Refractory hepatic hydrothorax is associated with increased mortality with death occurring at lower MELD-Na compared to cirrhosis and refractory ascites.

BACKGROUND AND AIMS: Although refractory hepatic hydrothorax (RH) is a serious complication of cirrhosis, waitlisted patients do not receive standardized Model for End-stage Liver Disease (MELD) exemption because of inadequate evidence suggesting mortality above biochemical MELD. This study aimed to examine liver-related death (LRD) associated with RH compared to refractory ascites (RA). APPROACH AND RESULTS: This was a retrospective cohort study of Veterans with cirrhosis. Eligibility criteria included participants with RH or RA, followed from their first therapeutic thoracentesis/second paracentesis until death or transplantation. The primary outcome was LRD with non-LRD or transplantation as competing risk. Of 2552 patients with cirrhosis who underwent therapeutic thoracentesis/paracentesis, 177 met criteria for RH and 422 for RA. RH was associated with a significantly higher risk of LRD (adjusted HR [aHR] 4.63, 95% CI 3.31-6.48) than RA overall and within all MELD-sodium (MELD-Na) strata (<10 aHR 4.08, 95% CI 2.30-7.24, 10-14.9 aHR 5.68, 95% CI 2.63-12.28, 15-24.9 aHR 4.14, 95% CI 2.34-7.34, ≥25 aHR 7.75, 95% CI 2.99-20.12). LRD was higher among participants requiring 1 (aHR 3.54, 95% CI 2.29-5.48), 2-3 (aHR 4.39, 95% CI 2.91-6.63), and ≥4 (aHR 7.89, 95% CI 4.82-12.93) thoracenteses relative to RA. Although participants with RH and RA had similar baseline MELD-Na, LRD occurred in RH versus RA at a lower MELD-Na (16.5 vs. 21.82, p =0.002) but higher MELD 3.0 (27.85 vs. 22.48, p <0.0001). CONCLUSIONS: RH was associated with higher risk of LRD than RA at equivalent MELD-Na. By contrast, MELD 3.0 may better predict risk of LRD in RH.

Ursodeoxycholic acid is associated with a reduction in SARS-CoV-2 infection and reduced severity of COVID-19 in patients with cirrhosis.

BACKGROUND AND AIMS: Studies have demonstrated that reducing farnesoid X receptor activity with ursodeoxycholic acid (UDCA) downregulates angiotensin-converting enzyme in human lung, intestinal and cholangiocytes organoids in vitro, in human lungs and livers perfused ex situ, reducing internalization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into the host cell. This offers a potential novel target against coronavirus disease 2019 (COVID-19). The objective of our study was to compare the association between UDCA exposure and SARS-CoV-2 infection, as well as varying severities of COVID-19, in a large national cohort of participants with cirrhosis. METHODS: In this retrospective cohort study among participants with cirrhosis in the Veterans Outcomes and Costs Associated with Liver cohort, we compared participants with exposure to UDCA, with a propensity score (PS) matched group of participants without UDCA exposure, matched for clinical characteristics, and vaccination status. The outcomes included SARS-CoV-2 infection, symptomatic, at least moderate, severe, or critical COVID-19, and COVID-19-related death. RESULTS: We compared 1607 participants with cirrhosis who were on UDCA, with 1607 PS-matched controls. On multivariable logistic regression, UDCA exposure was associated with reduced odds of developing SARS-CoV-2 infection (adjusted odds ratio [aOR] 0.54, 95% confidence interval [CI] 0.41-0.71, p < 0.0001). Among patients who developed COVID-19, UDCA use was associated with reduced disease severity, including symptomatic COVID-19 (aOR 0.54, 95% CI 0.39-0.73, p < 0.0001), at least moderate COVID-19 (aOR 0.51, 95% CI 0.32-0.81, p = 0.005), and severe or critical COVID-19 (aOR 0.48, 95% CI 0.25-0.94, p = 0.03). CONCLUSIONS: In participants with cirrhosis, UDCA exposure was associated with both a decrease in SARS-CoV-2 infection, and reduction in symptomatic, at least moderate, and severe/critical COVID-19.

Role of patient and public involvement in implementation research: a consensus study.

BACKGROUND: Patient and public involvement (PPI) is often an essential requirement for research funding. Distinctions can be drawn between clinical research, which generally focuses on patients, and implementation research, which generally focuses on health professional behaviour. There is uncertainty about the role of PPI in this latter field. We explored and defined the roles of PPI in implementation research to inform relevant good practice guidance. METHODS: We used a structured consensus process using a convenience sample panel of nine experienced PPI and two researcher members. We drew on available literature to identify 21 PPI research roles. The panel rated their agreement with roles independently online in relation to both implementation and clinical research. Disagreements were discussed at a face-to-face meeting prior to a second online rating of all roles. Median scores were calculated and a final meeting held to review findings and consider recommendations. RESULTS: Ten panellists completed the consensus process. For clinical research, there was strong support and consensus for the role of PPI throughout most of the research process. For implementation research, there were eight roles with consensus and strong support, seven roles with consensus but weaker support and six roles with no consensus. There were more disagreements relating to PPI roles in implementation research compared with clinical research. PPI was rated as contributing less to the design and management of implementation research than for clinical research. CONCLUSIONS: The roles of PPI need to be tailored according to the nature of research to ensure authentic and appropriate involvement. We provide a framework to guide the planning, conduct and reporting of PPI in implementation research, and encourage further research to evaluate its use.

Cycloastragenol is a potent telomerase activator in neuronal cells: implications for depression management.

Cycloastragenol (CAG) is an aglycone of astragaloside IV. It was first identified when screening Astragalus membranaceus extracts for active ingredients with antiaging properties. The present study demonstrates that CAG stimulates telomerase activity and cell proliferation in human neonatal keratinocytes. In particular, CAG promotes scratch wound closure of human neonatal keratinocyte monolayers in vitro. The distinct telomerase-activating property of CAG prompted evaluation of its potential application in the treatment of neurological disorders. Accordingly, CAG induced telomerase activity and cAMP response element binding (CREB) activation in PC12 cells and primary neurons. Blockade of CREB expression in neuronal cells by RNA interference reduced basal telomerase activity, and CAG was no longer efficacious in increasing telomerase activity. CAG treatment not only induced the expression of bcl2, a CREB-regulated gene, but also the expression of telomerase reverse transcriptase in primary cortical neurons. Interestingly, oral administration of CAG for 7 days attenuated depression-like behavior in experimental mice. In conclusion, CAG stimulates telomerase activity in human neonatal keratinocytes and rat neuronal cells, and induces CREB activation followed by tert and bcl2 expression. Furthermore, CAG may have a novel therapeutic role in depression.

Astragaloside IV and cycloastragenol stimulate the phosphorylation of extracellular signal-regulated protein kinase in multiple cell types.

Two Chinese herb-derived small molecule telomerase activators, astragaloside IV (AG-IV) and cycloastragenol (CAG), have recently been shown to improve the proliferative response of CD8+ T lymphocytes from HIV-infected patients by upregulating telomerase activity. Here, we examined the signaling mechanism of AG-IV and CAG. Telomerase activity in human embryonic kidney HEK293 fibroblasts was increased upon treatment with increasing concentrations of AG-IV or CAG. Both compounds induced the phosphorylation of extracellular signal-regulated protein kinase (ERK) in a time- and dose-dependent manner in HEK293 cells and HEK-neo keratinocytes. AG-IV and CAG also stimulated ERK phosphorylation in other cell lines of lung, brain, mammary, endothelial, and hematopoietic origins. Use of selective inhibitors and dominant negative mutants revealed the involvement of c-Src, MEK (ERK kinase), and epidermal growth factor receptor in CAG-induced ERK phosphorylation. Our data indicate that AG-IV and CAG may exert their cellular effects through the activation of the Src/MEK/ERK pathway.

In vitro intestinal absorption and first-pass intestinal and hepatic metabolism of cycloastragenol, a potent small molecule telomerase activator.

Cycloastragenol (CAG) is the aglycone derivative of astragaloside IV which has recently been demonstrated to activate telomerase and represents a potential drug candidate for the treatment of degenerative diseases. In the present study, intestinal absorption and metabolism of CAG were examined using the Caco-2 model and liver microsomes, respectively. The results showed that CAG rapidly passes through the Caco-2 cell monolayer by passive diffusion. Four different glucuronide conjugates and two oxidized CAG metabolites were found in the apical and basolateral sides of Caco-2 monolayer, suggesting that first-pass intestinal metabolism of CAG might occur upon passage through the intestinal epithelium. CAG underwent extensive metabolism in rat and human liver microsomes with only 17.4% and 8.2%, respectively, of the starting amount of CAG remaining after 30 min of incubation. Monohydroxylation of the parent and oxidization of the hydroxylated CAG were found in the liver samples. The present study indicates that CAG is efficiently absorbed through intestinal epithelium. However, extensive first-pass hepatic metabolism would limit the oral bioavailability of this compound.

Renew, refuel, and rebuild.

We can renew, refuel, and rebuild America with millions of green jobs, but they must be good jobs. Participants at the conference understand that environmental and economic stability go hand-in-hand, that the challenges of global warming are urgent and that huge opportunities exist for building a clean energy economy.

Telomerase-based pharmacologic enhancement of antiviral function of human CD8+ T lymphocytes.

Telomerase reverse transcribes telomere DNA onto the ends of linear chromosomes and retards cellular aging. In contrast to most normal somatic cells, which show little or no telomerase activity, immune cells up-regulate telomerase in concert with activation. Nevertheless, during aging and chronic HIV-1 infection, there are high proportions of dysfunctional CD8(+) CTL with short telomeres, suggesting that telomerase is limiting. The present study shows that exposure of CD8(+) T lymphocytes from HIV-infected human donors to a small molecule telomerase activator (TAT2) modestly retards telomere shortening, increases proliferative potential, and, importantly, enhances cytokine/chemokine production and antiviral activity. The enhanced antiviral effects were abrogated in the presence of a potent and specific telomerase inhibitor, suggesting that TAT2 acts primarily through telomerase activation. Our study is the first to use a pharmacological telomerase-based approach to enhance immune function, thus directly addressing the telomere loss immunopathologic facet of chronic viral infection.

Telomerase antagonists GRN163 and GRN163L inhibit tumor growth and increase chemosensitivity of human hepatoma.

Most cancer cells have an immortal growth capacity as a consequence of telomerase reactivation. Inhibition of this enzyme leads to increased telomere dysfunction, which limits the proliferative capacity of tumor cells; thus, telomerase inhibition represents a potentially safe and universal target for cancer treatment. We evaluated the potential of two thio-phosphoramidate oligonucleotide inhibitors of telomerase, GRN163 and GRN163L, as drug candidates for the treatment of human hepatoma. GRN163 and GRN163L were tested in preclinical studies using systemic administration to treat flank xenografts of different human hepatoma cell lines (Hep3B and Huh7) in nude mice. The studies showed that both GRN163 and GRN163L inhibited telomerase activity and tumor cell growth in a dose-dependent manner in vitro and in vivo. The potency and efficacy of the lipid-conjugated antagonist, GRN163L, was superior to the nonlipidated parent compound, GRN163. Impaired tumor growth in vivo was associated with critical telomere shortening, induction of telomere dysfunction, reduced rate of cell proliferation, and increased apoptosis in the treatment groups. In vitro, GRN163L administration led to higher prevalence of chromosomal telomere-free ends and DNA damage foci in both hepatoma cell lines. In addition, in vitro chemosensitivity assay showed that pretreatment with GRN163L increased doxorubicin sensitivity of Hep3B. In conclusion, our data support the development of GRN163L, a novel lipidated conjugate of the telomerase inhibitor GRN163, for systemic treatment of human hepatoma. In addition to limiting the proliferative capacity of hepatoma, GRN163L might also increase the sensitivity of this tumor type to conventional chemotherapy.

Lipid modification of GRN163, an N3'-->P5' thio-phosphoramidate oligonucleotide, enhances the potency of telomerase inhibition.

The vast majority of human cancers express telomerase activity, while most human somatic cells do not have detectable telomerase activity. Since telomerase plays a critical role in cell immortality, it is an attractive target for a selective cancer therapy. Oligonucleotides complementary to the RNA template region of human telomerase (hTR) have been shown to be effective inhibitors of telomerase and, subsequently, cancer cell growth in vitro. We show here that a lipid-modified N3'-->P5' thio-phosphoramidate oligonucleotide (GRN163L) inhibits telomerase more potently than its parental nonconjugated thio-phosphoramidate sequence (GRN163). Cells were treated with both the first- (GRN163) and second-generation (GRN163L) oligonucleotides, including a mismatch control, with or without a transfection enhancer reagent. GRN163L inhibited telomerase activity effectively in a dose-dependent manner, even without the use of a transfection reagent. The IC50 values for GRN163 in various cell lines were on average sevenfold higher than for GRN163L. GRN163L inhibition of telomerase activity resulted in a more rapid loss of telomeres and cell growth than GRN163. This report is the first to show that lipid modification enhanced the potency of the novel GRN163 telomerase inhibitor. These results suggest that the lipid-conjugated thio-phosphoramidates could be important for improved pharmacodynamics of telomerase inhibitors in cancer therapy.

Telomerase inhibition with an oligonucleotide telomerase template antagonist: in vitro and in vivo studies in multiple myeloma and lymphoma.

The effects of telomerase inhibition with an oligonucleotide N3' --> P5' thiophosphoramidate (GRN163) complementary to the telomerase template region were examined on human multiple myeloma (MM) and non-Hodgkin lymphoma (NHL) cell lines, primary MM cells, and tumor xenografts. GRN163 treatment reduced telomerase levels in all cells and induced more rapid telomeric shortening. Continuous GRN163 treatment for 7 to 14 days resulted in proliferative arrest, morphologic changes, and apoptosis characteristic of cell crisis in tumor cell lines with short (1.7-5.4 kb) but not long (9-11 kb) telomeres. Intratumoral administration of GRN163 also inhibited the growth of MM and NHL xenografts established from cell lines with short telomeres (Hs602 lymphoma, 2.7 kb; CAG myeloma, 2.7 kb) and increased tumor apoptosis. However, GRN163 therapy of NHL xenografts established from cells with long telomeres (11.0 kb) had equivocal effects on tumor growth and did not induce apoptosis during this time frame. Systemic daily intraperitoneal administration of GRN163 in myeloma xenografts with short telomere lengths also decreased tumor telomerase levels and reduced tumor volumes. These data demonstrate that telomerase is important for the replication of mature B-cell neoplasia by stabilizing short telomeres, and they suggest that telomerase inhibition represents a novel therapeutic approach to MM and NHL.

Oligonucleotide N3' --> P5' thio-phosphoramidate telomerase template antagonists as potential anticancer agents.

Human telomerase is a reverse transcriptase that is expressed in essentially all cancer cells, but not in the vast majority of normal somatic cells. Therefore, the specific inhibition of telomerase activity in tumors might have significant beneficial therapeutic effects. We have designed and evaluated oligonucleotide N3' --> P5' thio-phosphoramidates as telomerase template antagonists. In biochemical cell-free assays 11-13-mer thio-phosphoramidate oligonucleotides demonstrated sequence specific and dose dependent inhibition of telomerase with pico-molar IC50 values. Optimization of the oligonucleotide sequence and length resulted in the identification of a 13-mer-oligonucleotide thio-phosphoramidate GRN163 as a drug development candidate. In cell cultures GRN163 was able to inhibit telomerase activity in the absence of cationic lipid with approximately 1 microM IC50 values. Telomerase inhibition by GRN163 produced gradual telomere shortening, followed by cellular senescence and/or apoptosis of cancer derived cell lines.

Novel short oligonucleotide conjugates as inhibitors of human telomerase.

A series of oligonucleotide conjugates were designed and synthesized as novel inhibitors of human telomerase. These compounds contain a relatively short (6-7-mer) oligonucleotide domain, with an N3'-->P5' phosphoramidate (np) or thio-phosphoramidate (nps) backbone, targeted to the template region of the RNA component of the enzyme and various pendant groups attached to either their 5'- or preferably to the 3'-termini. The most potent compounds in the series inhibited telomerase with low nM IC50 values in biochemical assays whereas the cognate oligonucleotides without the pendant groups were significantly less active having IC50 values 100-1000-fold higher.

A novel telomerase template antagonist (GRN163) as a potential anticancer agent.

Telomerase, the enzyme responsible for proliferative immortality, is expressed in essentially all cancer cells, but not in most normal human cells. Thus, specific telomerase inhibition is potentially a universal anticancer therapy with few side effects. We designed N3'-->P5' thio-phosphoramidate (NPS) oligonucleotides as telomerase template antagonists and found that their ability to form stable duplexes with the telomerase RNA subunit was the key factor for antitelomerase activity. In biochemical assays 11-13-mer NPS oligonucleotides demonstrated sequence- and dose-dependent inhibition of telomerase with IC(50) values <1 nM. Optimization of the sequence, length, and bioavailability resulted in the selection of a 13-mer NPS oligonucleotide, GRN163, as a drug development candidate. GRN163 inhibited telomerase in a cell-free assay at 45 +/- 7 pM, and in various tumor cell lines at approximately 1 nM and approximately 0.3-1.0 micro M in the presence and absence of carriers, respectively. GRN163 was competitive with telomeric primer binding, primarily because of hybridization to human telomerase RNA (hTR) component. Tumor cells treated with GRN163 in culture underwent telomere shortening, followed by cellular senescence or apoptosis after a period of time that generally correlated with initial telomere length. In a flank DU145 (prostate cancer) xenograft model, parenterally administered GRN163 caused suppression of tumor growth in the absence of gross toxicity. These data demonstrate that GRN163 has significant potential for additional development as an anticancer agent.

Electrophysiologic properties of channels induced by Abeta25-35 in planar lipid bilayers.

Abeta25-35, a fragment of the neurotoxic amyloid beta protein Abeta1-42 found in the brain of Alzheimer patients, possesses amyloidogenic, neurotoxins and channel forming abilities similar to that of Abeta1-42. We have previously reported that Abeta25-35 formed voltage-dependent, relatively nonselective, ion-permeable channels in planar lipid bilayers. Here, we show that Abeta25-35 formed channels in both solvent-containing and solvent-free bilayers. We also report that for Abeta25-35, channel forming activity was dependent on ionic strength, membrane lipid composition, and peptide concentration, but not on pH. Lower ionic strength and negatively charged lipids increased channel formation activity, while cholesterol decreased activity. The nonlinear function relating [Abeta25-35] and membrane activity suggests that aggregation of at least three monomers is required for channel formation.