Alleviation of Hepatic Steatosis by Alpha-Defensin Is Associated with Enhanced Lipolysis.

Background and Objectives: The neutrophilic peptide, alpha-defensin, is considered an evolving risk factor intimately linked with lipid mobilization. It was previously linked to augmented liver fibrosis. Here, we assess a potential association between alpha-defensin and fatty liver. Materials and Methods: A cohort of transgenic C57BL/6JDef+/+ male mice that overexpress the human neutrophil-derived alpha-defensin in their polymorphonuclear neutrophils (PMNs) were assessed for liver steatosis and fibrosis development. Wild type (C57BL/6JDef.Wt) and transgenic (C57BL/6JDef+/+) mice were maintained on a standard rodent chow diet for 8.5 months. At the termination of the experiment, systemic metabolic indices and hepatic immunological cell profiling were assessed. Results: The Def+/+ transgenic mice exhibited lower body and liver weights, lower serum fasting glucose and cholesterol, and significantly lower liver fat content. These results were associated with impaired liver lymphocytes count and function (lower CD8, NK cells, and killing marker CD107a). The metabolic cage demonstrated dominant fat utilization with a comparable food intake in the Def+/+ mice. Conclusions: Chronic physiological expression of alpha-defensin induces favorable blood metabolic profile, increased systemic lipolysis, and decreased hepatic fat accumulation. Further studies are needed to characterize the defensin net liver effect.

Sodium+/taurocholate cotransporting polypeptide as target therapy for liver fibrosis.

OBJECTIVE: Sodium+/ taurocholate cotransporting polypeptide (NTCP) is a membrane transporter affecting the enterohepatic circulation of bile acids (BAs). We aimed to evaluate NTCP's roles in humans and animal models of liver fibrosis (LF). DESIGN: Primary hepatic stellate cells (pHSCs) isolated from livers biopsies of patients with LF with different fibrosis grading were stained for NTCP. NTCP gene silencing, taurocholic acid (TCA), obeticholic acid (OCA), epigallocatechin gallate (EGCG) and HA-100 dihydrochloride (HA-100) were used as tools to modulate NTCP expression on human HSC line (LX2). BA trafficking/uptake were assessed extracellularly (LX2 culture medium) and intracellularly following treatment with/without NTCP neutralizing antibody. LF models of C57/BL6 mice of carbon tetrachloride (CCl4) and leptin-deficient (Ob/Ob) fed with high-fat diet (Ob/Ob HFD ) were evaluated for pHSCs-NTCP expressions, metabolic and LF profiles following intraperitoneal injections of NTCP neutralizing antibody. RESULTS: pHSCs from F3/F4-scored patients of LF exhibit threefold increased NTCP expressions compared with F0-scored patients (p<0.0001). Sorted-activated HSCs (LX2αSMA+) showed high expressions of NTCP and high TCA uptake in vitro and triggered a further increase in their activations. This phenomenon was inhibited with NTCP small interfering RNA and the NTCP neutralizing antibody. Sorted LX2NTCP+ (high alpha smooth muscle actin (αSMA)/high NTCP) cells showed high phosphorylated pathways of AKT/mTOR and protein kinase C (PKC) accompanied with a decrease in farnesoid X receptor expression. Moreover, LX2NTCP+ cells treated with EGCG, OCA and PKC inhibitor HA-100 significantly decreased NTCP and αSMA. NTCP neutralizing antibody inhibited NTCP (less TCA uptake); it attenuated LF in both CCl4 and Ob/Ob HFD animal models with ameliorated metabolic profile. CONCLUSION: NTCP expression is linearly correlated with fibrosis severity. Modulated BA trafficking could be an important step in LF pathogenesis. Antagonising BA uptake may suggest a therapeutic strategy for preventing disease progression.

The impact of filtered water-pipe smoke on healthy versus cancer cells and their neurodegenerative role on AMPA receptor.

Water pipe smoking is highly prevalent in developing countries, especially in Eastern Mediterranean regions. Research finds that more than 100 million people smoke a water pipe. Furthermore, tobacco smoking is one of the leading behavioral factors related to an increased risk of cancer, a leading cause of death globally. We aim to introduce a novel filtration system for water-pipe smoking and evaluate cytotoxic effects of common water pipe condensed smoke in comparison with our novel filtration system on normal (HEK293t) and cancer cell lines (Hep3B and MCF7) by MTS assay, alpha-fetoprotein (aFP), and apoptosis/necrosis effects. More so, the smoke substituents' neurotoxicity effect was evaluated by analyzing the depressive property on AMPA receptors (AMPARs). Our results showed that the silica filtration system was more effective than the water filtration system. The number of toxic compounds was reduced from 145 mg in distilled water extract (DWE) to 57.5 mg in silica solution extract (SSE). The SSE method also showed lower toxicity impacts on normal and cancerous cell lines (HEK293t, Hep3B, and MCF7) with CC50 values 149.9, 10.14, and 8.9 µg/ml, relative to the DWE method (CC50 values 77.1, 3.1, and 5.24 µg/ml, respectively). SSE extraction also reduced the α-FP (tumor marker test) to 2273.3 ng/ml which was closer in value to untreated cells (4066.7 ng/ml) in comparison with DWE which reduced it greatly to 1658.7 ng/ml, and the biophysical properties of AMPAR subunits demonstrate a reduced effect on desensitization rates of GluA2 homomer and GluA1/2 heteromer, using SSE relative to DWE. In conclusion, the condensed smoke of ordinary water pipe (DWE) has cytotoxic and neurotoxic impacts on various cell lines, while our newly developed system (SSE) was less toxic.

Agonist of RORA Attenuates Nonalcoholic Fatty Liver Progression in Mice via Up-regulation of MicroRNA 122.

BACKGROUND & AIMS: Development of nonalcoholic steatohepatitis (NASH) is associated with reductions in hepatic microRNA122 (MIR122); the RAR related orphan receptor A (RORA) promotes expression of MIR122. Increasing expression of RORA in livers of mice increases expression of MIR122 and reduces lipotoxicity. We investigated the effects of a RORA agonist in mouse models of NASH. METHODS: We screened a chemical library to identify agonists of RORA and tested their effects on a human hepatocellular carcinoma cell line (Huh7). C57BL/6 mice were fed a chow or high-fat diet (HFD) for 4 weeks to induce fatty liver. Mice were given hydrodynamic tail vein injections of a MIR122 antagonist (antagomiR-122) or a control antagomiR once each week for 3 weeks while still on the HFD or chow diet, or intraperitoneal injections of the RORA agonist RS-2982 or vehicle, twice each week for 3 weeks. Livers, gonad white adipose, and skeletal muscle were collected and analyzed by reverse-transcription polymerase chain reaction, histology, and immunohistochemistry. A separate group of mice were fed an atherogenic diet, with or without injections of RS-2982 for 3 weeks; livers were analyzed by immunohistochemistry, and plasma was analyzed for levels of aminotransferases. We analyzed data from liver tissues from patients with NASH included in the RNA-sequencing databases GSE33814 and GSE89632. RESULTS: Injection of mice with antagomiR-122 significantly reduced levels of MIR122 in plasma, liver, and white adipose tissue; in mice on an HFD, antagomiR-122 injections increased fat droplets and total triglyceride content in liver and reduced ß-oxidation and energy expenditure, resulting in significantly more weight gain than in mice given the control microRNA. We identified RS-2982 as an agonist of RORA and found it to increase expression of MIR122 promoter activity in Huh7 cells. In mice fed an HFD or atherogenic diet, injections of RS-2982 increased hepatic levels of MIR122 precursors and reduced hepatic synthesis of triglycerides by reducing expression of biosynthesis enzymes. In these mice, RS-2982 significantly reduced hepatic lipotoxicity, reduced liver fibrosis, increased insulin resistance, and reduced body weight compared with mice injected with vehicle. Patients who underwent cardiovascular surgery had increased levels of plasma MIR122 compared to its levels before surgery; increased expression of plasma MIR122 was associated with increased levels of plasma free fatty acids and levels of RORA. CONCLUSIONS: We identified the compound RS-2982 as an agonist of RORA that increases expression of MIR122 in cell lines and livers of mice. Mice fed an HFD or atherogenic diet given injections of RS-2982 had reduced hepatic lipotoxicity, liver fibrosis, and body weight compared with mice given the vehicle. Agonists of RORA might be developed for treatment of NASH.

25(OH) D3 alleviate liver NK cytotoxicity in acute but not in chronic fibrosis model of BALB/c mice due to modulations in vitamin D receptor.

BACKGROUND: Low 25-Hydroxy-vitamin-D; "25(OH)-D3" serum and vitamin D receptor (VDR) levels were recently correlated to advanced fibrosis. However, VDR mechanism in liver fibrosis modulations is not well understood. In this study, we aimed to evaluate changes in liver NK cells cytotoxicity due to modulations in VDR in CCl4 fibrosis model following 25(OH) D3 injections. METHODS: Carbon-tetrachloride (CCl4) hepatic-fibrosis was induced in BALB/c mice for 1 and 4 weeks as an acute and chronic fibrosis model, respectively. Along 1th to 4th weeks, vitamin D were i.p injected/2x week. Liver were assessed histologically and for proteins quantification for VDR and αSMA expressions. In vitro, potential killing of NK cells were evaluated following co-culture with primary-hepatic-stellate-cells (pHSCs) obtained from BALB/c WT-mice. RESULTS: Systemic inflammation and hepatic-fibrosis increased along 4 weeks of CCl4 as indicated by serum ALT and αSMA expressions (P < 0.02) as well as histological assessments, respectively. These results were associated with increased NK1.1 activations and hypercalcemia. While vitamin D administrations delayed fibrosis of early stages, vitamin D worsen hepatic-fibrosis of late stages of CCl4. In week 4, no further activations of NK cells were seen following vitamin D injections and were associated with down-expressions of VDR (1.7 Fold, P < 0.004) indicating the inability of vitamin D to ameliorate hepatic fibrosis. In vitro, NK cells from the chronic model of CCl4 did not affect pHSCs killing and fail to reduce fibrosis. CONCLUSION: Vitamin D alleviate liver NK cytotoxicity in acute but not in chronic fibrosis model due to modulations in vitamin D receptor and calcium. Hypercalcemia associated with late fibrosis may inhibited VDR levels, however, may not explain the profibrogenic effects of vitamin D.

A novel flow cytometry tool for fibrosis scoring through hepatic stellate cell differentiation.

Hepatic stellate cells (HSCs) are a central fibrogenic cell type that contributes to collagen accumulation during chronic liver disease. Peripheral blood lymphocytes from HCV patients are phagocytized by HSCs and induce their differentiation. This study aimed to characterize HSCs differentiation using a flow cytometry tool for fibrosis scoring. NK cells from healthy donors and from patients with chronic HCV with various severities of fibrosis were co-cultured with a human HSC line (LX2). LX2 phagocytosis of NK cells were stained for NK cells (CD45/CD56/CD3) and NK activation marker (CD107a) as well as INF-γ, apoptosis (Annexin-V) and α-smooth-muscle-actin (αSMA, as a marker of LX2 activation). In addition, reactive oxygen species (ROS) and the senescence marker P15 were analyzed prior to flow cytometry analysis. LX2 mono-cultures demonstrated a homogenous cell-population according to size (forward-scattered; FSC), granularity and αSMA expressions. However, on their co-culture with NK cells, the HSCs formed four subpopulations, which were stratified by αSMA intensities and cell size. NK cells isolated from heathy donors did not activate LX2-cells. In contrast, HCV exposed to NK cells from both F1 and F4 fibrosis grade patients, showed elevated CD107a and INF-γ levels and increased αSMA intensities in two of the four cell populations, with fibrosis scoring showing a linear correlation with αSMA intensities and NK phagocytosis. The αSMAintermediate /SizeLow HSCs sub-population showed higher proliferation following F4-NK cells with higher phagocytosis ability, suggesting an active/regulatory population. The αSMAhigh /Sizehigh subpopulations showed low proliferation and phagocytosis capacity, and were correlated with higher apoptosis, increased ROS and P15 intensities, suggesting senescing cells. Taken together, NK cells lead to heterogeneous differentiation of HSCs. Flow-cytometry may provide a novel means of characterizing HSCs in relation to the severity of liver fibrosis. © 2017 International Society for Advancement of Cytometry.

Covalent functionalization of SWCNT with combretastatin A4 for cancer therapy.

Single walled carbon nanotubes (SWCNT) are currently under intensive investigation by many labs all over the world for being promising candidates for cancer chemotherapy delivery. On the other hand, combretastatin A4 (CA4) is an anticancer drug that induces cell apoptosis by inhibiting tubulin polymerization. However, it has the disadvantage of low water solubility and the non-selective targeting. Therefore, we aim to create nano-drug from the functionalization of SWCNT covalently with CA4 through click reaction in the presence of tetraethylene glycol linker in order to improve its dispersibility. Scanning electron microscopy and transmission electron microscopy showed good dispersibility of the functionalized SWCNT with diameters of 5-15 nm. Moreover, thermogravometric analysis showed that the efficiency of SWCNT functionalization was around 45%. The in vitro release profile of CA4 at physiological conditions showed that approximately 90% of the loaded drug was released over 50 h. After that MTS test was used to determine the suitable concentration range for the in vitro investigation of the SWCNT-CA4. After that the cytotoxic activity of the SWCNT-CA4 was evaluated by flow cytometry using annexin V/propidium iodide (PI) test. In comparison with free CA4, SWCNT-CA4 treatment demonstrated a significant increase in necrotic cells (around 50%) at the expense of the proportion of the apoptotic cells. Moreover, cell cycle PI test demonstrated that free CA4 and SWCNT-CA4 caused G2/M arrest. However with CA4 treatment higher proportion of cells were in the S-phase while with SWCNT-CA4 treatment greater proportion of cells appeared to be in the G1-phase. Taken together, the provided data suggest that the novel SWCNT-CA4 has a significant anticancer activity that might be superior to that of free CA4.

The pro-fibrotic effects of pregnancy in a carbon-tetrachloride-induced liver injury in mouse model.

BACKGROUND & AIMS: Immune cells interact with hepatic-stellate-cells (HSCs) in the development of liver fibrosis. Little is known about the influence of pregnancy on the development and progression of hepatic-fibrosis. In this study, we explored the influence of pregnancy on progression of hepatic fibrosis. METHODS: Female mice (C57Blc) were induced by 4 injections of peritoneal carbon-tetrachloride (CCl4) within 10 days, starting at day 10 of documented pregnancy. At end of experiment, serum samples were obtained for ALT and estradiol determination. Harvested livers were histological evaluated for liver injury and for protein αSMA expressions. Isolated intra-hepatic lymphocytes were assessed by flow cytometry. Isolated lymphocytes and serum samples were in- vitro co-cultured for 48 h with primary isolated naïve HSCs. Washed cells were analyzed for adherence (anti-αSMA+/anti-CD45 + ) and proliferations (CSFE). RESULTS: CCl4-model for liver injury was well tolerated when induced in pregnancy similar to non-pregnant state. Hepatic-fibrosis (Masson Trichrome Stain, Sirius red stain and αSMA expressions) and necro-inflammation (H&E stain and serum ALT levels) significantly increased in pregnancy. Increased liver injury was accompanied with pro-fibrotic lymphocyte profile; CD8 subsets increased and NK cells decreased. HSCs activation significantly increased when in-vitro cultured with lymphocytes from pregnant as compared to non-pregnant fibrotic ones. Pro-fibrotic profile was also explained by decreased NK activity (CD107a marker) and of their phagocytosis. Serum estradiol levels although elevated in fibrosis conditions of pregnancy was not associated with the pHSCs activations. CONCLUSION: Liver fibrosis in our murine model was severe in pregnant model; via pro-fibrotic lymphocyte and serum alterations.

E-cigarette use among university students in Palestine: Prevalence, knowledge, and determinant factors.

BACKGROUND: Recent reports indicated accelerated rates of e-cigarette use, especially among youth in various Middle Eastern countries, including Palestine. Nevertheless, little is known about knowledge, attitudes, and perceptions regarding this topic in Palestine. This study aimed to assess the prevalence of e-cigarette use among Palestinian university students, along with their knowledge, attitudes, and perceptions about e-cigarette use. METHODS: An observational cross-sectional study, utilizing an online self-administered questionnaire, was conducted on Palestinian students from five universities between 17/04/2023 and 04/11/2023. RESULTS: A total of 1002 Palestinian university students completed the questionnaire. The prevalence of e-cigarette use among students was 18.1%. The mean knowledge score about e-cigarettes was significantly lower among the users of e-cigarettes compared to non-users. E-cigarette use was significantly associated with the participants' smoking status. Among e-cigarette users, 43.6% were also current traditional cigarette users, and 66.9% were current waterpipe users. E-cigarette use was significantly associated with having a friend who is a smoker and/ or a smoking mother. Binary logistic regression revealed a significant positive effect between the participant's smoking status, the mother's smoking status, knowledge about e-cigarettes, and the use of e-cigarettes (p-value < 0.05). Among e-cigarette users, 18.8% used them in indoor places at the university, and 25% reported using them daily in the past month. Affordability of e-cigarettes was the most reported reason for their use (47.5%). CONCLUSION: This study concluded that e-cigarette use is prevalent and rapidly rising among university students in Palestine. This is worrisome as it is significantly associated with insufficient knowledge about the adverse health effects of E-cigarette use, and its addictive nature. These findings focus on the importance of improving the students' knowledge about e-cigarette use by implementing educational campaigns and considering age regulations on e-cigarette availability and use.

Evaluation of knowledge and attitudes regarding Alzheimer's disease and related dementia among medical students in Palestine: A cross-sectional study.

INTRODUCTION: Dementia, a major global health concern, is an acquired disorder that causes a progressive decline in cognitive abilities, affecting learning and memory, language, executive function, complex attention, perceptual-motor skills, and social cognition. Our study aims to evaluate the knowledge and attitudes regarding dementia and Alzheimer's disease among medical students. METHODS: This cross-sectional study was conducted among 393 medical students in Palestine from August 2023 to November 2023. The assessment of knowledge and attitude toward dementia was measured using the Alzheimer's Disease Knowledge Scale (ADKS) and Dementia Attitude Scale (DAS). The data were analyzed using SPSS version 26, and the Mann-Whitney U-test and the Kruskal-Wallis test were used to compare the mean between the groups with a 5% significance level. RESULTS: The overall mean score of the student's knowledge of dementia measured by the ADKS was 18.91 (±3.32 SD) out of 30. The mean score of the student's attitude toward dementia measured by the DAS was 91.68 (±3.32 SD). Clinical students had higher ADKS scores than pre-clinical students (p-value < 0.001). No significant differences in the knowledge and attitudes toward dementia were found between males and females. The medical students' knowledge and attitude scores were positively correlated (ρ = 0.227, p-value <0.001). CONCLUSION: Palestinian medical student's knowledge about Alzheimer's disease and dementia is insufficient, with students in the clinical phase showing better understanding than pre-clinical students. The findings highlight a necessity for enhancing the dementia curriculum and conducting further studies to evaluate training's impact on students' knowledge and attitudes.

Total phenolic contents, cytotoxic, free radicals, porcine pancreatic α-amylase, and lipase suppressant activities of Artemisia dracunculus plant from Palestine.

Artemisia dracunculus: L. (A. dracunculus) is a popular vegetable and spice cultivated across many Middle Eastern countries. The herb's aqueous extract has significant folkloric medicinal importance for treating various disorders. Hence, the present investigation aimed to investigate A. dracunculus hydrophilic extract phytochemical constituents and pleiotropic biological potentials, as no previous studies have investigated the antilipase and anti-α-amylase effects of the A. dracunculus plant. Total phenol content and phytochemical screening assays were performed utilizing standard analytical methods. While the α-amylase inhibition, free radical-scavenging, antilipase, and cytotoxic activities were determined using dinitrosalicylic acid (DNSA), DPPH, p-nitrophenyl butyrate (PNPB), and MTS assays, respectively. The standard phytochemical analysis of A. dracunculus aqueous extract shows that this extract contains only a phenolic group. The total phenol content was 0.146 ± 0.012 mg GAE/g of the plant dry extract. The A. dracunculus aqueous extract exhibited potent DPPH free radical inhibitory (IC50 dose of 10.71 ± 0.01 µg/mL) and anti-lipase activities (IC50 dose of 60.25 ± 0.33 µg/mL) compared with Trolox (IC50 = 5.7 ± 0.92 µg/mL) and Orlistat (IC50 = 12.3 ± 0.35 µg/mL), respectively. However, it showed a weak anti-α-amylase effect (IC50 value > 1,000 µg/mL) compared with Acarbose (IC50 = 28.18 ± 1.27 µg/mL). A. dracunculus has a cytotoxic effect against the HeLa cancer cell line compared with the chemotherapeutic agent Doxorubicin. The extract has the same percent of inhibition as Doxorubicin (99.9%) at 10 mg/mL. Overall, these results pointed out for the first time the importance of considering A. dracunculus effects as a favorite candidate for preventing and treating metabolic disorders. Also, our results confirm the findings of previous reports on the role of A. dracunculus in the management of cancer and disorders resulting from the accumulation of harmful free radicals. On the contrary, the current study concluded that the antidiabetic role of A. dracunculus could be minimal. Further in-depth investigations are urgently warranted to explore the importance of A. dracunculus in pharmaceutical production.

The immune and metabolic treatment approach of using testosterone on mice models of liver injury.

Background: Natural killer (NK) cells showed an anti-fibrotic effect; however, their function is thought to be impaired in advanced liver injury. In the current study, we aimed to assess the immune and metabolic impact of testosterone on mice models of liver injury. Methods: Carbon-tetrachloride induced liver fibrosis male mice models was i.p injected for 2 weeks (acute) and 4 weeks (chronic) (n = 36). Testosterone (4 mg/kg mouse body weight) was injected i.p. following the first week of the acute model of CCl4 and following the second week of the chronic model of CCl4. At the end of the experiments, mice were sacrificed, and serum was collected for assessing liver enzymes of ALT and AST, as well as inflammatory markers of IL-6, metabolic makers of C-peptide levels, and lipid and glucose profiles. Livers were harvested and used for histological assessments for inflammation and fibrosis. Fibrosis profiles from liver extracts, αSMA and Collagen III, were assessed by RT-PCR. Moreover, liver tissue-resident NK cells were isolated and evaluated for their activity by assessing INF-γ and IL-6 receptors using ELISA and flow cytometry, respectively. Results: Serum ALT, AST, and IL-6, as well as metabolic assessments of cholesterol, triglyceride, C-peptide, fasting blood sugar, and fibrotic profiles, were linearly correlated with disease progressions. Histological characterization of the liver was worsened in the chronic model of liver injury. Testosterone-treated mice exhibit a significant reduction in collagen depositions with less dense fibrosis tissue associated with reduced liver injury enzymes and metabolic markers in both the acute and chronic CCl4 mice models in favor of the latter one (p < 0.05). Moreover, testosterone treatments displayed a significant decrease in serum IL-6 of 2.4-fold (p = 0.0001) and 2.3-fold (p = 0.0003) in the acute and chronic models, respectively (p = 0.002), and data showed an increase in INF-γ release from NK associated with a reduction in their IL-6 receptor expressions (p < 0.05). Conclusion: Our results indicated effects of testosterone on mediating a decreased expressions of NK IL-6 receptors and consequently inducing their activation; which in part, could explain the amelioration of liver injury. Our data suggest an anti-inflammatory and anti-fibrotic treatment approach of using testosterone for delaying disease progressions.

Factor VII Deficiency in an End-Stage Renal Disease Patient With Recurrent Thrombosis: A Case Report.

Congenital factor VII deficiency is a rare bleeding disorder with variable presentations. Thromboembolism is a well-established complication of this heterogeneous disease. As it is a rare disease, there is no information regarding its treatment when it is present with other comorbidities such as end-stage renal disease. This study describes a 47-year-old male with multiple comorbidities who was recently diagnosed with end-stage renal disease. He had recurrent admissions to the hospital due to thrombotic arteriovenous access failure as well as acute coronary syndrome, despite a high international normalized ratio that was resistant to replacement therapy. Eventually, apixaban became his main treatment regimen. This case needs to be reported because it is rare in terms of including a factor VII deficiency patient with end-stage renal disease, as well as to emphasize the unclear recommendations available for patients with factor VII deficiency and end-stage renal disease. International collaboration may be the best course of action to study enough patients and come up with effective recommendations.

Assessment of Perceptions and Predictors Towards Consanguinity: A Cross-Sectional Study from Palestine.

Background/Aim: Consanguinity represents a biological relationship between two individuals. In clinical genetics, it specifically refers to the marriage between individuals who are second cousins or closer. The aim of the study is to assess perceptions and their predictors among the Palestinian population towards consanguinity. Methods: A survey-based cross-sectional study was conducted. The sample was collected using convenience and snowball sampling methods, yielding a sample of 1008 participants. The perceptions towards consanguinity and its predictors were assessed using Chi-square test. Results: The prevalence of consanguinity among married participants was 18.7% (N = 81/432), while it was 28.8% among their parents. Consanguinity rate was significantly low among the young age group (ie, <47 years old) and among participants whose mothers have undergraduate educational levels (P < 0.05). Other factors like parental consanguinity, educational level of participants, their father's educational level, and residency place showed no significant associations (P > 0.05). Rejection of consanguinity was significantly noted among young age participants, absence of parental consanguinity, the presence of children or family members with genetic disorders and female gender (P < 0.001). Furthermore, participants who indicated that they are governmental employees, those with 2000-5000 ILS monthly income, those who are married (P < 0.01), and those who indicated that their mothers are holders of postgraduate degree were significantly more likely to reject the idea of consanguinity (P < 0.05). Also, medical and/or scientific reasons were significantly associated with rejecting the idea of consanguinity (P < 0.001). Conclusion: Consanguinity prevalence has decreased among recent generations in Palestine, but it remains a significant challenge in Palestine. Therefore, educational and awareness programs about consanguinity and its health effects are effective strategies for reducing the consanguinity rate, especially for persons who are at the age of marriage.

Arum palaestinum delays hepatocellular carcinoma proliferation through the PI3K-AKT-mTOR signaling pathway and exhibits anticoagulant effects with antimicrobial properties.

Background: Arum palaestinum Boiss (AP) is a wild plant in Palestine whose leaves have a long history as food and medicine in Middle Eastern countries. The current study aimed to evaluate the biological characteristics of AP flower extract, including its antimicrobial and coagulation cascade activities and its effects on anticancer molecular pathways. Methods: The antimicrobial activity of the aqueous extract of AP flowers was assessed using a microdilution assay against eight pathogens. The coagulation properties were assessed by prothrombin time (PT), activated partial thromboplastin time (aPTT), and thrombin time (TT) tests using standard hematological methods. The biological effects of AP on hepatocellular carcinoma were measured by assessing the impact of AP on cell cycle, proliferation (CFSE), apoptosis (annexin-v+/PI), and tumorigenicity (αFP and HBsAg), as well as its effects on the PI3K-AKT-mTOR molecular signaling pathway. Results: The antimicrobial screening results revealed that the aqueous extract of AP had potent antibacterial effects against P. vulgaris and E. faecium compared to ampicillin, with MIC values of 6.25, 6.25, and 18 µg/mL, respectively. Moreover, the AP aqueous extract exerted anticoagulant activity, with significant prolonged results in the aPTT and TT tests (25 µg/mL and 50 µg/mL, respectively) and slightly prolonged results in the PT test (50 µg/mL). The anticancer results indicated a delay in the cell cycle through decreased cell proliferation rates following incubation with AP fractions. The effect of the aqueous fraction was most evident in a delay in the S phase. The aqueous and DMSO fractions maintained the cells in the G2-M phase, similar to the DOX, while the flower extract in methanol accelerated the cells in the G2-M phase, suggesting that AF flower extracts may have anti-cancer properties. The aqueous extract of AP 1) reduced secretions of HCC αFP by 1.55-fold and 3.3-fold at the 50 and 100 µg/mL concentrations, respectively (p = 0.0008); 2) decreased phosphorylation in the PI3K-AKT-mTOR signaling pathway (p < 0.05); and 3) shifted cells from necrosis to apoptosis by 50% and 70% at the 50 and 100 µg/mL concentrations, respectively (p < 0.05). Conclusion: The results of this study showed the activities of the bioactive components for the treatment of infectious diseases and blood coagulation disorders, which could also be a potential therapeutic approach for delaying HCC tumorigenicity.

SARS-CoV-2 vaccine alleviates disease burden and severity in liver transplant recipients even with low antibody titers.

BACKGROUND AND AIMS: We retrospectively assessed the clinical Pfizer's mRNA SARS-CoV-2 BNT162b2 vaccination outcomes and the serologic impact on liver transplant (LT) recipients. PATIENTS AND METHODS: One hundred and sixty-seven LT cases followed between March 1, 2020 and September 25, 2021, and were stratified into two groups: (1) 37 LT recipients after SARS-CoV-2 infection before vaccine era and (2) 130 LT recipients vaccinated with 2 doses without earlier SARS-CoV-2 exposure. Serum SARS-CoV-2 spike immunoglobulins (anti-S) were assessed 7 days following vaccination (Liaison assay). RESULTS: In addition to the 37 nonvaccinated cases (22.2% of total group) who experienced SARS-CoV-2 infection (34 symptomatic and 3 asymptomatic), another 8 vaccinated symptomatic recipients (4.8%) were infected (5 from the third and three from the fourth waves). Three of the 45 infected cases died (6.7%) before the vaccine program. Vaccinated group: of the 130 LT vaccinated recipients, 8 (6.2%) got infected postvaccination (added to the infected group) and were defined as clinical vaccine failure; 38 (29.2%) were serological vaccine failure (total failure 35.4%), and 64.6% cases were serological vaccine responders (anti-S≥19 AU/mL). Longer post-LT interval and lower consumption of immunosuppressants (steroids, FK506, and mycophenolate mofetil) correlated with favorable SARS-CoV-2 vaccine response. Mammalian target of rapamycin inhibitors improved vaccine outcomes associated with lower FK506 dosages and serum levels. Patients with anti-S levels <100 AU/mL risked losing serologic response or being infected with SARS-CoV-2. A booster dose achieved an effective serologic response in a third of failures and most responders, securing better and possibly longer protection. CONCLUSION: Pfizer's BNT162b2 vaccine seems to lessen SARS-CoV-2 morbidity and mortality of LT recipients even with weak serological immunogenicity. Switching mycophenolate mofetil to mammalian target of rapamycin inhibitors might be effective before boosters in vaccine failure cases. A booster vaccine should be considered for nonresponders and low-responders after the second dose.

Weaker SARS-CoV-2 vaccine responses in nonalcoholic fatty liver disease with advanced liver fibrosis.

Background: SARS-CoV-2 vaccine responses that could harbor potential risks to chronic liver diseased patients. Aims: To assess immune response following Pfizer's SARS-CoV-2 vaccine in patients with different liver fibrosis severities of nonalcoholic fatty liver disease (NAFLD). Methods: Clinical and histological (NAS-score and fibrosis stage) characteristics of NAFLD patients before vaccine were correlated with serologic vaccine responses of two doses of the BNT162b2. Serum SARS-CoV-2 spike immunoglobulins (anti-S) were assessed on day seven following immunization (Liaison assay). Results: The mean-age of patients (n = 157) was 56.9 ± 13.2 years (46.5 % males). 94.8 % had a positive response (anti-S levels ≥ 19 AU/ml). The anti-S cutoff of 200 AU/ml used to separate strong vs. weak responses. A strong response (anti-S titers ≥ 200 AU/ml) was observed in 93/157 (59.2 %) patients with a mean-age of 53.1 ± 13.8 years (45.2 % males). A weak response (anti-S titers < 200 AU/ml) was observed in 64/157 (40.8 %) cases with a mean-age of 62.3 ± 10.2 years (p < 0.0001). The strong response subgroup had lower metabolic comorbidities, including glucose hemostasis, hypertension, and dyslipidemia (p < 0.04). Moreover, the strong response subgroup had fibrosis stages F0-F2 (75.3 % vs. 56.3 %) and lower rates of advanced stages F3-F4 (24.7 % vs. 43.8 %). The F0-F2 subgroups had significantly higher rates of strong responses than the F3-F4 stages. The anti-S ≥ 200 and anti-S ≥ 400 AU/ml response achieved in 66 % and 36.8 % of the F0-F2 population was significantly higher than the 45.1 % (p = 0.006) and 23.5 % (p = 0.05) in the F3-F4 population, respectively. The Fib-4 calculations and Fibroscan evaluations were consistent with histologic fibrosis assessment. Conclusion: Advanced liver fibrosis (assessed by histology, Fib-4, or Fibroscan) is a risk factor for lower response to Pfizer's BNT162b2 vaccine, and patients should be prioritized for the vaccine booster against SARS-CoV-2.

CCL24 regulates biliary inflammation and fibrosis in primary sclerosing cholangitis.

ˆCCL24 is a pro-fibrotic, pro-inflammatory chemokine expressed in several chronic fibrotic diseases. In the liver, CCL24 plays a role in fibrosis and inflammation, and blocking CCL24 led to reduced liver injury in experimental models. We studied the role of CCL24 in primary sclerosing cholangitis (PSC) and evaluated the potential therapeutic effect of blocking CCL24 in this disease. Multidrug resistance gene 2-knockout (Mdr2-/-) mice demonstrated CCL24 expression in liver macrophages and were used as a relevant experimental PSC model. CCL24-neutralizing monoclonal antibody, CM-101, significantly improved inflammation, fibrosis, and cholestasis-related markers in the biliary area. Moreover, using spatial transcriptomics, we observed reduced proliferation and senescence of cholangiocytes following CCL24 neutralization. Next, we demonstrated that CCL24 expression was elevated under pro-fibrotic conditions in primary human cholangiocytes and macrophages, and it induced proliferation of primary human hepatic stellate cells and cholangiocytes, which was attenuated following CCL24 inhibition. Correspondingly, CCL24 was found to be highly expressed in liver biopsies of patients with PSC. CCL24 serum levels correlated with Enhanced Liver Fibrosis score, most notably in patients with high alkaline phosphatase levels. These results suggest that blocking CCL24 may have a therapeutic effect in patients with PSC by reducing liver inflammation, fibrosis, and cholestasis.

The direct profibrotic and indirect immune antifibrotic balance of blocking the cannabinoid 2 receptor.

Cannabinoid 2 (CB2) receptors expressed on immune cells are considered to be antifibrogenic. Hepatic stellate cells (HSCs) directly interact with phagocytosis lymphocytes, but the nature of this interaction is obscure. We aimed to study the effects of CB2 receptors on hepatic fibrosis via their role in mediating immunity. Hepatic fibrosis was induced by carbon-tetrachloride (CCl(4)) administration in C57BL/6 wild-type (WT) and CB2 knockout (CB2(-/-)) mice. Irradiated animals were reconstituted with WT or CB2(-/-) lymphocytes. Lymphocytes from naïve/fibrotic WT animals and healthy/cirrhotic hepatitis C virus were preincubated in vitro with or without CB2 antagonist, evaluated for proliferation and apoptosis, and then cocultured with primary mouse HSCs or a human HSC line (LX2), respectively. Lymphocyte phagocytosis was then evaluated. Following CCl(4)-administration, CB2(-/-) mice developed significant hepatic fibrosis but less necroinflammation. WT mice harbored decreased liver CD4(+) and NK(+) cells but increased CD8(+) subsets. Naïve CB2(-/-) mice had significantly decreased T cell subsets. Adoptive transfer of CB2(-/-) lymphocytes led to decreased fibrosis in the irradiated WT recipient compared with animals receiving WT lymphocytes. Moreover, necroinflammation also tended to decrease. In vitro, a CB2-antagonist directly increased human HSC activation and increased apoptosis and decreased proliferation of mice/human T cells (healthy/fibrotic) and their phagocytosis. We concluded that CB2(-/-) lymphocytes exert an antifibrotic activity, whereas lack of CB2 receptor in HSCs promotes fibrosis. These findings broaden our understanding of cannabinoid signaling in hepatic fibrosis beyond their activity solely in HSCs.

Amelioration of hepatic fibrosis by NK cell activation.

BACKGROUND AND AIMS: Interactions between hepatic stellate cells (HSCs) and immune cell subsets have emerged as important determinants of liver fibrosis progression and regression. Natural killer (NK) cells have an antifibrotic activity through killing of activated HSCs. In liver injury NK cell expression of activating/inhibitory killer immunoglobulin-related receptors (aKIR/iKIR) and their ratio are significantly increased, while class I major histocompatibilty (MHC) expression by activated HSCs is decreased. The aim of this study was to amplify the antifibrotic activity of NK cells and ameliorate hepatic fibrosis by iKIR silencing. METHODS: Human lymphocytes from patients with hepatitis C virus (HCV) infection were transfected with specific iKIR small interfering RNAs (siRNAs) or non-silencing control siRNAs, then co-cultured with a human HSC line and assessed for fibrogenic activity. To induce hepatic fibrosis, carbon tetrachloride was administrated to BALBc SCID-Beige male mice (lacking B/T/NK cells) for 4 weeks. Splenocytes from naive SCID donors (lacking B/T cells but with preserved NK cells) were transfected in vitro with either iKIR siRNA or non-silencing control siRNA, and then were transferred to the fibrotic SCID-Beige recipients. RESULTS: Transfection with iKIR or positive control siRNAs (mice and human) decreased mRNA expression of iKIR and mitogen-activated protein kinase 1 (MAPK1). Consequently, total NK cells and NK cell degranulation were increased (p=0.01), consistent with NK cell stimulation. Compared with healthy lymphocytes, when HCV lymphocytes were transfected with non-silencing control siRNA and co-cultured with HSCs there was increased α-smooth muscle actin (αSMA) expression, reflecting HSC activation. Expression of αSMA in co-cultures was attenuated when HCV lymphocytes were transfected with iKIR siRNA. In SCID-Beige recipients, hepatic fibrosis and serum alanine aminotransferase (ALT) levels were significantly attenuated as a result of receiving iKIR siRNA. CONCLUSIONS: iKIR knockdown stimulates NK cells and promotes their antifibrogenic activity in mice and human co-cultures. These findings have implications for possible immune therapeutic strategies in patients with advanced liver disease.