Rationale and Design of a Phase 2, Double-blind, Placebo-Controlled, Randomized Trial Evaluating AMP Kinase-Activation by Metformin in Focal Segmental Glomerulosclerosis.

Introduction: Focal segmental glomerulosclerosis (FSGS), the most common primary glomerular disease leading to end-stage kidney disease (ESKD), is characterized by podocyte injury and depletion, whereas minimal change disease (MCD) has better outcomes despite podocyte injury. Identifying mechanisms capable of preventing podocytopenia during injury could transform FSGS to an "MCD-like" state. Preclinical data have reported conversion of an MCD-like injury to one with podocytopenia and FSGS by inhibition of AMP-kinase (AMPK) in podocytes. Conversely, in FSGS, AMPK-activation using metformin (MF) mitigated podocytopenia and azotemia. Observational studies also support beneficial effects of MF on proteinuria and chronic kidney disease (CKD) outcomes in diabetes. A randomized controlled trial (RCT) to test MF in podocyte injury with FSGS has not yet been conducted. Methods: We report the rationale and design of phase 2, double-blind, placebo-controlled RCT evaluating the efficacy and safety of MF as adjunctive therapy in FSGS. By randomizing 30 patients with biopsy-confirmed FSGS to MF or placebo (along with standard immunosuppression), we will study mechanistic biomarkers that correlate with podocyte injury or depletion and evaluate outcomes after 6 months. We specifically integrate novel urine, blood, and tissue markers as surrogates for FSGS progression along with unbiased profiling strategies. Results and Conclusion: Our phase 2 trial will provide insight into the potential efficacy and safety of MF as adjunctive therapy in FSGS-a crucial step to developing a larger phase 3 study. The mechanistic assays here will guide the design of other FSGS trials and contribute to understanding AMPK activation as a potential therapeutic target in FSGS. By repurposing an inexpensive agent, our results will have implications for FSGS treatment in resource-poor settings.

A Rare Case of Methotrexate-Associated Lymphoproliferative Disease in the Orbit.

An 80-year-old Caucasian female with a history of rheumatoid arthritis presented with a 6-month history of progressive right upper eyelid ptosis, edema, erythema, and pain. MRI demonstrated a superior orbital mass. An incisional biopsy was performed, and pathologic analysis revealed an atypical lymphoid infiltrate, co-expressing both B and T-cell markers, with a low proliferation rate. Flow cytometry and IgH rearrangement study did not demonstrate any B- or T-cell monoclonal proliferation. Based on these findings, she was diagnosed with an iatrogenic immunodeficiency-associated lymphoproliferative disorder. Discontinuation of methotrexate resulted in the complete resolution of her symptoms, and she remains in remission 18 months later. Given the increased risk of lymphoproliferative disease in patients with rheumatoid arthritis, careful evaluation and close monitoring upon immunosuppressive medication withdrawal is necessary to confirm the diagnosis.

Recent Advances in Genitourinary Tumors Updates From the 5th Edition of the World Health Organization Blue Book Series.

CONTEXT.­: Urinary and Male Genital Tumours is the 8th volume of the World Health Organization Classification of Tumours series, 5th edition. Released in hard copy in September 2022, it presents an update to the classification of male genital and urinary tumors in the molecular age. Building upon previous volumes in this series, significant effort has been made to harmonize terminology across organ systems for biologically similar tumors (eg, neuroendocrine tumors). Genomic terminology has been standardized and genetic syndromes covered more comprehensively. This review presents a concise summary of this volume highlighting new entities, notable modifications relative to the 4th edition, and elements of relevance to routine clinical practice. OBJECTIVE.­: To provide a comprehensive update on the World Health Organization classification of urinary and male genital tumors, highlighting updated diagnostic criteria and terminology. DATA SOURCES.­: The 4th and 5th editions of the World Health Organization Classification of Tumours: Urinary and Male Genital Tumours. CONCLUSIONS.­: The World Health Organization has made several changes in the 5th edition of the update on urinary and male genital tumors that pathologists need to be aware of for up-to-date clinical practice.

Profilin1 is required for prevention of mitotic catastrophe in murine and human glomerular diseases.

The progression of proteinuric kidney diseases is associated with podocyte loss, but the mechanisms underlying this process remain unclear. Podocytes reenter the cell cycle to repair double-stranded DNA breaks. However, unsuccessful repair can result in podocytes crossing the G1/S checkpoint and undergoing abortive cytokinesis. In this study, we identified Pfn1 as indispensable in maintaining glomerular integrity - its tissue-specific loss in mouse podocytes resulted in severe proteinuria and kidney failure. Our results suggest that this phenotype is due to podocyte mitotic catastrophe (MC), characterized histologically and ultrastructurally by abundant multinucleated cells, irregular nuclei, and mitotic spindles. Podocyte cell cycle reentry was identified using FUCCI2aR mice, and we observed altered expression of cell-cycle associated proteins, such as p21, p53, cyclin B1, and cyclin D1. Podocyte-specific translating ribosome affinity purification and RNA-Seq revealed the downregulation of ribosomal RNA-processing 8 (Rrp8). Overexpression of Rrp8 in Pfn1-KO podocytes partially rescued the phenotype in vitro. Clinical and ultrastructural tomographic analysis of patients with diverse proteinuric kidney diseases further validated the presence of MC podocytes and reduction in podocyte PFN1 expression within kidney tissues. These results suggest that profilin1 is essential in regulating the podocyte cell cycle and its disruption leads to MC and subsequent podocyte loss.

IRF8 may be a useful marker for blastic plasmacytoid dendritic cell neoplasm, especially with weak CD123 expression.

We highlight the utility of interferon regulatory factor 8 (IRF8), a novel marker of monocytic and dendritic cell lineages, in the diagnosis of a case of blastic plasmacytoid dendritic cell neoplasm (BPDCN) presenting initially in the skin. A 60-year-old male with a previous history of myelodysplastic syndrome presented with cutaneous nodules on chest and scalp. A punch biopsy specimen of a skin nodule showed a diffuse dermal infiltrate of atypical mononuclear cells. The neoplastic cells expressed CD4, CD56, CD43, and TdT but showed minimal reaction for TCL-1 and CD123, and were negative for CD34, CD117, and MPO, confounding the diagnosis. IRF8 performed in retrospect was strongly positive. A new punch biopsy specimen of a chest nodule showed the blastoid tumor cells were positive for TCL-1, CD4, and CD56, but dim CD123. Subsequent bone marrow involvement showed blastoid tumor cells with intense positivity for CD123, CD4, and CD56, which was supportive of the BPDCN diagnosis. BPDCN cases with weak or variable CD123 and TCL-1 expression represent a potential diagnostic pitfall. In a recent study, 15 cases of BPDCN showed uniformly strong staining for IRF8, while CD123 was dim or negative in 4 of these 15 cases. We suggest IRF8 may be a useful marker for BPDCN, especially in cases with weak or variable expression of CD123 and TCL1.

Downregulation of a tumor suppressor gene LKB1 in lung transplantation as a biomarker for chronic murine lung allograft rejection.

BACKGROUND: We recently demonstrated decreased tumor suppressor gene liver kinase B1 (LKB1) level in lung transplant recipients diagnosed with bronchiolitis obliterans syndrome. STE20-related adaptor alpha (STRADα) functions as a pseudokinase that binds and regulates LKB1 activity. METHODS: A murine model of chronic lung allograft rejection in which a single lung from a B6D2F1 mouse was orthotopically transplanted into a DBA/2J mouse was employed. We examined the effect of LKB1 knockdown using CRISPR-CAS9 in vitro culture system. RESULTS: Significant downregulation of LKB1 and STRADα expression was found in donor lung compared to recipient lung. STRADα knockdown significantly inhibited LKB1, pAMPK expression but induced phosphorylated mammalian target of rapamycin (mTOR), fibronectin, and Collagen-I, expression in BEAS-2B cells. LKB1 overexpression decreased fibronectin, Collagen-I, and phosphorylated mTOR expression in A549 cells. CONCLUSIONS: We demonstrated that downregulation of LKB1-STRADα pathway accompanied with increased fibrosis, results in development of chronic rejection following murine lung transplantation.

Cell Cycle and Senescence Regulation by Podocyte Histone Deacetylase 1 and 2.

SIGNIFICANCE STATEMENT: The loss of integrity of the glomerular filtration barrier results in proteinuria that is often attributed to podocyte loss. Yet how damaged podocytes are lost remains unknown. Germline loss of murine podocyte-associated Hdac1 and Hdac2 ( Hdac1/2 ) results in proteinuria and collapsing glomerulopathy due to sustained double-stranded DNA damage. Hdac1/2 deletion induces loss of podocyte quiescence, cell cycle entry, arrest in G1, and podocyte senescence, observed both in vivo and in vitro . Through the senescence secretory associated phenotype, podocytes secrete proteins that contribute to their detachment. These results solidify the role of HDACs in cell cycle regulation and senescence, providing important clues in our understanding of how podocytes are lost following injury. BACKGROUND: Intact expression of podocyte histone deacetylases (HDAC) during development is essential for maintaining a normal glomerular filtration barrier because of its role in modulating DNA damage and preventing premature senescence. METHODS: Germline podocyte-specific Hdac1 and 2 ( Hdac1 / 2 ) double-knockout mice were generated to examine the importance of these enzymes during development. RESULTS: Podocyte-specific loss of Hdac1 / 2 in mice resulted in severe proteinuria, kidney failure, and collapsing glomerulopathy. Hdac1 / 2 -deprived podocytes exhibited classic characteristics of senescence, such as senescence-associated ß-galactosidase activity and lipofuscin aggregates. In addition, DNA damage, likely caused by epigenetic alterations such as open chromatin conformation, not only resulted in podocyte cell-cycle entry as shown in vivo by Ki67 expression and by FUCCI-2aR mice, but also in p21-mediated cell-cycle arrest. Through the senescence secretory associated phenotype, the damaged podocytes secreted proinflammatory cytokines, growth factors, and matrix metalloproteinases, resulting in subsequent podocyte detachment and loss, evidenced by senescent podocytes in urine. CONCLUSIONS: Hdac1 / 2 plays an essential role during development. Loss of these genes in double knockout mice leads to sustained DNA damage and podocyte senescence and loss.

Role for exosomes with self-antigens and immune regulatory molecules in allo- and auto-immunity leading to chronic immune injury following murine kidney transplantation.

OBJECTIVE: Antibodies against donor human leukocyte antigen are a risk factor for chronic immune injury (CII) following renal transplantation; however, it is often not detectable. The main goal of this study is to gain new insights into the kinetics of exosome release and content in sensitized vs non-sensitized recipients. Towards this, we investigated the role for circulating exosomes with allo and self-antigens as well as immunoregulatory molecules in the development of CII and acute rejection. METHODS: Using murine kidney allograft rejection models, we investigated the role of exosomes on immune responses leading to allo- and auto-immunity to self-antigens resulting in rejection. Exosomes were analyzed for kidney self-antigens (Collagen-IV, fibronectin, angiotensin II receptor type 1), and immune-regulatory molecules (PD-L1, CD73) using western blot. Antibodies to donor MHC in serum samples were detected by immunofluorescence, self-antigens by enzyme-linked immunosorbent assay and kidney tissue infiltrating cells were determined by immunohistochemistry. RESULTS: BALB/c; H2d to C57BL/6; H2b renal transplantation (BALB/c), resulted in tubulitis and cellular infiltration by day 14, suggestive of acute inflammation, that was self-limiting with functioning graft. This contributed to CII on post-transplant day >100, which was preceded by induction of exosomes with donor and self-antigens leading to antibodies and immune-regulatory molecules. The absence of acute rejection in this allogenic transplant model is likely due to the induction of splenic and, graft-infiltrating CD4 + FoxP3+ T regulatory cells. In contrast, prior sensitization by skin graft followed by kidney transplantation induced antibodies to MHC and self-antigens leading to acute rejection. CONCLUSION: We demonstrate a pivotal role for induction of exosomes with immune-regulatory molecules, allo- and auto-immunity to self-antigens leading to chronic immune injury following murine kidney transplantation.

Cellular recovery after prolonged warm ischaemia of the whole body.

After cessation of blood flow or similar ischaemic exposures, deleterious molecular cascades commence in mammalian cells, eventually leading to their death1,2. Yet with targeted interventions, these processes can be mitigated or reversed, even minutes or hours post mortem, as also reported in the isolated porcine brain using BrainEx technology3. To date, translating single-organ interventions to intact, whole-body applications remains hampered by circulatory and multisystem physiological challenges. Here we describe OrganEx, an adaptation of the BrainEx extracorporeal pulsatile-perfusion system and cytoprotective perfusate for porcine whole-body settings. After 1 h of warm ischaemia, OrganEx application preserved tissue integrity, decreased cell death and restored selected molecular and cellular processes across multiple vital organs. Commensurately, single-nucleus transcriptomic analysis revealed organ- and cell-type-specific gene expression patterns that are reflective of specific molecular and cellular repair processes. Our analysis comprises a comprehensive resource of cell-type-specific changes during defined ischaemic intervals and perfusion interventions spanning multiple organs, and it reveals an underappreciated potential for cellular recovery after prolonged whole-body warm ischaemia in a large mammal.

Restrictive allograft syndrome vs bronchiolitis obliterans syndrome: Immunological and molecular characterization of circulating exosomes.

BACKGROUND: Chronic lung allograft dysfunction in lung transplant recipients (LTxRs) has 2 phenotypes: obstructive bronchiolitis obliterans syndrome (BOS) and restrictive allograft syndrome (RAS). Our goal was to define distinct immunologic markers of exosomes from LTxRs with BOS or RAS. METHODS: Plasma was collected from LTxRs with BOS (n = 18), RAS (n = 13), and from stable LTxRs (n = 5). Antibodies to lung self-antigens (SAgs) were determined by ELISA. Exosomes were isolated by ultracentrifugation. Donor specific antibodies to HLA were quantified using Luminex. Exosomes were characterized for lung SAgs, transcription factors, 20S proteasome, HLA class I and II, and polymeric immunoglobulin receptor protein using western blot. Exosome miRNA was analyzed using NanoString. The exosome-induced immune response was determined in mice. RESULTS: LTxRs with RAS, but not BOS, had donor specific antibodies at diagnosis. CIITA, NFkB, polymeric immunoglobulin receptor protein, 20S proteasome, HLA-DQ, and HLA-DR were significantly higher in RAS exosomes than in BOS exosomes. RAS plasma had high levels of proinflammatory cytokines and distinct exosomal miRNA. Immunization of C57BL/6 mice with RAS exosomes showed severe inflammation and peribronchial fibrosis, whereas BOS exosomes induced patchy inflammation and fibrosis. CONCLUSION: LTxRs with BOS or RAS had exosomes with distinct molecular and immunologic profiles. RAS samples had a higher concentration of proinflammatory factors, HLA class II, lung SAgs, and antibodies to HLA class II molecules, indicating severe allograft injury. Mice immunized with RAS exosomes developed lesions in airways, pleura, interlobular septum, and alveoli, whereas BOS exosomes induced mild to patchy inflammation with lung fibrosis.

Tumor Microenvironment of Lymphomas and Plasma Cell Neoplasms: Broad Overview and Impact on Evaluation for Immune Based Therapies.

Lymphomas and plasma cell neoplasms are a heterogenous group of malignancies derived from lymphocytes. They are a significant cause of patient morbidity and mortality. Advances in morphologic, immunophenotypic and molecular techniques have led to better understanding of the pathogenesis and diagnosis of these neoplasms. Advances in treatment, particularly immune-based therapies, increasingly allow for targeted therapies of these diseases. Mechanistic studies using animal models and clinical trials have revealed the importance of the tumor microenvironment on disease pathogenesis, progression, and response to therapy in these malignancies. Simultaneous progress in diagnostic techniques has made it feasible to generate high-resolution, high-throughput data from the tumor microenvironment with spatial context. As the armamentarium of targeted therapies and diagnostic techniques grows, there is potential to harness these advances to better stratify patients for targeted therapies, including immune-based therapies, in hematologic malignancies.

Cutting Edge: Circulating Exosomes with COVID Spike Protein Are Induced by BNT162b2 (Pfizer-BioNTech) Vaccination prior to Development of Antibodies: A Novel Mechanism for Immune Activation by mRNA Vaccines.

Severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) causes severe acute respiratory syndrome. mRNA vaccines directed at the SARS-CoV-2 spike protein resulted in development of Abs and protective immunity. To determine the mechanism, we analyzed the kinetics of induction of circulating exosomes with SARS-CoV-2 spike protein and Ab following vaccination of healthy individuals. Results demonstrated induction of circulating exosomes expressing spike protein on day 14 after vaccination followed by Abs 14 d after the second dose. Exosomes with spike protein, Abs to SARS-CoV-2 spike, and T cells secreting IFN-γ and TNF-α increased following the booster dose. Transmission electron microscopy of exosomes also demonstrated spike protein Ags on their surface. Exosomes with spike protein and Abs decreased in parallel after four months. These results demonstrate an important role of circulating exosomes with spike protein for effective immunization following mRNA-based vaccination. This is further documented by induction of humoral and cellular immune responses in mice immunized with exosomes carrying spike protein.

Perianeurysmal retroperitoneal fibrosis presenting as acute kidney injury: A case report.

Retroperitoneal fibrosis and chronic periaortitis describe overlapping groups of rare diseases characterized by inflammation and fibrosis involving the aorta. The presentation is often non-specific, and while obstructive nephropathy is a common complication, these entities are an uncommon cause of renal failure necessitating dialysis. A 57-year-old man presented multiple times with acute kidney injury, even requiring hemodialysis, with repeated abrupt resolution. Renal ultrasound repeatedly did not reveal acute hydronephrosis. Renal biopsy on his first admission showed acute tubular injury attributed to hypovolemia. Computed tomography finally revealed a retroperitoneal soft tissue mass encasing the infrarenal abdominal aorta and partially encasing the bilateral ureters. Bilateral nephrostomy tubes were placed, steroids were initiated, and the patient experienced rapid and remarkable improvement in renal function. Chronic periaortitis should be considered in older patients with acute kidney injury, even in the absence of ultrasonographic evidence of obstruction. Additional studies are needed to describe the test characteristics of renal sonography for periaortitis, the long-term sequelae of acute kidney injury secondary to periaortitis, and the optimal management to preserve long-term renal function.

An independent assessment of an artificial intelligence system for prostate cancer detection shows strong diagnostic accuracy.

Prostate cancer is a leading cause of morbidity and mortality for adult males in the US. The diagnosis of prostate carcinoma is usually made on prostate core needle biopsies obtained through a transrectal approach. These biopsies may account for a significant portion of the pathologists' workload, yet variability in the experience and expertise, as well as fatigue of the pathologist may adversely affect the reliability of cancer detection. Machine-learning algorithms are increasingly being developed as tools to aid and improve diagnostic accuracy in anatomic pathology. The Paige Prostate AI-based digital diagnostic is one such tool trained on the digital slide archive of New York's Memorial Sloan Kettering Cancer Center (MSKCC) that categorizes a prostate biopsy whole-slide image as either "Suspicious" or "Not Suspicious" for prostatic adenocarcinoma. To evaluate the performance of this program on prostate biopsies secured, processed, and independently diagnosed at an unrelated institution, we used Paige Prostate to review 1876 prostate core biopsy whole-slide images (WSIs) from our practice at Yale Medicine. Paige Prostate categorizations were compared to the pathology diagnosis originally rendered on the glass slides for each core biopsy. Discrepancies between the rendered diagnosis and categorization by Paige Prostate were each manually reviewed by pathologists with specialized genitourinary pathology expertise. Paige Prostate showed a sensitivity of 97.7% and positive predictive value of 97.9%, and a specificity of 99.3% and negative predictive value of 99.2% in identifying core biopsies with cancer in a data set derived from an independent institution. Areas for improvement were identified in Paige Prostate's handling of poor quality scans. Overall, these results demonstrate the feasibility of porting a machine-learning algorithm to an institution remote from its training set, and highlight the potential of such algorithms as a powerful workflow tool for the evaluation of prostate core biopsies in surgical pathology practices.

Initial Evaluation of Rapid, Direct-to-Digital Prostate Biopsy Pathology.

CONTEXT.­: Pathologist interobserver discordance is significant in grading of prostate cancer, limiting reliability. Diagnostic reproducibility may be improved with digital images, but adoption faces workflow, cost, and quality challenges. A novel digital method using an alternative tissue processing approach and novel laser microscopy system potentially addresses these issues. OBJECTIVE.­: To evaluate the capability of this new method for primary diagnostic interpretation in clinical prostate biopsy specimens. DESIGN.­: Forty patients with a high likelihood of prostate cancer based on magnetic resonance imaging consented to investigational core biopsy. A subset of samples was used for direct comparison of physical slide preparation effects and time-tracking determination with multiphoton microscopy. Twenty samples were processed for diagnostic comparison between multilevel digital slides and subsequently produced physical slides. A reference diagnosis based on all data was established using grade groups. Level of diagnostic match and requests for immunohistochemistry were compared between physical and digital diagnoses. Immunohistochemical staining and length measurements were secondary outcomes. RESULTS.­: Interpretations based on direct multiphoton imaging yielded diagnoses that were at least as accurate as standard histology; cancer diagnosis correlation was 89% (51 of 57) by physical slides and 95% (53 of 56) by multiphoton microscopy. Grade-level concordance was 73% (44 of 60) by either method. Immunohistochemistry for routine prostate cancer-associated markers on these alternatively processed tissues was unaffected. Alternatively processed tissues resulted in longer measured core and cancer lengths, suggestive of improved orientation and visualization. CONCLUSIONS.­: Findings support high potential for complete interpretation of prostate core biopsies using solely multiphoton microscopy of intact specimens, with potential diagnostic benefits as well as reduced processing time and reduced processing complexity.