Long‐term follow‐up of 11 protease inhibitor (PI)‐naïve and PI‐treated HIV‐infected patients harbouring virus with insertions in the HIV‐1 protease gene

Objectives

Amino acid insertions in the protease gene have been reported rarely, and mainly in patients receiving protease inhibitors (PIs). The aim of the study was to assess the long‐term viro‐immunological follow‐up of HIV‐infected patients harbouring virus with protease insertions.

Methods

Cases of virus exhibiting protease insertions were identified in routine resistance genotyping tests. Therapeutic, immunological and virological data were retrospectively collected.

Results

Eleven patients harbouring virus with a protease gene insertion were detected (prevalence 0.24%), including three PI‐naïve patients. The insertions were mainly located between codons 33 and 39 and associated with surrounding mutations (M36I/L and R41K). The three PI‐naïve patients were infected with an HIV‐1 non‐B subtype. Follow‐up of these PI‐naïve patients showed that the insert‐containing virus persisted for several years, was archived in HIV DNA, and displayed a reduced viral replicative capacity with no impact on resistance level. Of the eight PI‐experienced patients, 63% were infected with HIV‐1 subtype B; one had been antiretroviral‐free for 5 years and seven were heavily PI‐experienced (median duration of follow‐up 24 months; range 10–62 months). The protease insertion was selected under lopinavir in four patients and under darunavir in one, in the context of major PI‐resistance mutations, and following long‐term exposure to PIs. The insert‐containing virus persisted for a median of 32 months (range 12–62 months) and displayed no specific impact on phenotypic resistance level or viral replicative capacity.

Conclusion

Our data, obtained during long‐term follow‐up, show that insertions in the protease gene do not seem to have an impact on resistance level. This finding supports the recommendation of PI‐based regimens, although further work is required to confirm it.     

Important role of 5‐hydroxytryptamine in glucocorticoid‐induced insulin resistance in liver and intra‐abdominal adipose tissue of rats

Aim/Introduction

Both glucocorticoids and 5‐hydroxytryptamine (5‐HT) have been shown to induce insulin resistance (IR) in hepatocytes and adipocytes. Here, we explore whether there is a correlation between them.

Materials and Methods

Except for the control group, male rats were exposed to dexamethasone treated with or without para‐chlorophenylalanine (pCPA), or carbidopa for 20 days. Except for the control group, buffalo rat liver 3A (BRL‐3A) cells were exposed to dexamethasone for 24 h, treated with or without pCPA, carbidopa, or clorgiline for 48 h, or exposed to 5‐HT treated with or without fluoxetine for 48 h. Whole‐body IR was determined by both glucose tolerance test and measurement of fasting blood glucose and insulin, whereas hepatocytes or adipocytes IR was determined by examining either hepatic gluconeogenesis, steatosis and glucose transporter 2 expression or lipolysis.

Results

Dexamethasone‐induced whole‐body IR, liver and intraabdominal adipose IR were accompanied by upregulated expressions of tryptophan hydroxylase‐1 and aromatic amino acid decarboxylase with increased 5‐HT level in both tissues, which were attenuated significantly by pCPA, inhibiting tryptophan hydroxylase‐1, or carbidopa, inhibiting aromatic amino acid decarboxylase. [Correction added on 22 September 2015, after first online publication: ‘inhibiting aromatic amino acid decarboxylase’ was duplicated and has been replaced by ‘tryptophan hydroxylase‐1’.] In the BRL‐3A cells, dexamethasone‐induced IR was also accompanied by upregulated 5‐HT synthesis in dose‐ and time‐dependent manners, and was attenuated by pCPA or carbidopa, but exacerbated by clorgiline, inhibiting monoamine oxidase‐A to further increase 5‐HT level. Dexamethasone also enhanced 5‐HT 2A and 2B receptor expressions in both tissues and BRL‐3A cells. Additionally, blocking 5‐HT transporter with fluoxetine significantly suppressed 5‐HT‐induced IR in BRL‐3A cells.

Conclusion

Enhancement of 5‐HT synthesis in liver and intra‐abdominal adipose is an important reason for glucocorticoids‐induced IR.     

Immunometabolic role of long‐chain omega‐3 fatty acids in obesity‐induced inflammation

Inflammation links obesity with the development of insulin resistance. Macrophages and phagocytic immune cells communicate with metabolic tissues to direct an inflammatory response caused by overnutrition and expanding adipose tissue. Marine‐derived omega‐3 fatty acids, specifically eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), modulate inflammatory signalling events, providing various anti‐inflammatory and cardioprotective benefits. Moreover, EPA and DHA may improve insulin sensitivity by generating proresolving lipid mediators and promoting alternatively activated macrophages. This review will assess the role of EPA and DHA in ameliorating obesity‐induced inflammation, evaluating clinical evidence and mechanisms of action. The pathophysiology of insulin resistance resulting from obesity‐induced inflammation will be discussed, highlighting the relationship between metabolism and immunity, and in particular, how EPA and DHA work with both systems to modulate immunometabolic complications and chronic disease. Copyright © 2013 John Wiley & Sons, Ltd.     

Unique anti‐myeloma activity by thiazolidine‐2,4‐dione compounds with Pim inhibiting activity

Proviral Integrations of Moloney virus 2 (PIM2) is overexpressed in multiple myeloma (MM) cells, and regarded as an important therapeutic target. Here, we aimed to validate the therapeutic efficacy of different types of PIM inhibitors against MM cells for their possible clinical application. Intriguingly, the thiazolidine‐2,4‐dione‐family compounds SMI‐16a and SMI‐4a reduced PIM2 protein levels and impaired MM cell survival preferentially in acidic conditions, in contrast to other types of PIM inhibitors, including AZD1208, CX‐6258 and PIM447. SMI‐16a also suppressed the drug efflux function of breast cancer resistance protein, minimized the sizes of side populations and reduced in vitro colony‐forming capacity and in vivo tumourigenic activity in MM cells, suggesting impairment of their clonogenic capacity. PIM2 is known to be subject to ubiquitination‐independent proteasomal degradation. Consistent with this, the proteasome inhibitors bortezomib and carfilzomib increased PIM2 protein levels in MM cells without affecting its mRNA levels. However, SMI‐16a mitigated the PIM2 protein increase and cooperatively enhanced anti‐MM effects in combination with carfilzomib. Collectively, the thiazolidine‐2,4‐dione‐family compounds SMI‐16a and SMI‐4a uniquely reduce PIM2 protein in MM cells, which may contribute to their profound efficacy in addition to their immediate kinase inhibition. Their combination with proteasome inhibitors is envisioned.     

Analysis of N1‐acetyl‐N2‐formyl‐5‐methoxykynuramine/N1‐acetyl‐5‐methoxy‐kynuramine formation from melatonin in mice

Abstract: The interactions of melatonin, a potent endogenous antioxidant, with reactive oxygen species generate several products that include N¹‐acetyl‐N²‐formyl‐5‐methoxykynuramine (AFMK) and N¹‐acetyl‐5‐methoxy‐kynuramine (AMK). The physiological or pathological significance of AFMK/AMK formation during the process of melatonin metabolism in mammals has not been clarified. Using a metabolomic approach in the current study, the AFMK/AMK pathway was thoroughly investigated both in mice and humans. Unexpectedly, AFMK and AMK were not identified in the urine of humans nor in the urine, feces or tissues (including liver, brain, and eyes) in mice under the current experimental conditions. Metabolomic analysis did identify novel metabolites of AMK, i.e. hydroxy‐AMK and glucuronide‐conjugated hydroxy‐AMK. These two newly identified metabolites were, however, not found in the urine of humans. In addition, oxidative stress induced by acetaminophen in the mouse model did not boost AFMK/AMK formation. These data suggest that AFMK/AMK formation is not a significant pathway of melatonin disposition in mice, even under conditions of oxidative stress.     

Antiviral response and resistance analysis of treatment‐naïve HCV‐infected patients receiving single and multiple doses of GS‐9190

GS‐9190 is a NS5B non‐nucleoside analogue with demonstrated effectiveness in a Phase 1 monotherapy study and in combination with other DAAs for treatment of chronic HCV infection. Here, the resistance profile of GS‐9190 monotherapy in a Phase 1b study was investigated. Resistance analysis was performed by population sequencing and allele‐specific PCR (AS‐PCR) for Y448H with an assay cut‐off of 0.5%. Phenotypic susceptibility analyses were performed on patient isolates as well as site‐directed mutagenesis of mutations selected during monotherapy. No resistance‐associated variants were observed in patients before or after receiving single doses of GS‐9190 by population sequencing. In contrast, in patients who received GS‐9190 for 8 days, mutations Y448H and Y452H in NS5B were observed by population sequencing in 21/36 (58%) and 2/36 (5.6%) patients, respectively, at Day 8 or Day 14. Among the remaining 15 patients who had no detectable Y448H at Day 8 or Day 14 by population sequencing, low frequencies of Y448H ranging from 1.3 to 9.7% were detected in 14 of 15 patients by AS‐PCR. By AS‐PCR, Y448H remained detectable at reduced frequency in the majority of patients analysed through 4–6 months of follow‐up. Chimeric HCV replicons constructed with the NS5B sequence from patients with Y448H and Y448H + Y452H/Y demonstrated 27‐fold and 78.5‐fold reduced susceptibility to GS‐9190. In conclusion, Y448H was rapidly selected in the majority of patients receiving multiple doses of GS‐9190 as monotherapy, despite undetectable levels in pretreatment samples. Y448H confers reduced susceptibility to GS‐9190 and other NNIs and persisted in most patients for months post‐treatment.     

Effects of non‐cytotoxic drugs on the growth of multidrug‐resistance human gastric carcinoma cell line

OBJECTIVE: To investigate the effects of the non‐cytotoxic drug (cyclooxygenase‐2 (COX‐2) inhibitor and octreotide) on growth of the multidrug‐resistant human gastric carcinoma cell line SGC‐7901/ADR. METHODS: The effects of non‐cytotoxic drug on the growth of SGC‐7901 and SGC‐7901/ADR cells were evaluated by ³H‐thymidine incorporation assay. The apoptosis of cells was measured by the TdT‐mediated dUTP nick end‐labeling assay (TUNEL) and flow cytometric assay. Western blotting was used to analysis the expression of cyclooxygenase (COX‐2) protein in SGC‐7901 cells and SGC‐7901/ADR cells and P‐glycoprotein (P‐gp) from SGC‐7901/ADR cells with variable treatments. Activator protein‐1 binding activity was examined by electrophoretic mobility shift assay. RESULTS: ³H‐thymidine incorporation into SGC‐7901/ADR cells treated with celecoxib was significantly lower than that of control group (471.3 ± 79.7 cpm vs 917.5 ± 130.8 cpm, P < 0.05). When combined with octreotide, celecoxib presented lower ³H‐thymidine incorporations than its used alone and decreased to 53.3% of that amount original. Either celecoxib or the combination group markedly induced apoptosis in SGC‐7901/ADR cells. COX‐2 protein in the SGC‐7901/ADR cells was higher than in that of the SGC‐7901 cells (1.543 ± 0.052 vs 0.564 ± 0.021, P < 0.05). The inhibition of P‐gp could be achieved with celecoxib alone and combination with octreotide (0.486 ± 0.012, 0.252 ± 0.014 vs 0.941 ± 0.033, P < 0.05). Moreover, AP‐1 binding activity could be suppressed by non‐cytotoxic drug and showed a synergistic effect. CONCLUSION: The combination of non‐cytotoxic drug significantly improved the inhibitive effects on the growth of multidrug‐resistant human gastric cancer cells.