TRAF3 loss-of-function reveals the noncanonical NF-κB pathway as a therapeutic target in diffuse large B cell lymphoma.

Here, we report recurrent focal deletions of the chr14q32.31-32 locus, including TRAF3, a negative regulator of NF-κB signaling, in de novo diffuse large B cell lymphoma (DLBCL) (24/324 cases). Integrative analysis revealed an association between TRAF3 copy number loss with accumulation of NIK, the central noncanonical (NC) NF-κB kinase, and increased NC NF-κB pathway activity. Accordingly, TRAF3 genetic ablation in isogenic DLBCL model systems caused upregulation of NIK and enhanced NC NF-κB downstream signaling. Knockdown or pharmacological inhibition of NIK in TRAF3-deficient cells differentially impaired their proliferation and survival, suggesting an acquired onco-addiction to NC NF-κB. TRAF3 ablation also led to exacerbated secretion of the immunosuppressive cytokine IL-10. Coculturing of TRAF3-deficient DLBCL cells with CD8+ T cells impaired the induction of Granzyme B and interferon (IFN) γ, which were restored following neutralization of IL-10. Our findings corroborate a direct relationship between TRAF3 genetic alterations and NC NF-κB activation, and highlight NIK as a potential therapeutic target in a defined subset of DLBCL.

Pralatrexate inhibited the replication of varicella zoster virus and vesicular stomatitis virus: An old dog with new tricks.

Varicella zoster virus (VZV) is associated with herpes zoster (HZ) or herpes zoster ophthalmicus (HZO). All antiviral agents currently licensed for the management of VZV replication via modulating different mechanisms, and the resistance is on the rise. There is a need to develop new antiviral agents with distinct mechanisms of action and adequate safety profiles. Pralatrexate (PDX) is a fourth-generation anti-folate agent with an inhibitory activity on folate (FA) metabolism and has been used as an anti-tumor drug. We observed that PDX possessed potent inhibitory activity against VZV infection. In this study, we reported the antiviral effects and the underlying mechanism of PDX against VZV infection. The results showed that PDX not only inhibited VZV replication in vitro and in mice corneal tissues but also reduced the inflammatory response and apoptosis induced by viral infection. Furthermore, PDX treatment showed a similar anti-VSV inhibitory effect in both in vitro and in vivo models. Mechanistically, PDX inhibited viral replication by interrupting the substrate supply for de novo purine and thymidine synthesis. In conclusion, this study discovered the potent antiviral activity of PDX with a novel mechanism and presented a new strategy for VZV treatment that targets a cellular metabolic mechanism essential for viral replication. The present study provided a new insight into the development of broad-spectrum antiviral agents.

FOLR1-induced folate deficiency reduces viral replication via modulating APOBEC3 family expression.

Folate receptor alpha (FOLR1) is vital for cells ingesting folate (FA). FA plays an indispensable role in cell proliferation and survival. However, it is not clear whether the axis of FOLR1/FA has a similar function in viral replication. In this study, we used vesicular stomatitis virus (VSV) to investigate the relationship between FOLR1-mediated FA deficiency and viral replication, as well as the underlying mechanisms. We discovered that FOLR1 upregulation led to the deficiency of FA in HeLa cells and mice. Meanwhile, VSV replication was notably suppressed by FOLR1 overexpression, and this antiviral activity was related to FA deficiency. Mechanistically, FA deficiency mainly upregulated apolipoprotein B mRNA editing enzyme catalytic subunit 3B (APOBEC3B) expression, which suppressed VSV replication in vitro and in vivo. In addition, methotrexate (MTX), an FA metabolism inhibitor, effectively inhibited VSV replication by enhancing the expression of APOBEC3B in vitro and in vivo. Overall, our present study provided a new perspective for the role of FA metabolism in viral infections and highlights the potential of MTX as a broad-spectrum antiviral agent against RNA viruses.

Potent Neutralizing Humanized Antibody With Topical Therapeutic Potential Against HPV18-Related Cervical Cancer.

Cervical cancer caused by human papillomavirus (HPV) infections is the fourth most common cancer in women worldwide. Current prophylactic HPV vaccines have achieved promising success in preventing HPV infection. However, still 570,000 new cases were reported in 2018. The current primary treatment for the patient with cervical cancer is either surgery or chemoradiotherapy. Cervical cancer still lacks standard medical therapy. HPV18 induced cervical cancer has the worst prognosis and high mortality compared to other HPV infections. The development of HPV18 related with cervical malignancy requires the persistent infection of cervical-vaginal epithelium by HPV18 subtype, which can take years to transform the epithelium. This period of repeated infection provides a window for therapeutic intervention. Neutralizing antibodies formulated as topical agents that inhibit HPV18 infection should reduce the chance of cervical malignancy. We previously demonstrated that potent neutralizing anti-sera against HPV18 infection were induced by HPV18 viral like particle (VLP) generated in mammalian cells. We, therefore, isolated two potent neutralizing antibodies, 2A12 and 8H4, from over 3,810 hybridomas prepared from mice immunized with HPV18 VLP. 2A12 and 8H4 exhibited excellent potency, with 50% virus-inhibitory concentrations (IC50) of 0.4 and 0.9 ng/ml, respectively. Furthermore, 2A12 and 8H4 recognized distinct and non-overlapping quaternary epitopes and bound specifically with HPV18. Humanized 2A12 (Hu2A12) retained comparable neutralizing activity against HPV18 infection in various acidic pH settings and in hydrogel formulation with IC50 values of 0.04 to 0.77 ng/ml, indicating that Hu2A12 will be a promising candidate for clinical development as a topical vaginal biopharmaceutical agent against HPV18 infection.

AAV-mediated in vivo CAR gene therapy for targeting human T-cell leukemia.

Chimeric antigen receptor (CAR) T-cell therapy is the most active field in immuno-oncology and brings substantial benefit to patients with B cell malignancies. However, the complex procedure for CAR T-cell generation hampers its widespread applications. Here, we describe a novel approach in which human CAR T cells can be generated within the host upon injecting an Adeno-associated virus (AAV) vector carrying the CAR gene, which we call AAV delivering CAR gene therapy (ACG). Upon single infusion into a humanized NOD.Cg-Prkdcscid Il2rgem26/Nju tumor mouse model of human T-cell leukemia, AAV generates sufficient numbers of potent in vivo CAR cells, resulting in tumor regression; these in vivo-generated CAR cells produce antitumor immunological characteristics. This instantaneous generation of in vivo CAR T cells may bypass the need for patient lymphodepletion, as well as the ß processes of traditional CAR T-cell production, which may make CAR therapy simpler and less expensive. It may allow the development of intricate, individualized treatments in the form of on-demand and diverse therapies.

Infection of humanized mice with a novel phlebovirus presented pathogenic features of severe fever with thrombocytopenia syndrome.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a tick-borne emerging phlebovirus with high mortality rates of 6.0 to 30%. SFTSV infection is characterized by high fever, thrombocytopenia, leukopenia, hemorrhage and multiple organ failures. Currently, specific therapies and vaccines remain elusive. Suitable small animal models are urgently needed to elucidate the pathogenesis and evaluate the potential drug and vaccine for SFTSV infection. Previous models presented only mild or no pathogenesis of SFTS, limiting their applications in SFTSV infection. Therefore, it is an urgent need to develop a small animal model for the investigation of SFTSV pathogenesis and evaluation of therapeutics. In the current report, we developed a SFTSV infection model based on the HuPBL-NCG mice that recapitulates many pathological characteristics of SFTSV infection in humans. Virus-induced histopathological changes were identified in spleen, lung, kidney, and liver. SFTSV was colocalized with macrophages in the spleen and liver, suggesting that the macrophages in the spleen and liver could be the principle target cells of SFTSV. In addition, histological analysis showed that the vascular endothelium integrity was severely disrupted upon viral infection along with depletion of platelets. In vitro cellular assays further revealed that SFTSV infection increased the vascular permeability of endothelial cells by promoting tyrosine phosphorylation and internalization of the adhesion molecule vascular endothelial (VE)-cadherin, a critical component of endothelial integrity. In addition, we found that both virus infection and pathogen-induced exuberant cytokine release dramatically contributed to the vascular endothelial injury. We elucidated the pathogenic mechanisms of hemorrhage syndrome and developed a humanized mouse model for SFTSV infection, which should be helpful for anti-SFTSV therapy and pathogenesis study.

A single-domain antibody inhibits SFTSV and mitigates virus-induced pathogenesis in vivo.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a novel tick-borne bunyavirus that recently emerged in East Asian countries. SFTS is characterized by high fever, thrombocytopenia, leukopenia, multiorgan failure, and hemorrhage with case fatality rates of 6.3% to 30%. Neither antivirals nor vaccines are available at present. We previously demonstrated that neutralizing antibodies specific for SFTSV glycoprotein (Gn) played a vital role in the survival of patients with SFTS. Nanobodies from camels present unique properties, such as thermostability, high affinity, and low immunogenicity. In the current study, mammalian expressed SFTSV Gn was used to immunize a camel, and functional nanobodies were isolated from the B cell nanobody library constructed from the immunized animal. Clone SNB02 was selected for in-depth analysis for its inhibition of SFTSV replication both in vitro and in vivo. We showed that SNB02 potently inhibited SFTSV infection and prevented thrombocytopenia in a humanized mouse model and is a potential candidate for therapeutics.

Nanotechnology and immunoengineering: How nanotechnology can boost CAR-T therapy.

Chimeric antigen receptor (CAR) therapy has achieved remarkable clinical efficacy against hematological cancers and has been approved by FDA for treatment of B-cell tumors. However, the complex manufacturing process and limited success in solid tumors hamper its widespread applications, thus prompting the development of new strategies for overcoming the abovementioned hurdles. In the last decade, nanotechnology has provided sustainable strategies for improving cancer immunotherapy through vaccine development and delivery of immunomodulatory drugs. Nanotechnology can boost CAR-T therapy and may overcome the existing challenges by emerging as a carrier for CAR-T therapy or in combination with CAR-T, it may inhibit solid tumors more effectively than conventional approaches. The revealing of cellular mechanisms, barriers and potential strategies that could be used to manipulate and/or modify cells would enable unprecedented advances in nanotechnology for biologics delivery. This review outlines the journey and barriers of nanoparticles (NPs) across the cell. Subsequently, the approaches to tackle the barriers and strategies to modulate NPs as a carrier for CAR-T therapy are discussed. Finally, the role of NPs in CAR-T therapy and the potential challenges are summarized. This review aims to provide the readers with a detailed overview of NP-based CAR-T therapy research and distil this information into an accessible form conducive to design desired CAR-T therapy using NP approach. STATEMENT OF SIGNIFICANCE: Chimeric antigen receptor (CAR) T-cell therapy is the most vibrant field in immuno-oncology today, with enormous benefits to patients with B-cell malignancies. However, a rapid and straightforward procedure for CAR-T generation is an exigent need to broaden its therapeutic avenue. Nanotechnology has emerged as a novel alternative approach for CAR-T generation. To the best of our knowledge, this is the first in-depth review that briefly highlights the various aspects of nanotechnology in CAR-T therapy, including the strategies to brand NPs as an effective carrier for CAR cargo, its potential advantages, challenges, and future roadmap. It provides readers with a detailed overview of NP-based CAR-T therapy research, and researchers would be able to distill this information into an accessible form conducive to design the desired CAR therapy using the nanotechnology approach.

Induction of neutralizing antibodies by human papillomavirus vaccine generated in mammalian cells.

BACKGROUND: Cervical cancer caused by human papillomavirus (HPV) infections is one of the most common cancers affecting women worldwide. Current preventative HPV vaccines on the market are composed of HPV L1 protein produced either in the yeast such as Gardasil or in the insect cells such as Cervarix. The duration of efficacy and cross-protection remain highly desirable for the improvement of current prophylactic HPV vaccine. Given that HPV carries out infection and replicates in mammalian cells, L2 protein, which is not included in the current licensed vaccines, is included in the third generation of HPV vaccine in pursuing of providing broader prevention. We hypothesize that a virus-like particle (VLP) consisting of HPV L1 plus L2 proteins generated in mammalian cells will present conformations more closely to native HPV, thus it will provide more durable and broader efficacy of prevention. METHODS: We took advantage of 293TT cells to produce VLP containing L1 and L2 proteins of HPV16 and HPV18, respectively. RESULTS: VLP particles of uniformed size and morphology were observed, and potent and broadly neutralizing antibodies were induced in mice and rabbits. In addition, compared to bivalent HPV vaccine of Cervarix, our HPV L1-L2 VLPs elicited higher titer of anti-sera, and the anti-sera also presented comparable neutralization potency against HPV16 and HPV18 infections even a much less potent adjuvant was used in our case. CONCLUSION: Our VLPs were capable of eliciting stronger and more broadly neutralizing activities against various HPV subtypes and were potential candidate HPV vaccines.

Therapeutic Versatility of Resveratrol Derivatives.

Resveratrol, a natural phytoalexin, exhibits a remarkable range of biological activities, such as anticancer, cardioprotective, neuroprotective and antioxidant properties. However, the therapeutic application of resveratrol was encumbered for its low bioavailability. Therefore, many researchers focused on designing and synthesizing the derivatives of resveratrol to enhance the bioavailability and the pharmacological activity of resveratrol. During the past decades, a large number of natural and synthetic resveratrol derivatives were extensively studied, and the methoxylated, hydroxylated and halogenated derivatives of resveratrol received particular more attention for their beneficial bioactivity. So, in this review, we will summarize the chemical structure and the therapeutic versatility of resveratrol derivatives, and thus provide the related structure activity relationship reference for their practical applications.

Establishment of a rat model of chronic Prostatitis/Chronic Pelvic Pain Syndrome (CP/CPPS) induced by immunization with a novel peptide T2.

BACKGROUND: The exact etiological mechanism of Chronic Prostatitis/chronic pelvic pain syndrome (CP/CPPS) is still unclear however autoimmunity is the most valid theory. We developed a rat model of Chronic Prostatitis/chronic pelvic pain syndrome by using a novel peptide (T2) isolated from TRPM8. This model might be beneficial in elucidating mechanisms involved in the pathogenesis of Chronic Prostatitis/Chronic Pelvic Pain Syndrome (CP/CPPS). METHODS: 40 male Sprague-Dawley rats with an average weight of 180-220g were equally distributed into five groups. The normal control group was injected with normal saline (.9% NACL), the CFA group with CFA, AL(OH)3 group was given AL(OH)3 injection, T2 group using a novel peptide T2 and T2+AL(OH)3+CFA group was injected with T2+AL(OH)3+CFA. Dosing to all rat groups were injected subcutaneously. Hematoxylin and eosin staining and Immunohistochemistry were used to investigate inflammatory cell infiltration and IL-1ß in the prostate tissue respectively. ELISA technique was used to measure the serum level of CRP and TNF-α. T-test was used to analyze the results. RESULTS: Maximum infiltration of inflammatory cells and the highest level of IL-1ß in the prostate tissue was observed in T2+AL(OH)3+CFA group as revealed by histopathology and Immunohistochemistry, respectively. Furthermore, T2+AL(OH)3+CFA group attained the peak value of serum TNF-α and CRP as determined by ELISA technique. CONCLUSION: Our results demonstrated that T2 in combination with AL(OH)3 and CFA induced severe Prostatitis in rats. We believe that our present model will be highly beneficial for investigation of the pathophysiology of Chronic Prostatitis/Chronic Pelvic Pain Syndrome.

A novel mouse model of chronic prostatitis/chronic pelvic pain syndrome induced by immunization of special peptide fragment with aluminum hydroxide adjuvant.

OBJECTIVES: CP/CPPS is a commonly observed distress in male patients. Because of its little-known etiology, no effective therapy has been developed which has promising outcomes. Therefore, there is a need to develop a valid model which can mimic the etiology of CP/CPPS. MATERIALS AND METHODS: Fifty male C57BL/6 mice were randomly and averagely divided into 5 groups of 10 mice each. The control group was injected with 0.9% NaCl solution. Aluminum hydroxide and T2 groups were injected with aluminum hydroxide adjuvant and T2 peptide. T2 plus complete Freund adjuvant (CFA) with aluminum hydroxide group was injected with a mixture of T2, CFA and aluminum hydroxide adjuvant. At the same time, CFA group was injected with complete Freund adjuvant. Hematoxylin-eosin stain and immunohistochemistry were used to investigate inflammatory lesion and expression of IL-ß1. Furthermore, TNF-α and CRP protein levels were evaluated by using commercially available ELISA kits. The ANOVA test was used to compare the statistical differences among groups. RESULTS: Prostates from a mixture of T2 plus CFA with aluminum hydroxide immunized mice showed elevated lesions and high level of inflammatory cells infiltration compared to the other groups. In addition, the levels of TNF-α, IL-ß1, and CRP were also higher in the T2 plus CFA with aluminum hydroxide group as compared to the other groups. CONCLUSION: Our results showed that T2 with CFA plus aluminum hydroxide adjuvant injection could successfully induce CP/CPPS in mice. This autoimmune novel model provides a useful, economic, safer, and easy tool for exploring the etiology and pathophysiology of CP/CPPS which will improve the therapeutic outcomes.

Antioxidant therapy for management of oxidative stress induced hypertension.

Hypertension is considered as the most common risk factor for cardiovascular diseases, also is regarded as a leading cause of the mortality and morbidity worldwide. The mechanisms underlying the pathological process of hypertension are not completely explained. However, there is growing evidence that increased oxidative stress plays an important role in the pathophysiology of hypertension. Several preclinical studies and clinical trials have indicated that antioxidant therapy is important for management of hypertension, using antioxidants compounds such as alpha tocopherol (Vit E) and ascorbic acid (Vit C), polyphenols with others and some antihypertensive drugs that are now in clinical use (e.g. ACEIs, ARBs, novel B-blockers, dihydropyridine CCBs) which have antioxidative pleiotropic effects. The purpose of this review is to highlight the importance of antioxidant therapy for management of oxidative stress induced hypertension. Furthermore, we review the current knowledge in the oxidative stress and its significance in hypertension.

Sugar-based novel niosomal nanocarrier system for enhanced oral bioavailability of levofloxacin.

CONTEXT: Vesicular systems have attracted great attention in drug delivery because of their amphiphilicity, biodegradability, non-toxicity and potential for increasing drug bioavailability. OBJECTIVE: A novel sugar-based double-tailed surfactant containing renewable block was synthesized for preparing niosomal vesicles that could be exploited for Levofloxacin encapsulation, aiming to increase its oral bioavailability. MATERIALS AND METHODS: The surfactant was characterized by 1H NMR, mass spectroscopy and Fourier transform infrared spectroscopy (FT-IR). Its biocompatibility was studied against cell cultures and human blood hemolysis. In vivo acute toxicity was evaluated in mice. The vesicle morphology, size, drug-excipients interaction and entrapment efficiency (EE) were examined using atomic force microscope (AFM), dynamic light scattering (DLS), FT-IR and HPLC. Oral bioavailability studies of Levofloxacin in surfactant-based niosomal formulation were carried out using rabbits and plasma samples were analyzed using HPLC. RESULTS AND DISCUSSION: Vesicles were spherical in shape and the size was 190.31 ± 4.51 nm with a polydispersity index (PDI) of 0.29 ± 0.03. The drug EE in niosomes was 68.28 ± 3.45%. When applied on cell lines, high cell viability was observed even after prolonged exposure at high concentrations. It caused 5.77 ± 1.34% hemolysis at 1000 µg/mL and was found to be safe up to 2000 mg/kg. Elevated Levofloxacin plasma concentration was achieved when delivered with novel vesicles. CONCLUSION: The surfactant was demonstrated to be safe and effective as carrier of Levofloxacin. The study suggests that this sugar-based double-tailed nonionic surfactant could be promising nano-vesicular system for delivery and enhancing oral bioavailability of the hydrophobic Levofloxacin.