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Viral and/or host factors that are directly responsible for the acute versus chronic outcome of hepatitis B virus (HBV) infection have not been identified yet. Information on immune response during the early stages of HBV infection in humans is mainly derived from blood samples of patients with acute hepatitis B (AHB), which are usually obtained after the onset of clinical symptoms. Features of intrahepatic immune response in these patients are less studied due to the difficulty of obtaining multiple liver biopsies. Woodchuck hepatitis virus (WHV) infection in woodchucks is a model for HBV infection in humans. In the present study, five adult woodchucks were experimentally infected with WHV and then followed for 18 weeks. Blood and liver tissues were frequently collected for assaying markers of WHV replication and innate and adaptive immune responses. Liver tissues were further analyzed for pathological changes and stained for important immune cell subsets and cytokines. The increase and subsequent decline of viral replication markers in serum and liver, the elicitation of antibodies against viral proteins, and the induction of virus-specific T-cell responses indicated eventual resolution of acute WHV infection in all animals. Intrahepatic innate immune makers stayed unchanged immediately after the infection, but increased markedly during resolution, as determined by changes in transcript levels. The presence of interferon-gamma and expression of natural killer (NK) cell markers suggested that a non-cytolytic response mechanism is involved in the initial viral control in liver. This was followed by the expression of T-cell markers and cytolytic effector molecules, indicating the induction of a cytolytic response mechanism. Parallel increases in regulatory T-cell markers suggested that this cell subset participates in the overall immune cell infiltration in liver and/or has a role in regulating AHB induced by the cytolytic response mechanism. Since the transcript levels of immune cell markers in blood, when detectable, were lower than in liver, and the kinetics, except for NK-cells and interferon-gamma, did not correlate well with their intrahepatic expression, this further indicated enrichment of immune cells within liver. Conclusion: The coordinated interplay of innate and adaptive immunity mediates viral clearance in the woodchuck animal model of HBV infection. The initial presence of NK-cell associated interferon-gamma response points to an important role of this cytokine in HBV resolution.

Amyloidosis is a protein deposition disorder caused by pathologic accumulation of fibrils, leading to organ dysfunction. The newest protein is Leukocyte Cell-derived Chemotaxin 2 Amyloidosis (ALECT2), which shows ethnic predilection for Hispanics, Middle Eastern, and other minority groups. We report a case of 71-year-old Persian male with history of hepatitis B and resected hepatocellular carcinoma who presented with acute onset jaundice and abnormal liver function tests. Liver biopsy performed for diagnostic workup revealed hepatic ALECT2 with distinct signet-ring like globular proteinaceous deposits infiltrating hepatic parenchyma, mimicking epithelioid/histiocytic neoplasm. The infiltrative spherular material was positive for Congo red and negative for pan-keratin stains. Amyloid protein analysis by Liquid Chromatography tandem Mass Spectrometry (LC MS/MS) identified a peptide profile indicative of ALECT2. Although, ALECT2 accounts for the second most common cause of hepatic amyloidosis, only limited cases of hepatic ALECT2 are reported in the literature and little is known about patient management, especially the role of transplant as curative option. In summary, hepatic ALECT2 is an emerging disorder with relative high prevalence that deserves morphologic recognition. The report intends to emphasise distinctive morphology for accurate diagnosis and understanding its pathogenesis, clinical significance, and therapeutic strategies.

Abstract Genomic instability is associated with late stage carcinomas and the epithelial mesenchymal transition (EMT). Of note is chromosome 8q24 amplification that has been documented in many epithelial-derived carcinomas. On this amplified region is the potent oncogene, c-myc. Not only does MYC overexpression activate targets that promote cell proliferation, it also activates transcription factors that drive EMT, including ZEB1. Further reinforcing EMT, overexpressed MYC also represses tumor suppressors involved in promoting the epithelial phenotype, including MIZ1. We predict that as carcinomas progress, chromosome 8q24 is amplified leading to high MYC levels that leads to ZEB1 expression and MIZ1 repression driving cells through EMT. To interrogate this clinically, limited cohorts of human epithelial-derived carcinomas were examined for MYC/ZEB1/MIZ1 expression patterns across increasing carcinoma grades. Interestingly, the predicted temporal patterns were only observed in hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinomas. Yet MIZ1 proved to be an excellent marker to assess carcinoma progression across types. We expanded the HCC cohort and determined that c-myc amplification was restricted to grade III/IV HCC that also exhibited increased MYC and ZEB1 nuclear expression whereas cytosolic MIZ1 expression was lost and only nuclear expression retained. These same resections were obtained from only individuals who had histories of alcohol consumption that were also diagnosed with cirrhosis, metastasis and had viral hepatitis suggesting etiology-specific mechanisms of cancer progression. Finally, analysis performed in Hep3B cells determined that alterations in MYC expression promoted the predicted changes in ZEB1 and MIZ1 expression and/or distributions and in markers for EMT further suggesting a relationship among these three transcription factors in HCC and their correlation to driving EMT.

SB 9200, an orally bioavailable dinucleotide, activates the viral sensor proteins, retinoic acid-inducible gene 1 (RIG-I) and nucleotide-binding oligomerization domain-containing protein 2 (NOD2) causing the induction of the interferon (IFN) signaling cascade for antiviral defense. The present study evaluated the overall antiviral response in woodchucks upon induction of immune response, first with SB 9200 followed by Entecavir (ETV) versus reduction of viral burden with ETV followed by SB 9200 immunomodulation. Woodchucks chronically infected with woodchuck hepatitis virus (WHV) were treated orally with SB 9200 (30 mg/kg/day) and ETV (0.5 mg/kg/day). Group 1 received ETV for 4 weeks followed by SB 9200 for 12 weeks. Group 2 received SB 9200 for 12 weeks followed by ETV for 4 weeks. At the end of treatment in Group 2, average reductions of 6.4 log10 in serum WHV DNA and 3.3 log10 in WHV surface antigen were observed whereas in Group 1, average reductions of 4.2 log10 and 1.1 log10 in viremia and antigenemia were noted. Both groups demonstrated marked reductions in hepatic WHV nucleic acid levels which were more pronounced in Group 2. Following treatment cessation and the 8-week follow-up, recrudescence of viral replication was observed in Group 1 while viral relapse in Group 2 was significantly delayed. The antiviral effects observed in both groups were associated with temporally different induction of IFN-α, IFN-β, and IFN-stimulated genes in blood and liver. These results suggest that the induction of host immune responses by pretreatment with SB 9200 followed by ETV resulted in antiviral efficacy that was superior to that obtained using the strategy of viral reduction with ETV followed by immunomodulation.

SB 9200, an oral prodrug of the dinucleotide SB 9000, is being developed for the treatment of chronic hepatitis B virus (HBV) infection and represents a novel class of antivirals. SB 9200 is thought to activate the viral sensor proteins, retinoic acid-inducible gene 1 (RIG-I) and nucleotide-binding oligomerization domain-containing protein 2 (NOD2) resulting in interferon (IFN) mediated antiviral immune responses in virus-infected cells. Additionally, the binding of SB 9200 to these sensor proteins could also sterically block the ability of the viral polymerase to access pre-genomic RNA for nucleic acid synthesis. The immune stimulating and direct antiviral properties of SB 9200 were evaluated in woodchucks chronically infected with woodchuck hepatitis virus (WHV) by daily, oral dosing at 15 and 30 mg/kg for 12 weeks. Prolonged treatment resulted in 2.2 and 3.7 log10 reductions in serum WHV DNA and in 0.5 and 1.6 log10 declines in serum WHV surface antigen from pretreatment level with the lower or higher dose of SB 9200, respectively. SB 9200 treatment also resulted in lower hepatic levels of WHV nucleic acids and antigen and reduced liver inflammation. Following treatment cessation, recrudescence of viral replication was observed but with dose-dependent delays in viral relapse. The antiviral effects were associated with dose-dependent and long-lasting induction of IFN-α, IFN-β and IFN-stimulated genes in blood and liver, which correlated with the prolonged activation of the RIG-I/NOD2 pathway and hepatic presence of elevated RIG-I protein levels. These results suggest that in addition to a direct antiviral activity, SB 9200 induces antiviral immunity during chronic hepadnaviral infection via activation of the viral sensor pathway.

Current treatment options for patients infected with hepatitis B virus (HBV) are suboptimal, because the approved drugs rarely induce cure due to the persistence of the viral DNA genome in the nucleus of infected hepatocytes, and are associated with either severe side effects (pegylated interferon-alpha) or require life-long administration (nucleos(t)ide analogs). We report here the evaluation of the safety and therapeutic efficacy of a novel, humanized antibody (hzVSF) in the woodchuck model of HBV infection. hzVSF has been shown to act as a viral entry inhibitor, most likely by suppressing vimentin-mediated endocytosis of virions. Targeting the increased vimentin expression on liver cells by hzVSF after infection with HBV or woodchuck hepatitis virus (WHV) was demonstrated initially. Thereafter, hzVSF safety was assessed in eight woodchucks naïve for WHV infection. Antiviral efficacy of hzVSF was evaluated subsequently in 24 chronic WHV carrier woodchucks by monotreatment with three ascending doses and in combination with tenofovir alafenamide fumarate (TAF). Consistent with the proposed blocking of WHV reinfection, intravenous hzVSF administration for 12 weeks resulted in a modest but transient reduction of viral replication and associated liver inflammation. In combination with oral TAF dosing, the antiviral effect of hzVSF was enhanced and sustained in half of the woodchucks with an antibody response to viral proteins. Thus, hzVSF safely but modestly alters chronic WHV infection in woodchucks; however, as a combination partner to TAF, its antiviral efficacy is markedly increased. The results of this preclinical study support future evaluation of this novel anti-HBV drug in patients.

As current interventions for chronic hepatitis B (CHB) rarely induce cure, more effective drugs are needed. Short-term treatment of woodchucks with the novel immunomodulator AIC649, a parapoxvirus-based stimulator of toll-like receptor 9 dependent and independent pathways, has been shown to reduce viral DNA and surface antigen via a unique, biphasic response pattern. The present study evaluated long-term AIC649 treatment in combination with Entecavir for potency and safety in woodchucks. AIC649 monotreatment induced modest reductions in serum viral DNA and surface and e antigens that were associated with the same biphasic response pattern previously observed. Entecavir monotreatment reduced transiently viremia but not antigenemia, while AIC649/Entecavir combination treatment mediated superior viral control. Undetectability of viral antigens and elicitation of antibodies in AIC649/Entecavir-treated woodchucks correlated with the expression of interferons and suppression of viral replication in liver. Combination treatment was well tolerated, and liver enzyme elevations were minor and transient. It was concluded that the AIC649-mediated effects were most likely based on an improvement and/or reconstitution of antiviral immune responses that are typically deficient in CHB. As a combination partner to Entecavir, the antiviral efficacy of AIC649 was markedly enhanced. This preclinical study supports future evaluation of AIC649 for treatment of human CHB.

Lymphadenopathy in chronic myeloid leukemia (CML) is usually due to extramedullary involvement with accelerated or blast phases of the disease. The occurrence of non-Hodgkin lymphoma (NHL) as a synchronous malignancy with CML is rare. We report a case of a 73-year-old male who presented with dyspnea and right-sided lower extremity edema in the setting of leukocytosis. Bone marrow evaluation indicated a chronic phase chronic myeloid leukemia (CML), confirmed by molecular testing. Imaging of the chest for persistent dyspnea revealed supraclavicular and mediastinal lymphadenopathy. Biopsy of the cervical node showed expanded lymphoid follicles with atypical germinal centers that were positive for CD10, BCL-2, and BCL-6, consistent with follicular lymphoma (FL). Nodal PCR demonstrated clonal IGH and IGK gene rearrangements, and FISH analysis was positive for IGH-BCL-2 fusion. Together, these tests supported the diagnosis of FL. Additionally, the lymph node showed paracortical expansion by maturing pan-hematopoietic elements, no blastic groups, and positive RT-PCR analysis for BCR-ABL1, indicating concomitant involvement by chronic phase-CML. To our knowledge, this is the first reported case of a patient with a concurrent diagnosis of CML and FL.

RG7834 is a small‐molecule inhibitor of hepatitis B virus (HBV) gene expression that significantly reduces the levels of hepatitis B surface antigen (HBsAg) and HBV DNA in a humanized liver HBV mouse model. In the current study, we evaluated the potency of RG7834 in the woodchuck model of chronic HBV infection, alone and in combination with entecavir (ETV) and/or woodchuck interferon‐α (wIFN‐α). RG7834 reduced woodchuck hepatitis virus (WHV) surface antigen (WHsAg) by a mean of 2.57 log10 from baseline and WHV DNA by a mean of 1.71 log10. ETV + wIFN‐α reduced WHsAg and WHV DNA by means of 2.40 log10 and 6.70 log10, respectively. The combination of RG7834, ETV, and wIFN‐α profoundly reduced WHsAg and WHV DNA levels by 5.00 log10 and 7.46 log10, respectively. However, both viral parameters rebounded to baseline after treatment was stopped and no antibody response against WHsAg was observed. Effects on viral RNAs were mainly seen with the triple combination treatment, reducing both pregenomic RNA (pgRNA) and WHsAg RNA, whereas RG7834 mainly reduced WHsAg RNA and ETV mainly affected pgRNA. When WHsAg was reduced by the triple combination, peripheral blood mononuclear cells (PBMCs) proliferated significantly in response to viral antigens, but the cellular response was diminished after WHsAg returned to baseline levels during the off‐treatment period. Consistent with this, Pearson correlation revealed a strong negative correlation between WHsAg levels and PBMC proliferation in response to peptides covering the entire WHsAg and WHV nucleocapsid antigen. Conclusion: A fast and robust reduction of WHsAg by combination therapy reduced WHV‐specific immune dysfunction in the periphery. However, the magnitude and/or duration of the induced cellular response were not sufficient to achieve a sustained antiviral response.

Current therapeutics for chronic infection with hepatitis B virus (HBV) rarely induce functional cure due to the immunotolerant status of patients. Small molecule agonists targeting toll‐like receptor 7 (TLR7) have been shown to elicit a functional cure in animal models of HBV but sometimes with poor tolerability due to immune‐related toxicities. In an effort to increase the therapeutic window of TLR7 agonists to treat chronic hepatitis B (CHB), we developed an oral TLR7 agonist, APR002, designed to act locally in the gastrointestinal tract and liver, thus minimizing systemic exposure and improving tolerability. Here, we describe the pharmacokinetic/pharmacodynamic (PK/PD) profile of APR002 in mice and uninfected woodchucks as well as the safety and antiviral efficacy in combination with entecavir (ETV) in woodchucks with CHB. Treatment of woodchucks chronically infected with woodchuck hepatitis virus (WHV) with weekly oral doses of APR002 was well‐tolerated. While APR002 and ETV single agents did not elicit sustained viral control, combination therapy resulted in durable immune‐mediated suppression of the chronic infection. These woodchucks also had detectable antibodies to viral antigens, enhanced interferon‐stimulated gene expression, and loss of WHV covalently closed circular DNA. Conclusion: APR002 is a novel TLR7 agonist exhibiting a distinct PK/PD profile that in combination with ETV can safely attain a functional cure in woodchucks with chronic WHV infection. Our results support further investigation of liver‐targeted TLR7 agonists in human CHB.

Sulfation of heparan sulfate proteoglycans (HSPG) regulates signaling of growth factor receptors via specific interactions with the sulfate groups. 6-O-Sulfation of HSPG is an impactful modification regulated by the activities of dedicated extracellular endosulfatases. Specifically, extracellular sulfatase Sulf-2 (SULF2) removes 6-O-sulfate from HS chains, modulates affinity of carrier HSPG to their ligands, and thereby influences activity of the downstream signaling pathway. In this study, we explored the effect of SULF2 expression on HSPG sulfation and its relationship to clinical outcomes of patients with head and neck squamous cell carcinoma (HNSCC). We found a significant overexpression of SULF2 in HNSCC tumor tissues which differs by tumor location and etiology. Expression of SULF2 mRNA in tumors associated with human papillomavirus (HPV) infection was two-fold lower than in tumors associated with a history of tobacco and alcohol consumption. High SULF2 mRNA expression is significantly correlated with poor progression-free interval and overall survival of patients (n = 499). Among all HS-related enzymes, SULF2 expression had the highest hazard ratio in overall survival after adjusting for clinical characteristics. SULF2 protein expression (n = 124), determined by immunohistochemical analysis, showed a similar trend. The content of 6-O-sulfated HSPG, measured by staining with the HS3A8 antibody, was higher in adjacent mucosa compared to tumor tissue but revealed no difference based on SULF2 staining. LC-MS/MS analysis showed low abundance of N-sulfation and O-sulfation in HS but no significant difference between SULF2-positive and SULF2-negative tumors. Levels of enzymes modifying 6-O-sulfation, measured by RT-qPCR in HNSCC tumor tissues, suggest that HSPG sulfation is carried out by the co-regulated activities of multiple genes. Imbalance of the HS modifying enzymes in HNSCC tumors modifies the overall sulfation pattern, but the alteration of 6-O-sulfate is likely non-uniform and occurs in specific domains of the HS chains. These findings demonstrate that SULF2 expression correlates with survival of HNSCC patients and could potentially serve as a prognostic factor or target of therapeutic interventions.

Restricted availability of cell and animal models is a rate-limiting step for investigation of salivary gland neoplasm pathophysiology and therapeutic response. Conditionally reprogrammed cell (CRC) technology enables establishment of primary epithelial cell cultures from patient material. This study tested a translational workflow for acquisition, expansion and testing of CRC-derived primary cultures of salivary gland neoplasms from patients presenting to an academic surgical practice. Results showed that cultured cells were sufficient for epithelial cell-specific transcriptome characterization to detect candidate therapeutic pathways and fusion genes, and for screening for cancer risk-associated single nucleotide polymorphisms (SNPs) and driver gene mutations through exome sequencing. Focused study of primary cultures of a low-grade mucoepidermoid carcinoma demonstrated amphiregulin-mechanistic target of rapamycin-protein kinase B (AKT; AKT1) pathway activation, identified through bioinformatics and subsequently confirmed as present in primary tissue and preserved through different secondary 2D and 3D culture media and xenografts. Candidate therapeutic testing showed that the allosteric AKT inhibitor MK2206 reproducibly inhibited cell survival across different culture formats. By contrast, the cells appeared resistant to the adenosine triphosphate competitive AKT inhibitor GSK690693. Procedures employed here illustrate an approach for reproducibly obtaining material for pathophysiological studies of salivary gland neoplasms, and other less common epithelial cancer types, that can be executed without compromising pathological examination of patient specimens. The approach permits combined genetic and cell-based physiological and therapeutic investigations in addition to more traditional pathologic studies, and can be used to build sustainable bio-banks for future inquiries. This article has an associated First Person interview with the first author of the paper.