Investigation of parenteral nutrition-induced hepatotoxicity using human liver spheroid co-cultures.

Parenteral nutrition (PN) is typically administered to individuals with gastrointestinal dysfunction, a contraindication for enteral feeding, and a need for nutritional therapy. When PN is the only energy source in patients, it is defined as total parenteral nutrition (TPN). TPN is a life-saving approach for different patient populations, both in infants and adults. However, despite numerous benefits, TPN can cause adverse effects, including metabolic disorders and liver injury. TPN-associated liver injury, known as intestinal failure-associated liver disease (IFALD), represents a significant problem affecting up to 90% of individuals receiving TPN. IFALD pathogenesis is complex, depending on the TPN components as well as on the patient's medical conditions. Despite numerous animal studies and clinical observations, the molecular mechanisms driving IFALD remain largely unknown. The present study was set up to elucidate the mechanisms underlying IFALD. For this purpose, human liver spheroid co-cultures were treated with a TPN mixture, followed by RNA sequencing analysis. Subsequently, following exposure to TPN and its single nutritional components, several key events of liver injury, including mitochondrial dysfunction, endoplasmic reticulum stress, oxidative stress, apoptosis, and lipid accumulation (steatosis), were studied using various techniques. It was found that prolonged exposure to TPN substantially changes the transcriptome profile of liver spheroids and affects multiple metabolic and signaling pathways contributing to liver injury. Moreover, TPN and its main components, especially lipid emulsion, induce changes in all key events measured and trigger steatosis.

Determination of structural features that underpin the pannexin1 channel inhibitory activity of the peptide 10Panx1.

Pannexin1 channels facilitate paracrine communication and are involved in a broad spectrum of diseases. Attempts to find appropriate pannexin1 channel inhibitors that showcase target-selective properties and in vivo applicability remain nonetheless scarce. However, a promising lead candidate, the ten amino acid long peptide mimetic 10Panx1 (H-Trp1-Arg2-Gln3-Ala4-Ala5-Phe6-Val7-Asp8-Ser9-Tyr10-OH), has shown potential as a pannexin1 channel inhibitor in both in vitro and in vivo studies. Nonetheless, structural optimization is critical for clinical use. One of the main hurdles to overcome along the optimization process consists of subduing the low biological stability (10Panx1 t1/2 = 2.27 ± 0.11 min). To tackle this issue, identification of important structural features within the decapeptide structure is warranted. For this reason, a structure-activity relationship study was performed to proteolytically stabilize the sequence. Through an Alanine scan, this study demonstrated that the side chains of Gln3 and Asp8 are crucial for 10Panx1's channel inhibitory capacity. Guided by plasma stability experiments, scissile amide bonds were identified and stabilized, while extracellular adenosine triphosphate release experiments, indicative of pannexin1 channel functionality, allowed to enhance the in vitro inhibitory capacity of 10Panx1.

Massive Release of CD9+ Microvesicles in Human Immunodeficiency Virus Infection, Regardless of Virologic Control.

BACKGROUND: The role of extracellular vesicles (EVs) in human immunodeficiency virus (HIV) pathogenesis is unknown. We examine the cellular origin of plasma microvesicles (MVs), a type of ectocytosis-derived EV, the presence of mitochondria in MVs, and their relationship to circulating cell-free mitochondrial deoxyribonucleic acid (ccf-mtDNA) in HIV-infected patients and controls. METHODS: Five participant groups were defined: 30 antiretroviral therapy (ART)-naive; 30 ART-treated with nondetectable viremia; 30 elite controllers; 30 viremic controllers; and 30 HIV-uninfected controls. Microvesicles were quantified and characterized from plasma samples by flow cytometry. MitoTrackerDeepRed identified MVs containing mitochondria and ccf-mtDNA was quantified by real-time polymerase chain reaction. RESULTS: Microvesicle numbers were expanded at least 10-fold in all HIV-infected groups compared with controls. More than 79% were platelet-derived MVs. Proportions of MVs containing mitochondria (22.3% vs 41.6%) and MV mitochondrial density (706 vs 1346) were significantly lower among HIV-infected subjects than controls, lowest levels for those on ART. Microvesicle numbers correlated with ccf-mtDNA levels that were higher among HIV-infected patients. CONCLUSIONS: A massive release of platelet-derived MVs occurs during HIV infection. Some MVs contain mitochondria, but their proportion and mitochondrial densities were lower in HIV infection than in controls. Platelet-derived MVs may be biomarkers of platelet activation, possibly reflecting pathogenesis even in absence of HIV replication.

Effects of Drugs Formerly Suggested for COVID-19 Repurposing on Pannexin1 Channels.

Although many efforts have been made to elucidate the pathogenesis of COVID-19, the underlying mechanisms are yet to be fully uncovered. However, it is known that a dysfunctional immune response and the accompanying uncontrollable inflammation lead to troublesome outcomes in COVID-19 patients. Pannexin1 channels are put forward as interesting drug targets for the treatment of COVID-19 due to their key role in inflammation and their link to other viral infections. In the present study, we selected a panel of drugs previously tested in clinical trials as potential candidates for the treatment of COVID-19 early on in the pandemic, including hydroxychloroquine, chloroquine, azithromycin, dexamethasone, ribavirin, remdesivir, favipiravir, lopinavir, and ritonavir. The effect of the drugs on pannexin1 channels was assessed at a functional level by means of measurement of extracellular ATP release. Immunoblot analysis and real-time quantitative reversetranscription polymerase chain reaction analysis were used to study the potential of the drugs to alter pannexin1 protein and mRNA expression levels, respectively. Favipiravir, hydroxychloroquine, lopinavir, and the combination of lopinavir with ritonavir were found to inhibit pannexin1 channel activity without affecting pannexin1 protein or mRNA levels. Thusthree new inhibitors of pannexin1 channels were identified that, though currently not being used anymore for the treatment of COVID-19 patients, could be potential drug candidates for other pannexin1-related diseases.

Effects of Drugs Formerly Proposed for COVID-19 Treatment on Connexin43 Hemichannels.

Connexin43 (Cx43) hemichannels form a pathway for cellular communication between the cell and its extracellular environment. Under pathological conditions, Cx43 hemichannels release adenosine triphosphate (ATP), which triggers inflammation. Over the past two years, azithromycin, chloroquine, dexamethasone, favipiravir, hydroxychloroquine, lopinavir, remdesivir, ribavirin, and ritonavir have been proposed as drugs for the treatment of the coronavirus disease 2019 (COVID-19), which is associated with prominent systemic inflammation. The current study aimed to investigate if Cx43 hemichannels, being key players in inflammation, could be affected by these drugs which were formerly designated as COVID-19 drugs. For this purpose, Cx43-transduced cells were exposed to these drugs. The effects on Cx43 hemichannel activity were assessed by measuring extracellular ATP release, while the effects at the transcriptional and translational levels were monitored by means of real-time quantitative reverse transcriptase polymerase chain reaction analysis and immunoblot analysis, respectively. Exposure to lopinavir and ritonavir combined (4:1 ratio), as well as to remdesivir, reduced Cx43 mRNA levels. None of the tested drugs affected Cx43 protein expression.

Expression and Functionality of Connexin-Based Channels in Human Liver Cancer Cell Lines.

Liver cancer cell lines are frequently used in vitro tools to test candidate anti-cancer agents as well as to elucidate mechanisms of liver carcinogenesis. Among such mechanisms is cellular communication mediated by connexin-based gap junctions. The present study investigated changes in connexin expression and gap junction functionality in liver cancer in vitro. For this purpose, seven human liver cancer cell lines, as well as primary human hepatocytes, were subjected to connexin and gap junction analysis at the transcriptional, translational and activity level. Real-time quantitative reverse transcription polymerase chain reaction analysis showed enhanced expression of connexin43 in the majority of liver cancer cell lines at the expense of connexin32 and connexin26. Some of these changes were paralleled at the protein level, as evidenced by immunoblot analysis and in situ immunocytochemistry. Gap junctional intercellular communication, assessed by the scrape loading/dye transfer assay, was generally low in all liver cancer cell lines. Collectively, these results provide a full scenario of modifications in hepatocyte connexin production and gap junction activity in cultured liver cancer cell lines. The findings may be valuable for the selection of neoplastic hepatocytes for future mechanistic investigation and testing of anti-cancer drugs that target connexins and their channels.

In Vitro Liver Toxicity Testing of Chemicals: A Pragmatic Approach.

The liver is among the most frequently targeted organs by noxious chemicals of diverse nature. Liver toxicity testing using laboratory animals not only raises serious ethical questions, but is also rather poorly predictive of human safety towards chemicals. Increasing attention is, therefore, being paid to the development of non-animal and human-based testing schemes, which rely to a great extent on in vitro methodology. The present paper proposes a rationalized tiered in vitro testing strategy to detect liver toxicity triggered by chemicals, in which the first tier is focused on assessing general cytotoxicity, while the second tier is aimed at identifying liver-specific toxicity as such. A state-of-the-art overview is provided of the most commonly used in vitro assays that can be used in both tiers. Advantages and disadvantages of each assay as well as overall practical considerations are discussed.

Targeting gap junctional intercellular communication by hepatocarcinogenic compounds.

Gap junctions in liver, as in other organs, play a critical role in tissue homeostasis. Inherently, these cellular constituents are major targets for systemic toxicity and diseases, including cancer. This review provides an overview of chemicals that compromise liver gap junctions, in particular biological toxins, organic solvents, pesticides, pharmaceuticals, peroxides, metals and phthalates. The focus in this review is placed upon the mechanistic scenarios that underlie these adverse effects. Further, the potential use of gap junctional activity as an in vitro biomarker to identify non-genotoxic hepatocarcinogenic chemicals is discussed.

Cholestasis Differentially Affects Liver Connexins.

Connexins are goal keepers of tissue homeostasis, including in the liver. As a result, they are frequently involved in disease. The current study was set up to investigate the effects of cholestatic disease on the production of connexin26, connexin32 and connexin43 in the liver. For this purpose, bile duct ligation, a well-known trigger of cholestatic liver injury, was applied to mice. In parallel, human hepatoma HepaRG cell cultures were exposed to cholestatic drugs and bile acids. Samples from both the in vivo and in vitro settings were subsequently subjected to assessment of mRNA and protein quantities as well as to in situ immunostaining. While the outcome of cholestasis on connexin26 and connexin43 varied among experimental settings, a more generalized repressing effect was seen for connexin32. This has also been observed in many other liver pathologies and could suggest a role for connexin32 as a robust biomarker of liver disease and toxicity.

Prevalence of drug resistance mutations among ART-naive and -experienced HIV-infected patients in Sierra Leone.

OBJECTIVES: The aim of this study was to assess the prevalence of HIV drug resistance (HIVDR) in HIV-infected ART-naive and -experienced patients in Sierra Leone. PATIENTS AND METHODS: We conducted a cross-sectional study of HIV-positive adults aged ≥18 years at Connaught Hospital in Freetown, Sierra Leone in November 2017. Sequencing was performed in the reverse transcriptase, protease and integrase regions, and interpreted using the Stanford HIVDR database and WHO 2009 mutation list. RESULTS: Two hundred and fifteen HIV-infected patients were included (64 ART naive and 151 ART experienced). The majority (66%) were female, the median age was 36 years and the median ART exposure was 48 months. The majority (83%) were infected with HIV-1 subtype CRF02_AG. In the ART-naive group, the pretreatment drug resistance (PDR) prevalence was 36.7% (14.2% to NRTIs and 22.4% to NNRTIs). The most prevalent PDR mutations were K103N (14.3%), M184V (8.2%) and Y181C (4.1%). In the ART-experienced group, 64.4% harboured resistance-associated mutations (RAMs) and the overall prevalence of RAMs to NRTIs and NNRTIs was 85.2% (52/61) and 96.7% (59/61), respectively. The most prevalent RAMs were K103N (40.7%), M184V (28.8%), D67N (15.3%) and T215I/F/Y (15.3%). Based on the genotypic susceptibility score estimates, 22.4% of ART-naive patients and 56% of ART-experienced patients were not susceptible to first-line ART used in Sierra Leone. CONCLUSIONS: A high prevalence of circulating NRTI- and NNRTI-resistant variants was observed in ART-naive and -experienced HIV-1-infected patients in Sierra Leone. This necessitates the implementation of HIVDR surveillance programmes to inform national ART guidelines for the treatment and monitoring of HIV-infected patients in Sierra Leone.

Prevalence of NS5A resistance associated substitutions in patients with hepatitis C virus genotypes 1a and 3: Impact on current therapeutic strategies.

The presence of resistance-associated substitutions (RASs) at NS5A region might compromise the efficacy of Direct Acting Antiviral agents (DAAs). HCV resistance at NS5A region is mainly focused on patients with hepatitis C virus (HCV) genotypes 1a (G1a) and 3 (G3) with other factors of poor treatment response (ie cirrhosis, prior treatment-exposure, or HCV-RNA >800 000 IU/mL). Herein, we evaluated in a cohort of HCV G1a and G3 infected patients the prevalence of RASs at domain I NS5A using population-based sequencing and the impact of RASs on the optimization of current therapeutic strategies. The RASs considered as clinically relevant were: M28A/G/T, Q30D/E/H/G/K/L/R, L31M/V/F, H58D, and Y93C/H/N/S for G1a and Y93H for G3. A total of 232 patients naïve to NS5A inhibitors were included (166 G1a, 66 G3). The overall prevalence of NS5A RASs for G1a and G3 patients was low (5.5%) or null, respectively. A high proportion of patients harbored, at least, one factor of poor response (78.9% for G1a, and 75.8% for G3). Overall, the rates of patients harboring NS5A RASs in combination with any of the other factors were low and the vast majority of patients (G1a> 94% and G3 100%) could be treated with standard treatments of 12 weeks without ribavirin. In conclusion, testing NS5A RASs in specific HCV-infected populations (ie G1a & G3, cirrhosis, prior treatment experienced, HCV-RNA >800 000 IU/mL) might be useful to optimize current NS5A-based therapies avoiding ribavirin-related toxicities, and shortening treatment duration in the majority of patients.

Plasma mitochondrial DNA levels are inversely associated with HIV-RNA levels and directly with CD4 counts: potential role as a biomarker of HIV replication.

OBJECTIVES: To evaluate plasma mitochondrial DNA (mtDNA) levels among HIV-infected patients and its potential role as a biomarker of residual viral replication. METHODS: HIV-infected patients on follow-up at a reference hospital in north-west Spain were selected. DNA was isolated from plasma samples and mtDNA levels were assessed using a quantitative real-time PCR assay. HIV-RNA levels and CD4+ cell counts were evaluated in the same blood samples used for plasma mtDNA quantification. Epidemiological and clinical variables were included for the analysis. RESULTS: A total of 235 HIV-infected patients were included. Mean plasma mtDNA levels were 217 ±âŸ656 copies/µL for naive (31.9%) and 364 ±âŸ939 copies/µL for HIV-infected patients receiving ART and with suppressed viraemia (P = 0.043). Among the latter, mean plasma mtDNA levels were 149 ±âŸ440 copies/µL for those with low-level viraemia (LLV; HIV-RNA 20-200 copies/mL), 265 ±âŸ723 copies/µL for those with detected-not-quantified (DNQ) viraemia (HIV-RNA <20 copies/mL) and 644 ±âŸ1310 copies/µL for those with not-detected (ND) viraemia. Of note, a linear trend (P = 0.006) was observed among virologically suppressed (LLV, DNQ and ND) patients. ND patients had higher mtDNA levels compared with LLV patients (P = 0.057). Moreover, mtDNA levels were inversely associated with HIV-RNA levels (Spearman's rho -0.191, P = 0.003) and directly associated with CD4+ counts (Spearman's rho 0.131, P = 0.046). CONCLUSIONS: Increased plasma mtDNA levels are associated with lower HIV-RNA levels and higher CD4+ cell counts. Among ART-suppressed patients, mtDNA levels were significantly higher in those with complete virological suppression (ND) than in those with LLV. These data suggest that plasma mtDNA levels might serve as a biomarker of residual HIV replication.

Characterization of chronic HCV infection in Northwest Spain: Impact of the treatment strategic plan of the Spanish National Health Service on HCV cure.

The aim of the study was to characterize HCV infection in Northwest Spain and assess the impact of the Spanish Strategic Plan to cure HCV infection. Overall, 387 patients were included (60.9% HIV/HCV coinfected and 28.2% cirrhotic). Of these, 72.9% of patients that were recognized as priority for HCV treatment according to the Spanish Strategic Plan (≥F2, transplant or extrahepatic manifestations), initiated treatment during 2015. Globally, SVR12 was achieved in 96.5% of patients. The implementation of the Spanish Strategic Plan has been critical to advance in HCV cure, but 27.1% of priority patients still remain awaiting HCV treatment initiation.

Any impact of blips and low-level viraemia episodes among HIV-infected patients with sustained virological suppression on ART?

OBJECTIVES: The objective of this study was to evaluate the prevalence of blips and risk of virological failure (VF) among HIV-infected patients with sustained virological suppression (HIV-RNA <50 copies/mL) on ART. METHODS: Newly diagnosed (2004-13) HIV-infected patients with sustained virological suppression on ART (minimum follow-up of 3 months) were identified. Risk of VF was evaluated according to different plasma HIV-RNA quantification values based on the limits of quantification/detection of current commercial assays (20 copies/mL). Kaplan-Meier and Cox proportional hazards models were used to compare the cumulative incidence of VF. RESULTS: A total of 565 newly diagnosed HIV-infected patients were identified: 453 started ART and 354 achieved virological suppression. Prevalence of blips (isolated HIV-RNA ranging from 50 to 200 copies/mL) and VF (HIV-RNA ≥50 copies/mL) was 22.7% and 8.8%, respectively (mean follow-up of 42 months). Multivariate analysis identified differences between HIV-RNA values as an independent predictor of VF (P = 0.008); risk of VF was higher for patients with blips [HR 2.500 (95% CI 0.524-11.926)] and for those with at least three consecutive detected, but not quantified, HIV-RNA determinations (HIV-RNA <20 copies/mL) [HR 3.813 (95% CI 0.675-21.535)]. Moreover, only HIV-infected patients with at least three consecutive detected, but not quantified, HIV-RNA determinations showed a higher probability of virological rebound with >200 copies/mL [33.7% at 24 and 60 months versus <5% for other HIV-RNA values; HR 6.943 (0.728-66.261), P = 0.092]. CONCLUSIONS: Blips are frequent (22.7%) among HIV-infected patients with sustained virological suppression on ART. HIV patients with blips and at least three consecutive detected, but not quantified, HIV-RNA determinations (<20 copies/mL) had a higher risk of VF. These findings highlight the relevance of maintaining HIV-RNA levels below the limits of quantification of current assays (<20 copies/mL).

Long-term clinical experience with darunavir (2007-2015) in a large cohort of HIV-infected patients in Spain.

The clinical experience with the protease inhibitor darunavir/ritonavir (DRV/r) was retrospectively evaluated in a cohort of 173 HIV+ patients who initiated antiretroviral treatment including DRV/r (period 2007-2015). The 43.2% had a CD4 nadir ≤100 cells/mm3 , 64.1% were treatment-experienced, and 36.5% had failed to >3 lines of antiretroviral therapy. Nonetheless, the rate of virological suppression (HIV-RNA <50 copies/ml) in naïve patients was 63%, 66.7%, and 63.6% at 48, 96, and 144 weeks, respectively. The rate of virological suppression in treatment-experienced patients was 62.7%, 78.7%, and 79.1% at 48, 96, and 144 weeks, respectively. No differences were observed according to the immunovirological status neither dosage of DRV/r. Most of them (82.6%) maintained DRV/r treatment. Causes for DRV/r discontinuation were mainly gastrointestinal and cutaneous adverse events (10.5%), switch to simplification treatment strategies (3.5%) and virological failure (1.7%). These findings demonstrate the prolonged efficacy and tolerability of DRV/r even in multi-treated HIV+ patients with an unfavorable immunovirological status. J. Med. Virol. 88:2125-2131, 2016. © 2016 Wiley Periodicals, Inc.

Assessment of Connexin43 Hemichannel Functionality Based on Cytosolic Uptake of Yo-Pro1.

Connexin proteins are the building blocks of gap junctions and connexin hemichannels. Both provide a pathway for cellular communication. Gap junctions support intercellular communication mechanisms and regulate homeostasis. In contrast, open connexin hemichannels connect the intracellular compartment and the extracellular environment, and their activation fuels inflammation and cell death. The development of clinically applicable connexin hemichannel blockers for therapeutic purposes is therefore gaining momentum. This chapter describes a well-established protocol optimized for assessing connexin hemichannel activity by using the reporter dye Yo-Pro1.

Pannexin1 channels in the liver: an open enemy.

Pannexin1 proteins form communication channels at the cell plasma membrane surface, which allow the transfer of small molecules and ions between the intracellular compartment and extracellular environment. In this way, pannexin1 channels play an important role in various cellular processes and diseases. Indeed, a plethora of human pathologies is associated with the activation of pannexin1 channels. The present paper reviews and summarizes the structure, life cycle, regulation and (patho)physiological roles of pannexin1 channels, with a particular focus on the relevance of pannexin1 channels in liver diseases.

Structure-Based Design and Synthesis of Stapled 10Panx1 Analogues for Use in Cardiovascular Inflammatory Diseases.

Following a rational design, a series of macrocyclic ("stapled") peptidomimetics of 10Panx1, the most established peptide inhibitor of Pannexin1 (Panx1) channels, were developed and synthesized. Two macrocyclic analogues SBL-PX1-42 and SBL-PX1-44 outperformed the linear native peptide. During in vitro adenosine triphosphate (ATP) release and Yo-Pro-1 uptake assays in a Panx1-expressing tumor cell line, both compounds were revealed to be promising bidirectional inhibitors of Panx1 channel function, able to induce a two-fold inhibition, as compared to the native 10Panx1 sequence. The introduction of triazole-based cross-links within the peptide backbones increased helical content and enhanced in vitro proteolytic stability in human plasma (>30-fold longer half-lives, compared to 10Panx1). In adhesion assays, a "double-stapled" peptide, SBL-PX1-206 inhibited ATP release from endothelial cells, thereby efficiently reducing THP-1 monocyte adhesion to a TNF-α-activated endothelial monolayer and making it a promising candidate for future in vivo investigations in animal models of cardiovascular inflammatory disease.

Connexin-Based Channels in the Liver.

Connexin proteins oligomerize in hexameric structures called connexin hemichannels, which then dock to form gap junctions. Gap junctions direct cell-cell communication by allowing the exchange of small molecules and ions between neighboring cells. In this way, hepatic gap junctions support liver homeostasis. Besides serving as building blocks for gap junctions, connexin hemichannels provide a pathway between the intracellular and the extracellular environment. The activation of connexin hemichannels is associated with acute and chronic liver pathologies. This article discusses the role of gap junctions and connexin hemichannels in the liver. © 2022 American Physiological Society. Compr Physiol 12:1-17, 2022.

Characterizing HIV-1 Genetic Subtypes and Drug Resistance Mutations among Children, Adolescents and Pregnant Women in Sierra Leone.

Human immunodeficiency virus (HIV) drug resistance (HIVDR) is widespread in sub-Saharan Africa. Children and pregnant women are particularly vulnerable, and laboratory testing capacity remains limited. We, therefore, used a cross-sectional design and convenience sampling to characterize HIV subtypes and resistance-associated mutations (RAMs) in these groups in Sierra Leone. In total, 96 children (age 2-9 years, 100% ART-experienced), 47 adolescents (age 10-18 years, 100% ART-experienced), and 54 pregnant women (>18 years, 72% ART-experienced) were enrolled. Median treatment durations were 36, 84, and 3 months, respectively, while the sequencing success rates were 45%, 70%, and 59%, respectively, among children, adolescents, and pregnant women. Overall, the predominant HIV-1 subtype was CRF02_AG (87.9%, 95/108), with minority variants constituting 12%. Among children and adolescents, the most common RAMs were M184V (76.6%, n = 49/64), K103N (45.3%, n = 29/64), Y181C/V/I (28.1%, n = 18/64), T215F/Y (25.0%, n = 16/64), and V108I (18.8%, n = 12/64). Among pregnant women, the most frequent RAMs were K103N (20.6%, n = 7/34), M184V (11.8%, n = 4/34), Y181C/V/I (5.9%, n = 2/34), P225H (8.8%, n = 3/34), and K219N/E/Q/R (5.9%, n = 2/34). Protease and integrase inhibitor-RAMs were relatively few or absent. Based on the genotype susceptibility score distributions, 73%, 88%, and 14% of children, adolescents, and pregnant women, respectively, were not susceptible to all three drug components of the WHO preferred first-line regimens per 2018 guidelines. These findings suggest that routine HIVDR surveillance and access to better ART choices may improve treatment outcomes in Sierra Leone.