Evaluating intra-action reviews at points of entry: ongoing learning opportunities during the COVID-19 pandemic.

BACKGROUND: Long-lasting crises, such as the COVID-19 pandemic, require proper interim evaluation in order to optimize response. The World Health Organization and the European Center for Disease Control have recently promoted the in(tra)-action review (IAR) method for this purpose. We systematically evaluated the added value of two IARs performed in the Dutch point of entry (PoE) setting. METHODS: Two online, 4-hour IAR meetings were organized in March 2021, for ports and airports respectively, to reflect on the ongoing COVID-19 response. Topics discussed were selected through a survey among participants. Participants were mainly self-selected by the (air)port public health service. Evaluation of the IAR method consisted of participant evaluation through a questionnaire, and hot and cold debriefs of the organizing team. Evaluation of the impact of the IAR was done through analysis of the meeting results, and a 3-month follow-up of the actions proposed during the meetings. RESULTS: Thirty-nine professionals joined the IAR meetings. In the participant evaluation (n = 18), 89% agreed or totally agreed the IAR made it possible to identify challenges and problems in the COVID-19 response at PoE. Participants especially appreciated the resulting insight in regional and national partners. Regarding the online setting of the meeting, participants suggested to choose accessible and familiar online tools. After 3 months, all national actions and actions for ports had been executed; some regional actions for airports required further attention. A major result was a new meeting structure for all ports and the participating national authorities in which remaining and newly occurring issues were discussed. CONCLUSIONS: Based on the evaluations, we conclude that the IAR method can be of value during long-term crises, such as the COVID-19 pandemic response. Although it is challenging to dedicate time and effort to the organization and attendance of IAR meetings during crisis, the IAR method is feasible in an online setting if appropriate organizing and technical capacity is available. A participatory set-up supports the IAR method as a starting point for continuous exchange and learning during ongoing crises.

Phospholipid flippases attenuate LPS-induced TLR4 signaling by mediating endocytic retrieval of Toll-like receptor 4.

P4-ATPases are lipid flippases that catalyze the transport of phospholipids to create membrane phospholipid asymmetry and to initiate the biogenesis of transport vesicles. Here we show, for the first time, that lipid flippases are essential to dampen the inflammatory response and to mediate the endotoxin-induced endocytic retrieval of Toll-like receptor 4 (TLR4) in human macrophages. Depletion of CDC50A, the ß-subunit that is crucial for the activity of multiple P4-ATPases, resulted in endotoxin-induced hypersecretion of proinflammatory cytokines, enhanced MAP kinase signaling and constitutive NF-κB activation. In addition, CDC50A-depleted THP-1 macrophages displayed reduced tolerance to endotoxin. Moreover, endotoxin-induced internalization of TLR4 was strongly reduced and coincided with impaired endosomal MyD88-independent signaling. The phenotype of CDC50A-depleted cells was also induced by separate knockdown of two P4-ATPases, namely ATP8B1 and ATP11A. We conclude that lipid flippases are novel elements of the innate immune response that are essential to attenuate the inflammatory response, possibly by mediating endotoxin-induced internalization of TLR4.

Disruption of HNF1α binding site causes inherited severe unconjugated hyperbilirubinemia.

Crigler-Najjar syndrome presents as severe unconjugated hyperbilirubinemia and is characteristically caused by a mutation in the UGT1A1 gene, encoding the enzyme responsible for bilirubin glucuronidation. Here we present a patient with Crigler-Najjar syndrome with a completely normal UGT1A1 coding region. Instead, a homozygous 3 nucleotide insertion in the UGT1A1 promoter was identified that interrupts the HNF1α binding site. This mutation results in almost complete abolishment of UGT1A1 promoter activity and prevents the induction of UGT1A1 expression by the liver nuclear receptors CAR and PXR, explaining the lack of a phenobarbital response in this patient. Although animal studies have revealed the importance of HNF1α for normal liver function, this case provides the first clinical proof that mutations in its binding site indeed result in severe liver pathology stressing the importance of promoter sequence analysis.

Characterization and treatment of persistent hepatocellular secretory failure.

BACKGROUND & AIMS: Hepatocellular secretory failure induced by drugs, toxins or transient biliary obstruction may sometimes persist for months after removal of the initiating factor and may then be fatal without liver transplantation. We characterized patients with severe persistent hepatocellular secretory failure (PHSF) and treated them with the pregnane X receptor (PXR) agonist, rifampicin. We also studied the effect of rifampicin on PXR-dependent expression of genes involved in biotransformation and secretion in vitro. METHODS: Thirteen patients (age 18-81 years, 6 male) with hepatocellular secretory failure that persisted after removal of the inducing factor (drugs/toxin: 9) or biliary obstruction (4) were identified over 6 years. Six of these patients were screened for ATP8B1 or ABCB11 mutations. All were treated with rifampicin (300 mg daily) for 1-10 weeks. Expression of genes involved in biotransformation and secretion was determined by rtPCR in human hepatocytes and intestinal cells incubated with rifampicin (10 µmol/L). RESULTS: Serum bilirubin of patients with PHSF ranged from 264 to 755 µmol/L. Normal γGT was found in 10/13 patients of whom 3/6 tested positive for ATP8B1/ABCB11 mutations. Serum bilirubin declined to <33 µmol/L after 1-10 weeks of rifampicin treatment. In vitro, rifampicin PXR-dependently upregulated biotransformation phase 1 (CYP3A4), phase 2 (UGT1A1) and phase 3 (MRP2) enzymes/carriers as well as the basolateral bile salt exporter OSTß. CONCLUSION: Persistent hepatocellular secretory failure may develop in carriers of transporter gene mutations. In severe cases, rifampicin may represent an effective therapeutic option of PHSF. PXR-dependent induction of CYP3A4, UGT1A1, MRP2 and OSTß could contribute to the anticholestatic effect of rifampicin in PHSF.

Advances in pathogenesis and management of pruritus in cholestasis.

Chronic pruritus is a burdensome feature of numerous hepatobiliary disorders such as primary biliary cirrhosis, primary sclerosing cholangitis, cholangiocarcinoma, inherited forms of cholestasis and intrahepatic cholestasis of pregnancy. Bile salts, µ-opioids, serotonin, histamine and steroids have been controversially discussed in the pathogenesis of cholestatic pruritus. However, for these substances neither a correlation with itch severity nor a causative link has ever been established. Recent findings indicate that the potent neuronal activator lysophosphatidic acid and autotaxin, the enzyme forming lysophosphatidic acid, may play a key element in the pathogenesis of cholestatic pruritus. Serum activity of autotaxin correlated with itch intensity and response to antipruritic treatment in patients with cholestatic pruritus, but not other forms of pruritus. Autotaxin activity thereby represents the first biomarker for pruritus and had a positive predictive value of 70% in differentiating cholestatic pruritus from other forms of pruritus. Treatment options for patients with cholestatic pruritus include the anion exchange resin colestyramine, the PXR agonist rifampicin, the µ-opioid antagonist naltrexone, and the serotonin reuptake inhibitor sertraline. These drugs are recommended by evidence-based guidelines as a stepwise therapeutic approach. Patients unresponsive to these drugs should be referred to specialized centers to receive experimental approaches such as UVB phototherapy, albumin dialysis, plasmapheresis or nasobiliary drainage. This review discusses pruritogen candidates in cholestasis, gives novel insights into the neuronal signaling pathway of pruritus and summarizes evidence-based treatment options for patients suffering from pruritus in cholestasis.

Serum autotaxin is increased in pruritus of cholestasis, but not of other origin, and responds to therapeutic interventions.

UNLABELLED: Pruritus is a seriously disabling symptom accompanying many cholestatic liver disorders. Recent experimental evidence implicated the lysophospholipase, autotaxin (ATX), and its product, lysophosphatidic acid (LPA), as potential mediators of cholestatic pruritus. In this study, we highlight that increased serum ATX levels are specific for pruritus of cholestasis, but not pruritus of uremia, Hodgkin's disease, or atopic dermatitis. Treatment of patients with cholestasis with the bile salt sequestrant, colesevelam, but not placebo, effectively reduced total serum bile salts and fibroblast growth factor 19 levels, but only marginally altered pruritus intensity and ATX activity. Rifampicin (RMP) significantly reduced itch intensity and ATX activity in patients with pruritus not responding to bile salt sequestrants. In vitro, RMP inhibited ATX expression in human HepG2 hepatoma cells and hepatoma cells overexpressing the pregnane X receptor (PXR), but not in hepatoma cells in which PXR was knocked down. Treatment of severe, refractory pruritus by the molecular adsorbents recirculation system or nasobiliary drainage improved itch intensity, which, again, correlated with the reduction of ATX levels. Upon reoccurrence of pruritus, ATX activity returned to pretreatment values. CONCLUSION: Serum ATX activity is specifically increased in patients with cholestatic, but not other forms of, systemic pruritus and closely correlates with the effectiveness of therapeutic interventions. The beneficial antipruritic action of RMP may be explained, at least partly, by the PXR-dependent transcriptional inhibition of ATX expression. Thus, ATX likely represents a novel therapeutic target for pruritus of cholestasis.

[Diabetes and developments in data-intensive care: self-regulation turns patients into professionals]. / Gaat data-intensieve zorg onze omgang met ziekte veranderen?

In addition to progress, medical-technological innovations often involve transformations, for example in the conceptualization and experience of disease(s). A historical example is the rise of self-regulation by diabetes patients in the 1970s and 1980s. Textual analysis of Diabc, the monthly magazine of the Diabetes Vereniging Nederland (DVN), shows that the introduction of self-testing and self-regulation was not only very welcome for many DVN members, but also brought about a qualitative change in their views of diabetes and themselves. They continued to present themselves as 'healthy', 'normal' and 'independent', but that was no longer based on their disciplined adherence to prescriptions from doctors and dietitians. They had now become 'professionals' themselves, who practiced 'diabetes as a profession'. Some interesting parallels can be drawn between this historical case and current data-intensive care. This illustrates that historical reflection, by analogical reasoning, can be helpful when questioning current medical-technological developments.

Activation-induced cytidine deaminase splice variants are defective because of the lack of structural support for the catalytic site.

Recently, conflicting results were reported on the hypermutation activity of activation-induced cytidine deaminase (AID) splice variants. With the generation of single point mutations, we studied the structure-function relationship of the amino acids that are commonly absent from all described splice variants. The results from this analysis pointed to several amino acids that are required for class switch recombination (CSR), without perturbing cellular localization or nucleocytoplasmic shuttling. A defect in deaminase activity was found to underlie this CSR deficiency. Interestingly, the most debilitating mutations concentrated on hydrophobic amino acids, suggesting a structural role for this part of the protein. Indeed, by generating homologous amino acid replacements, CSR activity could be restored. These results are in agreement with recent reports on the protein structure of the AID homolog APOBEC3G, suggesting a similar protein composition. In addition, the findings underscore that AID splice variants are unlikely to have preservation of catalytic activity.

The majority of cutaneous marginal zone B-cell lymphomas expresses class-switched immunoglobulins and develops in a T-helper type 2 inflammatory environment.

Extranodal marginal zone B-cell lymphomas (MZBCLs) arise on a background of chronic inflammation resulting from organ-specific autoimmunity, infection, or by unknown causes. Well-known examples are salivary gland MZBCL in Sjögren's sialadenitis and gastric MZBCL in Helicobacter pylori gastritis. MZBCLs express CXCR3, a receptor for interferon-gamma-induced chemokines highly expressed in the chronic inflammatory environment. The immunoglobulin (Ig) variable heavy/light chain (IgV(H)/IgV(L)) gene repertoire of salivary gland and gastric MZBCL appears restricted and frequently encodes B-cell receptors with rheumatoid factor reactivity. Primary cutaneous marginal zone B-cell lymphomas (PCMZLs) are regarded as the skin-involving counterparts of extranodal MZBCLs. Although PCMZLs have been associated with Borrelia burgdorferi dermatitis, PCMZLs generally arise because of unknown causes. We studied an extensive panel of PCMZLs and show that PCMZLs do not conform to the general profile of extranodal MZBCL. Whereas most noncutaneous MZBCLs express IgM, PCMZLs in majority express IgG, IgA, and IgE and do not show an obvious immunoglobulin repertoire bias. Furthermore, the isotype-switched PCMZLs lack CXCR3 and seem to arise in a different inflammatory environment, compared with other extranodal MZBCLs.

A Quantitative In Vitro Potency Assay for Adeno-Associated Virus Vectors Encoding for the UGT1A1 Transgene.

Potency assessment of clinical-grade vector lots is crucial to support adeno-associated virus (AAV) vector release and is required for future marketing authorization. We have developed and validated a cell-based, quantitative potency assay that detects both transgenic expression and activity of an AAV8-hUGT1A1 vector, which is currently under clinical evaluation for the treatment of Crigler-Najjar syndrome. Potency of AAV8-hUGT1A1 was evaluated in vitro. After transduction of human hepatoma 7 (Huh7) cells, transgene-positive cells were quantified using flow cytometry and transgenic activity by a bilirubin conjugation assay. The in vitro potency of various AAV8-hUGT1A1 batches was compared with their potency in vivo. After AAV8-hUGT1A1 transduction, quantification of UGT1A1-expressing cells shows a linear dose-response relation (R2 = 0.98) with adequate intra-assay and inter-day reproducibility (coefficient of variation [CV] = 11.0% and 22.6%, respectively). In accordance, bilirubin conjugation shows a linear dose-response relation (R2 = 0.99) with adequate intra- and inter-day reproducibility in the low dose range (CV = 15.7% and 19.7%, respectively). Both in vitro potency assays reliably translate to in vivo efficacy of AAV8-hUGT1A1 vector lots. The described cell-based potency assay for AAV8-hUGT1A1 adequately determines transgenic UGT1A1 expression and activity, which is consistent with in vivo efficacy. This novel approach is suited for the determination of vector lot potency to support clinical-grade vector release.

Biliverdin Reductase inhibitors did not improve severe unconjugated hyperbilirubinemia in vivo.

We aimed to identify potent biliverdin reductase (BVRA) inhibitors as a novel concept for the treatment of severe unconjugated hyperbilirubinemia. 1280 FDA-approved compounds were screened in vitro for their ability to inhibit human and rat BVRA activity and 26 compounds were identified as BVRA inhibitors. Montelukast and Disulfiram were selected as potentially clinically applicable drugs and tested to reduce serum unconjugated bilirubin (UCB) levels in the Ugt1a1-deficient rat, a model for chronic unconjugated hyperbilirubinemia. Oral administration of Disulfiram was toxic in the Ugt1a1-deficient rat (weight loss, transaminase elevation). Oral Montelukast administration led to low serum concentrations and did not alter serum UCB levels. Intraperitoneal injections of Montelukast resulted in concentrations up to 110 µmol/L in serum and 400 µmol/L in the liver. Still, serum UCB levels remained unaltered. This first study on biliverdin reductase inhibition as a novel concept for treatment of unconjugated hyperbilirubinemia identified putative in vitro BVRA inhibitors. Montelukast, the clinically most suitable inhibitor, did not result in reduction of serum UCB in the Ugt1a1-deficient rat. The proposed treatment strategy will not result in amelioration of severe unconjugated hyperbilirubinemia in humans without the identification or development of more potent BVRA inhibitors.

A translationally optimized AAV-UGT1A1 vector drives safe and long-lasting correction of Crigler-Najjar syndrome.

Crigler-Najjar syndrome is a severe metabolic disease of the liver due to a reduced activity of the UDP Glucuronosyltransferase 1A1 (UGT1A1) enzyme. In an effort to translate to the clinic an adeno-associated virus vector mediated liver gene transfer approach to treat Crigler-Najjar syndrome, we developed and optimized a vector expressing the UGT1A1 transgene. For this purpose, we designed and tested in vitro and in vivo multiple codon-optimized UGT1A1 transgene cDNAs. We also optimized noncoding sequences in the transgene expression cassette. Our results indicate that transgene codon-optimization is a strategy that can improve efficacy of gene transfer but needs to be carefully tested in vitro and in vivo. Additionally, while inclusion of introns can enhance gene expression, optimization of these introns, and in particular removal of cryptic ATGs and splice sites, is an important maneuver to enhance safety and efficacy of gene transfer. Finally, using a translationally optimized adeno-associated virus vector expressing the UGT1A1 transgene, we demonstrated rescue of the phenotype of Crigler-Najjar syndrome in two animal models of the disease, Gunn rats and Ugt1a1-/- mice. We also showed long-term (>1 year) correction of the disease in Gunn rats. These results support further translation of the approach to humans.

Gene replacement therapy for genetic hepatocellular jaundice.

Jaundice results from the systemic accumulation of bilirubin, the final product of the catabolism of haem. Inherited liver disorders of bilirubin metabolism and transport can result in reduced hepatic uptake, conjugation or biliary secretion of bilirubin. In patients with Rotor syndrome, bilirubin (re)uptake is impaired due to the deficiency of two basolateral/sinusoidal hepatocellular membrane proteins, organic anion-transporting polypeptide 1B1 (OATP1B1) and OATP1B3. Dubin-Johnson syndrome is caused by a defect in the ATP-dependent canalicular transporter, multidrug resistance-associated protein 2 (MRP2), which mediates the export of conjugated bilirubin into bile. Both disorders are benign and not progressive and are characterised by elevated serum levels of mainly conjugated bilirubin. Uridine diphospho-glucuronosyl transferase 1A1 (UGT1A1) is responsible for the glucuronidation of bilirubin; deficiency of this enzyme results in unconjugated hyperbilirubinaemia. Gilbert syndrome is the mild and benign form of inherited unconjugated hyperbilirubinaemia and is mostly caused by reduced promoter activity of the UGT1A1 gene. Crigler-Najjar syndrome is the severe inherited form of unconjugated hyperbilirubinaemia due to mutations in the UGT1A1 gene, which can cause kernicterus early in life and can be even lethal when left untreated. Due to major disadvantages of the current standard treatments for Crigler-Najjar syndrome, phototherapy and liver transplantation, new effective therapeutic strategies are under development. Here, we review the clinical features, pathophysiology and genetic background of these inherited disorders of bilirubin metabolism and transport. We also discuss the upcoming treatment option of viral gene therapy for genetic disorders such as Crigler-Najjar syndrome and the possible immunological consequences of this therapy.

Polyinosinic acid blocks adeno-associated virus macrophage endocytosis in vitro and enhances adeno-associated virus liver-directed gene therapy in vivo.

Adeno-associated virus serotype 8 (AAV8) has been demonstrated to be effective for liver-directed gene therapy in humans. Although hepatocytes are the main target cell for AAV8, there is a loss of the viral vector because of uptake by macrophages and Kupffer cells. Reducing this loss would increase the efficacy of viral gene therapy and allow a dose reduction. The receptor mediating this uptake has not been identified; a potential candidate seems the macrophage scavenger receptor A (SR-A) that is involved in the endocytosis of, for instance, adenovirus. In this study we show that SR-A can mediate scAAV8 endocytosis and that blocking it with polyinosinic acid (poly[i]) reduces endocytosis significantly in vitro. Subsequently, we demonstrate that blocking this receptor improves scAAV-mediated liver-directed gene therapy in a model for inherited hyperbilirubinemia, the uridine diphospho-glucuronyl transferase 1A1-deficient Gunn rat. In male rats, preadministration of poly[i] increases the efficacy of a low dose (1×10¹¹ gc/kg) but not of a higher dose (3×10¹¹ gc/kg) scAAV8-LP1-UT1A1. Administration of poly[i] just before the vector significantly increases the correction of serum bilirubin in female rats. In these, the effect of poly[i] is seen by both doses but is more pronounced in the females receiving the low vector, where it also results in a significant increase of bilirubin glucuronides in bile. In conclusion, this study shows that SR-A mediates the endocytosis of AAV8 in vitro and in vivo and that blocking this receptor can improve the efficacy of AAV-mediated liver-directed gene therapy.