Tanabe Y, Maekawa S, Itakura J. Mutations in the NS5B region of the hepatitis C virus genome correlate with clinical outcomes of interferon-alpha plus ribavirin combination therapy. J Gastroenterol Hepatol. 2005;20:1401-1409.
|Pawlotsky JM, Pellerin M, Bouvier M, Roudot-Thoraval F, Germanidis G, Bastie A, Darthuy F, Remire J, Soussy CJ, Dhumeaux D. Genetic complexity of the hypervariable region 1 (HVR1) of hepatitis C virus (HCV): influence on the characteristics of the infection and responses to interferon alfa therapy in patients with chronic hepatitis C. J Med Virol. 1998;54:256-264.
|Rothman AL, Morishima C, Bonkovsky HL, Polyak SJ, Ray R, Di Bisceglie AM, Lindsay KL, Malet PF, Chang M, Gretch DR. Associations among clinical, immunological, and viral quasispecies measurements in advanced chronic hepatitis C. Hepatology. 2005;41:617-625.
|Eckels DD, Zhou H, Bian TH, Wang H. Identification of antigenic escape variants in an immunodominant epitope of hepatitis C virus. Int Immunol. 1999;11:577-583.
|De Moliner L, Pontisso P, De Salvo GL, Cavalletto L, Chemello L, Alberti A. Serum and liver HCV RNA levels in patients with chronic hepatitis C: correlation with clinical and histological features. Gut. 1998;42:856-860.


text:
- Beaches Waterways
- Guide To Adventure
- Stay With Us
- Concierge Program
- Adventure Map
- Nature Watch
- Go Local
Stop in for a refreshment and let us help you with directions and things to do. Want to know the best places to find scallops? Just ask! Want to know how to clean them? Stop on by and we will tell you. Need help booking a fishing charter or renting a boat? We can help with that, too.
Open year-round, the Gulf County Welcome Center has free maps and brochures and a stunning view of St. Joseph Bay. Our huge, elevated deck provides one of the best vantage points in town for planning your trip around the Bay.
The back porch offers free WiFi so, if you need to reconnect with reality for any reason, this is the perfect spot. We must warn you, though... the view of the Bay is spectacular, so you might get distracted. The Welcome Center is a great place to park for a stroll along the BayWalk Trail or out to the gazebo for a closer view of St. Joseph Bay.
No matter whether you are taking a day trip, hanging out or just happened upon us, stop in for a visit.
No worries, our Visitor Information Kiosks are located throughout Gulf County and provide the most up-to-date information you need to make your visit a great one.
Drop by, give us a call or send us an email. We're easy to find and ready to help.
We have Visitor Guides available at the Welcome Center. But if you haven't made your way to Port St. Joe yet, we'll be happy to send you one. Just fill out the form and it will land in your mailbox in no time.


text:
News By Tag
* Mma
* Mayhem
* More Tags...
Industry News
News By Location
Industry News
UrFight has been Confirmed for Mansfield Mayhem 13
UrFight is proud to announce that it will be attending the premier MMA event in Mansfield Ohio. This will be the second Mansfield Mayhem that UrFight will be at
Josh Longood vs Cory Ruthsatz
John Gunther vs Niles Hootman
Rob Deweese vs TBD
Chris Trippi vs Ronald Mello
Levi Gwinup vs noel rodriguez
Daniel Haubiel vs Desmond Carroll
chris reed vs Darnell Pettis
brian haubiel vs Art Ludwick
Brandon Odom vs Kyle Booth
Joey Rine vs Ronnie Shoemaker
Mansfield, Ohio
United States
UrFight is a Mixed Martial Arts branding and marketing company. We special in branding, marketing and promotional products, services a consultation for MMA fights, gyms, promoters and other businesses and professionals that would like to get involved with the fastest growing sport qfxwm in the world.
UrFight is a branding and marketing company for Mixed Martial Arts fighters, MMA gyms, fight promoters, and other Mixed Martial Arts related businesses. We special in branding, marketing and promotional products, services and consultation for MMA fighters, gyms, promoters and other businesses and professionals that would like to get involved with the fastest growing sport in the world. UrFight has extensive experience in small business marketing and branding and can provide the following services: web design, graphic design, full color printing, consultative services, and business and financial advisory services.
UrFightGear is a brand of MMA apparel, products, training aids, gear, and other mixed martial arts products. UrFightGear has MMA inspired and designed t-shirts and apparel that is designed and printed in the USA. Our MMA style, gear, and clothing brings fans original and classic MMA Designs to help capture the essence for the fans, fighters and sport of Mixed Martial Arts. UrFightGear is now offering MMA board shorts, as well as vale tudo shorts, rashguards and other customize training gear and products.
UrFight is one of the fastest growing businesses in Mixed Martial Arts. We have a team of professionals that will take care of all your Mixed Martial Arts branding and Marketing needs. Give us a call today at (423) 297-1223 and get your career and business NOTICED!
Please take a look at our following links:
Like us on Facebook: www.facebook.com/
Follow us on Twitter: www.twitter.com/
Account Email Address Account Phone Number Disclaimer Report Abuse


text:
CALGARY - A year ago at this time, their best receiver was recovering from knee surgery, the offensive line was a jumbled mess, and they were adopting a new system.
Even with all those challenges, the Calgary Stampeders had the eighth-best offence in terms of yardage all-time.
Heading into next weekends training camp, the Stamps will get a full year from Ken-Yon Rambo, enjoy some continuity on the line and are more comfortable with offensive co-ordinator Dave Dickensons play-calling.
In the off-season, Stamps GM-head coach John Hufnagel didnt rest on those laurels of 2010.
The team did lose left tackle Ben Archibald to free-agency, but it added the dynamic Larry Taylor as well as intriguing Canadian Anthony Parker to the receiving corps.
Oh, and the Stamps do have the reigning league most outstanding player at quarterback in Henry Burris, who just seems to be getting better.
The fact we have everybody coming back except for Archie, it does mean that we will have a great year, Burris said.
You add guys like Larry Taylor, who is one of the most dangerous guys with the balls in his hands in the league, there will be some excitement with our offence.
There are so many things we can throw at opposing defences. Dont forget about Joffrey (Reynolds) and Corndog (Jon Cornish) running the ball.
In building a 9-1 record to start 2010, Burris and the Stamps offence was on fire, scoring more than 35 points in five straight games.
They cruised to the West Division title after scoring a league-high 626 points, the most for the franchise since 1995.
After one season as the quarterbacks coach and play-caller, Dickenson gained some buzz around the West Division as a possible head coach, even interviewing in Edmonton.
He stayed with the Stamps to take the promotion to offensive co-ordinator. With another year together, Burris expects the creativity to keep flowing from Dickensons brain and onto the field.
Weve been together for just that much longer now, and we all understand how Dave thinks, Burris said.
To go through Year One, it will be much better for us to communicate and have things running smoothly compared with last year.
Burris and several other teammates greeted fans inside the Viper Dome Saturday during the annual Fanfest.
Of course, Burris had plenty of smiles, but it was a long off-season thinking what could have been again.
Losing to the Saskatchewan Roughriders for the fourth times in five seasons, this one stung a little longer.
It hurts because we had a better team than Saskatchewan, Burris said about the 13-5 Stamps compared with the 10-8 Riders. Thats all everybody talked about because we knew we were a better team than them. Im looking forward to seeing the hunger with our guys when they arrive at camp.
The new guys will pick up on the energy and focus of the team and thats what were expecting. I dont think its all about beating Saskatchewan. This is a new chapter. They are the defending (West Division) champions, and if you want to be the champs, you must beat the champs.


text:
Rep. Grimm accused of threatening reporter
From our report on Rep. Michael Grimm's threats to a reporter late Tuesday night:
New York Rep. Michael Grimm was accused of physically and verbally threatening a reporter in the U.S. Capitol following President Obama's State of the Union address on Tuesday.
In an interview with NY1 reporter Michael Scotto, the Staten Island Republican was asked about allegations regarding a campaign finance probe at which he has been the center since 2012.
"I'm not talking about anything that's off-topic, this is only about the president," Grimm said before walking off camera, according to a transcript provided by NY1, which posted a video of the exchange on its website.
Grimm returned moments later and leaned into Scotto in what appeared to be an angry exchange that partially took place off camera. According to the transcript provided by NY1, Grimm said to Scotto, "Let me be clear to you, you ever do that to me again I'll throw you off this f——-g balcony."
Scotto defended his line of questioning several times, according to the transcript, at one point saying: "It's a valid question."
Grimm then said, according to the transcript, "No, no, you're not man enough, you're not man enough. I'll break you in half. Like a boy."
In a statement shortly after the incident, the lawmaker didn't deny that it happened.
"I was extremely annoyed because I was doing NY1 a favor by rushing to do their interview first in lieu of several other requests. The reporter knew that I was in a hurry and was only there to comment on the State of the Union, but insisted on taking a disrespectful and cheap shot at the end of the interview, because I did not have time to speak off-topic," Grimm said in the statement.
"I verbally took the reporter to task and told him off, because I expect a certain level of professionalism and respect, especially when I go out of my way to do that reporter a favor. I doubt that I am the first Member of Congress to tell off a reporter, and I am sure I won't be the last.
Scotto, who covers Capitol Hill for the New York-based news channel, referred a request for comment to NY1 Political Director Robert Hardt.
"It is extremely disturbing when anyone threatens one of our reporters – let alone a U.S. Congressman," Hardt told POLITICO in an email. "The NY1 family is extremely alarmed and disappointed by the behavior of Representative Grimm and demands a full apology from him. The behavior is unacceptable."
Burgess Everett contributed to this report.


text:
No one is bad in The Big Wedding, but no one is remotely believable, either. show more
Zackham's film feels as plastic as a cake topper — and just as hard to digest. show more
The fact that the three actors who do most of the fooling around — Robert De Niro, Diane Keaton and Susan Sarandon — have a combined age of 202 pegs this as a sex romp for the Viagra crowd. show more


text:
Wine and Cheese Graduation Reception
June 4, 2013
The end of the semester Wine and Cheese reception was a huge success! Thanks to everyone who turned up, dressed up ready to enjoy some delicious food. Special thanks to Prof. Mary Boyce for joining us and congratulations again to all this year's graduates! Be sure to check out more more photos from the event!


text: Food Storage Updated May 2, 2012 Updated August 16, 2012 Updated December 1, 2012 Updated February 27, 2013 Updated December 22, 2014 On this page I will show you how I use (used) my Vacuum SealerI will try to keep this up to date.August 16, 2012 I am looking into a new supplier and will post the results later,. December 1, 2012 NEW SUPPLIER FOUND Here s a link if you want to try them yourself Vacuum Bags 05/01/2011 UPDATE ON SORBENT SYSTEMS BAGS, SEE BELOW Read before purchasing from SorbentSystems Vacuum sealers are truly a must have when barbecuing. You can freeze into meal sized portions. I have used a vacuum sealer for 15 years or so, I am on my 3rd Food Saver Sealer. I just picked up the V3485 was $199.00, but they had a 50% off and free ship promotion What I will sometimes do is seal in different portions of the same food, so for example if you seal all that pork butt you just made in serving portions of 2 and serving portions of 4, you will have the option of reheating just enough food if you want to eat solo or with the family. Here is where I used to get my bags Sorbent Systems. Food Saver has pretty neat Stand Up Steamer Bags, however I find Food Saver bags to rich for my blood, unless you catch a good sale. I am not endorsing Food Saver or Vacuum Sealers Unlimited, that is just what I am currently using and they both work well. Some Quick Tips. Partially Freeze - If the item you are vacuum sealing has a lot of liquid in it, you can place it in the bag, opened and place in the freezer for a couple hours, remove from refrigerator then seal it. If you seal warm, you may pull the moisture out of the bag and this can be messy. Partially Freeze - Soft Foods, the same thing applies to soft food, something like pulled pork, you don't want to crush the meat so do a Partial Freeze before sealing. Moisture Barrier - Another trick I have learned is to use a paper towel the length of the opening of the bag and fold it up so it is about 1/2" wide then place it at the top of the bag about an inch below where you plan to seal it, as an example a 10" wide bag (inside measurement) would have a strip 10" x 1/2" at 1 inch below the seal area. The bag will start to seal before the moisture gets past this "make shift barrier". Some of the newer models have "Auto Liquid Detection". Another method is to make the bag extra long (tail) and when sealing, drape the food end over the edge of the table, if the vacuum sealer starts to pull the liquid, hit the manual seal button. Sealing - Make sure to give yourself a lot of extra head space when sealing items that may be opened and sealed several times, like bulk cheeses and such. Double seal - I seal twice, once to get the air out then a 2nd seal above that one or directly on top, plenty of times I have had a poor seal and after a week or so in the fridge its full of air, however, this is a rarer occurrence in the newer model, the seal is much thicker, but I still double seal most of the time. Also rotate sealed foods and inspect for broken seals. Protect from sharp foods - When sealing something like soft pretzels, make sure to partially freeze then place in a paper bag then into the vacuum bag, this will prevent the salt from cutting through the bag, the same goes for dehydrated foods like spaghetti or pastas that may puncture Date and Label - This is very important, make sure to date and label the food accurately. Reheat - To reheat, thaw in refrigerator overnight, boil some water then reduce heat to a simmer or turn off burner completely, place the bag in the water 15 minutes or so. Make sure to use bags that can be placed in boiling water. You can reheat from frozen but reheating time will be longer. The Sorbent bags I used to get are not made to place in boiling water, technically speaking. The Food Saver Bags are designed for Simmering and Microwave use as well as Vacuum Sealers Unlimited. Open / Reseal - If it is something like lunch meat or cheese and you only want a few pieces, make sure to open the bag closest to the seal then you can reseal.There are also Ziploc Vacuum bags on the market that are great for making dehydrated meals for Back packing. Reuse - I will sometime wash and reuse my bags, and they are dishwasher safe, This is fine for cooked foods but I wouldn't advise this with raw foods unless you plan to reuse the bags for garbage. 12/13/10 UPDATE ON SORBENT SYSTEMS (IMPAK) BAGS IMPAK IS HAVING TROUBLE WITH THEIR CHANNEL BAGS NOT WORKING PROPERLY AND WILL ISSUE FULL REFUNDS IN THE FORM OF A CHECK, THEY ARE AWARE OF THE PROBLEM AND ARE WORKING ON IT. I will repost when they have resolved the issue. Update 05/01/2011 Ok here's the deal, after 5 months of contacting these people and dozens of non returned phone calls and multiple promises I finally received my refund and of course, they neglected to reimburse me for the $24.63 I paid in shipping. Total was $188.83 and I received $164.20. I would steer clear of this company until they get their act together. IMPAK Corporation 2460 E. 57th Street Los Angeles,CA 90058 (323) 277-4700 ext 113 (323) 389-2200 EFAX www.sorbentsystems.com 2010 (outdated info, but gives you an idea of "Cost Comparison", If I have time I will update this with Vacuum Sealers Unlimited - Foodsaver) Bags - Sorbent System Bags are much cheaper, averaging about 0.17- 0.29 cents per bag depending on the quantity ordered, whereas the Foodsaver Bags are 0.60 - 0.92 cents per bag best price. If you are not going to use the bags for reheating, get the Sorbent Bags, If you are going to use the bags for reheating, get the Foodsaver Bags. If you do both, order both bags and only use the Food Saver Bags for foods that will be reheated in a microwave or boiling water, however, I have been using the Sorbent bags for near ten years and although Sorbent Systems does not recommend their bags in boiling water or microwave, I have yet to have a problem. Breakdown of bags Sorbent - 500 Bags (8" X 11.5" 3.5 MIL) [Quart] channel bags ($85.70) 0.17 each Tilia Foodsaver - 160 Bags (?" X ?" ? MIL) [Quart] channel bags ($11.99 x 8 = $95.92) 0.60 each (This is the price after the 30% discounted price for bulk orders) $0.43 difference per bag So you will spend $10.22 less and get 340 more bags ordering from Sorbent Systems. Sorbent - 300 Bags (10" X 14.5" 3.5 MIL) [Gallon] channel bags ($85.70) 0.29 each Tilia Foodsaver - 104 Bags (?" X ?" ? MIL) [Gallon] channel bags ($11.99 x 8 = $95.92) 0.92 each (This is the price after the 30% discounted price for bulk orders) $0.63 difference per bag So you will spend $10.22 less and get 196 more bags ordering from Sorbent Systems. Seems like a no brainer to me, but don't take my word for it, do your own homework and see for yourself. I also believe that Sorbent Systems makes bags and rolls for the Foodsaver and I am not sure if the specs on their bags are any different than the ones labeled Sorbent Systems, this I can not verify for sure. Tilia Foodsaver also offers a 40 pack of Quart food savers bags at $19.99 but there is no option to get discounts at bulk, so it's better to order the smaller pack size in bulk to get the discount. December 1st, 2012 I had recently ordered some bags from Vacuum Sealers Unlimited and will post my results on their product in the near future. February 27, 2013 So far so good using the Vacuum Sealers Unlimited Bags, I have used a few with no problems and Just ordered their Quart Sized Ziploc Vacuum Sealer Bags for using for BackPacking and Camping trips December 18, 2014 Vacuum Sealers Unlimited Bags are excellent, been using for backpacking foods, clothing storage for canoeing, and of course storing food. Pulled Pork. I suggest partially freeze this before sealing. Rib bits for Chili and Beans "Smoked Onions" Sometimes I will fill up the smoker with veggies and stuff to use for future cooks. Ribs; These ribs were Smoked, sealed, frozen and reheated a week later In a steamer pan and were perfect. Chukie,Sloppy Joe Chuckie and Pork Loin Pulled Pork, Chicken Cordon Bleu Fattie and Chicken Spinoccoli Fattie Manday 2014 Pulled Pork (large chunks can be pulled later or served in chunks), Ribs, Turkey/Bison Chili, Supreme Pizza Fattie, Reheating some pulled pork, very low heat Sous Vide Cooking Chicken Wings Venison Roast Top Round London Broil Chuck Roast Cured Foods Canadian Bacon, cured, sliced and packaged for the freezer. Corned Beef, in the Sous Vide. Buck Board Bacon, cured, cold smoked and packaged for the freezer. Nuts and Cheese Smoked Cheese Smoked Nuts, I do not seal these tight, keeps fresher and don't go stale. Camp Food Boil in Bag Omelet in a Bag, camping food. Boil in Bag Cheeseburger, Camping Food. Boil in Bag, Sausage and Peppers, Camping Food. Picnics and marinating Easy Picnic or party ready foods. Other Uses Clothing for Canoeing Dehydrated Meals Dehydrated meals for Backpacking Some more tips; Freeze broth or sauces at the bottom of a bag add your meat, pasta or whatever you like, then seal. Soft pretzels can be sealed but freeze them first then place in a paper bag then vacuum seal, if you seal without a paper bag, the salt will sometimes cut the bag, losing the seal. Bags larger than your sealer can be used, seal a corner on an angle. Remember to seal in various portions. Use bags to freeze ice cubes for freezer packs for the kids lunch boxes, or to place in your pitcher of beer or iced tea on a hot day. For dehydrated foods with sharp edges, place in a paper bag sleeve or two then seal to avoid punctures. I do not recommend this as standard practice, but if you are in a situation where the bag will not vacuum but rather seals before all the air is removed, you can place a small wrinkle in the bag in the clamp area then when all the air is vacuumed out, press the manual seal button, then seal once more above the seal. I was stuck with a bunch of bad bags but needed to seal 20 lbs. of sliced cheese, 30 lbs. of lunch meat and 15 lbs. of bacon. The Mold Technique, (Don't use all your freezer containers for storage) Place food in a plastic storage container and freeze Remove the next day run hot water on bottom of the container, pop out food and seal in Vac Seal bag. I do this a lot with soups in Tupperware to free up my Tupperware. Square containers are best for stacking I like to keep it no more than 2" thick, it freezes, seals, stacks and reheats much easier. There are just too many ideas to put in one place, but you get the idea.


text:
News Link • WAR
U.S. Marine Says He Will Fire on U.S. Citizens if Civil War Breaks Out
03/27/2010 • godlikeproductions.com
By Sgt C USMC It's been said that the military is always preparing for war. That is true. We prepare for combat every day. We ran 5 miles today to the rifle range and shot nearly 200 rounds a piece at targets and then ran back. However, we also pray for peace. I would love one day to be completely unnecessary. But alas, I am a realist, and I know that day will never come. The headlines of the last week have reminded me more of glimpsing at the S2 Daily Briefing Sheets while in theater or the Al-Jazeera than the NY Times or the Washington Post. Think about that for a moment, let it sink in. Before I get into the main premise of this article – I need to make two statements here. First and foremost , when it comes to the back and forth of who did what to whom and why – I don't give a @!$%#. It doesn't change the action.
In life we're judged by our actions, nothing more, nothing less. One of the greatest things of the military is when it comes to an enemy, the politics behind t
Read Full Story
Reported by Phennommennonn Glp


text:
Gysi, Gregor
|born on
|16 January 1948 at 00:30 (= 12:30 AM )
|Berlin, Germany, 52n29, 13e21
|MET h1e (is standard time)
|Data source
|Astrology data
|24°45' 15°20 Asc. 20°58'
German attorney and key politician of the socialist left-wing political party The Left (Die Linke). He was the last leader of the Socialist Unity Party of Germany, and led the effort that transformed it into the post-Communist Party of Democratic Socialism (PDS), forerunner of The Left. He is well known for his rhetoric talent and is considered one of the best German public speakers.
His father, Klaus Gysi, was a high-ranking official in East Germany, and had been Minister of Culture from 1966 to 1973. His mother, Irene, was the sister of political activist Gottfried Lessing, who was married to the British writer Doris Lessing during his exile in Southern Rhodesia. Doris Lessing is Gysi's aunt. The surname "Gysi" is of Swiss-German origin. Both of his parents were of part Jewish ancestry; his paternal grandmother was Jewish, as was one of his maternal great-grandfathers. One of his maternal great-grandmothers was Russian. His sister, Gabriele, is an actress, who left East Germany in 1985. Today, she is chief dramaturge at the Volksbühne in Berlin.
Gregor Gysi lives separated from his second wife. He has three children, one of them adopted.
- associate relationship with Lafontaine, Oskar (born 16 September 1943). Notes: political associates
Source Notes
Hoppmann published a letter from Gysi, authorising him for an inquiry at the birth registry. A handwritten note attached to this letter holds the date and time of birth.
- Vocation : Law : Attorney
- Vocation : Politics : Party Affiliation (German Left party)


text:
Philadelphia Police have located a missing 10-year-old boy in the Frankford section of the city.
Azhair Leach was found walking to his house in the 4600 block of Hawthorne Street with a friend.
Azhair was reported missing by his mother Friday and had last been seen on Thursday at approximately 7:00 p.m. Investigators say that a friend came to Azhair's house on Thursday evening and the two left together. It is not clear if that is the same friend with whom Azhair left his house.
Published at 11:00 AM EDT on Jul 7, 2012 | Updated at 11:54 AM EDT on Jul 7, 2012


text:
Photographer's Note
Shot early morning (6/7 am) from a road in the Charentes Maritime. The picture here does not completely reflect the mistic atmosphere of the reality there at that very moment. Magic!
Nobody has marked this note useful
Critiques | Translate
Hi Stephane,
i like the mystic athmosphere, colors and sharpness fine. I like the presentation.
Photo Information
- Copyright: Stephane Fauconnier (stf303) (861)
- Genre: Places
- Medium: Color
- Date Taken: 2008-08-06
- Categories: Nature
- Camera: CANON EOS 30 D, Canon 18-55 mm EF-S, Fujiyama C-PL
- Exposure: f/5.0, 1/160 seconds
- More Photo Info: view
- Photo Version: Original Version
- Date Submitted: 2010-06-14 7:26
Viewed: 791
Points: 2


text:
Artist description:
the mood ,the feeling,the inner side
Java Draw Created Oct 24, 2011 640 x 460px Unless noted Copyright 2011 shakeh.
We encourage you to submit a comment and let the artist know what you think of this drawing. Please log in or join to post a comment.
Post a comment
Wonderful drawing!!!!! 5/5 favs!!!
Excellent work!!
Very creative ") i like it alot!! +5 shows your artistic creativity
VERY beautiful and I can sure relate to her mood! 5h
What a beautiful portrait.. I love it.... Coll & fav's
Nice work!!


text:
You have chosen to publish your review anonymously. If You click 'OK' your review will be published under the pseudonym 'Perry376' which has been randomly generated by us. If you would prefer your review to be published as 'Guest' please select this option.Publish as 'Guest'
Item Name: No 5 Gaolmews Restaurant, Stafford
Category: Staffordshire Restaurants


text:
correction: my apologies to our friends at Hitwise: I had TV Web sites on the brain and referred to Hitwise as HitFix and worse, linked to Hitfix instead! D'oh! At least the proper branding was still in their chart. I apologize for any confusion.
A little while ago I wrote citing Deadline.com's press release announcing it was bigger than THR and Variety combined and that *we* were too when it comes to pageviews.
The kind folks at Hitwise sent this chart noting that they had us bigger than Variety and THR too online, but not as big as Deadline Hollywood. Though here, we're much closer to Variety and THR and further away from Deadline, but this is only measuring US traffic, and the numbers in the other post were global. About 25% of our visits are from outside the US lately.
I look at this and think "If that's reflective of Variety's traffic BEFORE the pay wall….YIKES!"
Update: I have asked Hitwise if they can produce this chart with James Hibberd's Live Feed (THRFEED.COM). Given all the great inbound linking he gets, I'm pretty sure it is not included below in the hollywoodreporter.com data. I will not be surprised if he is as big (or bigger) than Deadline Hollywood for November. He had some posts that had huge traffic that month. So look for "James Hibberd Bigger Than Hollywood Reporter Mother Ship Online" post soon 😉
Update 2: Hitwise produced the chart with thrfeed.com, which is now included below. I confess I am surprised by the results. I guess James gets some very high traffic posts, but we make it up in volume, at least according to Hitwise.


text:
GREENSBORO, NC -- When you watch the Wyndham coverage on tv this weekend, you'll notice the how lush the course looks. 2 Wants to Know what it takes to make the course look so good and what the rest of us can learn from the grass pros.
The game of golf is quiet game sort of. The course looks serene...but that takes a lot of serious work. Just ask the crew. Adam Brewer, 1st assistant superintendent at Sedgefield, says, "We might come in one morning and cut the greens and then look at them after lunch and say, 'Alright boys, let's do it again.'"
To say there's a lot of mowing going on wouldn't do this crew justice. The greens are mowed every day. The fairways two to three times a week. The surround areas two to three times a week and the tees...twice a week, even in the rain. Adam says, "During the tournament, everything will get cut at least once expect for the rough, every single day."
To get the job done, it it takes every tool including your run of the mill mower which brings us to your run of the mill lawn. Adam says, "For the homeowner with a tall fescue lawn, not scalping it down, keeping the height to cut up is very important especially during the summer."
Just like the game, keeping the course and your lawn looking good takes practice. And maybe even some sensitivity training. Adam says, "I look at grass every single day, so I can tell and look at something and tell it's not happy. And there is a lot of different reason why there is one grass that is happy or unhappy."
Adam says lawns are often unhappy because we choose the wrong kind of turf to grow in the area we're trying to cover. He says some grass likes shade, others die in it. So make sure you're picking the grass or plants that are right for your surroundings.


text:
Edit Article
wikiHow to Find a Bug by Isolating
This method is a very useful method for finding a bug, when it is hard to find, especially on HTML and XML (because there aren't debuggers or break points).
1Make a backup of your source. You will want a checkpoint that you can go back because you will go back later.
2Erase a small section, and put in the small something instead. Like a message box, a white space, a constant number variable (to erase an algorithm), etc. Sometimes, the placeholder will have to have the same size as the part you took out. You can do anything to it, because you have the backup.
3See the result. Does it work perfectly now? The bug was in the erased part, so go to the next step. Does it work even funkier than before? Undo the erased part and try again. Does it give the same results as last time? At least you know it isn't there.
4Go smaller. Undo the erased part, put markers around it, and try to find the exact place of the bug by the same method, in the markers.
Community Q&A
Ask a Question
200 characters leftSubmit
- Remember to erase the parts inside functions, tags, etc.
Article Info
Categories: Software Testing
Thanks to all authors for creating a page that has been read 5,118 times.
Is this article up to date?


text:
Savannah River Site's primary contractor will establish a "transition center" in Aiken to offer job placement and unemployment services to workers idled during upcoming layoffs that are part of a plan to cull 1,400 positions.
Although 328 people agreed to voluntary separations in December, Savannah River Nuclear Solutions will announce the details of its first round of involuntary separations by the end of this month, company spokesman Will Callicott said.
"The plan approved by the Department of Energy in November gives us the authority to decrease by up to 1,400 employees by the beginning of fiscal year 2012, which is in September," he said. "We have finished the voluntary phase and the involuntary phases will be on two occasions: the first is later in February and a second phase in August."
An exact date for announcing the layoffs hasn't been set, but it will be "within a fairly short window of time later in February," Callicott said.
Savannah River Nuclear Solutions is also arranging to open a transition center as soon as possible after the announcements.
"We haven't opened it yet, but there will be a transition center in downtown Aiken," he said. "People would have the opportunity to go there and meet with various state agencies, and we will also be inviting potential employers to come there to talk to people."
Savannah River Nuclear Solutions had 6,492 employees on Jan. 1, making it the largest of several employers at the site.


text: 2015-2016 Mexico Adidas Polo Shirt (Green). Official 2015-2016 Mexico Polo Shirt manufactured by Adidas. This green Mexico polo shirt is available to buy in adult sizes S, M, L, XL, XXL. El Tri are on the rise, and with the men's adidas Mexico Polo Shirt you can rise with them. The soccer shirt shows off the team badge on the chest and features a polo collar and contrast colors. Polo collar; Hidden buttons Contrast detailing Mexican Football Federation woven badge on left chest 55% cotton / 45% polyester pique ImportedProduct Code Z24368


text:
Police say an officer has shot and killed a pit bull that bit a fellow officer in a bullet-strewn Long Island backyard.
Nassau County police say officers were investigating around 12:10 a.m. Sunday after gunshots rang out in a backyard on Park Avenue in Roosevelt. They say the unleashed dog charged at them and bit one of the officers multiple times in both thighs.
After the officer broke free, another officer shot the dog in the torso.
The wounded officer has been treated and released from a hospital.
Authorities say they found many .380-caliber and shotgun shell casings in the yard.
Published at 12:38 PM EST on Jan 1, 2012 | Updated at 1:01 PM EST on Jan 1, 2012


text:
We are sorry to announce that this conference will not be offered in 2017.
Please check back for updates.
We leave the 2016 information here for your reference.
Evidence-based Research and Practices in Child Sexual Abuse Treatment
Oct 31-Nov 2, 2016
For more than 30 years, we've provided the very latest evidence-based information on the treatment, intervention, assessment, and prevention of child sexual abuse, and we are committed to maintaining the same standard of quality, value, and expertise for the next 30—and beyond.
This year we're offering 36 new institutes and workshops on a wide range of topics. Whether you are an experienced professional or are new to the field—and whether you work as a therapist, child protection worker, attorney, law enforcement officer, medical professional, or in any position with a need to know about child sexual abuse treatment—this highly regarded conference offers you the very latest information to best help those with whom you work.
At a glance
What: 32nd annual UW–Madison Conference on Child Sexual Abuse
Who: Therapists, child protection workers, attorneys, law enforcement officers, medical professionals
When: Oct 31-Nov 2, 2016
Where: Madison Marriott West, Middleton, WI (map)
Cost: Varies. Click for details»
CEUs: Up to 1.8 (18 hours)
or by phone at 800-725-9692
Questions? Email Denise Nolden or call 608-263-5130 (toll free 800-442-7107)
Why the UW-Madison Conference on Child Sexual Abuse?
The University of Wisconsin–Madison has a century-long tradition of successful development and innovation in continuing education for professionals. Whether you are an experienced professional or are new to the field, this highly regarded conference offers you the very latest evidence-based information on treatment, intervention, assessment, and prevention. Attendees include therapists, child protection workers, attorneys, law enforcement officers, medical professionals, and anyone with a need to know about child sexual abuse treatment.
Workshops and institutes are taught by highly qualified, credentialed experts in the fields of psychology, psychiatry, social work, law, training, and other disciplines relevant to child abuse and protection services.
Share conference benefits with your supervisor: printable handout»
Conference takeaways
At this conference you will:
- Get the most current information on working with sexually abused children, adults who were abused as children, and offenders
- Learn how to develop and apply effective assessment methods
- Explore best practices treatment methods
2016 plenary speakers
Sharon Cooper, MD, is the CEO of Developmental and Forensic Pediatrics, P.A., a consulting firm which provides medical care, research, training and expert witness experience in child maltreatment cases as well as medical care for children with disabilities. She works regularly with numerous national and international investigative agencies on Internet-facilitated crimes against children cases.
Dr. Cooper spent 21 years in the Armed Forces, retiring as a colonel, and has for the past several years, worked in both the civilian and military arenas in child abuse and developmental pediatrics. She holds a faculty position at the University of North Carolina Chapel Hill School of Medicine and the Uniformed Services University of Health Sciences in Bethesda, Maryland. She is an instructor at the Army Medical Education Department Center and School at Ft. Sam Houston, Texas where she provides multidisciplinary training in all forms of child maltreatment to health care providers, law enforcement, attorneys, judges, therapists, chaplains, and social workers.
For the past several years, Dr. Cooper has served as a consultant to and a Board member of the National Center for Missing & Exploited Children where she teaches about the victim aspects of Internet crimes against children and commercial sexual exploitation of children and youth. She has also provided numerous international training for the International Center for Missing and Exploited Children in Jordan, Russia, France, Germany, Turkey and other countries. Dr. Cooper has lectured both nationally and internationally in well over 300 conferences for the U.S. Department of Justice, the FBI, the North Carolina District Attorney's Association, several attorney general's conferences in various states, the American Academy of Pediatrics, the Royal Canadian Mounted Police, and Interpol, among others. She has published chapters in professional texts, and is the lead author of the first comprehensive textbook on the medical, legal, and social science aspects of child sexual exploitation and Internet-facilitated crimes against children.
Dr. Cooper has provided testimony to the U.S. Congress as well as to the Russian Duma (Parliament), the European Commission, and the Italian Senate. She is also a member of an international Expert Working Group on Children and Young Persons with Abusive and Violent Experiences Connected to Cyberspace hosted by the Council of the Baltic Sea States and the Swedish Children's Welfare Foundation.
She is a board member of several organizations and is a member of several professional associations including the American Academy of Pediatrics, the American Professional Society on the Abuse of Children, the International Association of Forensic Nurses, and the International Society for the Prevention of Child Abuse and Neglect.
Dr. Cooper was selected by Newsweek magazine as one of the 150 Women Who Shake the World (March 2011). She was also selected by the U.S. Attorney General for membership in the Defending Childhood Task Force: Children Exposed to Violence (2011-2012).
Mary Jo Barrett, MSW, is the Executive Director and co-founder of The Center for Contextual Change, Ltd. Previously, Ms. Barrett was the Director of Midwest Family Resource and has been working in the field of family violence since 1974.
Ms. Barrett has coauthored two books with Dr. Terry Trepper: Incest: A Multiple Systems Perspective and The Systemic Treatment of Incest: A Therapeutic Handbook. She co-created the Collaborative Stage Model (CSM), a highly successful contextual model of therapy used to transform the lives of those impacted by abuse and/or traumatic events.
Her trainings and published works focus on the teaching of the Collaborative Stage Model, systemic and feminist treatment of women, adult survivors of sexual abuse and trauma, eating disorders, couple therapy, Post Traumatic Stress Disorder, and Compassion Fatigue.
Ms. Barrett provides consultations, workshops, courses and other training opportunities nationally and internationally to parents, social service professionals, lawyers, mental health staffs, psychotherapists, residential treatment facilities and governmental agencies. Ms. Barrett founded the Family Dialogue Project, which strives to redefine relationships with families impacted by allegations of abuse and trauma.