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Background: Klebsiella quasipneumoniae (previously known as K. pneumoniae K6) strains are among the multidrug-resistant hypervirulent bacterial pathogens. Phage therapy can help treat infections caused by such pathogens. Here we report some aspects of virology and therapeutic potentials of vB_KquU_φKuK6, a bacteriophage that infects Klebsiella quasipneumoniae., Methods: K. quasipneumoniae (ATCC 700603) was used to screen wastewater lytic phages. The isolate vB_KquU_φKuK6 that consistently created large clear plaques was characterized using standard virological and molecular methods., Results: vB_KquU_φKuK6 has a complex capsid with an icosahedral head (~60 nm) and a slender tail (~140 nm × 10 nm). The phage has a 51% AT-rich linear dsDNA genome (51,251 bp) containing 121 open reading frames. The genome contains genes encoding spanin, endolysin, and holin proteins necessary for lytic infection and a recombinase gene possibly involved in lysogenic infection. vB_KquU_φKuK6 is stable at -80 to +67°C, pH 4-9, and brief exposure to one volume percent of chloroform. vB_KquU_φKuK6 has a narrow host range. Its lytic infection cycle involves a latency of 20 min and a burst size of 435 plaque-forming units. The phage can cause lysogenic infection, and the resulting lysogens are resistant to lytic infection by vB_KquU_φKuK6. vB_KquU_φKuK6 reduces the host cells' ability to form biofilm but fails to eliminate that ability. vB_KquU_φKuK6 demonstrates phage-antibiotic synergy and reduces the minimum inhibitory concentration of chloramphenicol and neomycin sulfate by about 8 folds., Conclusion: vB_KquU_φKuK6 cannot be directly used for phage therapy because it is a temperate bacteriophage. However, genetically modified strains of vB_KquU_φKuK6 alone or combined with antibiotics or other lytic Klebsiella phages can have therapeutic utilities in treating K. quasipneumoniae infections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Miller, Laney, Zahn, Sheehan, Whitley and Kuddus.)

BACKGROUND The COVID-19 pandemic had multifaceted and disproportionate impacts on various countries. We investigated the decline of heart and kidney transplantation in 2020 and recovery trends in 2020, 2021, and 2022 in 30 developed and developing countries, considering COVID-19 incidence and mortality and pandemic-time economic variables. MATERIAL AND METHODS Data were obtained from reliable open databases. Nations were grouped by hierarchical cluster analysis into high-gross domestic product (GDP), mid-GDP, and low-GDP countries. Expected transplant numbers for 2020 to 2021 were estimated by the artificial neural network method using data from 2015 to 2019. Effect size and its inference were determined through the Hodges-Lemann estimate and Wilcoxon signed-rank test, respectively. The possible disproportionate effect was estimated by the Jonckheere-Tersptra test. Associations between transplantation and economic variables, COVID-19 caseload, and mortality were examined using Kendall rank correlation analysis. RESULTS All nations experienced a decline in 2020 and some real recovery in 2020 to 2022. For high-GDP countries, decline was insignificant and recovery was marginal; for mid-GDP countries, decline was significant for heart and deceased kidneys and recovery was modest; for low-GDP countries, decline was significant for heart, live kidneys, and deceased kidneys and recovery was marginal. The low-GDP countries were disproportionally negatively impacted, although the associations between the impact and economic variables, COVID-19 incidence, and COVID-19 mortality were statistically insignificant. CONCLUSIONS More inclusive studies of socioeconomic and cultural factors that affected the impact of the COVID-19 pandemic in different countries can be useful for better preparedness and reducing disruption in healthcare in future global pandemics.

Background: Angiotensin I converting enzyme (ACE) insertion (I) and 287 bp Alu repeat DNA fragment deletion (D) polymorphisms have been indicated in various cancers. Here, we investigated I/D polymorphisms in prostate cancer (PCa) and benign prostate hyperplasia (BPH) among Lebanese men., Methods: Blood DNA extracted from 69 control subjects, 69 subjects with clinically confirmed PCa, and 69 subjects with clinical BPH, all the subjects were aged 50 years or older, was subjected to the polymerase chain reaction. The PCR products were resolved in polyacrylamide gels to determine II, ID, and DD genotypes. The odds ratios (OR), 95% confidence intervals (CI), and p values of the allele frequencies and genotype ratios were calculated for establishing possible association of the alleles and/or genotypes and PCa and/or BPH., Results: The proportions of II, ID, and DD genotypes were significantly different from Hardy-Weinberg equilibrium for BPH and PCa groups (but not the control group), mostly due to overabundance of the ID genotypes. There was no significant difference in the I and D allele frequencies between the control groups and the affected groups. The ratio of (DD + ID)/II is significantly lower among the control group compared to the BPH group (RR = 8.92, p values of the allele frequencies and genotype ratios were calculated for establishing possible association of the alleles and/or genotypes and PCa and/or BPH. p values of the allele frequencies and genotype ratios were calculated for establishing possible association of the alleles and/or genotypes and PCa and/or BPH., Conclusions: Our data indicate that the D allele of the I/D polymorphisms of the ACE gene is associated with increased risk of BPH, and the ID genotype is a risk factor for both BPH and PCa among Lebanese males., Competing Interests: The authors declare that there are no conflicts of interest regarding the publication of this work., (Copyright © 2020 Asmahan A. El Ezzi et al.)

Background: A severe shortage in donor organs is the major driver for organ transplantation-related crimes. The Declaration of Istanbul 2008 (DOI) was created to stop such crimes. We investigated the impact of DOI on Internet reporting of transplantation-related crimes., Methods: We conducted Google Advanced Searches to collect data on "kidney trade," "kidney sale," "organ trafficking," and "transplant tourism" in 15 original participant and 10 nonparticipant countries, 6 years prior through 8 years after the promulgation of DOI. The data were normalized for population and transformed to a logarithmic scale. Interrupted time series analysis (ITSA) was applied to estimate the changes in slopes of the outcome variables before and after DOI, and then the overall intervention impact was calculated by meta-analysis., Results: The combined results indicated that the overall impact of DOI on the reporting of "organ trafficking" and "transplant tourism" was statistically negative (reporting reduced significantly) as intended but on "kidney sale" and "kidney trade" was statistically positive (reporting increased significantly), and the increase was higher in the nonparticipant countries compared to the participant countries. The rate of reporting on "transplant tourism" declined in the participant countries more pronouncedly than in the nonparticipant countries., Conclusions: DOI has a positive impact on the reporting of "organ trafficking" and "transplant tourism" but not on the reporting of "kidney sale" and "kidney trade." The increased reporting of "kidney sale" and "kidney trade" can be indicative of an impact of DOI on public awareness and increased reporting of the residual transplantation-related crimes., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Aims: The goal of the study was to investigate possible association of some single nucleotide polymorphisms (SNPs) in the VDR gene (the FokI, BsmI, ApaI and TaqαI loci), and the CYP17 gene (the MspA1I locus), and 0 or 9 TA repeats in the SRD5A2 gene, and prostate cancer (PCa) among Lebanese men. Materials and Methods: Blood DNA of 69 subjects with confirmed PCa and 69 controls, all about 50 years of age or older, was subjected to PCR or PCR-restriction fragment-length polymorphism (PCR-RFLP) analyses, and the risk-bearing and the protective alleles were identified. The odds ratio (OR) of having a genotype and the relative risk (RR) of developing PCa were calculated. In addition, the distributions of homozygosis and heterozygosis in the risk-bearing alleles and the protective alleles among the control and the PCa groups were compared. Results: The f allele of the VDR FokI locus and the (TA) 9 repeat allele of the SRD5A2 gene were found to be associated with increased risks of PCa (p = 0.006 and 0.050, respectively). Homozygosis in the risk-bearing alleles was rare both in the control and the PCa groups. A higher fraction of the controls compared to the PCa group was double-homozygous in the two protective alleles (52.2% for controls, 24.6% for PCa group, p = <0.001). Conclusions: To the best of our knowledge, this is the first genetic study demonstrating the association of certain polymorphisms of the VDR gene and the SDR5A2 gene and increased risk of PCa among Lebanese men. Our study also indicates that the overall polymorphism profile of all genes involved in the prostate physiology is likely to be a better indicator for PCa risk than the polymorphisms in the individual genes., (Creative Commons Attribution License)

Objective: To assess associations between codon 72 polymorphisms (Pro or B and Arg or b alleles) of the TP53 gene and lung cancer risk among Bangladeshis., Materials and Methods: The distribution of the BB, Bb, and bb genotypes and the frequencies of the B and b alleles were determined by PCR-RFLP method using DNA extracted from leucocytes of 50 confirmed lung cancer patients and 50 age-matched controls and the data were analysed., Results: The ratio of BB, Bb, and bb genotypes were in Hardy-Weinberg equilibrium except for the male patients (χ2=4.6). The B allele is overrepresented among all patients (OR=2.0, p=0.02) and the female patients (OR=4.1, p≤0.01) compared to the controls. The BB/bb ratio was also higher among the patients (OR=3.0, p=0.03). The relative risk of cancer for having BB over bb genotype was 1.8 (p=0.04) but no effect was observed for the Bb genotype. The B allele was overrepresented among patients with adenocarcinomas (OR=2.4, p≤0.01) and squamous cell carcinomas (OR=2.7, p≤0.01) over the controls but the difference was not significant for those with small cell lung carcinomas (OR=1.1, p=0.66). The B allele was overrepresented among patients age 50 or younger (OR=2.7, p≤0.01), but not for older patients (OR=1.7, p=0.07), and among smokers compared to the controls (OR=1.8-10.0, p≤0.01-0.03). However, no correlation between increasing pack-years and lung cancer was observed., Conclusions: The Pro/Pro (BB) genotype and the B allele are risk factors for lung cancer among Bangladeshis, particularly for people under age 50, women and smokers.

Background: Muslims constitute about one-fourth of the human population, and a significant fraction of the organ recipients identify themselves as Muslims. A large fraction of the Muslim population is devout but unclear regarding the religious principles on organ donation and transplantation and is dependent on scholars' and imams' opinions., Methods: The Qur'an, the authentic Prophetic Traditions, and expert opinions on the subject were investigated., Results: The sources of the Islamic founding principles on organ donation and transplantation are the Qur'an, the Prophetic Traditions, Usulul Fiqh or expert opinions based on the Qur'an and Traditions, and Maslaha or the principles of public interest deduced by the scholars. Some Muslim scholars, mostly from the Indian subcontinent, opine that live organ donation, extraction of organs from dead persons, and transplantation are prohibited. Many Arab scholars and Muslim scholars settled in the western hemisphere opine that live organ donation, organ extraction from dead persons, and transplantation are permitted, but organ donation must be a voluntary act of charity. Of late, the Iranian imams/scholars have recognized that the national government may acquire live donor organs for a uniform compensation and equitably distribute the acquired organs to patients with failing organs., Conclusions: The current Islamic working principles on transplantation medicine are nonuniform, transitory, and somewhat detached from the bulk of the population. How such heterogeneity is affecting transplantation medicine, and organ donation in particular, among Muslim populations warrants further investigation., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: The majority of the patients presently waiting for an organ are waiting for a kidney. Living kidney donation by about 0.1% of the adult population of a nation may completely eliminate kidney shortage. We investigated the concerns of college students toward charitable and compensated organ donation., Methods: A 40-question survey was conducted. The respondents were students of the Biology Department of Utah Valley University, Orem, Utah, United States. The data were tabulated and analyzed. Tests of association among potentially linked attributes and the difference between two independent proportions were performed at the 0.05 level of significance and P-values were also calculated using XLSTAT software., Results: The participants (n = 321) were 47% male, 53% female, 89% Caucasian, and 93% healthy, and 7% of the respondents had some health conditions. Of the respondents, 55% were ages 18 to 25 and 40% were ages 26 to 50 years; 43% were unmarried or single, 57% were married, and 85% had health insurance. About 65% of the respondents lived in small cities and the rest lived in large cities (23%) or the countryside (9%). There was no significant association between gender, level of education, location of living, and household income in relation to belief in organ donation with or without compensation, except that males favored compensated organ donation over females (P = .004). Rumors on organ theft and extraction of organ from questionable brain-dead patients had not negatively affected the decision of participants on being listed as organ donors in their driver's license (P = .0001). Those who considered organ donation ethically acceptable also believed that a person has the right to sale a kidney (P = .015) and the donor party should be somehow compensated (P = .001)., Conclusions: A large percentage of college students supports compensated organ donation and considers that compensation will increase organ donation., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: The aim of the study was to investigate any associations between benign prostate hyperplasia (BPH) and single nucleotide polymorphisms (SNPs) in the VDR gene (FokI, BsmI, ApaI and TaqαI loci) and the CYP17 gene (MspA1I locus), as well as TA repeat polymorphism in SRD5A2 gene among Lebanese men., Materials and Methods: DNA extracted from blood of 68 subjects with confirmed BPH and 79 age-matched controls was subjected to PCR/PCR-restriction fragment length polymorphism analysis. The odds ra=tio (OR) of having a genotype and the relative risk (RR) of developing BPH for having the genotype were calculated and the alleles were designated risk-bearing or protective., Results: Our data indicated that the A and B alleles of the VDR ApaI and BsmI SNPs were highly associated with increased risk of BPH (p=0.0168 and 0.0002, respectively). Moreover, 63% of the controls compared to 43% of the subjects with BPH were homozygous for none of the risk-bearing alleles (p=0.0123) whereas 60% of the controls and 28% of the subjects with BPH were homozygous for two or more protective alleles (p<0.0001)., Conclusions: For the first time, our study demonstrated that ApaI and BsmI of the VDR gene are associated with risk of BPH among Lebanese men. Our study also indicated that overall polymorphism profile of all the genes involved in prostate physiology could be a better predictor of BPH risk.

Background: Congenital hypothyroidism is screened using blood spotted on filter paper that may be transported from remote areas to central testing facilities. However, storage conditions and transportation may affect sample quality., Methods: We examined long-term stability of thyroid-stimulating hormone (TSH) and thyroxin (TT4) in blood spotted on filter paper, which was stored at room temperature (RT), 4°C and -20°C under continuous or intermittent power supply (six hours on and six hours off around the clock.) Hormone levels in the discs were measured periodically for up to ten years. Extraction of DNA from blood spots and polymerase chain reaction were performed., Results: Our results showed that TT4 was stable for up to 6.1, 5.34 and 5.16 years when stored at -20°C, 4°C and RT, respectively. TSH was stable for up to 2.7 years at RT, and for up to 6.5 and 4.1 years when stored at -20°C and 4°C, respectively, under continuous power supply. However, under intermittent power supply, TSH was stable for up to 3.8 and 2.5 years when stored at 4°C and -20°C, respectively. Mitochondrial cytochrome oxidase and sex-determining region of Y chromosome genes were successfully amplified from DNA extracted from the blood spots., Conclusion: Our data indicate that TT4 and TSH are most stable in blood spots stored at -20°C under continuous power supply. However, they can be stored at RT or at 4°C and -20°C under interrupted power supply for at least 2.5 years. Moreover, the DNA extracted from the blood spots was intact and suitable for genetic studies., (© 2010 Japan Pediatric Society.)

The human herpes simplex virus type 1 regulatory protein ICP4 binds DNA as a dimer and forms a single protein-DNA complex (A complex) with short DNA probes. ICP4 oligomerized in a DNA-dependent manner, forming two or more protein-DNA complexes with longer DNA fragments containing a single DNA binding site. When resolved electrophoretically, one or more low-mobility DNA-protein complexes follow the fast-moving A complex. The major protein-DNA complex (B complex) formed by ICP4 with long DNA probes migrates just behind the A complex in the electric field, implying the oligomerization of ICP4 on the DNA. Binding experiments with circularly permutated DNA probes containing one ICP4 binding site revealed that about 70 bp of nonspecific DNA downstream of the cognate ICP4 binding site was required for efficient B complex formation. In addition, the C-terminal domain of ICP4 was found to be required for DNA-dependent oligomerization and B complex formation. Gel mobility shift analysis of protein-DNA complexes, combined with supershift analysis using different monoclonal antibodies, indicated that the B complex contained two ICP4 dimers. DNase I footprinting of ICP4-DNA complexes showed that one ICP4 dimer contacts the specific binding site and another ICP4 dimer contacts nonspecific DNA in the B complex. DNA-dependent oligomerization increased the affinity of ICP4 for relatively weak binding sites on large DNA molecules. The results of this study suggest how ICP4 may use multiple weak binding sites to aid in transcription activation.

Pigs are considered a suitable source of cells and organs for xenotransplantation. All known strains of pigs contain porcine endogenous retrovirus (PERV) and PERV released by porcine cells may infect human cells in vitro and severe-combined immunodeficient (SCID) mice in vivo. Humanized SCID (hu-SCID) mice develop immune response to porcine antigens. Here we investigated PERV transmission in humanized SCID-beige mice using porcine peripheral blood mononuclear cells (PBMC) as the donor tissue (and the source of PERV). Mice were infused in the peritoneal cavity with 1.5-3.0 x 10(7) unfractionated human PBMC. Unfractionated porcine PBMC (1.5-3.0 x 10(7) cell/mouse) were infused to the mice simultaneously with human PBMC or 3 weeks after human PBMC infusion. The treated mice were monitored for weight and skin changes, donor cell chimerism, anti-pig antibodies and PERV transmission. All humanized mice tested 5-12 weeks after human PBMC transplantation were macrochimeric (up to 40% of cells in blood) for human cells, where 99% of the human cells were T-lymphocytes. Although human B lymphocytes were very rare in the blood of humanized mice at that point, the mice were positive for human anti-pig natural antibodies. The control SCID-beige mice or mice treated with porcine PBMC alone were negative for anti-porcine antibodies. Approximately 70% of the humanized mice treated with porcine PBMC were also microchimeric for porcine cells. Although some tissue samples of these mice were positive for PERV DNA in the absence of porcine DNA indicating PERV infection, the infection was non-productive as PERV transcripts were not detectable in those tissues. PERV infection of human and mouse cells in vitro by co-culturing with porcine PBMC was also non-productive. Humanized SCID-beige mice suffered weight loss and occasional minor skin changes due to graft vs. host disease caused by human PBMC but none of the mice showed observable effect attributable to the apparent PERV infection alone.

Although bone marrow transplantation has been used to induce donor-specific tolerance in many allogeneic models, similar effort in xenogeneic transplantation is met with obstacles like more severe graft versus host disease (GVHD). We are currently engaged in developing a GVHD-free hamster-to-rat xenotransplantation model using splenectomy, total body irradiation, and donor bone marrow transplantation. To test donor cell chimerism, particularly in the solid tissues, we developed a semiquantitative polymerase chain reaction (PCR) method using primers specific for hamster beta-actin and mitochondrial cytochrome C oxidase I and II (MCO I and II) genes and rat sex determination region on the Y chromosome (SRY) gene. Using this method, we estimated the level of hamster cells chimerism in rats subjected to splenectomy, total body irradiation (10 Gy), and hamster bone marrow transplantation (3 x 10(8) cell/recipient) and observed high levels of donor cells in all recipient tissues tested.

A major concern in using porcine organs for transplantation is the potential of transmission of porcine endogenous retrovirus (PERV). To investigate the long-term effects of PERV infection on human cells, human embryonic kidney cell line HEK-293 infected with PERV PK-15 was maintained for up to 72 passages and samples were harvested at intervals for use in morphological, growth, and genomic analyses. Morphology, DNA content/cell, and doubling time of uninfected and infected cells were similar. Restriction fragment length polymorphism (RFLP) analysis of PCR-amplified nearly full-length PERV genome showed no alterations in band pattern. RFLP analysis of the long terminal repeats (LTR) showed some changes in band pattern, but not in length. Southern blot analysis of genomic DNA of infected cells indicated random integration of PERV without structural alterations in proviral genome. Semi-quantitative PCR demonstrated a gradual increase of proviral load in the infected cells. Sequence analysis of the LTR region of PERV from infected cells indicated a relatively low rate (6.0 x 10(-4)/bp or about 2 x 10(-6)/bp/generation) of mutation. There were also indications of recombination of PERV strains A and B. Finally, PERV infection had no effect on transcription of human endogenous retrovirus-K (HERV-K) genes. Together, no significant effect attributable to PERV infection was evident on chronically PERV-infected HEK-293 cells.

Random amplified polymorphic DNA (RAPD) analysis is a valuable tool in studying inter- and intra-specific genetic variations, patterns of gene expression, and for the identification of specific genes using nearly isogenic variants. Here we used RAPD analysis to study the genetic variation in Ginkgo biloba grown in the eastern United States. Our results support the evidence that Southern blot hybridization of RAPD using probes made from cloned DNA fragments allows a more accurate analysis of the RAPD pattern than dye-stained gels or Southern blot hybridization of RAPD blots using probes made from purified PCR products. Using these techniques, we observed a high degree of relatedness among plants grown in certain localities although significant genetic variation may exist in the species, and could be a possible explanation for the observed variations in the efficacy of medications derived from G. biloba extract.

Background: Mechanisms underlying hepatic microcirculatory failure during endotoxemia are incompletely understood. Because endothelin-1 (ET-1) has been implicated in endotoxin-induced liver injury, we investigated the hepatic ET-1 system in endotoxin-treated rats., Methods: Rats were treated with endotoxin (Escherichia coli lipopolysaccharide; 3 mg/kg, i.p.), and various determinations were made 24 h later., Results: Endotoxin treatment caused 11.2 +/- 1.6% weight loss, a decrease in mean arterial pressure (MAP; 96 +/- 5 mmHg vs 108 +/- 3 mmHg; P < 0.05) and an increase in portal pressure (11.6 +/- 1.3 mmHg vs 7.4 +/- 1 mmHg; P < 0.02). No significant changes in the serum levels of liver enzymes or hepatocellular necrosis were observed. Endotoxin caused increases in hepatic ET-1 (from 345 +/- 31 to 565 +/- 38 pg/g; P < 0.01), ET-1 receptor density (from 179 +/- 16 to 340 +/- 26 fmol/mg; P < 0.02), and mRNA expression of preproendothelin-1, and ET(A) and ET(B) receptors. While the serum nitric oxide (nitrite +/- nitrate) concentration was increased in endotoxin-treated rats, that of ET-1 remained unchanged. A mixed ET(A)/ET(B) receptor antagonist, TAK-044 (10 mg/kg, i.v.), reduced the weight loss from 11.2 +/- 1.6% to 5.9 +/- 2.9% (P < 0.05) and the portal pressure from 11.6 +/- 1.3 mmHg to 8.6 +/- 0.7 mmHg (P < 0.05) in endotoxin-treated rats. The mixed ET(A)/ET(B) receptor antagonist also caused an increase in serum ET-1 concentration, but did not affect serum nitric oxide and MAP in endotoxin-treated rats., Conclusions: These results suggest that the upregulated hepatic ET-1 system is an important mechanism of increased portal resistance and related complications of endotoxemia.

Background/aims: Hepatic concentration of endothelin-1 (ET-1) is increased in human and experimental liver cirrhosis. Because of its potent actions in the liver, ET-1 has been suggested to play an important role in the pathophysiology of cirrhosis. Since hepatocytes are the major cell type to metabolize ET-1, we investigated whether their reduced capacity to degrade ET-1 is a mechanism of its elevated levels in cirrhosis., Methods: The expression of ET-1 receptors, ET-1 and endothelin converting enzyme (ECE), and metabolism of ET-1 and ECE activity were compared in hepatocytes isolated from control and carbon tetrachloride-induced cirrhotic rats., Results: ET-1 receptor density and receptor-mediated internalization of ET-1 were significantly increased in cirrhotic hepatocytes relative to the control cells. However, compared to control hepatocytes, metabolism of ET-1 by the cirrhotic cells was reduced significantly. Interestingly, hepatocytes were found to contain preproET-1 mRNA, ECE-1 mRNA and ET-1. PreproET-1 mRNA and ET-1 levels were increased in cirrhotic hepatocytes but their ECE mRNA and ECE activity were not altered., Conclusions: These results provide the first evidence that hepatocytes have the ability to synthesize ET-1 and demonstrate that decreased metabolism and enhanced synthesis, of ET-1 in hepatocytes are an important mechanism of its elevated levels in cirrhosis.

Interactions between hepatic stellate cells and endothelin-1 are implicated in liver fibrosis. We determined endothelin-1, its receptors and its effects on the synthesis of a fibrogenic agent transforming growth factor (TGF)-beta1 and collagen in stellate cells from control and CCl(4)-induced cirrhotic rats. The basal synthesis of endothelin-1, TGF-beta1 and collagen was much higher in cirrhotic stellate cells than in control cells. Endothelin-1 stimulated TGF-beta1 and collagen synthesis via endothelin ET(A) and endothelin ET(B) receptors, respectively, in control stellate cells, but did not elicit these effects in the cirrhotic cells despite increased density of the respective receptor subtypes in them. These results indicate that the actions of endothelin-1 on stellate cells may be an important physiological mechanism in maintenance of hepatic architecture. However, inability of endothelin-1 to stimulate TGF-beta1 and collagen synthesis in cirrhotic stellate cells suggests that it does not influence fibrogenic activity by direct action on them probably because the processes are already maximally activated.

Background/aims: Hepatic endothelin-1 (ET-1) receptor density as well as the levels of both ET-1 and transforming growth factor beta1 (TGF-beta1) increase in liver cirrhosis. Considering their potent contractile (ET-1) and fibrogenic (TGF-beta1) actions on myofibroblastic stellate cells found in the fibrotic/cirrhotic liver, we aimed to investigate the effects of TGF-beta1 on ET-1 receptors and ET-1 synthesis in these cells., Methods: Stellate cells isolated from rat liver by enzymatic digestion were cultured and subjected to TGF-beta1 treatment. Cellular ET-1 receptors and ET-1 released in the medium were determined., Results: TGF-beta1 treatment produced time- and dose-dependent decrease in ET-1 binding sites, but did not affect the affinity of the receptors for ET-1. TGF-beta1 also stimulated the release of ET-1 from stellate cells. The extent of TGF-beta1-induced inhibition of [125I]ET-1 binding was much greater for ETB subtype (73+/-18% inhibition), which comprised a major portion (78+/-12%) of the total ET-1 receptors, than for ETA subtype (35+/-11% inhibition). The mRNA expression of the ET-1 receptors also was reduced by TGF-beta1 treatment. TGF-beta1-induced reduction in ET-1 receptor density was coupled to the inhibition of ET-1-stimulated release of [3Hlarachidonic acid from the prelabeled cells. The effects of TGF-beta1 were inhibited by a TGF-beta1 neutralizing monoclonal antibody., Conclusions: These results suggest that the TGF-beta1-induced decrease in ET-1 receptor density may be an important mechanism in limiting the pathologic actions of ET-1 on stellate cells in chronic liver disease.

Background/aims: Reactive oxygen species are mediators of various pathophysiologic events, including postischemic reperfusion injury and inflammation. Generation of reactive oxygen species and consequent organ injury are associated with increased levels of a powerful vasoconstrictor peptide endothelin-1. Current evidence suggests that actions of endothelin-1 on the contractile and fibrogenic transdifferentiated stellate cells may play a critical role in hepatic pathophysiology. The aim of this investigation was to determine whether reactive oxygen species modulate the synthesis of endothelin-1 and its receptors in stellate cells., Methods: Primary cultures of transdifferentiated stellate cells were exposed to reactive oxygen species-generating system, hypoxanthine/xanthine oxidase, before determination of endothelin-1 and its receptors., Results: The treatment caused an initial decrease in ET-1 receptor density (about 30% at 30 min), followed by a significant increase over the basal level at 6 h. The increase in the receptors, which occurred specifically in the ET(B) subtype, progressed thereafter up to 24 h and was accompanied by an augmented functional response, as indicated by an enhanced endothelin-1-induced release of [3H]arachidonic acid from the prelabeled cells. Furthermore, treatment of cells for 24 h but not 30 min caused increased expression of ET(B) mRNA as determined by semi-quantitative polymerase chain reaction. The release of endothelin-1 in the culture medium was also enhanced by hypoxanthine/xanthine oxidase treatment. These effects of hypoxanthine/xanthine oxidase were inhibited by superoxide dismutase and dimethyl sulfoxide. ET-1-induced [3H]arachidonic acid release was also inhibited by the ET(B) receptor antagonist BQ788, but not by the ET(A) receptor antagonist BQ123., Conclusions: These findings indicate that interactions between ET-1 and stellate cells during episodes of the generation of reactive oxygen species can be an important mechanism in the pathophysiology of hepatic disorders.