Diagnostic accuracy of serum matrix metalloproteinase-7 as a biomarker of biliary atresia in a large North American cohort.

BACKGROUND AND AIMS: High levels of serum matrix metalloproteinase-7 (MMP-7) have been linked to biliary atresia (BA), with wide variation in concentration cutoffs. We investigated the accuracy of serum MMP-7 as a diagnostic biomarker in a large North American cohort. APPROACH AND RESULTS: MMP-7 was measured in serum samples of 399 infants with cholestasis in the Prospective Database of Infants with Cholestasis study of the Childhood Liver Disease Research Network, 201 infants with BA and 198 with non-BA cholestasis (age median: 64 and 59 days, p = 0.94). MMP-7 was assayed on antibody-bead fluorescence (single-plex) and time resolved fluorescence energy transfer assays. The discriminative performance of MMP-7 was compared with other clinical markers. On the single-plex assay, MMP-7 generated an AUROC of 0.90 (CI: 0.87-0.94). At cutoff 52.8 ng/mL, it produced sensitivity = 94.03%, specificity = 77.78%, positive predictive value = 64.46%, and negative predictive value = 96.82% for BA. AUROC for gamma-glutamyl transferase = 0.81 (CI: 0.77-0.86), stool color = 0.68 (CI: 0.63-0.73), and pathology = 0.84 (CI: 0.76-0.91). Logistic regression models of MMP-7 with other clinical variables individually or combined showed an increase for MMP-7+gamma-glutamyl transferase AUROC to 0.91 (CI: 0.88-0.95). Serum concentrations produced by time resolved fluorescence energy transfer differed from single-plex, with an optimal cutoff of 18.2 ng/mL. Results were consistent within each assay technology and generated similar AUROCs. CONCLUSIONS: Serum MMP-7 has high discriminative properties to differentiate BA from other forms of neonatal cholestasis. MMP-7 cutoff values vary according to assay technology. Using MMP-7 in the evaluation of infants with cholestasis may simplify diagnostic algorithms and shorten the time to hepatoportoenterostomy.

Sarcopenia is associated with osteopenia and impaired quality of life in children with genetic intrahepatic cholestatic liver disease.

BACKGROUND: Sarcopenia occurs in pediatric chronic liver disease, although the prevalence and contributing factors in genetic intrahepatic cholestasis are not well-described. The objective of this study was to measure muscle mass in school-aged children with genetic intrahepatic cholestasis and assess relationships between sarcopenia, clinical variables, and outcomes. METHODS: Estimated skeletal muscle mass (eSMM) was calculated on dual-energy x-ray absorptiometry obtained in a Childhood Liver Disease Research Network study of children with bile acid synthesis disorders(BASD) alpha-1 antitrypsin deficiency (a1ATd), chronic intrahepatic cholestasis (CIC), and Alagille syndrome (ALGS). Relationships between eSMM, liver disease, and transplant-free survival were assessed. RESULTS: eSMM was calculated in 127 participants (5-18 y): 12 BASD, 41 a1ATd, 33 CIC, and 41 ALGS. eSMM z-score was lower in CIC (-1.6 ± 1.3) and ALGS (-2.1 ± 1.0) than BASD (-0.1 ± 1.1) and a1ATd (-0.5 ± 0.8, p < 0.001). Sarcopenia (defined as eSMM z-score ≤- 2) was present in 33.3% of CIC and 41.5% of ALGS participants. eSMM correlated with bone mineral density in the 4 disease groups (r=0.52-0.55, p < 0.001-0.07), but not serum bile acids, bilirubin, aspartate aminotransferase/platelet ratio index, or clinically evident portal hypertension. Of the 2 patients who died (1 with sarcopenia) and 18 who underwent liver transplant (LT, 4 with sarcopenia), eSMM z-score did not predict transplant-free survival. eSMM z-score correlated with the Physical Pediatric Quality of Life Inventory score (r=0.38-0.53, p = 0.007-0.04) in CIC and a1ATd. CONCLUSION: Severe sarcopenia occurs in some children with ALGS and CIC. The lack of correlation between eSMM and biochemical cholestasis suggests mechanisms beyond cholestasis contribute to sarcopenia. While sarcopenia did not predict transplant-free survival, LT and death were infrequent events. Future studies may define mechanisms of sarcopenia in genetic intrahepatic cholestasis.

Alpha-1 Antitrypsin Deficiency Liver Disease.

Liver disease in homozygous ZZ alpha-1 antitrypsin (AAT) deficiency occurs due to the accumulation of large quantities of AAT mutant Z protein polymers in the liver. The mutant Z protein folds improperly during biogenesis and is retained within the hepatocytes rather than appropriately secreted. These intracellular polymers trigger an injury cascade, which leads to liver injury. However, the clinical liver disease is highly variable and not all patients with this same homozygous ZZ genotype develop liver disease. Evidence suggests that genetic determinants of intracellular protein processing, among other unidentified genetic and environmental factors, likely play a role in liver disease susceptibility. Advancements made in development of new treatment strategies using siRNA technology, and other novel approaches, are promising, and multiple human liver disease trials are underway.

Alpha-1 antitrypsin deficiency liver disease.

The clinical presentation of liver disease is highly variable in homozygous ZZ alpha-1 antitrypsin (AAT) deficiency, and not all patients with the homozygous ZZ genotype develop liver disease. Although not fully identified, there is likely a strong influence of genetic and environmental modifiers of the intracellular injury cascade and fibrotic response. Most ZZ children are well and remain undiagnosed. Of those who come to medical attention, the most common pediatric presentation is neonatal cholestatic hepatitis, sometimes referred to as "neonatal hepatitis syndrome". The gold standard for diagnosis of AAT deficiency is analysis of the AAT protein phenotype in the patient serum or the genotype of their DNA genome. Careful follow up of all diagnosed children is important. Heterozygotes for S and Z alleles of AAT (SZ) may develop progressive liver disease similar to ZZ patients and also require close monitoring. There is no specific treatment for AAT deficiency induced liver disease and current therapy remains supportive with management of complications. Rarely, patients require liver transplant and typically the patient outcomes are excellent. With improved understanding of liver injury mechanisms, new strategies for treatment are now being explored, including siRNA technology, molecules to modulate secretion, and enhancers of proteolysis.

Up-regulation of miR-34b/c by JNK and FOXO3 protects from liver fibrosis.

α1-Antitrypsin (AAT) deficiency is a common genetic disease presenting with lung and liver diseases. AAT deficiency results from pathogenic variants in the SERPINA1 gene encoding AAT and the common mutant Z allele of SERPINA1 encodes for Z α1-antitrypsin (ATZ), a protein forming hepatotoxic polymers retained in the endoplasmic reticulum of hepatocytes. PiZ mice express the human ATZ and are a valuable model to investigate the human liver disease of AAT deficiency. In this study, we investigated differential expression of microRNAs (miRNAs) between PiZ and control mice and found that miR-34b/c was up-regulated and its levels correlated with intrahepatic ATZ. Furthermore, in PiZ mouse livers, we found that Forkhead Box O3 (FOXO3) driving microRNA-34b/c (miR-34b/c) expression was activated and miR-34b/c expression was dependent upon c-Jun N-terminal kinase (JNK) phosphorylation on Ser574 Deletion of miR-34b/c in PiZ mice resulted in early development of liver fibrosis and increased signaling of platelet-derived growth factor (PDGF), a target of miR-34b/c. Activation of FOXO3 and increased miR-34c were confirmed in livers of humans with AAT deficiency. In addition, JNK-activated FOXO3 and miR-34b/c up-regulation were detected in several mouse models of liver fibrosis. This study reveals a pathway involved in liver fibrosis and potentially implicated in both genetic and acquired causes of hepatic fibrosis.

Nonfasted Liver Stiffness Correlates with Liver Disease Parameters and Portal Hypertension in Pediatric Cholestatic Liver Disease.

Elastographic measurement of liver stiffness is of growing importance in the assessment of liver disease. Pediatric experiences with this technique are primarily single center and limited in scope. The Childhood Liver Disease Research Network provided a unique opportunity to assess elastography in a well-characterized multi-institutional cohort. Children with biliary atresia (BA), alpha-1 antitrypsin deficiency (A1ATD), or Alagille syndrome (ALGS) followed in a prospective longitudinal network study were eligible for enrollment in a prospective investigation of transient elastography (FibroScan). Studies were performed in participants who were nonfasted and nonsedated. Liver stiffness measurements (LSMs) were correlated with standard clinical and biochemical parameters of liver disease along with a research definition of clinically evident portal hypertension (CEPH) graded as absent, possible, or definite. Between November 2016 and August 2019, 550 participants with a mean age of 8.8 years were enrolled, 458 of whom had valid LSMs (BA, n = 254; A1ATD, n = 104; ALGS, n = 100). Invalid scans were more common in participants <2 years old. There was a positive correlation between LSM and total bilirubin, international normalized ratio (INR), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transpeptidase (GGT), GGT to platelet ratio (GPR), pediatric end-stage liver disease score, AST to platelet ratio index, and spleen size, and a negative correlation with albumin and platelet count in BA, with similar correlations for A1ATD (except AST, ALT, and albumin) and ALGS (except for INR, GGT, GPR, and ALT). Possible or definite CEPH was more common in BA compared to ALGS and A1ATD. LSM was greater in definite versus absent CEPH in all three diseases. Disease-specific clinical and biochemical characteristics of the different CEPH grades were observed. Conclusion: It is feasible to obtain LSMs in children, especially over the age of 2 years. LSM correlates with liver parameters and portal hypertension, although disease-specific patterns exist.

Development of an RNAi therapeutic for alpha-1-antitrypsin liver disease.

The autosomal codominant genetic disorder alpha-1 antitrypsin (AAT) deficiency (AATD) causes pulmonary and liver disease. Individuals homozygous for the mutant Z allele accumulate polymers of Z-AAT protein in hepatocytes, where AAT is primarily produced. This accumulation causes endoplasmic reticulum (ER) stress, oxidative stress, damage to mitochondria, and inflammation, leading to fibrosis, cirrhosis, and hepatocellular carcinoma. The magnitude of AAT reduction and duration of response from first-generation intravenously administered RNA interference (RNAi) therapeutic ARC-AAT and then with next-generation subcutaneously administered ARO-AAT were assessed by measuring AAT protein in serum of the PiZ transgenic mouse model and human volunteers. The impact of Z-AAT reduction by RNAi on liver disease phenotypes was evaluated in PiZ mice by measuring polymeric Z-AAT in the liver; expression of genes associated with fibrosis, autophagy, apoptosis, and redox regulation; inflammation; Z-AAT globule parameters; and tumor formation. Ultrastructure of the ER, mitochondria, and autophagosomes in hepatocytes was evaluated by electron microscopy. In mice, sustained RNAi treatment reduced hepatic Z-AAT polymer, restored ER and mitochondrial health, normalized expression of disease-associated genes, reduced inflammation, and prevented tumor formation. RNAi therapy holds promise for the treatment of patients with AATD-associated liver disease. ARO-AAT is currently in phase II/III clinical trials.

Hepatic Histology in Treatment-naïve Children With Chronic Hepatitis B Infection Living in the United States and Canada.

OBJECTIVES: Chronic hepatitis B virus infection is a major cause of morbidity and mortality. The aim of the study is to describe the hepatic histology in children chronically infected with hepatitis B virus living in the United States and Canada. METHODS: Liver biopsies of 134 treatment-naïve children with chronic hepatitis B virus infection were scored for inflammation, fibrosis, and other histological features, and correlated with clinical and laboratory data. RESULTS: Sixty percentage of subjects acquired the infection vertically, 51% were male, and 69% were hepatitis B e antigen-positive at the time of the biopsy. Hepatitis B DNA levels were generally high (mean 7.70 log IU/mL), as was serum alanine aminotransferase (median 120 U/L). Using the Ishak-modified histology activity index scoring system, interface hepatitis was mild in 31%, moderate in 61%, and severe in 6%. Lobular inflammation was mild in 54%, moderate in 29%, and marked in 7%. Portal inflammation was mild in 38% and moderate in 62% of subjects. Eighteen percentage had no fibrosis, 59% had portal expansion without bridging fibrosis, 19% had bridging fibrosis, and 4% had cirrhosis. Alanine aminotransferase positively correlated with inflammation and fibrosis. Neither age, duration of infection, nor Hepatitis B virus DNA levels correlated with fibrosis. Fibrosis-4 index did not correlate with fibrosis but correlated with inflammation. Aspartate aminotransferase/platelet ratio index correlated with both inflammation and fibrosis. CONCLUSIONS: Chronic hepatitis B virus infection results in significant inflammation and fibrosis during childhood. Serum alanine aminotransferase is a strong indicator of the severity and extent of hepatic inflammation and fibrosis.

Shear wave elastography correlates with liver fibrosis scores in pediatric patients with liver disease.

BACKGROUND: While liver biopsy remains the gold standard, given the procedure risks and sampling errors, there is a need for reliable noninvasive biomarkers of hepatic fibrosis. OBJECTIVE: Determine the accuracy of two-dimensional shear wave elastography (2-D SWE) in predicting the histological severity of liver fibrosis in pediatric patients with known or suspected liver disease. MATERIALS AND METHODS: Subjects 0-18 years old with known or suspected liver disease and liver biopsy within 30 days (n=70) were included. Comparisons by 2-D SWE were made to a control group (n=79). Two-dimensional SWE was performed using the GE LOGIQ E9 system. Liver biopsy specimens were scored according to METAVIR and Ishak scoring systems using Spearman's Rho correlation. Receiver operator characteristic (ROC) analysis, Kruskal-Wallis and Mann-Whitney U tests were conducted. RESULTS: Control group median 2-D SWE measurements were lower than in subjects with any degree of liver fibrosis (P<0.001). Those with METAVIR F0 and Ishak 0 scores had significantly lower median 2-D SWE measurements (1.35 m/s; 1.36 m/s) than those with more advanced liver disease (F1-F3: 1.49-1.62 m/s; 1-4: 1.45-1.63 m/s) (P<0.05 for all), whereas the 2-D SWE in the higher scores were similar. Results did not differ between METAVIR and Ishak scores for any degree of fibrosis. Fibrosis scores moderately correlated with median 2-D SWE measurements (rs=0.43). The area under the curve for F1 compared to combined control/F0 was 0.89 (95% confidence interval [CI] 0.83-0.95; P<0.001) with sensitivity of 94.6% and specificity of 78.6%. Results for Ishak score 1 were similar. The ideal cutoff value for identifying fibrosis was determined to be 1.29 m/s. CONCLUSION: The liver 2-D SWE measurements correlated with the histological liver fibrosis scores, regardless of the histopathological scoring system, although 2-D SWE was better at identifying patients with early fibrosis, not at distinguishing among the individual fibrosis levels. Two-dimensional SWE using the GE LOGIQ US system is useful for identifying pediatric patients at risk for liver fibrosis.

Age, race and viral genotype are associated with the prevalence of hepatitis B e antigen in children and adults with chronic hepatitis B.

Hepatitis B e antigen (HBeAg) is an important serological marker of hepatitis B virus (HBV) infection and is associated with higher levels of viraemia, increased risk of infectivity to others and increased risk of hepatocellular carcinoma. We analysed HBeAg status in a large cohort of adults and children enrolled in Cohort Studies of the Hepatitis B Research Network, long-term natural history studies of chronic HBV infection. A cross-sectional analysis examined factors associated with HBeAg positivity, including demographic and virologic data, across the age spectrum. Among 2241 enrolled participants who met criteria for this analysis, 825 (37%) were seropositive for HBeAg. The prevalence of HBeAg was lower in those with older age, ranging from 85% among those up to 10 years of age to only 12% among those older than 50 years. In addition to age, both race and HBV genotype were independently associated with HBeAg positivity. There was a significant interaction between age and race; the prevalence of HBeAg was significantly higher among Asians > 10-30 years old vs Whites or Blacks who were >10 to 30 years old and those infected with HBV genotype C. Conversely, the presence of the basal core promoter and precore variants was associated with significantly lower prevalence of HBeAg, even when adjusted for age, race and genotype. These data will provide a better understanding of factors associated with seropositivity for HBeAg and may lead to better strategies for preventing HBV infection and broader indications for antiviral therapy.

Diagnostic accuracy of non-contrast magnetic resonance enterography in detecting active bowel inflammation in pediatric patients with diagnosed or suspected inflammatory bowel disease to determine necessity of gadolinium-based contrast agents.

BACKGROUND: Pediatric patients with inflammatory bowel disease (IBD) are at increased risk of gadolinium deposition given the potential need for multiple contrast-enhanced magnetic resonance enterography (MRE) exams over their lifetime. OBJECTIVE: To determine whether gadolinium-based contrast agents are necessary in assessing active bowel inflammation on MRE in pediatric patients with known or suspected IBD. MATERIALS AND METHODS: We conducted a retrospective study of 77 patients (7-18 years; 68.8% male) with known (n=58) or suspected (n=19) IBD and endoscopy with biopsy performed within 30 days of MRE without and with contrast evaluated bowel and non-bowel findings. During three visual analysis sessions, two radiologists reviewed pre-, post-, and pre-/post-contrast MRE images. A third radiologist independently reviewed 27 studies to assess inter-reader reliability. We used Cohen kappa (κ), Fleiss kappa, (κF), McNemar test, and sensitivity and specificity to compare MRE readings to combined endoscopic/histopathological findings (the reference standard). RESULTS: The pre- and pre-/post-contrast-enhanced MRE vs. combined endoscopic/histopathological results had moderate agreement (85.7%; κ 0.713, P<0.001; P-value 0.549). Compared to combined endoscopy/histopathology, pre- vs. pre-/post-contrast sensitivity (67%, confidence interval [CI] 0.53-0.79 vs. 67%, CI 0.53-0.79) and specificity (80%, CI 0.59-0.92 vs. 68%, CI 0.46-0.84) varied little (κ 0.42, P<0.001 and κ 0.32, P=0.003, respectively). The three readers had moderate agreement (85.2%; κ 0.695, P=0.001; P-value 0.625). More penetrating complications were identified following contrast administration (P-value 0.04). CONCLUSION: Use of a contrast agent does not improve the detection of active inflammation in the terminal ileum and colon compared to non-contrast MRE, although use of a contrast agent does aid in the detection of penetrating disease.

Alpha-1-Antitrypsin Deficiency Liver Disease.

In homozygous ZZ alpha-1-antitrypsin (AAT) deficiency, the liver synthesizes large quantities of AAT mutant Z, which folds improperly during biogenesis and is retained within the hepatocytes and directed into intracellular proteolysis pathways. These intracellular polymers trigger an injury cascade, which can lead to liver injury. This is highly variable and not all patients develop liver disease. Although not fully described, there is likely a strong influence of genetic and environmental modifiers of the injury cascade and of the fibrotic response. With improved understanding of liver injury mechanisms, new strategies for treatment are now being explored.

NorUDCA promotes degradation of α1-antitrypsin mutant Z protein by inducing autophagy through AMPK/ULK1 pathway.

Alpha-1 Antitrypsin (α1AT) Deficiency is a genetic disease in which accumulation of α1AT mutant Z (α1ATZ) protein in the ER of hepatocytes causes chronic liver injury, liver fibrosis, and hepatocellular carcinoma. No effective medical therapy is currently available for the disease. We previously found that norUDCA improves the α1AT deficiency associated liver disease by promoting autophagic degradation of α1ATZ protein in liver in a mouse model of the disease. The current study unravels the novel underlying cellular mechanism by which norUDCA modulates autophagy. HTOZ cells, modified from HeLa Tet-Off cells by transfection with the resulting pTRE1-ATZ plasmid and expressing mutant Z proteins, were studied in these experiments. The role of norUDCA in inducing autophagy, autophagy-mediated degradation of α1ATZ and the role of AMPK in norUDCA-induced autophagy were examined in the current report. NorUDCA promoted disposal of α1ATZ via autophagy-mediated degradation of α1ATZ in HTOZ cells. Activation of AMPK was required for norUDCA-induced autophagy and α1ATZ degradation. Moreover, mTOR/ULK1 was involved in norUDCA-induced AMPK activation and autophagy in HTOZ cells. Our results provide novel mechanistic insights into the therapeutic action of norUDCA in promoting the clearance of α1ATZ in vitro and suggest a novel therapeutic approach for the treatment of α1ATZ deficiency disease and its associated liver diseases.

Gastrointestinal pathophysiology and nutrition in cystic fibrosis.

INTRODUCTION: Cystic fibrosis (CF) is a severe, progressive, multisystemic disease that is caused by mutations in the cystic fibrosis transmembrane conductance regulator gene. Optimizing nutrition is critical, as higher growth parameters are associated with better pulmonary function and outcomes, but unfortunately patients with this disease are prone to malnutrition, growth failure, and vitamin deficiencies. The purpose of this review is to provide a timely highlight of the physiologic processes and outcome data to support today's management strategies, as well as review these principles themselves. Areas covered: This review covers the background of the importance of vigilant attention to nutrition and growth in these patients, the underlying physiology leading to an abnormal gastrointestinal tract and its role in CF malnutrition, and current evaluation and management strategies to address nutrition in CF. Analysis of up-to-date relevant literature was performed using PubMed. Expert commentary: Advances in research and clinical developments over the years have improved knowledge of this disease as well as patient outcomes. Of particular importance is optimizing nutrition especially in the early stages of life, as well as accounting for the markedly abnormal CF intestinal milieu when addressing the gastrointestinal and nutritional needs of these patients.

Amelioration of Alpha-1 Antitrypsin Deficiency Diseases with Genome Editing in Transgenic Mice.

Alpha-1 antitrypsin deficiency (AATD) is a hereditary liver disease caused by mutations in the SERPINA1 serine protease inhibitor gene. Most severe patients are homozygous for PiZ alleles (PiZZ; amino acid E324K), which lead to protein aggregates in hepatocytes and reduced circulating levels of AAT. The liver aggregates typically lead to fibrosis, cirrhosis, and hepatocellular carcinoma, and the reduced circulating AAT levels can lead to emphysema and chronic obstructive pulmonary diseases. In this study, two CRISPR/Cas9 gene editing approaches were used to decrease liver aggregates and increase systemic AAT-M levels in the PiZ transgenic mouse. In the first approach, AAT expression in hepatocytes was reduced more than 98% following the systemic delivery of AAV8-CRISPR targeting exon 2 of hSERPINA1, leading to reduced aggregates in hepatocytes. In the second approach, a second adeno-associated virus, which provided the donor template to correct the Z mutation, was also administered. These treated mice had reduced AAT expression (> 98%) and a low level (5%) of wildtype AAT-M mRNA. Taken together, this study shows that CRISPR gene editing can efficiently reduce liver expression of AAT-Z and restore modest levels of wildtype AAT-M in a mouse model of AATD, raising the possibility of CRISPR gene editing therapeutic for AATD.

Pathophysiology of Alpha-1 Antitrypsin Deficiency Liver Disease.

Classical alpha-1 antitrypsin (a1AT) deficiency is an autosomal recessive disease associated with an increased risk of liver disease in adults and children, and with lung disease in adults (Teckman and Jain, Curr Gastroenterol Rep 16(1):367, 2014). The vast majority of the liver disease is associated with homozygosity for the Z mutant allele, the so-called PIZZ. These homozygous individuals synthesize large quantities of a1AT mutant Z protein in the liver, but the mutant protein folds improperly during biogenesis and approximately 85% of the molecules are retained within the hepatocytes rather than appropriately secreted. The resulting low, or "deficient," serum level leaves the lungs vulnerable to inflammatory injury from uninhibited neutrophil proteases. Most of the mutant Z protein molecules retained within hepatocytes are directed into intracellular proteolysis pathways, but some molecules remain in the endoplasmic reticulum for long periods of time. Some of these molecules adopt an unusual aggregated or "polymerized" conformation (Duvoix et al., Rev Mal Respir 31(10):992-1002, 2014). It is thought that these intracellular polymers trigger a cascade of intracellular injury which can lead to end-organ liver injury including chronic hepatitis, cirrhosis, and hepatocellular carcinoma (Lindblad et al., Hepatology 46(4):1228-1235, 2007). The hepatocytes with the largest accumulations of mutant Z polymers undergo apoptotic death and possibly other death mechanisms. This intracellular death cascade appears to involve ER stress, mitochondrial depolarization, and caspase cleavage, and is possibly linked to autophagy and redox injury. Cells with lesser burdens of mutant Z protein proliferate to maintain the liver cell mass. This chronic cycle of cell death and regeneration activates hepatic stellate cells and initiates the process of hepatic fibrosis. Cirrhosis and hepatocellular carcinoma then result in some patients. Since not all patients with the same homozygous PIZZ genotype develop end-stage disease, it is hypothesized that there is likely to be a strong influence of genetic and environmental modifiers of the injury cascade and of the fibrotic response.

Semiquantitation of Monomer and Polymer Alpha-1 Antitrypsin by Centrifugal Separation and Assay by Western Blot of Soluble and Insoluble Components.

Alpha-1 antitrypsin (a1AT) deficiency, in its classical form, is an autosomal recessive disease associated with an increased risk of liver disease in adults and children, and with lung disease in adults. The vast majority of liver disease is associated with homozygosity for the Z mutant allele, also called PiZZ. This homozygous allele synthesizes large quantities of a1AT mutant Z protein in the liver, but the mutant protein also folds improperly during biogenesis. As a result, approximately 85% of the molecules are retained within the hepatocytes instead of being appropriately secreted. The resulting low, or "deficient," serum level leaves the lungs vulnerable to inflammatory injury from uninhibited neutrophil proteases. Most of the mutant Z protein retained within hepatocytes is directed into intracellular proteolysis pathways, but some molecules remain in the endoplasmic reticulum for long periods of time and others adopt an unusual aggregated or "polymerized" conformation. It is thought that these intracellular polymers trigger a cascade of intracellular injury which can lead to end organ liver injury including chronic hepatitis, cirrhosis, and hepatocellular carcinoma. It is widely accepted that the disease causing factor in mutant Z-alpha-1 antitrypsin deficiency (AATD-Z) is the toxic build-up of the mutant Z protein. Since misfolding of some but not all of the Z protein during its maturation leads to homopolymerization, an assay to assess the amount of normally folded ATZ and accumulated polymeric ATZ would be very useful. Here we describe a method to semiquantitatively assess these two fractions in a tissue or cell culture source.

Bone marrow stem cell therapy partially ameliorates pathological consequences in livers of mice expressing mutant human α1-antitrypsin.

Alpha-1-antitrypsin (AAT) deficiency (AATD) is a genetic disease, caused by mutation of the AAT gene. Accumulation of mutated AAT protein aggregates in hepatocytes leads to endoplasmic reticulum stress, resulting in impairment of liver functions and, in some cases, hepatocellular carcinoma, whereas decline of AAT levels in sera is responsible for pulmonary emphysema. In advanced liver disease, the only option for treatment is liver transplantation, whereas AAT replacement therapy is therapeutic for emphysema. Given that hepatocytes are the primary affected cells in AATD, we investigated whether transplantation of bone marrow (BM)-derived stem cells in transgenic mice expressing human AATZ (the Z variant of AAT) confers any competitive advantages compared to host cells that could lead to pathological improvement. Mouse BM progenitors and human mesenchymal stem cells (MSCs) appeared to contribute in replacement of 40% and 13% host hepatocytes, respectively. Transplantation of cells resulted in decline of globule-containing hepatocytes, improvement in proliferation of globule-devoid hepatocytes from the host-derived hepatocytes, and apparently, donor-derived cells. Further analyses revealed that transplantation partially improves liver pathology as reflected by inflammatory response, fibrosis, and apoptotic death of hepatocytes. Cell therapy was also found to improve liver glycogen storage and sera glucose level in mice expressing human AATZ mice. These overall improvements in liver pathology were not restricted to transplantation of mouse BM cells. Preliminary results also showed that following transplantation of human BM-derived MSCs, globule-containing hepatocytes declined and donor-derived cells expressed human AAT protein. CONCLUSION: These results suggest that BM stem cell transplantation may be a promising therapy for AATD-related liver disease. (Hepatology 2017;65:1319-1335).

AFM Imaging Reveals Topographic Diversity of Wild Type and Z Variant Polymers of Human α1-Proteinase Inhibitor.

α1-Proteinase inhibitor (antitrypsin) is a canonical example of the serpin family member that binds and inhibits serine proteases. The natural metastability of serpins is crucial to carry out structural rearrangements necessary for biological activity. However, the enhanced metastability of the mutant Z variant of antitrypsin, in addition to folding defect, may substantially contribute to its polymerization, a process leading to incurable serpinopathy. The metastability also impedes structural studies on the polymers. There are no crystal structures of Z monomer or any kind of polymers larger than engineered wild type (WT) trimer. Our understanding of polymerization mechanisms is based on biochemical data using in vitro generated WT oligomers and molecular simulations. Here we applied atomic force microscopy (AFM) to compare topography of monomers, in vitro formed WT oligomers, and Z type polymers isolated from transgenic mouse liver. We found the AFM images of monomers closely resembled an antitrypsin outer shell modeled after the crystal structure. We confirmed that the Z variant demonstrated higher spontaneous propensity to dimerize than WT monomers. We also detected an unexpectedly broad range of different types of polymers with periodicity and topography depending on the applied method of polymerization. Short linear oligomers of unit arrangement similar to the Z polymers were especially abundant in heat-treated WT preparations. Long linear polymers were a prominent and unique component of liver extracts. However, the liver preparations contained also multiple types of oligomers of topographies undistinguishable from those found in WT samples polymerized with heat, low pH or guanidine hydrochloride treatments. In conclusion, we established that AFM is an excellent technique to assess morphological diversity of antitrypsin polymers, which is important for etiology of serpinopathies. These data also support previous, but controversial models of in vivo polymerization showing a surprising diversity of polymer topography.