Serum and tissue biomarkers of plasma cell-rich rejection in liver transplant recipients.

The distinction between autoimmune and alloimmune reactions in liver transplant recipients can be challenging. Plasma cell-rich rejection (PCRR), previously known as de novo autoimmune hepatitis or plasma cell hepatitis, is an atypical and underrecognized form of allograft rejection observed post-liver transplantation, often in conjunction with features of T cell-mediated and antibody-mediated rejection. If PCRR is not recognized and treated with prompt immunosuppressive augmentation, patients can develop advanced hepatic fibrosis, necroinflammation, and allograft failure. Given the significant morbidity and mortality associated with PCRR, there exists a need to develop noninvasive biomarkers which can be used in screening, diagnosis, and treatment monitoring of PCRR. This study is a literature review of candidate serum-based and tissue-based biomarkers in adult and pediatric liver transplant PCRR. We also discuss biomarkers from plasma cell-rich processes observed in other disease states and other organ transplant recipients that might be tested in liver transplant PCRR. We conclude with proposed future directions in which biomarker implementation into clinical practice could lead to advances in personalized management of PCRR.

Banff 2022 Liver Group Meeting report: Monitoring long-term allograft health.

The Banff Working Group on Liver Allograft Pathology met in September 2022. Participants included hepatologists, surgeons, pathologists, immunologists, and histocompatibility specialists. Presentations and discussions focused on the evaluation of long-term allograft health, including noninvasive and tissue monitoring, immunosuppression optimization, and long-term structural changes. Potential revision of the rejection classification scheme to better accommodate and communicate late T cell-mediated rejection patterns and related structural changes, such as nodular regenerative hyperplasia, were discussed. Improved stratification of long-term maintenance immunosuppression to match the heterogeneity of patient settings will be central to improving long-term patient survival. Such personalized therapeutics are in turn contingent on a better understanding and monitoring of allograft status within a rational decision-making approach, likely to be facilitated in implementation with emerging decision-support tools. Proposed revisions to rejection classification emerging from the meeting include the incorporation of interface hepatitis and fibrosis staging. These will be opened to online testing, modified accordingly, and subject to consensus discussion leading up to the next Banff conference.

A Novel Soluble ACE2 Protein Provides Lung and Kidney Protection in Mice Susceptible to Lethal SARS-CoV-2 Infection.

BACKGROUND: Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) uses full-length angiotensin converting enzyme 2 (ACE2) as a main receptor to enter target cells. The goal of this study was to demonstrate the preclinical efficacy of a novel soluble ACE2 protein with increased duration of action and binding capacity in a lethal mouse model of COVID-19. METHODS: A human soluble ACE2 variant fused with an albumin binding domain (ABD) was linked via a dimerization motif hinge-like 4-cysteine dodecapeptide (DDC) to improve binding capacity to SARS-CoV-2. This novel soluble ACE2 protein (ACE2-1-618-DDC-ABD) was then administered intranasally and intraperitoneally to mice before intranasal inoculation of SARS-CoV-2 and then for two additional days post viral inoculation. RESULTS: Untreated animals became severely ill, and all had to be humanely euthanized by day 6 or 7 and had pulmonary alveolar hemorrhage with mononuclear infiltrates. In contrast, all but one mouse infected with a lethal dose of SARS-CoV-2 that received ACE2-1-618-DDC-ABD survived. In the animals inoculated with SARS-CoV-2 that were untreated, viral titers were high in the lungs and brain, but viral titers were absent in the kidneys. Some untreated animals, however, had variable degrees of kidney proximal tubular injury as shown by attenuation of the proximal tubular brush border and increased NGAL and TUNEL staining. Viral titers in the lung and brain were reduced or nondetectable in mice that received ACE2-1-618-DDC-ABD, and the animals developed only moderate disease as assessed by a near-normal clinical score, minimal weight loss, and improved lung and kidney injury. CONCLUSIONS: This study demonstrates the preclinical efficacy of a novel soluble ACE2 protein, termed ACE2-1-618-DDC-ABD, in a lethal mouse model of SARS-CoV-2 infection that develops severe lung injury and variable degrees of moderate kidney proximal tubular injury.

Alpha-defensins: risk factor for thrombosis in COVID-19 infection.

The inflammatory response to SARS/CoV-2 (COVID-19) infection may contribute to the risk of thromboembolic complications. α-Defensins, antimicrobial peptides released from activated neutrophils, are anti-fibrinolytic and prothrombotic in vitro and in mouse models. In this prospective study of 176 patients with COVID-19 infection, we found that plasma levels of α-defensins were elevated, tracked with disease progression/mortality or resolution and with plasma levels of interleukin-6 (IL-6) and D-dimers. Immunohistochemistry revealed intense deposition of α-defensins in lung vasculature and thrombi. IL-6 stimulated the release of α-defensins from neutrophils, thereby accelerating coagulation and inhibiting fibrinolysis in human blood, imitating the coagulation pattern in COVID-19 patients. The procoagulant effect of IL-6 was inhibited by colchicine, which blocks neutrophil degranulation. These studies describe a link between inflammation and the risk of thromboembolism, and they identify a potential new approach to mitigate this risk in patients with COVID-19 and potentially in other inflammatory prothrombotic conditions.

Porto-Sinusoidal Vascular Disease in Congenital Erythropoietic Porphyria Needing Liver Transplantation.

Porphyria caused by inherited disorders in heme biosynthesis can lead to accumulation of porphyrins in various organs. Liver involvement due to porphyria mostly results in cholestasis leading to liver cirrhosis or hepatocellular carcinoma. Congenital erythropoietic porphyria (CEP), a rare porphyria due to deficiency of uroporphyrinogen III synthase, mostly results in cutaneous manifestations. There are reports of liver involvement including varying degree of fibrosis in patients with CEP. We report a unique case of a patient with CEP who developed porto-sinusoidal vascular disease with complications of portal hypertension that necessitated liver transplantation.

Alloimmunization Against RBC Antigens Is Not Associated With Decreased Survival in Liver Transplant Recipients.

BACKGROUND: Improvement of liver transplantation (LT) outcomes requires better understanding of factors affecting survival. The presence of RBC alloantibodies (RBCAs) on survival in LT recipients was evaluated. METHODS: This study was a single-center, retrospective cohort study reviewing transfusion records and all-cause mortality between 2002 and 2021. RESULTS: Between 2002 and 2021, 2079 LTs were completed, 1,396 of which met inclusion criteria (1,305 RBCA negative; 91 RBCA positive [6.5%]). The cohorts were similar in age (mean [range], 55.8 [17-79] years vs 56.8 [25-73] years; P = .41, respectively) or sex (RBCA negative, 859 [65%] men and 446 [35%] women vs RBCA positive, 51 [56%] men and 40 [44%] women; P = .0684). Of 132 RBCAs detected, 10 were most common were to E (27.27%), Jka (15.91%), K (9.09%), C (8.33%), M (6.06%), D (5.3%), Fya (4.55%), e (2.27%), c (2.27%), and Jkb (2.27%). Twenty-seven patients (29.7%) had more than 1 RBCA; the most common combinations were C with Jka (7.4%) and E with Dia (7.4%). All-cause mortality was increased in men (men, 14.45 years vs women, 17.27 years; P = .0266) and patients 65 years of age and older (≥65 years of age, 10.21 years vs <64 years of age, 17.22 years; P < .0001). The presence of RBCA (≥1) did not affect all-cause mortality (RBCA negative, 14.17 years vs RBCA positive, 15.29 years; P = .4367). The top 5 causes of death were infection (11.9%), primary malignancy (solid) (10.8%), recurrent malignancy (10.5%), cardiovascular arrest (7.1%), and pulmonary insufficiency/respiratory failure (5.7%). CONCLUSIONS: Survival in RBCA-positive LT recipients is no different from that in RBCA-negative LT recipients.

Intranasal soluble ACE2 improves survival and prevents brain SARS-CoV-2 infection.

A soluble ACE2 protein bioengineered for long duration of action and high affinity to SARS-CoV-2 was administered either intranasally (IN) or intraperitoneally (IP) to SARS-CoV-2-inoculated k18hACE2 mice. This decoy protein (ACE2 618-DDC-ABD) was given either IN or IP, pre- and post-inoculation, or IN, IP, or IN + IP but only post-inoculation. Survival by day 5 was 0% in untreated mice, 40% in the IP-pre, and 90% in the IN-pre group. In the IN-pre group, brain histopathology was essentially normal and lung histopathology significantly improved. Consistent with this, brain SARS-CoV-2 titers were undetectable and lung titers reduced in the IN-pre group. When ACE2 618-DDC-ABD was administered only post-inoculation, survival was 30% in the IN + IP, 20% in the IN, and 20% in the IP group. We conclude that ACE2 618-DDC-ABD results in markedly improved survival and provides organ protection when given intranasally as compared with when given either systemically or after viral inoculation, and that lowering brain titers is a critical determinant of survival and organ protection.

Superiority of intranasal over systemic administration of bioengineered soluble ACE2 for survival and brain protection against SARS-CoV-2 infection.

The present study was designed to investigate the effects of a soluble ACE2 protein termed ACE2 618-DDC-ABD, bioengineered to have long duration of action and high binding affinity to SARS-CoV-2, when administered either intranasally (IN) or intraperitoneally (IP) and before or after SARS-CoV-2 inoculation. K18hACE2 mice permissive for SARS-CoV-2 infection were inoculated with 2Ã-10 4 PFU wildtype SARS-CoV-2. In one protocol, ACE2 618-DDC-ABD was given either IN or IP, pre- and post-viral inoculation. In a second protocol, ACE2 618-DDC-ABD was given either IN, IP or IN+IP but only post-viral inoculation. In addition, A549 and Vero E6 cells were used to test neutralization of SARS-CoV-2 variants by ACE2 618-DDC-ABD at different concentrations. Survival by day 5 was 0% in infected untreated mice, and 40% in mice from the ACE2 618-DDC-ABD IP-pre treated group. By contrast, in the IN-pre group survival was 90%, histopathology of brain and kidney was essentially normal and markedly improved in the lungs. When ACE2 618-DDC-ABD was administered only post viral inoculation, survival was 30% in the IN+IP group, 20% in the IN and 0% in the IP group. Brain SARS-CoV-2 titers were high in all groups except for the IN-pre group where titers were undetectable in all mice. In cells permissive for SARS-CoV-2 infection, ACE2 618-DDC-ABD neutralized wildtype SARS-CoV-2 at high concentrations, whereas much lower concentrations neutralized omicron BA. 1. We conclude that ACE2 618-DDC-ABD provides much better survival and organ protection when administered intranasally than when given systemically or after viral inoculation and that lowering brain titers is a critical determinant of survival and organ protection.

IL-1ß-dependent extravasation of preexisting lung-restricted autoantibodies during lung transplantation activates complement and mediates primary graft dysfunction.

Preexisting lung-restricted autoantibodies (LRAs) are associated with a higher incidence of primary graft dysfunction (PGD), although it remains unclear whether LRAs can drive its pathogenesis. In syngeneic murine left lung transplant recipients, preexisting LRAs worsened graft dysfunction, which was evident by impaired gas exchange, increased pulmonary edema, and activation of damage-associated pathways in lung epithelial cells. LRA-mediated injury was distinct from ischemia-reperfusion injury since deletion of donor nonclassical monocytes and host neutrophils could not prevent graft dysfunction in LRA-pretreated recipients. Whole LRA IgG molecules were necessary for lung injury, which was mediated by the classical and alternative complement pathways and reversed by complement inhibition. However, deletion of Fc receptors in donor macrophages or mannose-binding lectin in recipient mice failed to rescue lung function. LRA-mediated injury was localized to the transplanted lung and dependent on IL-1ß-mediated permeabilization of pulmonary vascular endothelium, which allowed extravasation of antibodies. Genetic deletion or pharmacological inhibition of IL-1R in the donor lungs prevented LRA-induced graft injury. In humans, preexisting LRAs were an independent risk factor for severe PGD and could be treated with plasmapheresis and complement blockade. We conclude that preexisting LRAs can compound ischemia-reperfusion injury to worsen PGD for which complement inhibition may be effective.

Chronic liver disease not a significant comorbid condition for COVID-19.

To explore the role of chronic liver disease (CLD) in COVID-19. A total of 1439 consecutively hospitalized patients with COVID-19 from one large medical center in the United States from March 16, 2020 to April 23, 2020 were retrospectively identified. Clinical characteristics and outcomes were compared between patients with and without CLD. Postmortem examination of liver in 8 critically ill COVID-19 patients was performed. There was no significant difference in the incidence of CLD between critical and non-critical groups (4.1% vs 2.9%, p = 0.259), or COVID-19 related liver injury between patients with and without CLD (65.7% vs 49.7%, p = 0.065). Postmortem examination of liver demonstrated mild liver injury associated central vein outflow obstruction and minimal to moderate portal lymphocytic infiltrate without evidence of CLD. Patients with CLD were not associated with a higher risk of liver injury or critical/fatal outcomes. CLD was not a significant comorbid condition for COVID-19.

Subcellular Detection of SARS-CoV-2 RNA in Human Tissue Reveals Distinct Localization in Alveolar Type 2 Pneumocytes and Alveolar Macrophages.

The widespread coronavirus disease 2019 (COVID-19) is caused by infection with the novel coronavirus SARS-CoV-2. Currently, we have limited understanding of which cells become infected with SARS-CoV-2 in human tissues and where viral RNA localizes on the subcellular level. Here, we present a platform for preparing autopsy tissue for visualizing SARS-CoV-2 RNA using RNA fluorescence in situ hybridization (FISH) with amplification by hybridization chain reaction. We developed probe sets that target different regions of SARS-CoV-2 (including ORF1a and N), as well as probe sets that specifically target SARS-CoV-2 subgenomic mRNAs. We validated these probe sets in cell culture and tissues (lung, lymph node, and placenta) from infected patients. Using this technology, we observe distinct subcellular localization patterns of the ORF1a and N regions. In human lung tissue, we performed multiplexed RNA FISH HCR for SARS-CoV-2 and cell-type-specific marker genes. We found viral RNA in cells containing the alveolar type 2 (AT2) cell marker gene (SFTPC) and the alveolar macrophage marker gene (MARCO) but did not identify viral RNA in cells containing the alveolar type 1 (AT1) cell marker gene (AGER). Moreover, we observed distinct subcellular localization patterns of viral RNA in AT2 cells and alveolar macrophages. In sum, we demonstrate the use of RNA FISH HCR for visualizing different RNA species from SARS-CoV-2 in cell lines and FFPE (formalin fixation and paraffin embedding) autopsy specimens. We anticipate that this platform could be broadly useful for studying SARS-CoV-2 pathology in tissues, as well as extended for other applications, including investigating the viral life cycle, viral diagnostics, and drug screening. IMPORTANCE Here, we developed an in situ RNA detection assay for RNA generated by the SARS-CoV-2 virus. We found viral RNA in lung, lymph node, and placenta samples from pathology specimens from COVID patients. Using high-magnification microscopy, we can visualize the subcellular distribution of these RNA in single cells.

Multiplexed detection of SARS-CoV-2 genomic and subgenomic RNA using in situ hybridization.

The widespread Coronavirus Disease 2019 (COVID-19) is caused by infection with the novel coronavirus SARS-CoV-2. Currently, we have a limited toolset available for visualizing SARS-CoV-2 in cells and tissues, particularly in tissues from patients who died from COVID-19. Generally, single-molecule RNA FISH techniques have shown mixed results in formalin fixed paraffin embedded tissues such as those preserved from human autopsies. Here, we present a platform for preparing autopsy tissue for visualizing SARS-CoV-2 RNA using RNA FISH with amplification by hybridization chain reaction (HCR). We developed probe sets that target different regions of SARS-CoV-2 (including ORF1a and N) as well as probe sets that specifically target SARS-CoV-2 subgenomic mRNAs. We validated these probe sets in cell culture and tissues (lung, lymph node, and placenta) from infected patients. Using this technology, we observe distinct subcellular localization patterns of the ORF1a and N regions, with the ORF1a concentrated around the nucleus and the N showing a diffuse distribution across the cytoplasm. In human lung tissue, we performed multiplexed RNA FISH HCR for SARS-CoV-2 and cell-type specific marker genes. We found viral RNA in cells containing the alveolar type 2 (AT2) cell marker gene (SFTPC) and the alveolar macrophage marker gene (MARCO), but did not identify viral RNA in cells containing the alveolar type 1 (AT1) cell marker gene (AGER). Moreover, we observed distinct subcellular localization patterns of viral RNA in AT2 cells and alveolar macrophages, consistent with phagocytosis of infected cells. In sum, we demonstrate the use of RNA FISH HCR for visualizing different RNA species from SARS-CoV-2 in cell lines and FFPE autopsy specimens. Furthermore, we multiplex this assay with probes for cellular genes to determine what cell-types are infected within the lung. We anticipate that this platform could be broadly useful for studying SARS-CoV-2 pathology in tissues as well as extended for other applications including investigating the viral life cycle, viral diagnostics, and drug screening.

Strongyloides Hyperinfection Syndrome causing fatal meningitis and septicemia by Citrobacter koseri.

Citrobacter koseri is a gram-negative bacillius that belongs to the Enterobacteriaceae family. It is an uncommon pathogen that typically causes meningitis and brain abscesses in children, however central nervous system (CNS) infections are rarely found in adults. We present a case of C. koseri meningitis in an immunocompetent adult secondary to intestinal micro-perforation caused by Strongyloides A 76-year-old man admitted for asthma exacerbation developed septic shock. A lumbar puncture revealed bacterial meningitis. Blood and CSF cultures grew Citrobacter koseri with identical susceptibilities, suggesting infection by one strain. Despite broad-spectrum antibiotics, the patient expired of multi-organ failure. Autopsy identified diffuse alveolar hemorrhage as the immedi ate cause of death with a heavy burden of Strongyloides stercoralis in his gastrointestinal system, lungs, and meninges. Citrobacter koseri is a gram-negative bacillus of the Enterobacteriaceae family. It is an uncommon pathogen that typically causes meningitis and brain abscesses in children. Infections in adults occur in immunocompromised hosts or instances where an insult creates a port of entry. This is the first documented case of C.koseri sepsis in an immunocompetent host associated with Strongyloides Hyperinfection Syndrome (SHS), where massive parasitic intestinal invasion reaches pulmonary circulation and perforates the alveolar membrane. This case highlights that presence of rare enterobacterial infections should prompt consideration of differentials including SHS.