Kaposi's sarcoma herpesvirus exploits the DNA damage response to circularize its genome.

To establish lifelong, latent infection, herpesviruses circularize their linear, double-stranded, DNA genomes through an unknown mechanism. Kaposi's sarcoma (KS) herpesvirus (KSHV), a gamma herpesvirus, is tightly linked with KS, primary effusion lymphoma, and multicentric Castleman's disease. KSHV persists in latently infected cells as a multi-copy, extrachromosomal episome. Here, we show the KSHV genome rapidly circularizes following infection, and viral protein expression is unnecessary for this process. The DNA damage response (DDR) kinases, ATM and DNA-PKcs, each exert roles, and absence of both severely compromises circularization and latency. These deficiencies were rescued by expression of ATM and DNA-PKcs, but not catalytically inactive mutants. In contrast, γH2AX did not function in KSHV circularization. The linear viral genomic ends resemble a DNA double strand break, and non-homologous DNA end joining (NHEJ) and homologous recombination (HR) reporters indicate both NHEJ and HR contribute to KSHV circularization. Last, we show, similar to KSHV, ATM and DNA-PKcs have roles in circularization of the alpha herpesvirus, herpes simplex virus-1 (HSV-1), while γH2AX does not. Therefore, the DDR mediates KSHV and HSV-1 circularization. This strategy may serve as a general herpesvirus mechanism to initiate latency, and its disruption may provide new opportunities for prevention of herpesvirus disease.

Kaposi's sarcoma herpesvirus latency-associated nuclear antigen broadly regulates viral gene expression and is essential for lytic infection.

Kaposi's sarcoma herpesvirus (KSHV) is a leading cause of malignancy in AIDS and current therapies are limited. Like all herpesviruses, KSHV infection can be latent or lytic. KSHV latency-associated nuclear antigen (LANA) is essential for viral genome persistence during latent infection. LANA also maintains latency by antagonizing expression and function of the KSHV lytic switch protein, RTA. Here, we find LANA null KSHV is not capable of lytic replication, indicating a requirement for LANA. While LANA promoted both lytic and latent gene expression in cells partially permissive for lytic infection, it repressed expression in non-permissive cells. Importantly, forced RTA expression in non-permissive cells led to induction of lytic infection and LANA switched to promote, rather than repress, most lytic viral gene expression. When basal viral gene expression levels were high, LANA promoted expression, but repressed expression at low basal levels unless RTA expression was forcibly induced. LANA's effects were broad, but virus gene specific, extending to an engineered, recombinant viral GFP under control of host EF1α promoter, but not to host EF1α. Together, these results demonstrate that, in addition to its essential role in genome maintenance, LANA broadly regulates viral gene expression, and is required for high levels of lytic gene expression during lytic infection. Strategies that target LANA are expected to abolish KSHV infection.

Identification of unstable regulatory and autoreactive effector T cells that are expanded in patients with FOXP3 mutations.

Studies of the monogenic autoimmune disease immunodysregulation polyendocrinopathy enteropathy X-linked syndrome (IPEX) have elucidated the essential function of the transcription factor FOXP3 and thymic-derived regulatory T cells (Tregs) in controlling peripheral tolerance. However, the presence and the source of autoreactive T cells in IPEX remain undetermined. Here, we investigated how FOXP3 deficiency affects the T cell receptor (TCR) repertoire and Treg stability in vivo and compared T cell abnormalities in patients with IPEX with those in patients with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy syndrome (APECED). To study Tregs independently of their phenotype and to analyze T cell autoreactivity, we combined Treg-specific demethylation region analyses, single-cell multiomic profiling, and bulk TCR sequencing. We found that patients with IPEX, unlike patients with APECED, have expanded autoreactive T cells originating from both autoreactive effector T cells (Teffs) and Tregs. In addition, a fraction of the expanded Tregs from patients with IPEX lost their phenotypic and functional markers, including CD25 and FOXP3. Functional experiments with CRISPR-Cas9-mediated FOXP3 knockout Tregs and Tregs from patients with IPEX indicated that the patients' Tregs gain a TH2-skewed Teff-like function, which is consistent with immune dysregulation observed in these patients. Analyses of FOXP3 mutation-carrier mothers and a patient with IPEX after hematopoietic stem cell transplantation indicated that Tregs expressing nonmutated FOXP3 prevent the accumulation of autoreactive Teffs and unstable Tregs. These findings could be directly used for diagnostic and prognostic purposes and for monitoring the effects of immunomodulatory treatments.

Positron Emission Tomography (PET) Scan as a Diagnostic Tool for Giant Cell Arteritis.

Giant cell arteritis (GCA) is an inflammatory vasculitis that typically affects the elderly, preferentially involving large and medium-sized arteries and can potentially cause irreversible loss of vision. Early diagnosis and treatment are necessary to prevent this dreaded complication. Temporal artery biopsy has been the gold standard test in diagnosing GCA, however, false negative results due to presence of skip lesions, restricted inflammation, and early initiation of steroids have limited its diagnostic significance. We report a case of a 67-year-old female with headache, blurry vision, posterior scalp tenderness, feeble left temporal artery pulse on a physical exam with normal inflammatory markers. Temporal artery biopsy showed disruption and reduplication of internal elastic lamina without any evidence of giant cells or inflammatory cells. Owing to high clinical suspicion, fluorodeoxyglucose (FDG)-positron emission tomography (PET)/computed tomography (CT) was further done which revealed mildly increased uptake in the thoracic aorta, consistent with a diagnosis of large vessel vasculitis.

Epigenetic and immunological indicators of IPEX disease in subjects with FOXP3 gene mutation.

BACKGROUND: Forkhead box protein 3 (FOXP3) is the master transcription factor in CD4+CD25hiCD127lo regulatory T (Treg) cells. Mutations in FOXP3 result in IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) syndrome. Clinical presentation of IPEX syndrome is broader than initially described, challenging the understanding of the disease, its evolution, and treatment choice. OBJECTIVE: We sought to study the type and extent of immunologic abnormalities that remain ill-defined in IPEX, across genetic and clinical heterogeneity. METHODS: We performed Treg-cell-specific epigenetic quantification and immunologic characterization of severe "typical" (n = 6) and "atypical" or asymptomatic (n = 9) patients with IPEX. RESULTS: Increased number of cells with Treg-cell-Specific Demethylated Region demethylation in FOXP3 is a consistent feature in patients with IPEX, with (1) highest values in those with typical IPEX, (2) increased values in subjects with pathogenic FOXP3 but still no symptoms, and (3) gradual increase over the course of disease progression. Large-scale profiling using Luminex identified plasma inflammatory signature of macrophage activation and TH2 polarization, with cytokines previously not associated with IPEX pathology, including CCL22, CCL17, CCL15, and IL-13, and the inflammatory markers TNF-α, IL-1A, IL-8, sFasL, and CXCL9. Similarly, both Treg-cell and Teff compartments, studied by Mass Cytometry by Time-Of-Flight, were skewed toward the TH2 compartment, especially in typical IPEX. CONCLUSIONS: Elevated TSDR-demethylated cells, combined with elevation of plasmatic and cellular markers of a polarized type 2 inflammatory immune response, extends our understanding of IPEX diagnosis and heterogeneity.

Multiregional single-cell proteogenomic analysis of ccRCC reveals cytokine drivers of intratumor spatial heterogeneity.

Intratumor heterogeneity (ITH) represents a major challenge for anticancer therapies. An integrated, multidimensional, multiregional approach dissecting ITH of the clear cell renal cell carcinoma (ccRCC) tumor microenvironment (TME) is employed at the single-cell level with mass cytometry (CyTOF), multiplex immunofluorescence (MxIF), and single-nucleus RNA sequencing (snRNA-seq) and at the bulk level with whole-exome sequencing (WES), RNA-seq, and methylation profiling. Multiregional analyses reveal unexpected conservation of immune composition within each individual patient, with profound differences among patients, presenting patient-specific tumor immune microenvironment signatures despite underlying genetic heterogeneity from clonal evolution. Spatial proteogenomic TME analysis using MxIF identifies 14 distinct cellular neighborhoods and, conversely, demonstrated architectural heterogeneity among different tumor regions. Tumor-expressed cytokines are identified as key determinants of the TME and correlate with clinical outcome. Overall, this work signifies that spatial ITH occurs in ccRCC, which may drive clinical heterogeneity and warrants further interrogation to improve patient outcomes.

Precise reconstruction of the TME using bulk RNA-seq and a machine learning algorithm trained on artificial transcriptomes.

Cellular deconvolution algorithms virtually reconstruct tissue composition by analyzing the gene expression of complex tissues. We present the decision tree machine learning algorithm, Kassandra, trained on a broad collection of >9,400 tissue and blood sorted cell RNA profiles incorporated into millions of artificial transcriptomes to accurately reconstruct the tumor microenvironment (TME). Bioinformatics correction for technical and biological variability, aberrant cancer cell expression inclusion, and accurate quantification and normalization of transcript expression increased Kassandra stability and robustness. Performance was validated on 4,000 H&E slides and 1,000 tissues by comparison with cytometric, immunohistochemical, or single-cell RNA-seq measurements. Kassandra accurately deconvolved TME elements, showing the role of these populations in tumor pathogenesis and other biological processes. Digital TME reconstruction revealed that the presence of PD-1-positive CD8+ T cells strongly correlated with immunotherapy response and increased the predictive potential of established biomarkers, indicating that Kassandra could potentially be utilized in future clinical applications.

Clostridium tertium Bacteremia: A Marker of an Underlying Perforated Colonic Diverticular Disease in a Non-Neutropenic Patient With COVID-19.

Clostridium tertium (C. tertium) is an aero-tolerant, gram-positive, endospore-forming, and non-exotoxin-producing bacillus that has colonized the gastrointestinal tract of animals and humans. It is considered a rare pathogen of humans, possibly because of its low virulence. Most C. tertium infections in the reviewed literatures were predominately reported among neutropenic hosts with hematological malignancies. A 66-year-old female patient with a past medical history of type II diabetes mellitus and chronic obstructive pulmonary disease was admitted with coronavirus disease 2019 (COVID-19) that initially required non-invasive ventilation. The patient developed septic shock due to C. tertium bacteremia. Computed tomography of the abdomen depicted free intraperitoneal gas and sigmoid colon perforation. Exploratory laparotomy revealed perforated sigmoid diverticulitis, and Hartmann's procedure was performed. The patient received a prolonged course of susceptibility-guided antibiotics to clear C. tertium bacteremia. The authors described a rare case of C. tertium bacteremia as a marker of underlying perforated colonic diverticulitis in a non-neutropenic patient with COVID-19 that necessitated operative procedure intervention for primary source control and an extended course of targeted antibiotic therapy to treat the Clostridial infection. Our case reaffirmed the available literature that suggested the presence of C. tertium bacteremia in non-neutropenic patients raises suspicion of an associated gastrointestinal tract pathology that should warrant a diagnostic workup to identify the infection source culprit.

A Plausible Association Between the Use of Elderberry and Autoimmune Hepatitis.

Hepatic injury due to dietary and herbal supplements can often share similar clinical characteristics with autoimmune hepatitis (AIH). Sambucus species, commonly known as elderberry, have been used in traditional medicine for centuries to prevent and treat respiratory problems. Although there are no clear reports on the association of elderberry with AIH or drug-induced hepatitis, there have been concerns about negative health manifestations linked to elderberry and the overproduction of inflammatory cytokines. In this article, we discuss a case of a patient who developed autoimmune hepatitis while on long-term elderberry-containing supplements and a probable association between the two.

Rare Occurrence of Apical Hypertrophic Cardiomyopathy Among Hispanics.

Apical hypertrophic cardiomyopathy (ApHCM), also known as Yamaguchi syndrome represents an uncommon morphologic variant of hypertrophic cardiomyopathy (HCM) in which the myocardial hypertrophy predominantly involves the apex of the left ventricle (LV). It is exemplified by "giant" negative precordial T-waves on electrocardiography and a peculiar "spade-like" configuration of LV cavity on ventriculography historically, and more recently, on echocardiography with use of image enhancing agents. The disease entity was first described in 1976. Available literature reveals that it is prevalent largely among the East-Asian population but is rare among non-Asians. Here, we report a case of a 66-year-old Hispanic male with multiple cardiac histories including persistent atrial fibrillation, non-ST-elevation myocardial infarction (NSTEMI), and ventricular fibrillation cardiac arrest with multiple inconclusive evaluations, who later in life was found to have ApHCM. This case highlights the rare incidence of the disease among the Hispanic population and underlines the challenging diagnosis that requires a high index of suspicion in patients with cardiac symptoms, as ApHCM can masquerade as ischemic coronary heart disease. Our case also describes an unusual clinical course for ApHCM presenting with extreme clinical features, including ventricular arrhythmias and cardiac arrest, unlike the usual benign natural history of this disease.

Diagnostic Utility of RNA-Seq for Evaluation of PD-L1 Expression in Clear Cell Renal Cell Carcinoma.

BACKGROUND: Although there are immune checkpoint inhibitors (ICIs) available for the treatment of renal cell carcinoma (RCC), the utility of PD-L1 detection by immunohistochemistry (IHC) as a predictive biomarker in clear cell RCC (ccRCC) remains controversial. Nevertheless, alternative methods for PD-L1 detection, such as RNA sequencing (RNA-Seq), may be clinically useful in ccRCC; therefore, we sought to determine the ability of RNA-Seq to accurately and sensitively detect PD-L1 expression across different ccRCC clinical samples in comparison with IHC. PATIENTS AND METHODS: Patients with ccRCC (n=127) who received treatment from Washington University in St. Louis between 2018 and 2020 were identified. Tumors from these patients were analyzed using RNA-Seq and IHC. RESULTS: PD-L1 detection by RNA-Seq strongly correlated with IHC (P < .001), which was further validated using two independent datasets. Furthermore, RNA-Seq analysis identified an immune-enriched (higher PD-L1 positivity) and an immune-desert (lower PD-L1 positivity) microenvironment of ccRCC, which also correlated with IHC (P < .00001). CONCLUSION: The results demonstrate the ability of RNA-Seq to detect PD-L1 in various ccRCC clinical samples compared to IHC. Ultimately, these findings suggest that PD-L1 detection by RNA-Seq can be further developed to determine the clinical utility of this methodology in ccRCC.

Deubiquitination and Activation of the NLRP3 Inflammasome by UCHL5 in HCV-Infected Cells.

Chronic hepatitis C virus (HCV) infection induces liver inflammation that can lead to fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). Inflammation is the outcome of the action of proinflammatory cytokines and chemokines, including interleukin-1 beta (IL-1ß) and tumor necrosis factor alpha. Mature IL-1ß production and secretion are facilitated by active inflammasome complexes, including the NACHT-LRR pyrin domain-containing protein 3 (NLRP3) inflammasome. Our study shows that the NLRP3 inflammasome is activated in HCV-infected hepatocytes and that the activation is regulated by posttranslational modifications. NLRP3 is modified by lysine-63 ubiquitin chains in hepatocytes and is deubiquitinated during HCV infection. Inhibition of deubiquitinases (DUBs) with chemical inhibitors or blocking UCHL5 DUB expression with small interfering RNA (siRNA) abrogated NLRP3 inflammasome assembly and activation. Inhibition of inflammasome deubiquitination was correlated with a reduction in IL-1ß maturation, decrease in HCV protein expression, and reduction in release of HCV from the cells. Together, this study suggests that HCV-induced activation of the NLRP3 inflammasome through posttranslational modification is crucial for the HCV life cycle and pathogenesis. IMPORTANCE HCV infection induces inflammation leading to fibrosis, cirrhosis, and cancer. The current study identifies the mechanisms leading to the activation of the NLRP3 inflammasome in hepatocytes, which is an important site of viral replication. Deubiquitination of NLRP3 by UCHL5 is required for inflammasome activation. Inhibition of deubiquitination blocks NLRP3 inflammasome activation and IL-1ß maturation and also decreases HCV replication, suggesting the importance of the NLRP3 inflammasome in inflammation as well as other signaling pathways.

Single-cell Spatial Proteomic Revelations on the Multiparametric MRI Heterogeneity of Clinically Significant Prostate Cancer.

PURPOSE: Multiparametric MRI (mpMRI) has become an indispensable radiographic tool in diagnosing prostate cancer. However, mpMRI fails to visualize approximately 15% of clinically significant prostate cancer (csPCa). The molecular, cellular, and spatial underpinnings of such radiographic heterogeneity in csPCa are unclear. EXPERIMENTAL DESIGN: We examined tumor tissues from clinically matched patients with mpMRI-invisible and mpMRI-visible csPCa who underwent radical prostatectomy. Multiplex immunofluorescence single-cell spatial imaging and gene expression profiling were performed. Artificial intelligence-based analytic algorithms were developed to examine the tumor ecosystem and integrate with corresponding transcriptomics. RESULTS: More complex and compact epithelial tumor architectures were found in mpMRI-visible than in mpMRI-invisible prostate cancer tumors. In contrast, similar stromal patterns were detected between mpMRI-invisible prostate cancer and normal prostate tissues. Furthermore, quantification of immune cell composition and tumor-immune interactions demonstrated a lack of immune cell infiltration in the malignant but not in the adjacent nonmalignant tissue compartments, irrespective of mpMRI visibility. No significant difference in immune profiles was detected between mpMRI-visible and mpMRI-invisible prostate cancer within our patient cohort, whereas expression profiling identified a 24-gene stromal signature enriched in mpMRI-invisible prostate cancer. Prostate cancer with strong stromal signature exhibited a favorable survival outcome within The Cancer Genome Atlas prostate cancer cohort. Notably, five recurrences in the 8 mpMRI-visible patients with csPCa and no recurrence in the 8 clinically matched patients with mpMRI-invisible csPCa occurred during the 5-year follow-up post-prostatectomy. CONCLUSIONS: Our study identified distinct molecular, cellular, and structural characteristics associated with mpMRI-visible csPCa, whereas mpMRI-invisible tumors were similar to normal prostate tissue, likely contributing to mpMRI invisibility.

Anti-fibrotic activity of gold and platinum complexes - Au(I) compounds as a new class of anti-fibrotic agents.

Molecular gold(I) and platinum(II) species were examined for the inhibition of liver fibrosis and the hepatitis C virus (HCV). Determination of inhibition efficiency was conducted via morphological analysis, cell viability, western blot analysis, and quantitative reverse transcription polymerase chain reaction (RT-PCR). Auranofin and Ph3PAuCl demonstrated the greatest inhibition of liver fibrosis amongst the tested gold species in human hepatic stellate LX-2 cells. Western blot analysis indicated that auranofin and Ph3PAuCl prevent signal transducer and activator of transcription 3 (STAT3) phosphorylation, which may be a key connection to fibrosis and inflammation. Auranofin and Ph3PAuCl also reduced expression of HCV-nonstructural protein 3 (NS3) and HCV-NS5a proteins in a HCV subgenomic replicon system. These results demonstrate significant promise for the use of gold compounds in treating liver diseases such as HCV.

Osteopontin Regulates Hepatitis C Virus (HCV) Replication and Assembly by Interacting with HCV Proteins and Lipid Droplets and by Binding to Receptors αVß3 and CD44.

Hepatitis C virus (HCV) replication and assembly occur at the specialized site of endoplasmic reticulum (ER) membranes and lipid droplets (LDs), respectively. Recently, several host proteins have been shown to be involved in HCV replication and assembly. In the present study, we demonstrated the important relationship among osteopontin (OPN), the ER, and LDs. OPN is a secreted phosphoprotein, and overexpression of OPN in hepatocellular carcinoma (HCC) tissue can lead to invasion and metastasis. OPN expression is also enhanced in HCV-associated HCC. Our recent studies have demonstrated the induction, proteolytic cleavage, and secretion of OPN in response to HCV infection. We also defined the critical role of secreted OPN in human hepatoma cell migration and invasion through binding to receptors integrin αVß3 and CD44. However, the role of HCV-induced OPN in the HCV life cycle has not been elucidated. In this study, we showed a significant reduction in HCV replication, assembly, and infectivity in HCV-infected cells transfected with small interfering RNA (siRNA) against OPN, αVß3, and CD44. We also observed the association of endogenous OPN with HCV proteins (NS3, NS5A, NS4A/B, NS5B, and core). Confocal microscopy revealed the colocalization of OPN with HCV NS5A and core in the ER and LDs, indicating a possible role for OPN in HCV replication and assembly. Interestingly, the secreted OPN activated HCV replication, infectivity, and assembly through binding to αVß3 and CD44. Collectively, these observations provide evidence that HCV-induced OPN is critical for HCV replication and assembly.IMPORTANCE Recently, our studies uncovered the critical role of HCV-induced endogenous and secreted OPN in migration and invasion of hepatocytes. However, the role of OPN in the HCV life cycle has not been elucidated. In this study, we investigated the importance of OPN in HCV replication and assembly. We demonstrated that endogenous OPN associates with HCV NS3, NS5A, NS5B, and core proteins, which are in close proximity to the ER and LDs. Moreover, we showed that the interactions of secreted OPN with cell surface receptors αVß3 and CD44 are critical for HCV replication and assembly. These observations provide evidence that HCV-induced endogenous and secreted OPN play pivotal roles in HCV replication and assembly in HCV-infected cells. Taken together, our findings clearly demonstrate that targeting OPN may provide opportunities for therapeutic intervention of HCV pathogenesis.

Genomic analysis of 220 CTCLs identifies a novel recurrent gain-of-function alteration in RLTPR (p.Q575E).

Cutaneous T-cell lymphoma (CTCL) is an incurable non-Hodgkin lymphoma of the skin-homing T cell. In early-stage disease, lesions are limited to the skin, but in later-stage disease, the tumor cells can escape into the blood, the lymph nodes, and at times the visceral organs. To clarify the genomic basis of CTCL, we performed genomic analysis of 220 CTCLs. Our analyses identify 55 putative driver genes, including 17 genes not previously implicated in CTCL. These novel mutations are predicted to affect chromatin (BCOR, KDM6A, SMARCB1, TRRAP), immune surveillance (CD58, RFXAP), MAPK signaling (MAP2K1, NF1), NF-κB signaling (PRKCB, CSNK1A1), PI-3-kinase signaling (PIK3R1, VAV1), RHOA/cytoskeleton remodeling (ARHGEF3), RNA splicing (U2AF1), T-cell receptor signaling (PTPRN2, RLTPR), and T-cell differentiation (RARA). Our analyses identify recurrent mutations in 4 genes not previously identified in cancer. These include CK1α (encoded by CSNK1A1) (p.S27F; p.S27C), PTPRN2 (p.G526E), RARA (p.G303S), and RLTPR (p.Q575E). Last, we functionally validate CSNK1A1 and RLTPR as putative oncogenes. RLTPR encodes a recently described scaffolding protein in the T-cell receptor signaling pathway. We show that RLTPR (p.Q575E) increases binding of RLTPR to downstream components of the NF-κB signaling pathway, selectively upregulates the NF-κB pathway in activated T cells, and ultimately augments T-cell-receptor-dependent production of interleukin 2 by 34-fold. Collectively, our analysis provides novel insights into CTCL pathogenesis and elucidates the landscape of potentially targetable gene mutations.