Faculty Bibliography

The central tenet of scientific research is the rigorous application of the scientific method to experimental design, analysis, interpretation, and reporting of results. In order to confer validity to a hypothesis, experimental details must be transparent and results must be reproducible. Failure to achieve this minimum indicates a deficiency in rationale, design, and/or execution, necessitating further experimental refinement or hypothesis reformulation. More importantly, rigorous application of the scientific method advances scientific knowledge by enabling others to identify weaknesses or gaps that can be exploited by new ideas or technology that inevitably extend, improve, or refine a hypothesis. Experimental details, described in manuscript materials and methods, are the principal vehicle used to communicate procedures, techniques, and resources necessary for experimental reproducibility. Recent examination of the biomedical literature has shown that many published articles lack sufficiently detailed methodological information to reproduce experiments. There are few broadly established practice guidelines and quality assurance standards in basic biomedical research. The current paper provides a framework of best practices to address the lack of reporting of detailed materials and methods that is pervasive in histological slide-based assays. Our goal is to establish a structured framework that highlights the key factors necessary for thorough collection of metadata and reporting of slide-based assays.

Background: The progression of alcoholic liver disease is associated with genetic variants, including the 148M substitution in patatin-like phospholipase domain containing protein 3 (PNPLA3 148M). Mechanistic understanding of these associations has been hindered by species differences. Therefore, we set out to model alcoholic liver disease in human hepatocytes in chimeric mice and test whether genetic variants affected steatosis severity.
Method(s): Liver chimeric Fah-/- mice were created with primary human hepatocytes (PHH) from various donors, including donors homozygous for PNPLA3 148I or 148M. In addition, PNPLA3 148M was lentivirally overexpressed in a 148I homozygous PHH donor prior to re-transplantation into chimeric mice. Liver chimeras were then challenged with an ad lib Lieber-deCarli (LdC) +/-5% ethanol diet or a regular chow diet supplemented with 10% ethanol drinking water.
Result(s): The LdC diet without alcohol caused mild steatosis in human PNPLA3 148I hepatocytes but not mouse hepatocytes. Ethanol in LdC did not worsen steatosis in 148I hepatocytes, contrasting with 148M overexpressing hepatocytes, in which LdC with ethanol caused severe steatosis. Ten percent ethanol in drinking water with a chow diet did not cause steatosis in 148I hepatocytes from 3 different PHH donors yet caused mild to moderate steatosis in mice that were humanized with a 148M homozygous donor.
Conclusion(s): Human, but not mouse, hepatocytes in liver chimeras develop mild steatosis in response to the LdC diet, which is exacerbated by ethanol when PNPLA3 148M is overexpressed. Ethanol drinking water caused steatosis only in chimeras humanized with a PNPLA3 148M donor. These models illustrate the importance of genetic variants in PHH responses to ethanol and will facilitate research into the human genetics implicated in advanced alcoholic liver disease

Background: Hepatocyte transplantation is being pursued as an alternative to liver transplantation. This strategy requires hepatocyte proliferation following transplantation, but the extent to which transplanted hepatocytes can proliferate is unknown. Primary human hepatocytes (PHH) from some donors can efficiently humanize liver chimeric mice yet the relative contributions of engraftment and proliferation have not been quantified. We here aimed to define how many PHH engraft after transplantation in chimeric mice and test their proliferation limit.
Method(s): PHH were serially transplanted into immunodeficient Fah-/- mice with liver injury. PHH were transduced with lentiviruses to deliver fluorophores or barcodes prior to transplantation to quantify engraftment and proliferation.
Result(s): On a population level PHH expanded approximately 200-fold per round of transplantation resulting in ~10⁸ cells per liver. Fluorophore labeling showed PHH islands to be of clonal origin. Barcode labeling indicated that approximately 104 cells engrafted per mouse liver. After three rounds of efficient repopulation humanization deteriorated >10-fold per round.
Conclusion(s): PHH can efficiently expand approximately 1012 -fold (104 per transplantation for 3 serial transplantations) in liver chimeric mice, after which their engraftment and/or proliferation potential diminishes. This limit of ~40 cell divisions in mouse livers may guide therapeutic PHH transplantation protocols. (Figure Presented)

Advanced non-alcoholic fatty liver disease (NAFLD) is a rapidly emerging global health problem associated with pre-disposing genetic polymorphisms, most strikingly an isoleucine to methionine substitution in patatin-like phospholipase domain-containing protein 3 (PNPLA3-I148M). Here, we study how human hepatocytes with PNPLA3 148I and 148M variants engrafted in the livers of broadly immunodeficient chimeric mice respond to hypercaloric diets. As early as four weeks, mice developed dyslipidemia, impaired glucose tolerance, and steatosis with ballooning degeneration selectively in the human graft, followed by pericellular fibrosis after eight weeks of hypercaloric feeding. Hepatocytes with the PNPLA3-148M variant, either from a homozygous 148M donor or overexpressed in a 148I donor background, developed microvesicular and severe steatosis with frequent ballooning degeneration, resulting in more active steatohepatitis than 148I hepatocytes. We conclude that PNPLA3-148M in human hepatocytes exacerbates NAFLD. These models will facilitate mechanistic studies into human genetic variant contributions to advanced fatty liver diseases.

Background/Purpose: The skin is recognized as a window into the immunopathogenic mechanisms driving the vast phenotypic spectrum of psoriatic disease.
Method(s): To better decipher the cellular landscape of both healthy and psoriatic skin, we employed spatial transcriptomics (ST), a ground-breaking technology that precisely maps gene expression from histologically-intact tissue sections (Fig. 1A).
Result(s): Findings gleaned from computationally integrating our 23 matched lesional and non-lesional psoriatic and 7 healthy control samples with publicly-available single-cell ribonucleic acid (RNA) sequencing datasets established the ability of ST to recapitulate the tissue architecture of both healthy and inflamed skin (Fig. 1B) and highlighted topographic shifts in the immune cell milieu, from a predominantly perifollicular distribution in steady-state skin to the papillary and upper reticular dermis in psoriatic lesional skin. We also incidentally discovered that ST's ability to ascertain gene expression patterns from intact tissue rendered it particularly conducive to studying the transcriptome of lipid-laden cells such as dermal adipose tissue and sebaceous glands (Fig. 1C), whose expression profiles are typically lost in the process of tissue handling and dissociation for bulk and single-cell RNA seq. Unbiased clustering of pooled healthy and psoriatic samples identified two epidermal clusters and one dermal cluster that were differentially expanded in psoriatic lesional skin (p values <=0.05) (Fig. 1D); pathway analysis of these clusters revealed enrichment of known psoriatic inflammatory pathways (Fig. 1E). Unsupervised classification of skin-limited psoriasis and psoriatic arthritis samples revealed stratification by cutaneous disease severity or Psoriasis Area and Severity Index (PASI) score and not by presence or absence of concomitant systemic/synovial disease (Fig. 1F). Remarkably, this PASI-dependent segregation was also evident in distal, non-lesional samples and was driven by the dermal macrophage and fibroblast cluster and the lymphatic endothelium (Fig. 2A). Inquiry into the mechanistic drivers of this observed stratification yielded enrichment of pathways associated with key T cell and innate immune cell activation, B cells, and metabolic dysfunction (Fig. 2B). Finally, tissue scale computational cartography of gene expression revealed differences in regional enrichment of specific cell types across phenotypic groups, most notably upward extension of fibroblasts to the upper dermis in both lesional and non-lesional samples from mild psoriasis and restriction to the lower dermis in the moderate-to-severe psoriasis samples (Fig. 2C), suggesting that disease severity stratification may be driven by emergent cellular ecosystems in the upper dermis. Fig. 1. (A) Schematic of spatial transcriptomics study workflow. Four mm skin punch biopsies were obtained from healthy volunteers (n=3) and lesional and non-lesional skin from patients with psoriatic disease (n=11). Ten micron-thick sections were then placed on capture areas on the ST microarray slide, each containing molecularly barcoded, spatially encoded spots with a diameter of 50 microns and a center-to-center distance of 100 microns. (B) Side-by-side comparison of a hematoxylin-eosin (H&E) stained section of representative healthy, lesional, and non-lesional skin samples and the corresponding ST plots showed concordance of unbiased gene expression-based clustering with histologic tissue architecture. (C) Pathway analysis of the adipose cluster in healthy skin (cluster 2) confirmed upregulation of lipid-associated processes. Inset: Spots corresponding to the adipose cluster highlighted in yellow. (D) Wilcoxon rank sum test (results displayed as box plots) yielded statistically significant expansion of three clusters in lesional skin compared to both non-lesional and healthy skin-inflamed suprabasal epidermis (cluster 4), epidermis 2 (cluster 7), and inflamed dermis (cluster 10). HC=healthy control, L=lesional psoriatic skin, NL=non-lesional psoriatic skin. (E) Pathways enriched in clusters 4, 7, and 10. (F) Principal component analysis (PCA) plots demonstrating segregation of samples by severity of cutaneous disease in both lesional and non-lesional samples along the first principal component (right) that was not seen in the samples categorized according to presence or absence of arthritis (left). PsA=psoriatic arthritis, PsO=skin-limited psoriasis. Fig. 2. (A) PCA of lesional and non-lesional samples colored by disease severity in spatial clusters 1 (left) and 12 (right) revealed more discrete clustering. (B) Pathways significantly enriched in clusters 1 (left) and 12 (right) showed enrichment of pathways associated with key T cell and innate immune cell activation, B cells, and metabolic dysfunction (highlighted in red). (C) SpaceFold one dimension projection of cell distribution from an independently-generated single-cell RNA seq data set on aggregated ST lesional and non-lesional samples from mild (PASI-low) and moderate-severe (PASI-high) samples. Y-axis represents tissue position, starting with the lower dermis marked as position 0 to suprabasal epidermis marked as position 1. Dashed line represents epidermal-dermal junction, discerned by cell types in the basal epidermal layer (melanocytes and Langerhans cells). Fibroblast signatures (red arrows) were largely relegated to the lower dermis in the PASI-high group, but extended to the upper dermis in the PASI-low group. This striking difference in fibroblast localization was also noted in non-lesional PASI-high vs. PASI-low groups. In addition to fibroblasts, lymphatic, endothelial, myeloid, and T cells signatures (black arrows) were also observed in the upper dermis of lesional PASI-low samples, but were much lower in the dermis of PASI-low non-lesional and all samples in the PASI-high group. Interfollicular epidermis (IFE), hair follicle and infundibulum (HF/IFN), n= number of individual biopsies.
Conclusion(s): Thus, we have been able to successfully leverage ST integrated with independently-generated single-cell RNA seq data to spatially define the emergent cellular ecosystems of healthy and matched psoriatic lesional and non-lesional skin and in so doing, demonstrated the value of ST in unearthing the genetic groundwork at both the site of inflammation and in distal, clinically-uninvolved skin

OBJECTIVES/OBJECTIVE:Patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may develop end-stage lung disease requiring lung transplantation. We report the clinical course, pulmonary pathology with radiographic correlation, and outcomes after lung transplantation in three patients who developed chronic respiratory failure due to postacute sequelae of SARS-CoV-2 infection. METHODS:A retrospective histologic evaluation of explanted lungs due to coronavirus disease 2019 was performed. RESULTS:None of the patients had known prior pulmonary disease. The major pathologic findings in the lung explants were proliferative and fibrotic phases of diffuse alveolar damage, interstitial capillary neoangiogenesis, and mononuclear inflammation, specifically macrophages, with varying numbers of T and B lymphocytes. The fibrosis varied from early collagen deposition to more pronounced interstitial collagen deposition; however, pulmonary remodeling with honeycomb change was not present. Other findings included peribronchiolar metaplasia, microvascular thrombosis, recanalized thrombi in muscular arteries, and pleural adhesions. No patients had either recurrence of SARS-CoV-2 infection or allograft rejection following transplant at this time. CONCLUSIONS:The major pathologic findings in the lung explants of patients with SARS-CoV-2 infection suggest ongoing fibrosis, prominent macrophage infiltration, neoangiogenesis, and microvascular thrombosis. Characterization of pathologic findings could help develop novel management strategies.

Introduction: Gene rearrangements play a critical role in the development of brain tumors. RNA next-generation sequencing (NGS) panels cover a limited number of genes, are rarely successful in FFPE samples > 5 years old, and cannot detect rearrangements between genes and non-coding regulatory regions. We evaluated whole genome Hi-C NGS for detection of gene fusions and cryptic rearrangements.
Method(s): DNA was extracted from FFPE scrolls of 55 glial and non-glial brain tumors and processed using Arima-HiC+ FFPE Sample protocol, consisting of chromatin fragmentation, labeling, and re-ligation, followed by DNA purification and library preparation for paired-end Illumina sequencing with an average of 10X genome coverage (100M PE reads per sample). Data were analyzed using the Arima-SV pipeline using Juicer and HiCUP, SV detection using HiC-Breakfinder, loop calling using Juicer Tools, and integrative data visualization using Juicebox. Overexpression of putative driver genes was confirmed by immunohistochemistry.
Result(s): Hi-C libraries were prepared and sequenced from FFPE tissues including samples that failed RNA NGS. Hi-C successfully detected gene-gene fusions including actionable EML4-NTRK3, ETV6-NTRK3, fusions. We detected rearrangements missed by RNA NGS (i.e., complex MYBL1 rearrangement) or between non-coding regions and known cancer genes (i.e. PDL1, PAX5, NRAS, TERT, KAT6A, GATA6, and ARID1B). Since Hi-C data captures 3D genome structural features such as chromatin loops and topological domains, datasets were of high quality and capable of detecting up to 13,000 chromatin loops per tumor.
Conclusion(s): Genome-wide Hi-C NGS is successful in detecting gene fusions and cryptic rearrangements between coding and non-coding regions in archival FFPE tissue including degraded samples. Because Hi-C data captures 3D genome structures, these datasets simultaneously inform gene regulatory mechanisms that may play a role in oncogenesis or tumor progression. Whole-genome Hi-C NGS expands our ability to detect actionable and novel drivers, and potentially new therapeutic targets in a single NGS workflow

BACKGROUND:Hepatocellular carcinoma (HCC) patient-derived xenograft (PDX) models hold potential to advance knowledge in HCC biology to help improve systemic therapies. Beside hepatitis B virus-associated tumors, HCC is poorly established in PDX. METHODS: mice through cycling off nitisinone after HCC biopsy implantation, versus continuous nitisinone as non-liver injury controls. Mice with macroscopically detectable PDX showed rising human alpha1-antitrypsin (hAAT) serum levels, and conversely, no PDX was observed in mice with undetectable hAAT. RESULTS:Using rising hAAT as a marker for PDX formation, 20 PDX were established out of 45 HCC biopsy specimens (44%) reflecting the four major HCC etiologies most commonly identified at Memorial SloanKettering similar to many other institutions in the United States. PDX was established only in severely immunodeficient mice lacking lymphocytes and NK cells. Implantation under the renal capsule improved PDX formation two-fold compared to intrahepatic implantation. Two out of 18 biopsies required murine liver injury to establish PDX, one associated with hepatitis C virus and one with alcoholic liver disease. PDX tumors were histologically comparable to biopsy specimens and 75% of PDX lines could be passaged. CONCLUSIONS:Using cycling off nitisinone-induced liver injury, HCC biopsies implanted under the renal capsule of severely immunodeficient mice formed PDX with 57% efficiency as determined by rising hAAT levels. These findings facilitate a more efficient make-up of PDX for research into subset-specific HCC.

INTRODUCTION/BACKGROUND:Treatment failures for lupus nephritis (LN) are high with 10%-30% of patients progressing to end-stage renal disease (ESRD) within 10 years. Interstitial fibrosis/tubular atrophy (IFTA) is a predictor of progression to ESRD. Prior studies suggest that tubulointerstitial injury secondary to proteinuria in LN is mediated by complement activation in the tubules, specifically through the membrane attack complex (MAC). This study aimed to investigate the associations between tubular MAC deposition with IFTA and proteinuria. METHODS:In this cross-sectional study, LN kidney biopsies were assessed for MAC deposition by staining for Complement C9, a component of the MAC. Chromogenic immunohistochemistry was performed on paraffin-embedded human renal biopsy sections using unconjugated, murine anti-human Complement C9 (Hycult Biotech, clone X197). Tubular C9 staining intensity was analysed as present versus absent. IFTA was defined as minimal (<10%), mild (10%-24%), moderate (25%-50%) and severe (>50%). RESULTS:Renal biopsies from 30 patients with LN were studied. There were 24 (80%) female sex, mean age (SD) was 33 (12) years old and 23 (77%) had pure/mixed proliferative LN. Tubular C9 staining was present in 7 (23%) biopsies. 27 patients had minimal-to-mild IFTA and 3 patients had moderate IFTA. Among the C9 + patients, 3 (43%) had moderate IFTA as compared with none in the C9- group, p=0.009. C9 + patients had higher median (IQR) proteinuria as compared with C9- patients: 6.2 g (3.3-13.1) vs 2.4 g (1.3-4.6), p=0.001 at the time of biopsy. There was no difference in estimated glomerular filtration rate (eGFR) between the C9 + and C9- groups. CONCLUSION/CONCLUSIONS:This study demonstrated that tubular MAC deposition is associated with higher degree of IFTA and proteinuria, which are predictors of progression to ESRD. These results suggest that tubular MAC deposition may be useful in classification of LN. Understanding the role of complement in tubulointerstitial injury will also identify new avenues for LN treatment.