We Are Not Immune: Racial and Ethnic Disparities in Autoimmune Liver Diseases.

Autoimmune liver diseases are attributed to a complex interplay of biologic, acquired, and environmental factors. Increased prevalence, later stage at presentation, worse response to standard therapy, and transplant-related disparities have all been reported in racial and ethnic minorities such as Black and Latinx patients with autoimmune liver diseases. While biology and inherited genetic predispositions may partly explain these disparities, definitive and universal genetic variations underlying these differences in outcomes have not been defined. Nonetheless, socioeconomic status, access to health care, environmental and societal factors, and implicit provider bias can all contribute to poor patient outcomes. There remains an unmet need to understand and mitigate the factors contributing to health inequity in autoimmune liver diseases. In this review, we summarize the data on racial and ethnic disparities in presentation, treatment response, and outcomes pertaining to autoimmune liver diseases in minority populations, on the premise that understanding disparities is the first step toward reaching health equity.

Assessment of Selected Parameters of Liver Fibrosis and Inflammation in Patients with Diagnosed Cystic Fibrosis.

Changes in the liver and bile ducts observed in patients diagnosed with cystic fibrosis result from inflammatory processes as well as fibrosis, remodeling, apoptosis, and cholestasis. As a consequence, portal hypertension, cirrhosis, and hepatic failure may develop. So far, the complexity of these processes has not been elucidated. Study Objectives. The aim of the study was to evaluate the selected parameters of hepatitis and fibrosis (Fibrotest, Actitest, and APRI) in patients diagnosed with cystic fibrosis. Material and Methods. The study included 79 patients with cystic fibrosis, aged 1 to 20 years (mean age 9.8 years), 49 girls (62%) and 30 boys (38%). The analysis involved the following: age, sex, clinical manifestations, laboratory tests evaluating pancreas function, parameters of liver damage, and cholestasis. Fibrotest, Actitest, and APRI were performed in all subjects. Results. Elevated parameters of hepatic cell damage (hypertransaminasemia) were found in 31/79 (39.2%) patients, while abnormal cholestasis parameters in 21/79 (26.6%). The abnormal results of Fibrotest were reported in 15% of patients (12/79), while of Actitest in 10% (8/79). In contrast, elevated APRI values were found in only 7.6% (6/79) of subjects. There was a statistically significant correlation between APRI and age (higher values were observed in younger children) and between Fibrotest and Actitest and pancreatic insufficiency (higher values were found in subjects without this abnormality). Moreover, Fibrotest values were significantly higher in girls. There was no correlation between Fibrotest, Actitest, and APRI values and the type of mutation. Conclusion. It appears that Fibrotest may be used as an early marker of liver fibrosis in patients with cystic fibrosis. Increased APRI values were only found in subjects with advanced hepatic lesions, most often in the form of portal hypertension.

Assessment of the immunogenicity of residual host cell protein impurities of OsrHSA.

Human serum albumin (HSA) is the most abundant protein in human plasma and is widely used at high doses for treating various diseases. Recombinant HSA is an alternative approach to plasma-derived HSA, providing increased safety and an unlimited supply. However, the safety of the residual host cell proteins (HCPs) co-purified with Oryza sativa HSA (OsrHSA) remains to be determined. An animal system was used to assess the immunogenicity of OsrHSA and its residual HCPs. Low immunogenicity and immunotoxicity of the residual HCPs at a dose of 25 µg/kg, equivalent to 25 times the clinical dosage of HSA, were observed. An anti-drug-antibody (ADA) analysis revealed that anti-HSA, anti-OsrHSA or anti-HCP antibodies developed with a low frequency in pHSA and OsrHSA treatments, but the titers were as low as 1.0-2.0. Furthermore, the titer and the incidence of the specific antibodies were not significantly different between the pHSA and OsrHSA groups, indicating that OsrHSA presents similar immunogenicity to that of pHSA. More importantly, no cytokines were stimulated after the administration of OsrHSA and the residual HCPs, suggesting that there was no risk of a cytokine storm. These results demonstrated that the residual HCPs from OsrHSA have low immunogenicity, indicating that the rice endosperm is one of the best hosts for plant molecular pharming.

Assessment of novel vaccination regimens using viral vectored liver stage malaria vaccines encoding ME-TRAP.

Heterologous prime-boost vaccination with viral vectors simian adenovirus 63 (ChAd63) and Modified Vaccinia Ankara (MVA) induces potent T cell and antibody responses in humans. The 8-week regimen demonstrates significant efficacy against malaria when expressing the pre-erythrocytic malaria antigen Thrombospondin-Related Adhesion Protein fused to a multiple epitope string (ME-TRAP). We tested these vaccines in 7 new 4- and 8- week interval schedules to evaluate safety and immunogenicity of multiple ChAd63 ME-TRAP priming vaccinations (denoted A), multiple MVA ME-TRAP boosts (denoted M) and alternating vectors. All regimens exhibited acceptable reactogenicity and CD8+ T cell immunogenicity was enhanced with a 4-week interval (AM) and with incorporation of additional ChAd63 ME-TRAP vaccination at 4- or 8-weeks (AAM or A_A_M). Induction of TRAP antibodies was comparable between schedules. T cell immunity against the ChAd63 hexon did not affect T cell responses to the vaccine insert, however pre-vaccination ChAd63-specific T cells correlated with reduced TRAP antibodies. Vaccine-induced antibodies against MVA did not affect TRAP antibody induction, and correlated positively with ME-TRAP-specific T cells. This study identifies potentially more effective immunisation regimens to assess in Phase IIa trials and demonstrates a degree of flexibility with the timing of vectored vaccine administration, aiding incorporation into existing vaccination programmes.

Preclinical renal chemo-protective potential of Prunus amygdalus Batsch seed coat via alteration of multiple molecular pathways.

Prunus amygdalus Batsch (almond) is a classical nutritive traditional Indian medicine. Along with nutritive with anti-oxidant properties, it is, clinically, used in the treatment of various diseases with underlying anti-oxidant mechanism. This study is an effort to scrutinise the renal protective effect of P. amygdalus Batsch or green almond (GA) seed coat extract and its underlying mechanism in animal model of Ferric nitrilotriacetate (Fe-NTA) induced renal cell carcinoma (RCC). RCC was induced in Swiss Albino Wistar rats by intraperitoneal injection of Fe-NTA. The rats were then treated with ethanolic extract of GA (25, 50 and 100 mg/kg per oral) for 22 weeks. Efficacy of GA administration was evaluated by change in biochemical, renal, macroscopical and histopathological parameters and alterations. Additionally, interleukin-6 (IL-6), tumour necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and inflammatory mediator including prostaglandin E2 (PGE2), nuclear factor-kappa B (NF-κB) were also observed to explore the possible mechanisms. The oral administration of GA significantly (p < .001) altered the Fe-NTA induced RCC in rats by inhibition of renal nodules, decolourisation of tissues, tumour promoter marker including thymidine 3[H] incorporation, ornithine decarboxylase, renal parameters and anti-oxidant parameters in serum. Additionally, GA treatment significantly (p < .001) down-regulated the IL-6, IL-1ß, TNF-α, inflammatory mediators PGE2 and NF-κB in a dose-dependent manner. Histopathology observation supported the renal protective effect of GA by alteration in necrosis, size of Bowman capsules and inflammatory cells. Hence, it can be concluded that GA possesses observable chemo-protective action and effect on Fe-NTA induced RCC via dual inhibition mechanism one by inhibiting free radical generation and second by inhibiting inflammation.

Integrated Assessment of Diclofenac Biotransformation, Pharmacokinetics, and Omics-Based Toxicity in a Three-Dimensional Human Liver-Immunocompetent Coculture System.

In vitro hepatocyte culture systems have inherent limitations in capturing known human drug toxicities that arise from complex immune responses. Therefore, we established and characterized a liver immunocompetent coculture model and evaluated diclofenac (DCF) metabolic profiles, in vitro-in vivo clearance correlations, toxicological responses, and acute phase responses using liquid chromatography-tandem mass spectrometry. DCF biotransformation was assessed after 48 hours of culture, and the major phase I and II metabolites were similar to the in vivo DCF metabolism profile in humans. Further characterization of secreted bile acids in the medium revealed that a glycine-conjugated bile acid was a sensitive marker of dose-dependent toxicity in this three-dimensional liver microphysiological system. Protein markers were significantly elevated in the culture medium at high micromolar doses of DCF, which were also observed previously for acute drug-induced toxicity in humans. In this immunocompetent model, lipopolysaccharide treatment evoked an inflammatory response that resulted in a marked increase in the overall number of acute phase proteins. Kupffer cell-mediated cytokine release recapitulated an in vivo proinflammatory response exemplified by a cohort of 11 cytokines that were differentially regulated after lipopolysaccharide induction, including interleukin (IL)-1ß, IL-1Ra, IL-6, IL-8, IP-10, tumor necrosis factor-α, RANTES (regulated on activation normal T cell expressed and secreted), granulocyte colony-stimulating factor, macrophage colony-stimulating factor, macrophage inflammatory protein-1ß, and IL-5. In summary, our findings indicate that three-dimensional liver microphysiological systems may serve as preclinical investigational platforms from the perspective of the discovery of a set of clinically relevant biomarkers including potential reactive metabolites, endogenous bile acids, excreted proteins, and cytokines to predict early drug-induced liver toxicity in humans.

CD8+ T-cell mediated anti-malaria protection induced by malaria vaccines; assessment of hepatic CD8+ T cells by SCBC assay.

Malaria is a severe infectious disease with relatively high mortality, thus having been a scourge of humanity. There are a few candidate malaria vaccines that have shown a protective efficacy in humans against malaria. One of the candidate human malaria vaccines, which is based on human malaria sporozoites and called PfSPZ Vaccine, has been shown to protect a significant proportion of vaccine recipients from getting malaria. PfSPZ Vaccine elicits a potent response of hepatic CD8+ T cells that are specific for malaria antigens in non-human primates. To further characterize hepatic CD8+ T cells induced by the sporozoite-based malaria vaccine in a mouse model, we have used a cutting-edge Single-cell Barcode (SCBC) assay, a recently emerged approach/method for investigating the nature of T-cells responses during infection or cancer. Using the SCBC technology, we have identified a population of hepatic CD8+ T cells that are polyfunctional at a single cell level only in a group of vaccinated mice upon malaria challenge. The cytokines/chemokines secreted by these polyfunctional CD8+ T-cell subsets include MIP-1α, RANTES, IFN-γ, and/or IL-17A, which have shown to be associated with protective T-cell responses against certain pathogens. Therefore, a successful induction of such polyfunctional hepatic CD8+ T cells may be a key to the development of effective human malaria vaccine. In addition, the SCBC technology could provide a new level of diagnostic that will allow for a more accurate determination of vaccine efficacy.

Assessment of intrahepatic regulatory T cells in children with autoimmune hepatitis.

UNLABELLED:  Background. T-cell populations regulate the balance of immune responses. The CD (Cluster of differentiation) 4+CD25+ regulatory T cells (Tregs) are crucial for maintaining negative control of various immune responses. There are different T-cell subpopulations with regulatory functions, as natural killer T cells, CD8+ and CD28. The forkhead box P3 (FOXP3) regulates Treg development and is required for its suppressive function. AIM: To evaluate the hepatic expression of the intrahepatic Tregs, Ig (immunoglobulin) G and IgM plasma cells in autoimmune hepatitis (AIH) and other chronic liver diseases (CLDs). MATERIAL AND METHODS: This study included 100 pediatric patients; 50 AIH and 50 CLDs other than AIH. All patients were subjected to routine investigations of CLDs plus immune-staining of liver tissue for FOXp3, IgG and IgM plasma cells, CD4 and CD8 T-cells. RESULTS: The FOXP3+ T cells in patients with AIH (6.3 ± 5) were significantly higher than that in the non-AIH (2.1 ± 2.6). FOXP3+ T cells were abundant in liver tissue with marked inflammatory cellular infiltrate. CD4+ and CD8+ infiltrating the liver tissue and IgG positive cells were significantly higher in AIH group, while the expression of IgM positive cells showed no significant difference. The IgG/IgM was significantly higher in the AIH treatment responders (3 ± 3) than non-responders (1.6 ± 0.5), while there was no significant difference regarding the intrahepatic expression of FOXP3+, CD4+, CD8+ cells, T-cells, IgG and IgM plasma cells. CONCLUSION: Intrahepatic Tregs were increased in number in patients with AIH in the initial presentation, and their presence is associated with increased activity and inflammation in liver biopsy.

Diagnostic performance of T lymphocyte subpopulations in assessment of liver fibrosis stages in hepatitis C virus patients: simple noninvasive score.

BACKGROUND/AIMS: Evaluation of liver fibrosis in patients infected with hepatitis C virus is highly useful for the diagnosis of the disease as well as therapeutic decision. Our aim was to develop and validate a simple noninvasive score for liver fibrosis staging in chronic hepatitis C (CHC) patients and compare its performance against three published simple noninvasive indexes. MATERIALS AND METHODS: CHC patients were divided into two groups: an estimated group (n=70) and a validated group (n=52). Liver fibrosis was tested in biopsies using the Metavair score system. CD4 and CD8 count/percentage were assayed by fluorescence-activated cell sorting analysis. RESULTS: The multivariate discriminant analysis selects a function on the basis of absolute values of five biochemical markers: immune fibrosis index (IFI); score=3.07+3.06×CD4/CD8+0.02×α-fetoprotein (U/l)-0.07×alanine aminotransferase ratio-0.005×platelet count (10/l)-1.4×albumin (g/dl). The IFI score produced areas under curve of 0.949, 0.947, and 0.806 for differentiation of all patient categories [significant fibrosis (F2-F4), advanced fibrosis (F3-F4), and cirrhosis (F4)]. CONCLUSION: The IFI score, a novel noninvasive test, can be used easily for the prediction of liver fibrosis stage in CHC patients. Our score was more efficient than aspartate aminotransferase to platelet ratio index, fibrosis index, and fibroQ and more suitable for use in Egyptian hepatitis C virus patients.

Assessment of Parasite Liver-Stage Burden in Human-Liver Chimeric Mice.

Humanized mice with a chimeric liver are a promising tool to evaluate the "in vivo" efficacy of novel compounds or vaccine-induced antibodies directed against the pre-erythrocytic stages of Plasmodium falciparum. The absence of human red blood cells in these humanized mice precludes the transition from liver to blood stage. The qPCR-based method described below allows for a sensitive and reliable quantification of parasite DNA in the chimeric liver following a challenge via infected mosquito bite or intravenous injection of sporozoites. With this method approximately 25 % of the total chimeric liver is examined and a single infected hepatocyte can be detected in the analyzed tissue. The use of appropriate species-specific probes can also allow for the detection of other Plasmodium species in vivo.

Comparative assessment of vaccine vectors encoding ten malaria antigens identifies two protective liver-stage candidates.

The development of an efficacious Plasmodium falciparum malaria vaccine remains a top priority for global health. Vaccination with irradiated sporozoites is able to provide complete sterile protection through the action of CD8(+) T cells at the liver-stage of infection. However, this method is currently unsuitable for large-scale deployment and focus has instead turned to the development of sub-unit vaccines. Sub-unit vaccine efforts have traditionally focused on two well-known pre-erythrocytic antigens, CSP and TRAP, yet thousands of genes are expressed in the liver-stage. We sought to assess the ability of eight alternative P. falciparum pre-erythrocytic antigens to induce a high proportion of CD8(+) T cells. We show that all antigens, when expressed individually in the non-replicating viral vectors ChAd63 and MVA, are capable of inducing an immune response in mice. Furthermore, we also developed chimeric P. berghei parasites expressing the cognate P. falciparum antigen to enable assessment of efficacy in mice. Our preliminary results indicate that vectors encoding either PfLSA1 or PfLSAP2 are capable of inducing sterile protection dependent on the presence of CD8(+) T cells. This work has identified two promising P. falciparum liver-stage candidate antigens that will now undergo further testing in humans.

Th-1 biased immunomodulation and synergistic antileishmanial activity of stable cationic lipid-polymer hybrid nanoparticle: biodistribution and toxicity assessment of encapsulated amphotericin B.

To address issues related to Amphotericin B (AmpB) clinical applications, we developed macrophage targeted cationic stearylamine lipid-polymer hybrid nanoparticles (LPNPs) with complementary characteristics of both polymeric nanoparticles and liposomes, for enhancement of therapeutic efficacy and diminishing toxic effect of encapsulated AmpB. The LPNPs (size 198.3 ± 3.52 nm, PDI 0.135 ± 0.03, zeta potential +31.6 ± 1.91 mV) provide core-shell type structure which has the ability to encapsulate amphiphilic AmpB in higher amount (Encapsulation efficiency 96.1 ± 2.01%), sustain drug release and stabilize formulation tremendously. Attenuated erythrocytes and J774A.1 toxicity of LPNPs demonstrated safe applicability for parenteral administration. Elevated macrophage uptake of LPNPs, rapid plasma clearance and higher drug allocation in macrophage abundant liver and spleen illustrated admirable antileishmanial efficacy of AmpB-LPNPs in vitro (IC50, 0.16 ± 0.04 µg AmpB/ml) and in vivo (89.41 ± 3.58% parasite inhibition) against visceral leishmaniasis models. Augmentation in antileishmanial activity due to Th-1 biased immune-alteration mediated by drug-free LPNPs which elevated microbicidal mediators of macrophages. Moreover, minimal distribution to kidney tissues and low level of nephrotoxicity markers (creatinine and BUN) demonstrated the safety profile of AmpB-LPNPs. Conclusively, reliable safety and macrophage directed therapeutic performance of AmpB-LPNPs suggest it as promising alternative to commercial AmpB-formulations for the eradication of intra-macrophage diseases.

Noninvasive markers in the assessment and management of autoimmune liver diseases.

Historically, liver biopsy has been used to determine the etiology of liver disease, the degree of inflammation, the stage of liver fibrosis, and the response to treatments. In the last decade, the advent of noninvasive tests has improved the diagnosis and management of autoimmune liver diseases. For example, serum markers can identify hepatic inflammation, whereas ultrasound and MRI can diagnose liver fibrosis. Physicians now have a much larger repertoire of diagnostic tests to assess the liver parenchyma compared with liver biopsy alone. In some rare cases, noninvasive tests may provide an alternative to liver biopsy. In general, however, these noninvasive tests complement liver biopsy and provide quick, accurate, and reliable adjunctive data.

Model for in vivo assessment of humoral protection against malaria sporozoite challenge by passive transfer of monoclonal antibodies and immune serum.

Evidence from clinical trials of malaria vaccine candidates suggests that both cell-mediated and humoral immunity to pre-erythrocytic parasite stages can provide protection against infection. Novel pre-erythrocytic antibody (Ab) targets could be key to improving vaccine formulations, which are currently based on targeting antigens such as the circumsporozoite protein (CSP). However, methods to assess the effects of sporozoite-specific Abs on pre-erythrocytic infection in vivo remain underdeveloped. Here, we combined passive transfer of monoclonal Abs (MAbs) or immune serum with a luciferase-expressing Plasmodium yoelii sporozoite challenge to assess Ab-mediated inhibition of liver infection in mice. Passive transfer of a P. yoelii CSP MAb showed inhibition of liver infection when mice were challenged with sporozoites either intravenously or by infectious mosquito bite. However, inhibition was most potent for the mosquito bite challenge, leading to a more significant reduction of liver-stage burden and even a lack of progression to blood-stage parasitemia. This suggests that Abs provide effective protection against a natural infection. Inhibition of liver infection was also achieved by passive transfer of immune serum from whole-parasite-immunized mice. Furthermore, we demonstrated that passive transfer of a MAb against P. falciparum CSP inhibited liver-stage infection in a humanized mouse/P. falciparum challenge model. Together, these models constitute unique and sensitive in vivo methods to assess serum-transferable protection against Plasmodium sporozoite challenge.

Safety assessment of intraportal liver cell application in New Zealand white rabbits under GLP conditions.

Liver cell transplantation (LCT) is considered a new therapeutic strategy for the treatment of acute liver failure and inborn metabolic defects of the liver. Although minimally invasive, known safety risks of the method include portal vein thrombosis and pulmonary embolism. Since no systematic data on these potential side effects exist, we investigated the toxicological profile of repeated intraportal infusion of allogeneic liver cells in 30 rabbits under GLP conditions. Rabbit liver cells were administered once daily for 6 consecutive days at 3 different dose levels, followed by a 2-week recovery period. No test item-related mortality was observed. During cell infusion, clinical findings such as signs of apathy and hyperventilation, moderate elevations of liver enzymes ALT and AST and a slight decrease in AP were observed, all fully reversible. Cell therapy-related macroscopic and histological findings, especially in liver and lungs, were observed in animals of all dose groups. In conclusion, the liver and lungs were identified as potential toxicological target organs of intraportal allogeneic liver cell infusion. A NOAEL (no observed adverse effect level) was not defined because of findings observed also in the low-dose group. No unexpected reactions became apparent in this GLP study. Overall, LCT at total doses up to 12 % (2 % daily over 6 days) of the total liver cell count were tolerated in rabbits. Observed adverse effects are not considered critical for treatment in the intended patient populations provided that a thorough monitoring of safety relevant parameters is in place during the application procedure.

Behavioral assessment of cognitive function using a translational neonatal piglet model.

Pigs are used in myriad research disciplines related to human health, but no studies have employed the piglet to directly assess cognitive function during the neonatal period. Our objective was to develop a behavioral assay for neonatal piglets to assess learning and memory. At 2-wk of age, piglets were trained to locate a milk reward in an 8-arm radial maze using colored intra-maze cues (acquisition phase, 60-s trials with 8 trials per d for 4d). Cue colors were then reversed and pigs re-tested to assess learning and working memory (reversal phase). Piglets quickly learned the simple associative acquisition task, and proficiency greatly improved throughout reversal testing. To further assess the behavioral assay, piglets received an i.p. injection of saline or polyinosinic:polycytidylic acid (poly I:C; 5mg/kg body weight) immediately preceding reversal testing. Poly I:C-treated piglets exhibited acute sickness behaviors, but observationally, were asymptomatic 12-h post-injection. Pro-inflammatory cytokine mRNA expression was elevated 4-h post-injection in both peripheral and central compartments, and plasma cytokine protein levels were concurrently elevated. Specifically, poly I:C elicited the largest increases in interleukin (IL)-1ß mRNA in the liver, spleen, and hippocampus. At 24-, 48-, and 72-h post-injection (i.e., after acute sickness), poly I:C-treated piglets committed more incorrect arm entries, required more time to complete the reversal task, and moved a greater distance in the maze compared with control piglets. Collectively, these data demonstrate that neonatal piglets are capable of being trained in traditional learning and memory tests, and peripheral immune activation elicits alterations in cognitive processing in the neonate.

Production of polyclonal antibody against interleukin-33 and assessment of its distribution in murine liver and lung.

Interleukin (IL)-33 is the latest member of IL-1 cytokine family. In this study, the cloning, expression, purification, and polyclonal antibody preparation of mouse IL-33 were described. The coding region of IL-33 mature protein was cloned into the prokaryotic expression vector pET-44. The recombinant protein, IL-33 containing a hexahistidine tag in the C-terminal, was expressed in Escherichia coli. The expressed soluble protein was purified by immobilized metal-ion affinity chromatography using Ni2+-nitrilotriacetic acid agarose. The rabbits were immunized with the purified recombinant protein. The obtained antiserum was precipitated by saturated ammonium sulfate and then purified by Protein A affinity chromatography. The sensitivity and specificity of the antibodies were evaluated by enzyme-linked immunosorbent assay and immunohistochemistry. The high titer (1 : 32000) polyclonal antibodies with high specificity were obtained by immunizing rabbits with the purified recombinant protein. Significant expression of IL-33 was seen in mouse liver and lung tissues determined with the anti-IL-33. The production of the polyclonal antibody against IL-33 provides a good tool for studying the biofunctions of IL-33.

Histologic graft assessment after clinical islet transplantation.

BACKGROUND: An accurate monitoring would help understanding the fate of islet grafts after transplantation. METHODS: This work assessed the feasibility of needle biopsy monitoring after intraportal islet transplantation (n=16), and islet graft morphology was studied with the addition of autopsy samples (n=2). Pancreas autopsy samples from two nondiabetic individuals were used as control. RESULTS: Islet tissue was found in five needle samples (31%). Sampling success was related to size (100% sampling for the four biopsies of 1.8 cm in length or higher, P