Humanized mouse liver reveals endothelial control of essential hepatic metabolic functions.

Hepatocytes, the major metabolic hub of the body, execute functions that are human-specific, altered in human disease, and currently thought to be regulated through endocrine and cell-autonomous mechanisms. Here, we show that key metabolic functions of human hepatocytes are controlled by non-parenchymal cells (NPCs) in their microenvironment. We developed mice bearing human hepatic tissue composed of human hepatocytes and NPCs, including human immune, endothelial, and stellate cells. Humanized livers reproduce human liver architecture, perform vital human-specific metabolic/homeostatic processes, and model human pathologies, including fibrosis and non-alcoholic fatty liver disease (NAFLD). Leveraging species mismatch and lipidomics, we demonstrate that human NPCs control metabolic functions of human hepatocytes in a paracrine manner. Mechanistically, we uncover a species-specific interaction whereby WNT2 secreted by sinusoidal endothelial cells controls cholesterol uptake and bile acid conjugation in hepatocytes through receptor FZD5. These results reveal the essential microenvironmental regulation of hepatic metabolism and its human-specific aspects.

IL-7R licenses a population of epigenetically poised memory CD8+ T cells with superior antitumor efficacy that are critical for melanoma memory.

Recurrence of advanced melanoma after therapy is a major risk factor for reduced survival, and treatment options are limited. Antitumor immune memory plays a critical role in preventing melanoma recurrence and memory T cells could be a potent cell-based therapy, but the identity, and functional properties of the required immune cells are incompletely understood. Here, we show that an IL-7Rhi tumor-specific CD8+ population is critical for antitumor memory and can be epigenetically augmented to drive powerful antitumor immune responses. Using a model of functional antimelanoma memory, we found that high IL-7R expression selectively marks a CD8+ population in lymphoid organs that plays critical roles in maintaining tumor remission after immunotherapy or surgical resection. This population has intrinsic cytotoxic activity, lacks markers of exhaustion and has superior antitumor efficacy. IL-7Rhi cells have a functionally poised epigenetic landscape regulated by DNA methylation, which can be augmented by hypomethylating agents to confer improved survival and complete melanoma clearance in naive mice. Importantly, greater than 95% of tumor-specific T cells in draining lymph nodes after therapy express high levels of IL-7R. This overlap between IL-7Rhi and antigen-specific T cells allows for enrichment of a potent functional CD8+ population without determining antigen-specificity, which we demonstrate in a melanoma model without a known antigen. We identify that IL-7R expression in human melanoma is an independent prognostic factor of improved survival. These findings advance our basic understanding of antitumor memory and suggest a cell-based therapy using high IL-7R expression to enrich for a lymph node population with superior antitumor activity that can be augmented by hypomethylating agents.

An update on ductal plate malformations and fibropolycystic diseases of the liver.

A variety of cystic and fibrocystic lesions can occur in the liver, which may be single or multiple and etiologically can be acquired or have genetic underpinnings. Although the morphology of ductal plate development and various associated malformations has been well described, the genetic etiologies of many of these disorders are still poorly understood. Multiple clinical phenotypes in the liver are proposed to originate from ductal plate malformations: congenital hepatic fibrosis, Caroli's disease, Von Meyenburg complex, and the liver cysts of autosomal dominant polycystic kidney and liver diseases. Although many of the patients with these disorders, particularly with isolated liver involvement remain asymptomatic, some develop portal hypertension or symptoms from cyst enlargement. Development of hepatocellular malignancy is a risk in a small subset. Recent advances have made it now possible for some of these phenotypes to be genetically defined, and intriguingly animal models of adult polycystic liver disease suggest that abnormal organ development is not required. This review describes the current understanding, genetic underpinning, and key clinicopathologic and imaging features of these fibropolycystic liver diseases.

Inflammasome activation in infected macrophages drives COVID-19 pathology.

Severe COVID-19 is characterized by persistent lung inflammation, inflammatory cytokine production, viral RNA and a sustained interferon (IFN) response, all of which are recapitulated and required for pathology in the SARS-CoV-2-infected MISTRG6-hACE2 humanized mouse model of COVID-19, which has a human immune system1-20. Blocking either viral replication with remdesivir21-23 or the downstream IFN-stimulated cascade with anti-IFNAR2 antibodies in vivo in the chronic stages of disease attenuates the overactive immune inflammatory response, especially inflammatory macrophages. Here we show that SARS-CoV-2 infection and replication in lung-resident human macrophages is a critical driver of disease. In response to infection mediated by CD16 and ACE2 receptors, human macrophages activate inflammasomes, release interleukin 1 (IL-1) and IL-18, and undergo pyroptosis, thereby contributing to the hyperinflammatory state of the lungs. Inflammasome activation and the accompanying inflammatory response are necessary for lung inflammation, as inhibition of the NLRP3 inflammasome pathway reverses chronic lung pathology. Notably, this blockade of inflammasome activation leads to the release of infectious virus by the infected macrophages. Thus, inflammasomes oppose host infection by SARS-CoV-2 through the production of inflammatory cytokines and suicide by pyroptosis to prevent a productive viral cycle.

Inflammasome activation in infected macrophages drives COVID-19 pathology.

Severe COVID-19 is characterized by persistent lung inflammation, inflammatory cytokine production, viral RNA, and sustained interferon (IFN) response all of which are recapitulated and required for pathology in the SARS-CoV-2 infected MISTRG6-hACE2 humanized mouse model of COVID-19 with a human immune system 1-20 . Blocking either viral replication with Remdesivir 21-23 or the downstream IFN stimulated cascade with anti-IFNAR2 in vivo in the chronic stages of disease attenuated the overactive immune-inflammatory response, especially inflammatory macrophages. Here, we show SARS-CoV-2 infection and replication in lung-resident human macrophages is a critical driver of disease. In response to infection mediated by CD16 and ACE2 receptors, human macrophages activate inflammasomes, release IL-1 and IL-18 and undergo pyroptosis thereby contributing to the hyperinflammatory state of the lungs. Inflammasome activation and its accompanying inflammatory response is necessary for lung inflammation, as inhibition of the NLRP3 inflammasome pathway reverses chronic lung pathology. Remarkably, this same blockade of inflammasome activation leads to the release of infectious virus by the infected macrophages. Thus, inflammasomes oppose host infection by SARS-CoV-2 by production of inflammatory cytokines and suicide by pyroptosis to prevent a productive viral cycle.

A humanized mouse model of chronic COVID-19.

Coronavirus disease 2019 (COVID-19) is an infectious disease that can present as an uncontrolled, hyperactive immune response, causing severe immunological injury. Existing rodent models do not recapitulate the sustained immunopathology of patients with severe disease. Here we describe a humanized mouse model of COVID-19 that uses adeno-associated virus to deliver human ACE2 to the lungs of humanized MISTRG6 mice. This model recapitulates innate and adaptive human immune responses to severe acute respiratory syndrome coronavirus 2 infection up to 28 days after infection, with key features of chronic COVID-19, including weight loss, persistent viral RNA, lung pathology with fibrosis, a human inflammatory macrophage response, a persistent interferon-stimulated gene signature and T cell lymphopenia. We used this model to study two therapeutics on immunopathology, patient-derived antibodies and steroids and found that the same inflammatory macrophages crucial to containing early infection later drove immunopathology. This model will enable evaluation of COVID-19 disease mechanisms and treatments.

A humanized mouse model of chronic COVID-19 to evaluate disease mechanisms and treatment options.

Coronavirus-associated acute respiratory disease, called coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). More than 90 million people have been infected with SARS-CoV-2 and more than 2 million people have died of complications due to COVID-19 worldwide. COVID-19, in its severe form, presents with an uncontrolled, hyperactive immune response and severe immunological injury or organ damage that accounts for morbidity and mortality. Even in the absence of complications, COVID-19 can last for several months with lingering effects of an overactive immune system. Dysregulated myeloid and lymphocyte compartments have been implicated in lung immunopathology. Currently, there are limited clinically-tested treatments of COVID-19 with disparities in the apparent efficacy in patients. Accurate model systems are essential to rapidly evaluate promising discoveries but most currently available in mice, ferrets and hamsters do not recapitulate sustained immunopathology described in COVID19 patients. Here, we present a comprehensively humanized mouse COVID-19 model that faithfully recapitulates the innate and adaptive human immune responses during infection with SARS-CoV-2 by adapting recombinant adeno-associated virus (AAV)-driven gene therapy to deliver human ACE2 to the lungs 1 of MISTRG6 mice. Our unique model allows for the first time the study of chronic disease due to infection with SARS-CoV-2 in the context of patient-derived antibodies to characterize in real time the potential culprits of the observed human driving immunopathology; most importantly this model provides a live view into the aberrant macrophage response that is thought to be the effector of disease morbidity and ARDS in patients. Application of therapeutics such as patient-derived antibodies and steroids to our model allowed separation of the two aspects of the immune response, infectious viral clearance and immunopathology. Inflammatory cells seeded early in infection drove immune-patholgy later, but this very same early anti-viral response was also crucial to contain infection.

Mutations Affecting Keratin 10 Surface-Exposed Residues Highlight the Structural Basis of Phenotypic Variation in Epidermolytic Ichthyosis.

Epidermolytic ichthyosis (EI) due to KRT10 mutations is a rare, typically autosomal dominant, disorder characterized by generalized erythema and cutaneous blistering at birth followed by hyperkeratosis and less frequent blistering later in life. We identified two KRT10 mutations p.Q434del and p.R441P in subjects presenting with a mild EI phenotype. Both occur within the mutational "hot spot" of the keratin 10 (K10) 2B rod domain, adjacent to severe EI-associated mutations. p.Q434del and p.R441P formed collapsed K10 fibers rather than aggregates characteristic of severe EI KRT10 mutations such as p.R156C. Upon differentiation, keratinocytes from p.Q434del showed significantly lower apoptosis (P-value<0.01) compared with p.R156C as assessed by the TUNEL assay. Conversely, the mitotic index of the p.Q434del epidermis was significantly higher compared with that of p.R156C (P-value<0.01) as estimated by the Ki67 assay. Structural basis of EI phenotype variation was investigated by homology-based modeling of wild-type and mutant K1-K10 dimers. Both mild EI mutations were found to affect the surface-exposed residues of the K10 alpha helix coiled-coil and caused localized disorganization of the K1-K10 heterodimer. In contrast, adjacent severe EI mutations disrupt key intermolecular dimer interactions. Our findings provide structural insights into phenotypic variation in EI due to KRT10 mutations.

Bimodal Influence of Vitamin D in Host Response to Systemic Candida Infection-Vitamin D Dose Matters.

Vitamin D level is linked to susceptibility to infections, but its relevance in candidemia is unknown. We aimed to investigate the in vivo sequelae of vitamin D3 supplementation in systemic Candida infection. Implicating the role of vitamin D in Candida infections, we showed that candidemic patients had significantly lower 25-OHD concentrations. Candida-infected mice treated with low-dose 1,25(OH)2D3 had reduced fungal burden and better survival relative to untreated mice. Conversely, higher 1,25(OH)2D3 doses led to poor outcomes. Mechanistically, low-dose 1,25(OH)2D3 induced proinflammatory immune responses. This was mediated through suppression of SOCS3 and induction of vitamin D receptor binding with the vitamin D-response elements in the promoter of the gene encoding interferon γ. These beneficial effects were negated with higher vitamin D3 doses. While the antiinflammatory effects of vitamin D3 are well described, we found that, conversely, lower doses conferred proinflammatory benefits in Candida infection. Our study highlights caution against extreme deviations of vitamin D levels during infections.

Intra-subtype variation in enteroadhesion accounts for differences in epithelial barrier disruption and is associated with metronidazole resistance in Blastocystis subtype-7.

Blastocystis is an extracellular, enteric pathogen that induces intestinal disorders in a range of hosts including humans. Recent studies have identified potential parasite virulence factors in and host responses to this parasite; however, little is known about Blastocystis-host attachment, which is crucial for colonization and virulence of luminal stages. By utilizing 7 different strains of the parasite belonging to two clinically relevant subtypes ST-4 and ST-7, we investigated Blastocystis-enterocyte adhesion and its association with parasite-induced epithelial barrier disruption. We also suggest that drug resistance in ST-7 strains might result in fitness cost that manifested as impairment of parasite adhesion and, consequently, virulence. ST-7 parasites were generally highly adhesive to Caco-2 cells and preferred binding to intercellular junctions. These strains also induced disruption of ZO-1 and occludin tight junction proteins as well as increased dextran-FITC flux across epithelial monolayers. Interestingly, their adhesion was correlated with metronidazole (Mz) susceptibility. Mz resistant (Mzr) strains were found to be less pathogenic, owing to compromised adhesion. Moreover, tolerance of nitrosative stress was also reduced in the Mzr strains. In conclusion, the findings indicate that Blastocystis attaches to intestinal epithelium and leads to epithelial barrier dysfunction and that drug resistance might entail a fitness cost in parasite virulence by limiting entero-adhesiveness. This is the first study of the cellular basis for strain-to-strain variation in parasite pathogenicity. Intra- and inter-subtype variability in cytopathogenicity provides a possible explanation for the diverse clinical outcomes of Blastocystis infections.

Strain-dependent induction of human enterocyte apoptosis by blastocystis disrupts epithelial barrier and ZO-1 organization in a caspase 3- and 9-dependent manner.

Blastocystis is an emerging protistan parasite colonizing the human intestine. It is frequently reported to cause general intestinal symptoms of vomiting, diarrhea, and abdominal pain. We recently demonstrated that Blastocystis rearranged cytoskeletal proteins and induced intestinal epithelial barrier compromise. The effect of Blastocystis on enterocyte apoptosis is unknown, and a possible link between microbially induced enterocyte apoptosis and increased epithelial permeability has yet to be determined. The aim of this study is to assess if Blastocystis induces human enterocyte apoptosis and whether this effect influences human intestinal epithelial barrier function. Monolayers of polarized human colonic epithelial cell-line Caco-2 were incubated with Blastocystis subtype 7 and subtype 4. Assays for both early and late markers of apoptosis, phosphatidylserine externalization, and nuclear fragmentation, respectively, showed that Blastocystis ST-7, but not ST-4, significantly increased apoptosis in enterocytes, suggesting that Blastocystis exhibits host specificity and strain-to-strain variation in pathogenicity. ST-7 also activated Caco-2 caspases 3 and 9 but not 8. ST-7 induced changes in epithelial resistance, permeability, and tight junction (ZO-1) localization. Pretreatment of Caco-2 monolayers with a pan-caspase inhibitor z-VAD-fmk significantly inhibited these changes. This suggests a role for enterocyte apoptosis in Blastocystis-mediated epithelial barrier compromise in the human intestine.

Statin pleiotropy prevents rho kinase-mediated intestinal epithelial barrier compromise induced by Blastocystis cysteine proteases.

Blastocystis is an enteric parasite that causes acute and chronic intestinal infections, often non-responsive to conventional antibiotics. The effects of Blastocystis infections on human epithelial permeability are not known, and molecular mechanisms of Blastocystis-induced intestinal pathology remain unclear. This study was conducted to determine whether Blastocystis species alters human intestinal epithelial permeability, to assess whether these abnormalities are rho kinase (ROCK)-dependent, and to investigate the therapeutic potential of the HMG-CoA reductase inhibitor Simvastatin in altered intestinal epithelial barrier function. The effect of metronidazole resistant (Mz(r)) Blastocystis isolated from a symptomatic patient on human colonic epithelial monolayers (Caco-2) was assessed. Modulation of enterocyte myosin light chain phosphorylation, transepithelial fluorescein isothiocyanate-dextran fluxes, transepithelial resistance, cytoskeletal F-actin and tight junctional zonula occludens-1 (ZO-1) by parasite cysteine proteases were measured in the presence or absence of HMG-CoA reductase and ROCK inhibition. Blastocystis significantly decreased transepithelial resistance, increased epithelial permeability, phosphorylated myosin light chain and reorganized epithelial actin cytoskeleton and ZO-1. These alterations were abolished by inhibition of enterocyte ROCK, HMG-CoA reductase and parasite cysteine protease. Our findings suggest that cysteine proteases of Mz(r) Blastocystis induce ROCK-dependent disruption of intestinal epithelial barrier function and correlates with reorganization of cytoskeletal F-actin and tight junctional ZO-1. Simvastatin prevented parasite-induced barrier-compromise, suggesting a therapeutic potential of statins in intestinal infections.

A metronidazole-resistant isolate of Blastocystis spp. is susceptible to nitric oxide and downregulates intestinal epithelial inducible nitric oxide synthase by a novel parasite survival mechanism.

Blastocystis, one of the most common parasites colonizing the human intestine, is an extracellular, noninvasive, luminal protozoan with controversial pathogenesis. Blastocystis infections can be asymptomatic or cause intestinal symptoms of vomiting, diarrhea, and abdominal pain. Although chronic infections are frequently reported, Blastocystis infections have also been reported to be self-limiting in immunocompetent patients. Characterizing the host innate response to Blastocystis would lead to a better understanding of the parasite's pathogenesis. Intestinal epithelial cells produce nitric oxide (NO), primarily on the apical side, in order to target luminal pathogens. In this study, we show that NO production by intestinal cells may be a host defense mechanism against Blastocystis. Two clinically relevant isolates of Blastocystis, ST-7 (B) and ST-4 (WR-1), were found to be susceptible to a range of NO donors. ST-7 (B), a metronidazole-resistant isolate, was found to be more sensitive to nitrosative stress. Using the Caco-2 model of human intestinal epithelium, Blastocystis ST-7 (B) but not ST-4 (WR-1) exhibited dose-dependent inhibition of Caco-2 NO production, and this was associated with downregulation of inducible nitric oxide synthase (iNOS). Despite its higher susceptibility to NO, Blastocystis ST-7 (B) may have evolved unique strategies to evade this potential host defense by depressing host NO production. This is the first study to highlight a strain-to-strain variation in the ability of Blastocystis to evade the host antiparasitic NO response.

Epidemiology of rheumatoid arthritis in a tertiary care unit, Karachi, Pakistan.

OBJECTIVE: To determine the frequency, demographic characteristics, associated co- morbidities and extraarticular manifestations in patients with rheumatoid arthritis (RA) visiting the rheumatology clinic at a tertiary care hospital, Karachi. METHOD: A retrospective medical chart review of 4900 patients, who visited the rheumatology clinic at Liaquat National Hospital, from January 2005 to June 2007, was conducted. All patients with RA, of both gender and ages 16 years and above, who fulfilled the 1987 ACR criteria were included. Demographic characteristics, base line co-morbidity and extra-articular manifestations were recorded according to pre-defined criteria. RESULT: Among 4900 patients, 633 (12.9%) visited the rheumatology clinic with RA, female to male ratio being 4:1. The mean age of onset was 38.5 +/- 12.4 years in females and 44.8 +/- 13.12 years in males. Among the age group 16-29 years females were more affected, whereas greater number of males presented between 50-75 years of age. Co-morbidities were found in 35.38% of RA patients. Cardiovascular disease including hypertension (13.79%) and ischaemic heart disease (6.6%) were the most common co-morbidities. RA factor was positive in 85.05% of the patients with hypertension and 88.09% of the patients with ischaemic heart disease. Extra-articular manifestations were reported in 3.47% of patients. Interstitial lung disease (1.57%) was the most common extra-articular manifestation. CONCLUSION: Significant proportion of patients with female predominance visited the rheumatology clinic at a tertiary care hospital due to RA. Rheumatoid factor may predict the risk of developing cardiovascular disease in patients with RA. The frequency of extra-articular manifestation was lower than that reported in western population.

A rapid, high-throughput viability assay for Blastocystis spp. reveals metronidazole resistance and extensive subtype-dependent variations in drug susceptibilities.

Blastocystis is an emerging protistan parasite of controversial pathogenesis. Although metronidazole (Mz) is standard therapy for Blastocystis infections, there have been accumulating reports of treatment failure, suggesting the existence of drug-resistant isolates. Furthermore, very little is known about Blastocystis susceptibility to standard antimicrobials. In the present study, we established resazurin and XTT viability microassays for Blastocystis spp. belonging to subtypes 4 and 7, both of which have been suggested to represent pathogenic zoonotic subtypes. The optimized resazurin assay was used to screen a total of 19 compounds against both subtypes. Interestingly, subtype 7 parasites were resistant to Mz, a 1-position-substituted 5-nitroimidazole (5-NI), while subtype 4 parasites were sensitive. Some cross-resistance was observed to tinidazole, another 1-position 5-NI. Conversely, subtype 4 parasites were resistant to emetine, while subtype 7 parasites were sensitive. Position 2 5-NIs were effective against both subtypes, as were ornidazole, nitazoxanide, furazolidone, mefloquine, quinicrine, quinine, cotrimoxazole (trimethoprim-sulfamethoxazole), and iodoacetamide. Both subtypes were resistant to chloroquine, doxycycline, paromomycin, ampicillin, and pyrimethamine. This is the first study to report extensive variations in drug sensitivities among two clinically important subtypes. Our study highlights the need to reevaluate established treatment regimens for Blastocystis infections and offers clear new treatment options for Mz treatment failures.

Current Views on the Clinical Relevance of Blastocystis spp.

Blastocystis is an enteric protistan parasite of uncertain clinical relevance. Recent studies indicate that the parasite is a species complex and humans are potentially hosts to nine Blastocystis subtypes, most of which are zoonotic. Subtype 3 is the most common in prevalence studies, followed by subtype 1. Laboratory diagnosis is challenging; the currently recommended diagnostic approach is trichrome staining of direct smears coupled with stool culture. Polymerase chain reaction testing from stools or culture is useful for determining Blastocystis subtype information. The controversial pathogenesis of Blastocystis is attributed to subtype variations in virulence; although current studies seem to support this idea, evidence suggests other factors also contribute to the clinical outcome of the infection. Clinical signs and symptoms of blastocystosis include abdominal pain, diarrhea, bloating, and flatulence. Extraintestinal manifestations, predominantly cutaneous, also were reported. In vitro and animal studies shed new light on the pathobiology of Blastocystis.

Blastocystis exhibits inter- and intra-subtype variation in cysteine protease activity.

Blastocystis is an enteric protistan parasite of zoonotic potential and poorly understood pathogenesis. We have previously reported that Blastocystis cysteine proteases can degrade human secretory IgA and are also responsible for the induction of IL-8 response in colonic epithelial cells in vitro. Differences in virulence between Blastocystis subtypes have been reported recently in both animal models and clinical studies, although cellular mechanisms for these differences are currently unknown. Parasites such as Giardia intestinalis and Entamoeba histolytica have distinct virulent and non-virulent strains which may be attributable to variations in their cysteine proteases. In the present study, variations in cysteine protease activity was observed between avian (subtype 7) and rodent (subtype 4) isolates of Blastocystis with avian isolates exhibiting approximately two times higher peak cysteine protease activity than rodent isolates. Cysteine protease activity and parasite cell size varied over time within cultures of the same isolate. An association between parasite cell size and protease activity was observed.