8%). Additionally, it was well tolerated, and 100% of the patients had good compliance. The data indicate

that the novel 10-day quadruple therapy containing tetracycline and levofloxacin has great potential to become the choice of salvage treatment of sequential therapy. Clarithromycin resistance has been identified as the main reason for the failure of standard triple therapy [31]. According to the Maastricht IV/Florence Consensus Report [10], sequential treatment has been recommended for H. pylori infection in areas of high clarithromycin resistance. However, a significant number of patients fail to eradicate H. pylori by sequential therapy, Regorafenib cell line and the best rescue therapy for sequential therapy remains unclear. In clinical practice, patients with failed sequential therapy would have limited options for further treatment because they would already have received three commonly used antibiotics: amoxicillin, clarithromycin, and metronidazole. CHIR-99021 chemical structure Currently, the antibiotic resistance profiles of H. pylori strains following sequential therapy are still lacking. In this study, antibiotic

sensitivity data were available only in five patients. The frequencies of H. pylori resistance to tetracycline, levofloxacin, amoxicillin, clarithromycin, and metronidazole were 0, 0, 0, 80, and 100%, respectively. The antibiotic resistance profiles of H. pylori strains following sequential therapy were also available in three of five patients receiving PPI–bismuth–tetracycline–metronidazole quadruple therapy in this study period (data not shown). The frequencies of H. pylori resistance to tetracycline, levofloxacin, amoxicillin, clarithromycin, and metronidazole in the three patients were 0, 67, 0, 67, and 33%, respectively. Taken together, the drug resistant Megestrol Acetate rates to tetracycline, levofloxacin, amoxicillin, clarithromycin, and metronidazole following sequential therapy were 0, 25, 0, 75, and 75%, respectively. Currently, the studies investigating rescue treatment following sequential therapy are extremely rare. Although levofloxacin-containing

triple therapy (PPI, amoxicillin, and levofloxacin) has been recommended by the Maastricht IV/Florence Consensus Report as a rescue treatment of sequential therapy [10], the eradication rate of the rescue regimen is suboptimal (mean eradication rate: 77.5% (79 of 102), range: 50% (three of six) to 100% (seven of seven)) [19-23]. In addition, the sample size of previous studies was remarkably low [19-23]. In the current study, we employed a novel quadruple therapy containing tetracycline and levofloxacin to treat H. pylori infection following failure of sequential therapy. Bismuth salt was also applied in the salvage regimen because bismuth salts have a synergistic effect on antibiotics by destroying bacteria in the manner of an antiseptic [32].

8%). Additionally, it was well tolerated, and 100% of the patients had good compliance. The data indicate

that the novel 10-day quadruple therapy containing tetracycline and levofloxacin has great potential to become the choice of salvage treatment of sequential therapy. Clarithromycin resistance has been identified as the main reason for the failure of standard triple therapy [31]. According to the Maastricht IV/Florence Consensus Report [10], sequential treatment has been recommended for H. pylori infection in areas of high clarithromycin resistance. However, a significant number of patients fail to eradicate H. pylori by sequential therapy, Ixazomib mw and the best rescue therapy for sequential therapy remains unclear. In clinical practice, patients with failed sequential therapy would have limited options for further treatment because they would already have received three commonly used antibiotics: amoxicillin, clarithromycin, and metronidazole. Y-27632 ic50 Currently, the antibiotic resistance profiles of H. pylori strains following sequential therapy are still lacking. In this study, antibiotic

sensitivity data were available only in five patients. The frequencies of H. pylori resistance to tetracycline, levofloxacin, amoxicillin, clarithromycin, and metronidazole were 0, 0, 0, 80, and 100%, respectively. The antibiotic resistance profiles of H. pylori strains following sequential therapy were also available in three of five patients receiving PPI–bismuth–tetracycline–metronidazole quadruple therapy in this study period (data not shown). The frequencies of H. pylori resistance to tetracycline, levofloxacin, amoxicillin, clarithromycin, and metronidazole in the three patients were 0, 67, 0, 67, and 33%, respectively. Taken together, the drug resistant learn more rates to tetracycline, levofloxacin, amoxicillin, clarithromycin, and metronidazole following sequential therapy were 0, 25, 0, 75, and 75%, respectively. Currently, the studies investigating rescue treatment following sequential therapy are extremely rare. Although levofloxacin-containing

triple therapy (PPI, amoxicillin, and levofloxacin) has been recommended by the Maastricht IV/Florence Consensus Report as a rescue treatment of sequential therapy [10], the eradication rate of the rescue regimen is suboptimal (mean eradication rate: 77.5% (79 of 102), range: 50% (three of six) to 100% (seven of seven)) [19-23]. In addition, the sample size of previous studies was remarkably low [19-23]. In the current study, we employed a novel quadruple therapy containing tetracycline and levofloxacin to treat H. pylori infection following failure of sequential therapy. Bismuth salt was also applied in the salvage regimen because bismuth salts have a synergistic effect on antibiotics by destroying bacteria in the manner of an antiseptic [32].

Examination of scatter plots of focus area in individual recipient mice (Fig. 3A) indicated that focus growth varied considerably among recipients. Thus, we normalized the data by dividing each hPAP focus area by the mean area of lacZ foci for that mouse, obtaining a focus ratio distribution

for each recipient (Fig. 3B). Normalization is possible because we compare data between two cell populations in the same recipient mouse liver, which therefore have been exposed throughout the study to the same hepatic and systemic environments. The average of median focus ratio distribution values for all mice at each time posttransplantation should equal “one” if there is no difference in the size of hPAP versus lacZ foci (Table 3). At 1 week posttransplantation, hPAP foci appear larger than lacZ foci (P = 0.049; likely because of a measurement artifact, as noted above), but at subsequent times the values are very Luminespib close to 1. Note that Fig. 3 displays only representative data. All data are summarized in Table 3. We next examined growth of hepatocytes expressing transgenes that had been shown to increase the incidence of liver cancer in transgenic mice (Figs. 2B-D, 3C,D, and Table 3). Only TGFα Venetoclax concentration and c-myc significantly

increased the rate of focus growth during the growth phase in recipient livers compared with hPAP alone (Table 3). However, no single growth regulatory molecule induced continued focus growth during the quiescent phase, MycoClean Mycoplasma Removal Kit indicating that they were not sufficient to cause growth in an environment that was not growth-stimulatory. To determine whether focus growth was affected by immune recognition of donor cells expressing the viral simian virus 40 T antigen (TAg), we also transplanted TAg/hPAP donor cells into athymic nu/nu recipient mice and measured focus size at 4 and 8 weeks posttransplantation. We found no significant focus ratio differences between nu/nu and immune-competent recipients (data not shown), indicating that immune rejection was not a major factor in these experiments. In addition, hPAP-marked donor parenchyma is stable for

more than 18 months in recipient mice.14 Coexpression of growth regulatory molecules in donor hepatocytes produced dramatic differences in focus size at all times posttransplantation (Fig. 2E-G). Focus ratio distribution medians also were increased (Fig. 3F and Table 3), indicating that expression of each oncogene pair was sufficient to increase the rate of hepatocyte focus growth during the growth phase. Furthermore, TAg/TGFα donor focus growth continued during the quiescent phase (Table 3; compare weeks 8 and 12), so this combination of growth regulatory molecules induced cell-autonomous hepatocyte growth in the quiescent liver. The most dramatic growth was observed after coexpression of TAg and c-myc (Fig. 2G and Tables 2 and 3).

Twenty-one (35%) NAFLD patients had advanced fibrosis. From univariate analysis, age, NAS, hemoglobin A1C, omentin-1 levels were significantly elevated in advanced fibrosis patients. Only age and NAS were independently associated with advanced fibrosis.

Conclusions: Chemerin and omentin-1 levels are elevated in NAFLD patients. Age and NAFLD activity score, but not chemerin, omentin-1 and vaspin levels, are associated with NAFLD PF-02341066 solubility dmso with advanced fibrosis. The role of adipokines in NAFLD requires further study Disclosures: The following people have nothing to disclose: Akharawit Pulsombat, Daruneewan Warodomwichit, Pattana Sornmayura, Napat Angkathunyakul, Sasivimol Rattanasiri, Wathanee Chaiyaratana, Piyaporn Kaewdoung, Supanna Petraksa, Abhasnee Sobhonslidsuk Background: Nonalcoholic fatty liver disease (NAFLD), the hepatic manifestation of metabolic syndrome, is the most common cause of liver disease in our environment. The prevalence of NAFLD in patients with morbid obesity reaches 80%. click here It has been proposed that the small intestinal bacterial overgrowth (SIBO) and microbial translocation through the intestinal wall (MT) are related to NAFLD, in its initial form of simple steatosis and in a more advanced stage

of steatohepatitis (NASH). The aim of this study was to investigate in patients with morbid obesity and NAFLD the relationship between SIBO and MT measured by serum levels of lipopolysaccharide (LPS) and LPS binding protein (LBP), with NAFLD activity score (NAS) and the severity of steatosis. Patients and Methods: We included consecutive morbid obese patients (BMI >40 kg/m2 or >35 kg/m2 in association with comorbidities) prior to bariatric surgery Edoxaban intervention. Exclusion criteria

were: normal liver biopsy, other causes of liver disease or duodenal mucosal atrophy. Endoscopy was performed to obtain duodenal aspirate for culture and duodenal mucosa biopsy. We studied peripheral venous blood sampling for liver enzymes, viral hepatitis, LPS and LBP. The following values were considered pathological: 10000 CFU/mL (SIBO), LPS >5 EU/mL and LBP >10 mg/mL. Liver biopsy was performed during surgery. The severity of steatosis was quantified in three grades (1: 5-33% 2: 34-66% 3: >66%) and NAS >4 was associated with increased likelihood of having NASH. Results: 71 patients were initially included, but 26 were excluded because they had normal liver biopsy. Forty-five patients had NAFLD therefore. Eighteen men, mean age 45.88 years (22-69) and mean BMI 47.81 kg/m2 (37-58).25% had SIBO measured with a sensitive and specific method as the duodenal aspirate culture. Degree of steatosis: 20/17/8. NAS >4 in almost half of patients. Statistical significance was observed between LBP levels and SIBO with the severity of steatosis (p <0.05 and p =0.077, respectively).

pylori was cultured after failure of eradication. Minimal inhibitory concentration selleck chemical test was performed for amoxicillin, clarithromycin, metronidazole, tetracycline, azithromycin, levofloxacin, and moxifloxacin using agar dilution method. Primary and secondary resistance rates of H. pylori to 7 antibiotics were evaluated and risk

factors for the antibiotic resistance were analyzed. Increase in the primary resistance rate was found in amoxicillin (6.3–14.9%, p = .051), clarithromycin (17.2–23.7%, p = .323), and both of levofloxacin and moxifloxacin (4.7–28.1%, p = .002) during the study period. Secondary resistance rate significantly increased in metronidazole, levofloxacin, and moxifloxacin. Increase of resistance occurred after initial failure of eradication therapy in case of clarithromycin (p < .001), azithromycin (p < .001), levofloxacin (p = .011), and moxifloxacin (p = .020). Multivariable analyses showed that clarithromycin, azithromycin, levofloxacin, and moxifloxacin selleck resistance was associated with previous eradication treatment history. The increased primary and secondary antibiotic resistance of H. pylori in Korea is ongoing,

and it will become a significant limitation for effective eradication of H. pylori in the future. “
“Helicobacter pylori (H. pylori) infection plays an important role in the early stage of cancer development. However, various bacteria that promote the synthesis Phosphatidylinositol diacylglycerol-lyase of reactive oxygen and nitrogen species may be involved in the later stages. We aimed to determine the microbial composition of gastric mucosa from the patients with chronic gastritis, intestinal metaplasia, and gastric cancer using 454 GS FLX Titanium. Gastric mucosal biopsy samples were collected from 31 patients during endoscopy. After the extraction of genomic DNA, variable region V5 of the 16S rRNA gene was amplified. PCR products

were sequenced using 454 high-throughput sequencer. The composition, diversity, and richness of microbial communities were compared between three groups. The composition of H. pylori-containing Epsilonproteobacteria class appeared to be the most prevalent, but the relative increase in the Bacilli class in the gastric cancer group was noticed, resulting in a significant difference compared with the chronic gastritis group. By analyzing the Helicobacter-dominant group at a family level, the relative abundance of Helicobacteraceae family was significantly lower in the gastric cancer group compared with chronic gastritis and intestinal metaplasia groups, while the relative abundance of Streptococcaceae family significantly increased. In a UPGMA clustering of Helicobacter-dominant group based on UniFrac distance, the chronic gastritis group and gastric cancer group were clearly separated, while the intestinal metaplasia group was distributed in between the two groups.

pylori was cultured after failure of eradication. Minimal inhibitory concentration LY2157299 research buy test was performed for amoxicillin, clarithromycin, metronidazole, tetracycline, azithromycin, levofloxacin, and moxifloxacin using agar dilution method. Primary and secondary resistance rates of H. pylori to 7 antibiotics were evaluated and risk

factors for the antibiotic resistance were analyzed. Increase in the primary resistance rate was found in amoxicillin (6.3–14.9%, p = .051), clarithromycin (17.2–23.7%, p = .323), and both of levofloxacin and moxifloxacin (4.7–28.1%, p = .002) during the study period. Secondary resistance rate significantly increased in metronidazole, levofloxacin, and moxifloxacin. Increase of resistance occurred after initial failure of eradication therapy in case of clarithromycin (p < .001), azithromycin (p < .001), levofloxacin (p = .011), and moxifloxacin (p = .020). Multivariable analyses showed that clarithromycin, azithromycin, levofloxacin, and moxifloxacin selleckchem resistance was associated with previous eradication treatment history. The increased primary and secondary antibiotic resistance of H. pylori in Korea is ongoing,

and it will become a significant limitation for effective eradication of H. pylori in the future. “
“Helicobacter pylori (H. pylori) infection plays an important role in the early stage of cancer development. However, various bacteria that promote the synthesis Nabilone of reactive oxygen and nitrogen species may be involved in the later stages. We aimed to determine the microbial composition of gastric mucosa from the patients with chronic gastritis, intestinal metaplasia, and gastric cancer using 454 GS FLX Titanium. Gastric mucosal biopsy samples were collected from 31 patients during endoscopy. After the extraction of genomic DNA, variable region V5 of the 16S rRNA gene was amplified. PCR products

were sequenced using 454 high-throughput sequencer. The composition, diversity, and richness of microbial communities were compared between three groups. The composition of H. pylori-containing Epsilonproteobacteria class appeared to be the most prevalent, but the relative increase in the Bacilli class in the gastric cancer group was noticed, resulting in a significant difference compared with the chronic gastritis group. By analyzing the Helicobacter-dominant group at a family level, the relative abundance of Helicobacteraceae family was significantly lower in the gastric cancer group compared with chronic gastritis and intestinal metaplasia groups, while the relative abundance of Streptococcaceae family significantly increased. In a UPGMA clustering of Helicobacter-dominant group based on UniFrac distance, the chronic gastritis group and gastric cancer group were clearly separated, while the intestinal metaplasia group was distributed in between the two groups.

Based on retrospective analyses of the IDEAL trial,11 the sponsor proposed that an HCV-RNA decline ≤1.0 log10 at week 4 was predictive of the more traditionally defined null response on P/R (i.e., <2 log10 HCV RNA decline at week 12). SVR rates for SPRINT-II subjects administered BOC who had <1.0 log10 decline at week 4 were 28% in Arm 2 and 38% in Arm 3, compared to 4% in the P/R control arm. The FDA's SVR analysis Napabucasin purchase of treatment-naïve subjects in SPRINT-II demonstrated that 31% (26/83) of subjects with <1.0 log10 decline at week 4 in the P/R control

arm (Arm 1) would be incorrectly classified as null responders10 (the remaining subjects discontinued treatment, had a partial response, or relapsed). To obtain a more conservative estimate of the SVR rate in null responders, an alternative surrogate definition of <0.5 log10 HCV RNA decline at week 4 was investigated. Based on the end of study outcomes (i.e., null responder, partial responder, relapser,

or responder achieving SVR) for such subjects in SPRINT-II treated with P/R (n = 25), 22 subjects were null responders (88%), one was a partial responder, and two discontinued treatment. The SPRINT-II study design allowed us to analyze outcomes of treatment-naïve subjects who were treated with BOC (Arms 2 and 3) and had similar interferon responsiveness during the lead-in period. The SVR rates in these poorly interferon responsive subjects (defined here as <0.5 log10 decline in HCV RNA at week 4) who received BOC Ibrutinib nmr treatment was 28% (Arm 2: n/N = 13/47) and 30% (Arm 3: n/N = 11/37). By comparison, the observed SVR rate for poorly interferon responsive subjects treated with P/R (i.e., those with ≤0.5 log10 HCV RNA decline at week 4) was 0%. Whether a Mephenoxalone <1.0

or <0.5 log10 decline in HCV RNA at week 4 was used to categorize poorly interferon responsive subjects from SPRINT-II, a treatment benefit was demonstrated in the BOC regimens over treatment with P/R alone. Further, it is important to consider the reasons behind equating prior null responders and treatment-naïve subjects with poor interferon responsiveness. The previous analysis demonstrated that BOC provided meaningful benefit in treatment-naïve subjects with interferon response characteristics similar to prior null responders. However, to consider using data from treatment-naïve patients to predict response in P/R-experienced patients, one needs to demonstrate that P/R response remains similar after a second course of P/R treatment. To address this question we assessed the relationship between virologic responsiveness through week 4 and treatment outcome for P/R arm subjects in SPRINT-II (i.e., log10 decline in HCV RNA at week 4 grouped according to end of study outcome) (Fig. 2). Similarly, subjects from RESPOND-II were grouped according to previous treatment outcome (relapser or partial responders) (Fig. 2).

09% versus 1.21 ± 0.90%) (Fig. 6B,C). We also observed a higher percentage of IL-4-producing cells in addition to IL-17-producing cells, but not IL-10-producing cells (data not shown). In addition, NS3/5 treatment also resulted in a significant induction of IL-17 production, while they reduced the production of IFN-γ (Fig. 6D). Collectively, these data indicate that IL-17-producing CD4+ T cells are induced during HCV Ku-0059436 price infection. We next examined the possibility that the direct action of TSLP on CD4+ T cells is involved the differentiation of Th17 cells. CD4+ T cells from PBMC of HCV-infected

patients were stimulated with NS3/NS5 in the presence or absence of TSLP for 3 days. Stimulation with TSLP or NS3/5 significantly enhanced IL-17 mRNA and protein compared to control cells cultured medium only (Fig. 6E,F,G). Moreover, differentiation of IL-17 cells was increased following combined stimulation of TSLP and NS3/5 compared to either TSLP or NS3/5 alone. These results clearly

indicate that TSLP is an important factor in the differentiation of IL-17-producing CD4+ T cells during HCV infection and might play a role in the development of chronic liver diseases. In this Rucaparib clinical trial report we demonstrate that HCV infection of hepatocytes induces NFκB-dependent TSLP gene expression and protein production. Furthermore, TSLP mRNA and protein was increased selectively in liver tissues from chronic HCV patients. Intriguingly, TSLP released from HCV-infected hepatocytes activates human monocyte-derived DCs and conditions DCs to support the polarization of CD4+ T cells toward Th17 cells. The blockade of TSLP action by neutralizing antibody suppresses differentiation of Th17 cells. These results suggest a novel mechanism to account for the infiltration of TH17 cells

in the liver as next likely a result of the role of TSLP in promoting Th17 differentiation. However, it remains unclear whether hepatic TSLP accounts for facilitating the recruitment of Th17 infiltration in the infected liver. These results also raise the intriguing possibility that the crosstalk between HCV-infected hepatocytes and local (liver and/or liver draining lymph node) DCs may be a pivotal mechanism both in a defective antiviral (Th1) CD4+ T-cell response and also in an enhanced expression of an injury-provoking (Th17) CD4+ T-cell response. To our knowledge, our findings represent the first report identifying hepatic-secreted TSLP as a regulator of Th17 differentiation. Although CD4+ T cells have been reported to be critical to the antiviral function of CD8+ T cells in chronic infection, failure of CD4+ T cell help is associated with the inability to clear HCV infection and CD4+ T-cell responses in chronically infected HCV patients leans toward Th2 deviation and T regulatory (Treg) cells.

[6] In terms of drug metabolism enzymes and transporters, Tac is a substrate of cytochrome P-450 (CYP) 3A enzyme and drug transporter ATP-binding cassette sub-family B member 1 (ABCB1).[4] Both CYP3A4 and CYP3A5 are known to be involved in the metabolism of Tac,[7] and there are many reports on the relationship between

Tac pharmacokinetics and genetic polymorphisms of CYP3A4, CYP3A5, and ABCB1 in organ transplantation patients.[8-11] However, there has been no investigation of these genetic polymorphisms check details and Tac pharmacokinetics in inflammatory bowel disease (IBD) patients, and only one report on the response to Tac therapy.[12] Genetic polymorphisms are known to exist in CYP3A4, CYP3A5, and ABCB1, and there are also known to be large differences among ethnic groups.[9-11] In general, CYP3A5 genetic polymorphisms, namely, expressers (Exp) with *1 or non-expressers (Non-Exp) without *1, are thought to have the greatest effect on Tac pharmacokinetics.[13, 14] In the present study, CYP3A4, CYP3A5, and ABCB1 genetic polymorphisms and their potential associations with Tac pharmacokinetics

and efficacy were analyzed in Japanese IBD patients. In our department, therapy with Tac is indicated for UC patients with moderate-to-severe activity who are resistant to prednisolone (PSL) and other drugs. Many cases are severe, and inpatient therapy is the fundamental approach when starting Tac. As a rule, the initial dose is 0.05 mg/kg twice Stem Cell Compound Library daily for C1GALT1 patients ingesting food and 0.04 mg/kg twice daily for patients who are fasting. To monitor blood levels of Tac, trough levels are normally measured at least on days 2–5 and 7–10 during the early period of therapy. Measurement of Tac blood levels is contracted to SRL, Inc. (Tokyo, Japan), and ELISA is done using the PRO-TRAC

II TM FK 506 (Bio-Rad Laboratories, Inc., Los Angeles, CA, USA). Depending on the trough level results on days 2–5 and 7–10 during the remission induction period, the Tac dose is then adjusted to achieve the optimal trough level of 10–15 ng/mL. The equation (previous dose × 12.5 mg/mL/the blood trough level) was used for the dose adjustment of Tac.[2, 3] Patients with frequent diarrhea or severe abdominal pain are managed by fasting with total parenteral nutrition for about 2 weeks. Seventy patients with UC were treated by Tac in our department between February 2001 and February 2012. Of these patients, full explanations of the present study were given to 45 patients examined in our hospital between August 2011 and May 2012. There was no special selection; all 45 of these patients undergoing follow-up at our hospital during this period were the subjects of this study. Genotyping analysis of CYP3A5, CYP3A4, and ABCB1 was contracted to SRL, Inc., and gene analysis was done by fluorescence correlation spectroscopy.

4; Table 2). Besides substitutions at known resistance loci (NS3 amino acid positions 36, 41, 43, 138, 168), potential resistance mutations were also identified at positions 28, 77, 80, 85, 98, 133, 160, and 174, of which those at 77, 80, and 174 °Ccurred in both replicates of the same genotype and absent in the control passage, observations clearly indicative of resistance-induction. The mutation Q41R was found either as a double mutant with E168A or as a triple mutant with E28G and E168A, but not as a single mutant. P85L was

only observed in combination with T98R (Supporting Information Fig. S3). Previously described resistance loci found at position 36, 54, 155, 156 in genotype 1-infected patients treated with telaprevir were represented in one or several clones of virus passaged in vitro under PI selection, along with the additional V36A+A156S and T54A+N77S double Erismodegib datasheet mutations (Fig. 4; Supporting Information Fig. S4). Replacement of the wildtype codon was NVP-BEZ235 furthermore observed at position 174 (genotype 1b) and 77 (genotypes 3a and 6a). These latter

mutations have also been found during danoprevir therapy, indicating mutations potentially conferring cross-resistance. As previously shown for BILN 2061,16 the pattern of resistance-associated mutations under danoprevir and telaprevir was highly diverse among the different genotypes, indicating major mechanistic differences in invivo resistance development. To investigate the influence of the acquired mutations on the viral replication fitness of the individual intra- and intergenotypic recombinants, Dynein substitutions were reintroduced into the original recombinants. Mutants were passaged in Huh7.5 cells without antivirals by cell splitting and the spread within the cell culture compared to that of the wildtype (Supporting Information Fig. S2; Supporting Information Material). Most substitutions did not have an obvious effect on the spread of the intra- and intergenotypic recombinants within the cell culture. Interestingly, the same substitutions showed different effects on different genotypes; for example,

D168G and A156V reduced replication of genotype 1b but had no effect on 4a. Similarly, D168V dramatically reduced the replicative fitness of genotypes 2a and 6a recombinant viruses, but showed no obvious phenotype with genotypes 1b and 4a. In order to determine the precise phenotypic effect on PI susceptibility of mutations developing under antiviral pressure, recombinants with the individual substitutions inserted were assessed for their susceptibility towards BILN 2061 and danoprevir. As expected, those at previously described resistance loci conferred an increase in resistance towards the PIs (Fig. 5), as did each of the newly documented substitutions that developed under treatment pressure investigated in the current study.