Viral infections lead to caspase activation as a cellular defense response. Some viruses overcome this response by encoding viral proteins that undergo caspase cleavage and, by various mechanisms, aid in cell survival. Kaposi sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen acts as a pseudo-substrate for caspases-1 and -3, thereby interfering with the inflammatory and apoptotic response. Here, we screened the KSHV proteome for additional caspase cleavage sites. Using SitePrediction, many KSHV proteins with potential caspase cleavage sites were identified. Among the highest-scoring proteins was the early lytic protein, K5. Treatment of BJAB-K5-FLAG-expressing cells with ⍺Fas led to caspase processing of K5-FLAG. Using mass spectrometry, we determined that caspase cleavage of K5 occurs at D222. K5 was also cleaved by caspases when KSHV was induced to lytic replication. Interestingly, the expression of K5-FLAG significantly inhibited ⍺Fas-induced caspase-mediated cell death. To determine if K5 plays a protective role in KSHV-infected cells, iSLK cells infected with wild-type or K5-knockout BAC16 virus were induced to lytic replication. Although lytic induction showed no significant effect on the viability of wild-type-infected cells, the viability of K5-knockout cells decreased by 25%. Thus, K5 may protect KSHV-infected cells from caspase-mediated cell death during lytic replication. Interestingly, cleavage of K5 by caspases did not affect its previously reported ability to downregulate immune surface markers. These data suggest that K5 not only downregulates immune surface markers to help avoid immune recognition but plays an additional role in mitigating caspase-mediated cell death during lytic replication.IMPORTANCEKaposi sarcoma-associated herpesvirus (KSHV) is the etiological agent for Kaposi sarcoma, primary effusion lymphoma, multicentric Castleman's disease, and KSHV inflammatory cytokine syndrome. Understanding how KSHV thwarts host defense responses is necessary to help develop strategies to treat these rare, yet deadly, diseases. We profiled potential caspase cleavage sites in the viral proteome in silico and found many viral proteins with high-scoring caspase cleavage sites. We follow up on this by demonstrating that K5 is, in fact, a substrate for caspases in vitro and in cellulo and provide data to suggest that K5 plays a role in obviating caspase-mediated cell death during lytic replication. The work described here furthers our understanding of the roles that KSHV proteins, like K5, play to prevent or divert the host apoptotic defense responses that involve host caspase activation that normally would lead to cell death.

We previously reported that Kaposi sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) acts as a pseudo-substrate for caspases-1 and 3, thereby interfering with their inflammatory and apoptotic activity, respectively. To determine if other KSHV proteins undergo caspase cleavage, we screened the KSHV proteome for potential caspase cleavage sites. Using SitePrediction (SP), 30 KSHV proteins with potential caspase-cleavage sites were identified. Among those with highest SP score was an early lytic protein, K5. Treatment of BJAB K5-FLAG cells with ⍺Fas, an apoptotic stimulus, led to caspase-processing of full length K5-FLAG and generation of a C-terminal peptide fragment. Using mass spectrometry, we determined that K5-FLAG undergoes caspase cleavage at D222. K5 was also cleaved by caspases in KSHV infected cells induced to lytic replication. Importantly, the expression of K5-FLAG significantly inhibited ⍺Fas-induced caspase-mediated cell death. To determine if K5 plays a protective role in KSHV infected cells, iSLKK cells infected with wild type or K5 knockout BAC16 virus were induce to lytic replication to activate caspases. Although lytic induction showed little effect on the viability of WT infected cells, the viability of K5-knockout cells decreased by 25%. Thus, K5 may protect KSHV-infected cells from caspase-mediated cell death during lytic replication. Interestingly, cleavage of K5 by caspases did not affect its ability to downregulate MHC-1 surface expression. Overall, these data suggest that K5 not only downregulates immunologic surface marker expression to avoid immune recognition but may also play an additional role in mitigating caspase-mediated cell death during KSHV lytic replication.

Primary effusion lymphoma (PEL), which is caused by Kaposi sarcoma herpesvirus (KSHV), and Burkitt lymphoma (BL), a subset of which are associated with Epstein-Barr virus (EBV), are aggressive non-Hodgkin's lymphomas. Both have relatively poor survival compared to other lymphomas. Cereblon-binding immunomodulators (CBIs), such as pomalidomide (Pom), show in vitro efficacy and clinical activity against certain of these lymphomas. Next generation CBIs, such as golcadomide (Golc) and iberdomide (Iber), have increased affinity to the primary cellular target, cereblon, making them potentially better anticancer agents. Here, we report the in vitro activity of these novel CBIs against PEL and BL cell lines. Both Golc and Iber, but primarily Golc, caused substantial growth suppression of PEL and BL lines with much lower half-maximal inhibitory concentration (IC50) compared to Pom. This growth suppression was mediated, in part, by enhanced downregulation of interferon regulatory factor 4 (IRF4) in PEL cell lines. Additionally, both Golc and Iber increased immune surface markers such as ICAM-1, B7-2, and MHC-I in PEL and BL cells at lower concentrations than Pom; these increases led to enhanced recognition of both PEL and BL cells by T-cells. The novel CBIs had relatively little activity in Pom-resistant cell lines with low levels of cereblon, suggesting that binding to cereblon is also important for the functions of the novel CBIs. These data show that the newer CBIs are more potent and effective against PEL and BL lines than Pom, and therefore, are worth investigating clinically in patients with these tumors.

Kaposi sarcoma herpesvirus (KSHV)-associated diseases include Kaposi sarcoma (KS), primary effusion lymphoma (PEL), KSHV-associated multicentric Castleman disease (MCD), and KS inflammatory cytokine syndrome (KICS). PEL, MCD, and KICS are associated with elevated circulating inflammatory cytokines. However, activation of the inflammasome, which generates interleukin-1 beta (IL-1 beta) and IL-18 via active caspase-1/4/5, has not been evaluated in patients with KSHV-associated diseases (KADs). Herein we report that patients with HIV and >= 1 KAD present with higher plasma levels of IL-18 and increased caspase-1/4/5 activity in circulating monocytes compared with HIV-negative healthy volunteers (HVs) or people with HIV (PWH) without KAD. Within KAD subtypes, KICS and MCD shared enhanced caspase-1/4/5 activity and IL-18 production compared with HVs and PWH, whereas patients with PEL showed remarkably high levels of inflammasome complex formation (known as apoptosis-associated speck-like protein containing a caspase recruitment domain). Moreover, caspase-1/4/5 activity and IL-18 plasma levels correlated with KSHV viral load, indicating KSHV-driven inflammasome activation in KAD. Accordingly, factors released by cells latently infected with KSHV triggered inflammasome activation and cytokine production in bystander monocytes in vitro. Finally, both supervised and unsupervised analyses with inflammasome measurements and other inflammatory biomarkers demonstrate a unique inflammatory profile in patients with PEL, MCD, and KICS as compared with KS. Our data indicate that detrimental inflammation in patients with KAD is at least partially driven by KSHV-induced inflammasome activation in monocytes, thus offering novel approaches to diagnose and treat these complex disorders.

Coronaviruses rely on the viral-encoded chymotrypsin-like main protease (Mpro or 3CLpro) for replication and assembly. Our previous research on Mpro of SARS-CoV-2 identified cysteine 300 (Cys300) as a potential allosteric site of Mpro inhibition. Here, we identified tixocortol (TX) as a covalent modifier of Cys300 which inhibits Mpro activity in vitro as well as in a cell-based Mpro expression assay. Most importantly TX inhibited SARS-CoV-2 replication in ACE2 expressing HeLa cells. Biochemical analysis and kinetic assays were consistent with TX acting as a non-competitive inhibitor. By contrast, TX was a weaker inhibitor and modifier of C300S Mpro, confirming a role for Cys300 in inhibition of WT Mpro but also providing evidence for an additional Cys target. TX pivalate (TP), a prodrug for TX that was previously marketed as a nasal spray, also inhibited SARS-CoV-2 replication in HeLa-ACE2 cells at low micromolar IC50s. These studies suggest that TX and/or TP could possibly be repurposed for the prevention and/or treatment of SARS-CoV-2 infection.

The spindle cells of Kaposi sarcoma (KS) lesions primarily express Kaposi sarcoma herpesvirus (KSHV) latent genes with minimal expression of lytic genes. However, recent transcriptome analyses of KS lesions have shown high expression of KSHV open reading frame (ORF) 75, which is considered a late lytic gene based on analyses in primary effusion lymphoma (PEL) lines. ORF75 encodes a pseudo-amidotransferase that is part of the viral tegument, acts as a suppressor of innate immunity, and is essential for viral lytic replication. We assessed a representative KS lesion by RNAscope and found that ORF75 RNA was expressed in the majority of latency-associated nuclear antigen (LANA)-expressing cells. Luciferase fusion reporter constructs of the ORF75 promoter were analyzed for factors potentially driving its expression in KS. The ORF75 promoter construct showed high basal transcriptional activity in vitro in endothelial cells, mediated by a proximal consensus specificity protein 1 (Sp1) (GGGGCGGGGC) element along with two distal CCAAT boxes. Sp proteins formed complexes with the proximal consensus Sp1 element to activate ORF75 promoter transcription. We also found evidence that a repressive factor or factors in B cells, but not endothelial or epithelial cells, interacted with more distal elements in the ORF75 promoter region to repress constitutive ORF75 expression in B cells. Alternate forms of Sp1 were found to accumulate during latency and showed substantial enrichment during viral lytic replication in PEL cells and infected endothelial cells, but their functional significance is unclear. We also found that ORF75 can in turn upregulate its own expression and that of other KSHV genes. Thus, while ORF75 acts primarily as a lytic gene in PEL cell lines, Sp proteins induce substantial constitutive ORF75 transcription in infected endothelial cells and this can account for its high expression in KS lesions.

In this editorial, we highlight the potential use of inhibitors of hypoxia-inducible factors (HIFs) for the use in Kaposi's sarcoma associated herpesvirus (KSHV) (also known as human herpesvirus-8) related malignancies. The past 20 years has accumulated detailed knowledge of the role of these factors in ensuring the maintenance of the KSHV in infected cells, in aiding the growth of the virus infected cells and aiding in the spread of virus from infected cells by inducing lytic reactivation. Today, a wide range of inhibitors for HIFs are currently being clinically evaluated for use in treating a variety of cancers. We discuss the current state of this research area as it relates to KSHV malignancies and describe pre-clinical and clinical evidence of drugs that target HIF to back up the idea that these inhibitors could be a novel way to treat KSHV related diseases.

In 1981, a cluster of cases of Kaposi sarcoma (KS), a hitherto rare skin tumor, in previously healthy men who have sex with men in the US, often along with Pneumocystis pneumonia, heralded the onset of the HIV/AIDS pandemic. The frequent occurrence of KS in people with HIV (PWH) remained a puzzle until 1994 when Yuan Chang, Patrick Moore and colleagues showed that this tumor was caused by a novel gammaherpesvirus, Kaposi sarcoma herpesvirus (KSHV), and epidemiologic studies documented the simultaneous spread of KSHV and HIV among men who had sex with men during the early 1980s. Over the next several years, it became apparent that PWH had a substantially increased risk of certain other tumors, including aggressive B cell lymphomas. We now know that most of the tumors associated with HIV are caused by oncogenic viruses, including Epstein Barr virus (EBV), KSHV, and human papillomavirus (HPV). The subsequent development of anti-retroviral drugs, initially spearheaded by NCI scientists and collaborators, and the widespread use of these drugs converted AIDS from an almost universally lethal disease to a manageable condition. It also resulted in a substantial decline in the incidence of those tumors (such as primary central nervous system lymphoma) associated with very low CD4 counts. However, as PWH lived longer and the population of PWH increased, there was a substantial increase in the number of other tumors (such as anal carcinoma) that occur at higher CD4 counts. Also, we continue to see new cases of KS and other tumors associated with severe immunodeficiency. KS is increasingly a disease of minority communities in the US and its incidence is rising in young Black men in the rural South. Moreover, HIV-associated tumors continue to be a major public health issue in sub-Saharan Africa and other low- and middle-income countries (LMIC). In particular, KS continues to be the most common tumor among men 65 or younger in several countries in sub-Saharan Africa. Before the development and use of combination anti-retroviral therapy (cART), PWH who developed cancer often could not tolerate full doses of chemotherapy and treatment regimens employing lower doses of chemotherapy drugs were used, usually with relatively low rates of cure. This has changed, and in general cancers in PWH are best treated with standard-dose regimens that would be used in the general population. For some cancers, such as Hodgkin lymphoma and non-small cell lung cancer, data have shown that when PWH are treated with standard chemotherapy regimens, their outcomes are the same as the general population. In the past several years, there has been a revolution in the development and use of immunologic therapy for various cancers, such as anti-PD-(L)1 monoclonal antibodies. However, there has been a reluctance to enroll PWH on clinical trials using immunotherapy approaches because of concern that the agents may not be tolerated or may not be effective because of the CD4 T cell lymphopenia. Two recent trials, one by the Cancer Immunotherapy Trials Network with the NCI Intramural Program (TS Uldrick et al., JAMA Oncology, 2019) and one by the AIDS Malignancy Consortium (AMC) (TA Rasmussen et al., Clin. Infect. Dis., 2021) showed that these agents could be administered safely to PWH and can have activity. Anti-PD-(L)1 agents are generally most effective in tumors with high mutational burden resulting in neoantigens. Virus-induced tumors tend to have low mutational burdens. However, the oncogenic viruses themselves can provide novel antigens for attack, and these tumors are thus potentially attractive targets for such therapies. A problem, however, is that oncogenic viruses have evolved various mechanisms to suppress expression of surface immune markers (such as MHC-1, B7-2/CD86, or ICAM-1), making the tumor cells invisible to the immune system. Our group recently conducted a clinical trial showing that pomalidomide, a cereblon-binding immunomodulatory drug, was active against KS (MN Polizzotto et al., J. Clin, Oncol. 2016; R. Ramaswami et al., Clin Cancer Res., 2022). Based on this trial, pomalidomide was recently approved by the US Food and Drug Administration for KS. Also, the AMC is studying the feasibility of using pomalidomide against KS in sub-Saharan Africa and other LMIC. In investigating possible mechanisms of its activity, David Davis, Prabha Shrestha et al. in our group found that pomalidomide could reverse or prevent the KSHV-mediated downregulation of surface immune marker expression, at least in lymphoid cells (DA Davis et al, Oncoimmunology, 2018; P Shrestha et al., PLoS Path, 2021). Surprisingly, this upregulation was not found in KSHV-infected endothelial cells (a model for KS), and it isn’t clear if this accounts for the activity of pomalidomide in KS. At the same time, it suggested that this approach might be worth considering in other virus-induced tumors and moreover, that pomalidomide and related drugs might be worth combining with checkpoint inhibition (for example with anti-PD-(L)1 antibodies) in these tumors. We are now testing this in an ongoing study in the NCI HIV and AIDS Malignancy Branch (NCT04902443). In a limited retrospective study, Kathryn Lurain et al. in our group (K Lurain et al., J. Immunotherapy Cancer, 2021) have shown that pembrolizumab with pomalidomide can be effective in HIV-associated lymphomas caused by EBV. Also, we have been exploring the use of other agents that can enhance expression of surface immune markers in virus-infected cells. Yi-Quan Wu et al in our group recently showed that the CDK4/6 inhibitor abemaciclib could upregulate surface immune markers in KSHV-infected endothelial cells as well as in lymphoid cells (Y Wu et al., J. Translational Med., 2022) and we are now studying this in a clinical trial for KS (NCT04941274). In addition to KS, KSHV is the cause of several other severe diseases that occur predominantly in PWH, including a form of multicentric Castleman disease (MCD), primary effusion lymphoma (PEL), and KSHV-inflammatory cytokine syndrome (KICS). These diseases often occur together in the same patient. KSHV-MCD and KICS are characterized by severe inflammatory symptoms associated with increased levels of various cytokines, especially human interleukin-6 (IL-6), interleukin-10 (IL-10), and a KSHV-encoded form of IL-6 (vIL-6). Recent studies by our group and others have suggested that a key pathway in these diseases is the induction of human cytokines by vIL-6 and other factors produced by KSHV-infected cells. We are currently exploring mechanisms to specifically target these pathways. Thus, while we have made substantial progress in the prevention and therapy of HIV-associated malignancies, they continue to pose a major health problem. At the same time, our increasing understanding of the pathogenesis of these tumors is leading to novel approaches to their treatment. This work was supported by the Intramural Research program of the NIH, National Cancer Institute. Citation Format: Robert Yarchoan, Kathryn Lurain, Ramya Ramaswami, David A. Davis, Prabha Shrestha. Malignancies in People with HIV: Successes and Challenges at the Intersection of Virology, Immunology, and Oncology. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr PL02-04.

ABSTRACT:Efforts to optimize continuing professional development (CPD) are ongoing and include advocacy for the use of clinician performance data. Several educational and quality-based frameworks support the use of performance data to achieve intended improvement outcomes. Although intuitively appealing, the role of performance data for CPD has been uncertain and its utility mainly assumed. In this Scholarly Perspective, the authors briefly review and trace arguments that have led to the conclusion that performance data are essential for CPD. In addition, they summarize and synthesize a recent and ongoing research program exploring the relationship physicians have with performance data. They draw on Collins, Onwuegbuzie, and Johnson's legitimacy model and Dixon-Woods' integrative approach to generate inferences and ways of moving forward. This interpretive approach encourages questioning or raising of assumptions about related concepts and draws on the perspectives (i.e., interpretive work) of the research team to identify the most salient points to guide future work. The authors identify 6 stimuli for future programs of research intended to support broader and better integration of performance data for CPD. Their aims are to contribute to the discourse on data advocacy for CPD by linking conceptual, methodologic, and analytic processes and to stimulate discussion on how to proceed on the issue of performance data for CPD purposes. They hope to move the field from a discussion on the utility of data for CPD to deeper integration of relevant conceptual frameworks.

Nuclear localization signal (NLS) of HIV-1 integrase (IN) is implicated in nuclear import of HIV-1 preintegration complex (PIC). Here, we established a multiclass drug-resistant HIV-1 variant (HIV KGD ) by consecutively exposing an HIV-1 variant to various antiretroviral agents including IN strand transfer inhibitors (INSTIs). HIV KGD was extremely susceptible to a previously reported HIV-1 protease inhibitor, GRL-142, with IC 50 of 130 femtomolar. When cells were exposed to HIV KGD IN–containing recombinant HIV in the presence of GRL-142, significant decrease of unintegrated 2-LTR circular cDNA was observed, suggesting that nuclear import of PIC was severely compromised by GRL-142. X-ray crystallographic analyses revealed that GRL-142 interacts with NLS’s putative sequence (DQAEHLK) and sterically blocks the nuclear transport of GRL-142–bound HIV KGD ’s PIC. Highly INSTI-resistant HIV-1 variants isolated from heavily INSTI-experienced patients proved to be susceptible to GRL-142, suggesting that NLS-targeting agents would serve as salvage therapy agents for highly INSTI-resistant variant–harboring individuals. The data should offer a new modality to block HIV-1 infectivity and replication and shed light on developing NLS inhibitors for AIDS therapy.

Primary effusion lymphoma (PEL), an aggressive non-Hodgkin lymphoma caused by Kaposi sarcoma-associated herpesvirus (KSHV), lacks standard therapy and has a median survival of 10-22 months with combination chemotherapy. PEL is a tumor of plasmablast-like B cells generally expressing CD38, the target of daratumumab (Dara). Initially, we assessed PEL cells from eight patients and established that each expressed high levels of CD38 by flow cytometry. PEL cell lines were also evaluated and most had high CD38 expression. We then assessed Dara's effects on complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) of PEL cell lines as well as its clinical benefits on two patients with PEL. Despite high CD38 expression, Dara did not induce CDC of PEL cell lines, due in part to high levels of the complement-inhibitory proteins, CD55 and CD59. However, Dara induced significant and dose-dependent increases in ADCC, particularly in those lines with high CD38 levels. Two FDA-approved drugs, all trans-retinoic acid (ATRA) and pomalidomide (Pom), significantly increased surface CD38 levels in low-CD38 expressing PEL cell lines, resulting in increased Dara-induced ADCC. Two patients with refractory PEL were treated with Dara alone or in combination with Pom. One patient with leptomeningeal PEL had a complete response to Dara and Pom combination treatment. Others had improvement in performance status and resolution of malignant ascites with Dara alone. Together, these data support the use of Dara monotherapy or in combination with ATRA or Pom as a potential therapeutic option for PEL.

Epstein-Barr virus (EBV) downregulates immune surface markers to avoid immune recognition. Pomalidomide (Pom) was previously shown to increase immune surface marker expression in EBV-infected tumor cells. We explored the mechanism by which Pom leads to these effects in EBV-infected cells. Pom increased B7-2/CD86 mRNA, protein, and surface expression in EBV-infected cells but this was virtually eliminated in EBV-infected cells made resistant to Pom-induced cytostatic effects. This indicates that Pom initiates the upregulation of these markers by interacting with its target, cereblon. Interestingly, Pom increased the proinflammatory cytokines IP-10 and MIP-1∝/β in EBV infected cells, supporting a possible role for the phosphoinositide 3-kinase (PI3K)/AKT pathway in Pom’s effects. Idelalisib, an inhibitor of the delta subunit of PI3 Kinase, blocked AKT-Ser phosphorylation and Pom-induced B7-2 surface expression. PU.1 is a downstream target for AKT that is expressed in EBV-infected cells. Pom treatment led to an increase in PU.1 binding to the B7-2 promoter based on ChIP analysis. Thus, our data indicates Pom acts through cereblon leading to degradation of Ikaros and activation of the PI3K/AKT/PU.1 pathway resulting in upregulation of B7-2 mRNA and protein expression. The increased immune recognition in addition to the increases in proinflammatory cytokines upon Pom treatment suggests Pom may be useful in the treatment of EBV-positive lymphomas.

Kaposi sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi sarcoma and several other tumors and hyperproliferative diseases seen predominantly in human immunodeficiency virus-infected and other immunocompromised persons. There is an increasing body of evidence showing that hypoxia and hypoxia-inducible factors (HIFs) play important roles in the biology of KSHV and in the pathogenesis of KSHV-induced diseases. Hypoxia and HIFs can induce lytic activation of KSHV and KSHV can in turn lead to a hypoxic-like state in infected cells. In this review, we describe the complex interactions between KSHV biology, the cellular responses to hypoxia, and the pathogenesis of KSHV-induced diseases. We also describe how interference with HIFs can lead to decreased tumor growth and/or death of infected cells and KSHV-induced tumors. Finally, we show how these observations may lead to novel strategies for the treatment of KSHV-induced diseases.

Most viruses encode their own proteases to carry out viral maturation and these often require dimerization for activity. Studies on human immunodeficiency virus type 1 (HIV-1), type 2 (HIV-2) and human T-cell leukemia virus (HTLV-1) proteases have shown that the activity of these proteases can be reversibly regulated by cysteine (Cys) glutathionylation and/or methionine oxidation (for HIV-2). These modifications lead to inhibition of protease dimerization and therefore loss of activity. These changes are reversible with the cellular enzymes, glutaredoxin or methionine sulfoxide reductase. Perhaps more importantly, as a result, the maturation of retroviral particles can also be regulated through reversible oxidation and this has been demonstrated for HIV-1, HIV-2, Mason-Pfizer monkey virus (M-PMV) and murine leukemia virus (MLV). More recently, our group has learned that SARS-CoV-2 main protease (Mpro) dimerization and activity can also be regulated through reversible glutathionylation of Cys300. Overall, these studies reveal a conserved way for viruses to regulate viral polyprotein processing particularly during oxidative stress and reveal novel targets for the development of inhibitors of dimerization and activity of these important viral enzyme targets.

Human T cell leukemia virus type 1 (HTLV-1) persists in the host despite a vigorous immune response that includes cytotoxic T cells (CTL) and natural killer (NK) cells, suggesting the virus has developed effective mechanisms to counteract host immune surveillance. We recently showed that in vitro treatment of HTLV-1-infected cells with the drug pomalidomide (Pom) increases surface expression of MHC-I, ICAM-1, and B7-2, and significantly increases the susceptibility of HTLV-1-infected cells to NK and CTL killing, which is dependent on viral orf-I expression. We reasoned that by restoring cell surface expression of these molecules, Pom treatment has the potential to reduce virus burden by rendering infected cells susceptible to NK and CTL killing. We used the rhesus macaque model to determine if Pom treatment of infected individuals activates the host immune system and allows recognition and clearance of HTLV-1-infected cells. We administered Pom (0.2 mg/kg) orally to four HTLV-1-infected macaques over a 24 day period and collected blood, urine, and bone marrow samples throughout the study. Pom treatment caused immune activation in all four animals and a marked increase in proliferating CD4(+), CD8(+), and NK cells as measured by Ki-67(+) cells. Activation markers HLA-DR, CD11b, and CD69 also increased during treatment. While we detected an increased frequency of cells with a memory CD8(+) phenotype, we also found an increased frequency of cells with a Treg-like phenotype. Concomitant with immune activation, the frequency of detection of viral DNA and the HTLV-1-specific humoral response increased as well. In 3 of 4 animals, Pom treatment resulted in increased antibodies to HTLV-1 antigens as measured by western blot and p24Gag ELISA. Consistent with Pom inducing immune and HTLV-1 activation, we measured elevated leukotrienes LTB4 and LTE4 in the urine of all animals. Despite an increase in plasma LTB4, no significant changes in plasma cytokine/chemokine levels were detected. In all cases, however, cellular populations, LTB4, and LTE4 decreased to baseline or lower levels 2 weeks after cessation of treatment. These results indicated that Pom treatment induces a transient HTLV-1-specific immune activation in infected individuals, but also suggest Pom may not be effective as a single-agent therapeutic.

This chapter interprets original research and systematic reviews across disciplines including education, knowledge translation, implementation science, training, and organizational psychology. It presents a conceptual framework for measuring outcomes. The chapter organizes educational interventions, stressing theories and linkages to evidence to assure the effectiveness of continuing education across a spectrum of desirable effects. A more recent systematic review of interventions tailored to support transfer of training in health care showed that only six out of 26 studies reported use of theory to guide the instructional design of the tailored intervention. Cost perspectives also differ for a sponsoring institution concerned with changing physician performance or to a health care payer sensitive to the utilization of health care resources and patient outcomes. Cost-inclusive evaluation can take one or more of these perspectives. Leaders in education and health care are challenged to improve the quality of care, decrease the risk of adverse events, and reduce expenses.

Health care delivery has evolved from a variably connected collective of individually owned proprietorships and independent hospitals to an environment in which physicians increasingly contract with or are employed by health care enterprises. While continuing medical education (CME) that is focused on the dissemination and maintenance of medical knowledge and the development of skills plays a critical role in helping physicians keep up to date, the authors of this manuscript believe the structure and delivery of CME have not suffi ciently evolved to be broadly viewed by health enterprise leaders as a strategic or integral asset to improving health care delivery. Therefore, an evolution and a reconceptualization of the structure and function of CME are necessary to enable collaboration between leaders and improvement experts in health care enterprises and CME. In this paper, the authors describe models that better refl ect a more eff ective role of CME within learning health care delivery enterprises and the implications of such models for these enterprises and the CME profession.

Background & Aims: While certain nucleos(t)ide reverse transcriptase inhibitors (NRTIs) are efficacious in treating HBV infection, their effects are yet to be optimized and the emergence of NRTI-resistant HBV variants is an issue because of the requirement for lifelong treatment. The development of agents that more profoundly suppress wild-type and drug-resistant HBVs, and that have a long-acting effect, are crucial to improve patient outcomes. Methods: Herein, we synthesized a novel long-acting 4'-modified NRTI termed E-CFCP. We tested its anti-HBV activity in vitro, before evaluating its anti-HBV activity in HBV-infected humanliver-chimeric mice (PXB-mice). E-CFCP's long-acting features and E-CFCP-triphosphate's interactions with the HBV reverse transcriptase (HBV-RT) were examined. Results: E-CFCP potently blocked HBVWTD1 production (IC50qPCR_cell =1.8 nM) in HepG2.2.15 cells and HBVWTC2 (IC50SB_cell=0.7 nM), entecavir (ETV)-resistant HBVETV- RL180M/S202G/M204V (IC50SB_cell=77.5 nM), and adefovir-resistant HBVADV- RA181T/N236T production (IC50SB_cell=14.1 nM) in Huh7 cells. E-CFCP profoundly inhibited intracellular HBV DNA production to below the detection limit, but ETV and tenofovir alafenamide (TAF) failed to do so. E-CFCP also showed less toxicity than ETV and TAF. E-CFCP better penetrated hepatocytes and was better triphosphorylated; E-CFCP-triphosphate persisted intracellularly for longer than ETV-triphosphate. Once-daily peroral E-CFCP administration over 2 weeks (0.02 similar to 0.2 mg/kg/day) reduced HBVWTC2-viremia by 2-3 logs in PXB-mice without significant toxicities and the reduction persisted over 1-3 weeks following treatment cessation, suggesting once-weekly dosing capabilities. E-CFCP also reduced HBVETV- RL180M/S202G/M204V-viremia by 2 logs over 2 weeks, while ETV completely failed to reduce HBVETV- RL180M/S202G/M204V-viremia. E-CFCP's 4'-cyano and fluorine interact with both HBVWT-RT and HBVETV- RL180M/S202G-M204 -RT via Van der Waals and polar forces, being important for ECFCP-triphosphate's interactions and anti-HBV potency. Conclusion: E-CFCP represents the first reported potential longacting NRTI with potent activity against wild-type and treatment-resistant HBV. Lay summary: Although there are currently effective treatment options for HBV, treatment-resistant variants and the need for lifelong therapy pose a significant challenge. Therefore, the development of new treatment options is crucial to improve outcomes and quality of life. Herein, we report preclinical evidence showing that the anti-HBV agent, E-CFCP, has potent activity against wild-type and treatment-resistant variants. In addition, once-weekly oral dosing may be possible, which is preferrable to the current daily dosing regimens. Published by Elsevier B.V. on behalf of European Association for the Study of the Liver. This is an open access article under the CC BY-NCND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Pomalidomide (Pom) is an immunomodulatory drug that has efficacy against Kaposi’s sarcoma, a tumor caused by Kaposi’s sarcoma-associated herpesvirus (KSHV). Pom also induces direct cytotoxicity in primary effusion lymphoma (PEL), a B-cell malignancy caused by KSHV, in part through downregulation of IRF4, cMyc, and CK1α as a result of its interaction with cereblon, a cellular E3 ubiquitin ligase. Additionally, Pom can reverse KSHV-induced downregulation of MHCI and co-stimulatory immune surface molecules ICAM-1 and B7-2 on PELs. Here, we show for the first time that Pom-induced increases in ICAM-1 and B7-2 on PEL cells lead to an increase in both T-cell activation and NK-mediated cytotoxicity against PEL. The increase in T-cell activation can be prevented by blocking ICAM-1 and/or B7-2 on the PEL cell surface, suggesting that both ICAM-1 and B7-2 are important for T-cell co-stimulation by PELs. To gain mechanistic insights into Pom’s effects on surface markers, we generated Pom-resistant (PomR) PEL cells, which showed about 90% reduction in cereblon protein level and only minimal changes in IRF4 and cMyc upon Pom treatment. Pom no longer upregulated ICAM-1 and B7-2 on the surface of PomR cells, nor did it increase T-cell and NK-cell activation. Cereblon-knockout cells behaved similarly to the pomR cells upon Pom-treatment, suggesting that Pom’s interaction with cereblon is necessary for these effects. Further mechanistic studies revealed PI3K signaling pathway as being important for Pom-induced increases in these molecules. These observations provide a rationale for the study of Pom as therapy in treating PEL and other KSHV-associated tumors.