Hepatitis B RNA and Core-Related Antigen Provide Value Beyond DNA in Evaluating e But Not Surface Antigen Clearance.

In chronic hepatitis B virus (HBV) infection, hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg) clearance are important milestones toward immune control.1 A drop in HBV DNA is an established correlate of both HBeAg and HBsAg clearance.2 We evaluated changes in HBV RNA and hepatitis B core-related antigen (HBcrAg) levels, markers of transcriptional activity of covalently closed circular DNA (cccDNA),3,4 with HBeAg and HBsAg clearance, and compared them with changes in HBV DNA level among adult participants in the Hepatitis B Research Network (HBRN).

Liver-Related Mortality in Hepatitis B Virus Core Antibody+/Hepatitis B Virus Surface Antigen- Patients: Occult Hepatitis B Virus, Hepatitis B Virus Reactivation, and Hepatocellular Carcinoma Development.

ABSTRACT: Although hepatitis B virus surface antigen (HBsAg) serum clearance is an important milestone in the natural history of chronic hepatitis B virus (HBV) infection, HBsAg-negative patients are at risk of liver cancer and liver-related death, especially when progressive fibrosis is present. HBsAg-negative/anti-HBV core antibody-positive patients should be carefully evaluated and managed accordingly for the presence of significant liver fibrosis, other viral coinfections, occult HBV infection, risk of HBV reactivation, and hepatocellular carcinoma. Antiviral prophylaxis should be initiated in isolated anti-HBV core antibody patients receiving high-risk chemotherapy or biologics. Hepatocellular carcinoma surveillance with liver ultrasound and serum alpha-fetoprotein should be considered for patients with risk factors.

Screening of Phyllanthus niruri Leaves Phytoconstituents for Antiviral and Antibacterial Activity by Molecular Docking Studies.

The genus Phyllanthus belongs to one of the largest plant families, the Phyllantaceae (L.). Phyllanthus niruri is an annual perennial herb that grows in tropical Asia, America, China, and the islands of the Indian Ocean. Numerous alkaloids, steroids, flavonoids, lignans, coumarins, polyphenols, and lipids are present in Phyllanthus. The effects of plants have been studied for a variety of purposes, including their antioxidant (Giribabu et al., Evid Based Complement Alternat Med, 2014), anti-inflammatory (Porto et al., Revista Brasileira de Pharmacognosy, 2013), antinociceptive (Sathisha et al., Indian Drugs, 2009), analgesic (Mostofa et al., BMC Complement Altern Med, 2017), antiulcer (Mali et al., Biomed Aging Pathol, 2011), antiarthritic (Obidike and Salawu, Planta Medica, 2010), antiplasmodial (Shilpa et al., Environ Dis, 2018), immunomodulatory (Manikkoth et al., Anticonvulsant activity of Phyllanthus amarus in experimental animal models), anticonvulsant (Wasnik et al., Int J Pharm Sci Rev Res, 2014), antidepressant (Venkateswaran et al., Effects of an extract from Phyllanthus niruri on hepatitis B and woodchuck hepatitis viruses: In vitro and in vivo studies (antiviral agent/Marmota monax/DNA polymerase/hepatitis B surface antigen/woodchuck hepatitis surface antigen). In Hepatitis B and The Prevention of Primary Cancer of The Liver: Selected Publications of Baruch S Blumberg, pp 535-539), antiviral (Venkateswaran et al., Effects of an extract from Phyllanthus niruri on hepatitis B and woodchuck hepatitis viruses: In vitro and in vivo studies (antiviral agent/Marmota monax/DNA polymerase/hepatitis B surface antigen/woodchuck hepatitis surface antigen). In Hepatitis B and The Prevention of Primary Cancer of The Liver: Selected Publications of Baruch S Blumberg, pp 535-539), antitumor (Sharma et al., Asian Pac J Cancer Prev, 2009), hyperlipidemia (Khanna et al., J Ethnopharmacol, 2002), and antifertility (Ezeonwu, Inquiries J, 2011). For additional docking investigations with distinct proteins, the leaf chemicals are assessed, that is, the crystal structure of serine protease hepsin in complex with inhibitor [PDB ID:5 CE1] for antiviral activity human topoisomerase II beta in complex with DNA and etoposide [PDB ID:3QX3] and crystal structure of E. coli GyraseB 24 kDa in complex with 4-(4-bromo-1H-pyrazol-1-yl)-6-[(ethylcarbamoyl)amino]-N-(pyridin-3-yl) pyridine-3-carboxamide [PDB ID: 6F86] for antibacterial activity and have been selected. To evaluate the in silico results and grading of virtual screening, or molecular docking, ritonavir antiviral activity and ampicillin for antibacterial activity were used as a benchmark.

Neomorphic DNA-binding enables tumor-specific therapeutic gene expression in fusion-addicted childhood sarcoma.

Chimeric fusion transcription factors are oncogenic hallmarks of several devastating cancer entities including pediatric sarcomas, such as Ewing sarcoma (EwS) and alveolar rhabdomyosarcoma (ARMS). Despite their exquisite specificity, these driver oncogenes have been considered largely undruggable due to their lack of enzymatic activity.Here, we show in the EwS model that - capitalizing on neomorphic DNA-binding preferences - the addiction to the respective fusion transcription factor EWSR1-FLI1 can be leveraged to express therapeutic genes.We genetically engineered a de novo enhancer-based, synthetic and highly potent expression cassette that can elicit EWSR1-FLI1-dependent expression of a therapeutic payload as evidenced by episomal and CRISPR-edited genomic reporter assays. Combining in silico screens and immunohistochemistry, we identified GPR64 as a highly specific cell surface antigen for targeted transduction strategies in EwS. Functional experiments demonstrated that anti-GPR64-pseudotyped lentivirus harboring our expression cassette can specifically transduce EwS cells to promote the expression of viral thymidine kinase sensitizing EwS for treatment to otherwise relatively non-toxic (Val)ganciclovir and leading to strong anti-tumorigenic, but no adverse effects in vivo. Further, we prove that similar vector designs can be applied in PAX3-FOXO1-driven ARMS, and to express immunomodulatory cytokines, such as IL-15 and XCL1, in tumor entities typically considered to be immunologically 'cold'.Collectively, these results generated in pediatric sarcomas indicate that exploiting, rather than suppressing, the neomorphic functions of chimeric transcription factors may open inroads to innovative and personalized therapies, and that our highly versatile approach may be translatable to other cancers addicted to oncogenic transcription factors with unique DNA-binding properties.

Hepatitis B virus-specific CD4 T cell responses differentiate functional cure from chronic surface antigen+ infection.

BACKGROUND & AIMS: With or without antiviral treatment, few individuals achieve sustained functional cure of chronic hepatitis B virus (HBV) infection. A better definition of what mediates functional cure is essential for improving immunotherapeutic strategies. We aimed to compare HBV-specific T cell responses in patients with different degrees of viral control. METHODS: We obtained blood from 124 HBV-infected individuals, including those with acute self-limiting HBV infection, chronic infection, and chronic infection with functional cure. We screened for HBV-specific T cell specificities by ELISpot, assessed the function of HBV-specific T cells using intracellular cytokine staining, and characterized HBV-specific CD4 T cells using human leukocyte antigen (HLA) class II tetramer staining, all directly ex vivo. RESULTS: ELISpot screening readily identified HBV-specific CD4 and CD8 T cell responses in acute resolving infection compared with more limited reactivity in chronic infection. Applying more sensitive assays revealed higher frequencies of functional HBV-specific CD4 T cells, but not CD8 T cells, in functional cure compared to chronic infection. Function independent analysis using HLA multimers also identified more HBV-specific CD4 T cell responses in functional cure compared to chronic infection, with the emergence of CD4 T cell memory both after acute and chronic infection. CONCLUSIONS: Functional cure is associated with higher frequencies of functional HBV-specific CD4 memory T cell responses. Thus, immunotherapeutic approaches designed to induce HBV functional cure should also aim to improve CD4 T cell responses. LAY SUMMARY: Immunotherapy is a form of treatment that relies on harnessing the power of an individual's immune system to target a specific disease or pathogen. Such approaches are being developed for patients with chronic HBV infection, in an attempt to mimic the immune response in patients who control HBV infection spontaneously, achieving a so-called functional cure. However, what exactly defines protective immune responses remains unclear. Herein, we show that functional cure is associated with robust responses by HBV-specific CD4 T cells (a type of immune cell).

Long-term outcome of lamivudine/dolutegravir dual therapy in HIV-infected, virologically suppressed patients.

BACKGROUND: The use of DTG-containing two-drug regimens is one of the most promising solutions to the need to ease the management of HIV treatment without harming its efficacy and safety. We report long- term results in patients switched, while virologically suppressed, to the combination of dolutegravir (DTG) plus lamivudine (3TC). METHODS: This is a prospective, clinical, uncontrolled cohort enrolling ART-experienced people living with HIV (PLWH) with HIV-RNA < 50 copies/ml for 6 months or longer, negative hepatitis B virus surface antigen, and without known M184V/I mutations. Kaplan-Meiers curves are used to describe persistency of virological suppression on therapy and a Cox regression model to evaluate baseline characteristics and the risk of stopping therapy. RESULTS: 218 individuals switched their regimen since 2015. The mean estimated follow-up was of 64.3 months (95% CI 61.3-67.3) for approximately 1000 patient/years. After 5 years of follow-up, 77.1% were still on the DTG-3TC combination. No virologic failure was detected throughout the whole study period, and only 15 subjects presented single isolated viral blips above 50 copies/ml. Most patients stopped therapy because of reasons unrelated to study drugs (lost to follow-up; patients' decision; moved to other Centers), but due to the unselected nature of the casuistry; 11 subjects died in the 5 years of follow-up mostly because of pre-existing co-morbidities (6 neoplastic diseases and 2 end-stage liver disease). The median baseline CD4 count was 669 cells/mcl (IQR 483-927). After 5 years it raised to 899 cells/mcl (IQR 646-1160) (P < 0.001) without a significant change of CD8 counts that lowered from 767 cells/mcl (IQR 532-1034) to 683 cells/mcl (IQR 538-988). Consequently, the CD4/CD8 ratio varied from 0.93 (IQR 0.60-1.30) to 1.15 (IQR 0.77-1.45) (P < 0.0001). A non-significant (P = 0.320) increment of mean creatinine, 0.06 mg/dl in magnitude, was observed over the whole follow-up. CONCLUSION: These long-term results over 5 years reinforce the durability and good tolerability of DTG-3TC. Our results continue to support the recommended switch use of this 2DR as a well-accepted treatment option for ART-experienced PLWH.

SPAG1 promotes the development of AML by activating the ERK/MAPK signaling pathway and affects the chemotherapy sensitivity of venetoclax.

Sperm-associated antigen 1 (SPAG1) is considered to be associated with infertility and tumorigenesis. However, its function in acute myeloid leukemia (AML) remains unclear. In this study, we evaluated the expression level of SPAG1 and explored its clinical prognostic value in patients with AML, as well as its biological function in AML cells. SPAG1 is widely expressed in AML patients, resulting in a poor prognosis. However, its expression was not associated with Fms-related receptor tyrosine kinase 3 (FLT3) mutations. Utilizing the RNA interference knockdown tests, we found that SPAG1 could promote the proliferation and survival of AML cells and regulate the expression of structural maintenance of chromosomes protein 3 (SMC3), activating the ERK/MAPK signaling pathway. Furthermore, we discovered that inhibiting SPAG1 impacted AML cell susceptibility to venetoclax. In conclusion, SPAG1 may serve as a potential therapeutic target in AML.

Prostate-Specific Membrane Antigen (PSMA) Theranostics for Treatment of Oligometastatic Prostate Cancer.

Theranostics, a combination of therapy and diagnostics, is a field of personalized medicine involving the use of the same or similar radiopharmaceutical agents for the diagnosis and treatment of patients. Prostate-specific membrane antigen (PSMA) is a promising theranostic target for the treatment of prostate cancers. Diagnostic PSMA radiopharmaceuticals are currently used for staging and diagnosis of prostate cancers, and imaging can predict response to therapeutic PSMA radiopharmaceuticals. While mainly used in the setting of metastatic, castrate-resistant disease, clinical trials are investigating the use of PSMA-based therapy at earlier stages, including in hormone-sensitive or hormone-naïve prostate cancers, and in oligometastatic prostate cancers. This review explores the use of PSMA as a theranostic target and investigates the potential use of PSMA in earlier stage disease, including hormone-sensitive metastatic prostate cancer, and oligometastatic prostate cancer.

Incidence and Factors Associated With HBV Relapse After Cessation of Entecavir or Tenofovir in Patients With HBsAg Below 100 IU/mL.

BACKGROUND & AIMS: We investigated the incidence and factors associated with relapse of hepatitis B virus (HBV) infection in patients with levels of HB surface antigen (HBsAg) less than 100 IU/mL after cessation of entecavir or tenofovir disoproxil fumarate (TDF) treatment. METHODS: We collected data from patients with chronic HBV infection without cirrhosis treated with entecavir from 2007 through 2015 or TDF from 2011 through 2016 in Taiwan. We identified 135 patients with levels of HBsAg less than 100 IU/mL at the end of treatment (79 entecavir and 56 TDF) and collected data from them for a median of 87 weeks (interquartile range, 48-161 wk) for use as the development set. We collected data from 108 patients from separate medical centers in Taiwan, followed up for a median of 126 weeks (interquartile range, 61-214 wk), and used these as the validation group. Post-treatment virologic relapse was defined as a serum HBV DNA level greater than 2000 IU/mL, and clinical relapse was defined as an alanine aminotransferase level greater than 80 U/L and a HBV DNA level greater than 2000 IU/mL. RESULTS: In the development group, the 5-year incidences of virologic relapse, clinical relapse, and HBsAg loss were 40.9%, 32.5%, and 47%, respectively. The baseline HBV DNA and end-of-treatment levels of HBsAg were associated independently with relapse. In the development group, 17.3% of patients with end-of-treatment HBsAg levels less than 40 IU/mL had a virologic relapse within 5 years, whereas 67.6% of patients with a HBsAg level of 40 IU/mL or more had a virologic relapse within 5 years (P < .001); proportions of patients with clinical relapses were 10.2% (HBsAg <40 IU/mL) and 57.6% (HBsAg ≥40 IU/mL; P < .001). In the validation groups, for patients with end-of-treatment HBsAg levels less than 40 IU/mL or 40 IU/mL or more, the rates of virologic relapse at 5 years were 31.1% and 80.5% (P < .001), and rates of clinical relapse were 14.2% and 50.3% (P < .001), respectively. Rates of virologic and clinical relapse within 5 years were low (<10%) in patients with a combination of end-of-treatment HBsAg level less than 40 IU/mL and baseline HBV DNA level less than 5 × 104 IU/mL, or baseline hepatitis B core-related antigen level less than 4 log U/mL in the development group. CONCLUSIONS: An end-of-treatment HBsAg level of 40 IU/mL or less is optimal for stopping nucleos(t)ide analog therapy. Waiting to stop therapy until patients have a combination of baseline HBV DNA level of 5 × 104 IU/mL or hepatitis B core-related antigen of 4 log U/mL and end-of-treatment HBsAg level of 40 IU/mL might reduce the risk of HBV relapse.

Lactadherin promotes microvesicle clearance to prevent coagulopathy and improves survival of severe TBI mice.

Coagulopathy is common in patients with traumatic brain injury (TBI) and predicts poor clinical outcomes. We have shown that brain-derived extracellular microvesicles, including extracellular mitochondria, play a key role in the development of TBI-induced coagulopathy. Here, we further show in mouse models that the apoptotic cell-scavenging factor lactadherin, given at a single dose of 400 µg/kg 30 minutes before (preconditioning) or 30 minutes after cerebral fluid percussion injury, prevented coagulopathy as defined by clotting time, fibrinolysis, intravascular fibrin deposition, and microvascular bleeding of the lungs. Lactadherin also reduced cerebral edema, improved neurological function, and increased survival. It achieved these protective effects by enhancing the clearance of circulating microvesicles through phosphatidylserine-mediated phagocytosis. Together, these results identify the scavenging system for apoptotic cells as a potential therapeutic target to prevent TBI-induced coagulopathy and improve the outcome of TBI.

Treatment outcomes of metastasis-directed treatment using 68Ga-PSMA-PET/CT for oligometastatic or oligorecurrent prostate cancer: Turkish Society for Radiation Oncology group study (TROD 09-002).

PURPOSE: The aim of this study was to evaluate the outcomes of 68Ga prostate-specific membrane antigen (68Ga-PSMA) positron-emission tomography (PET)/CT-based metastasis-directed treatment (MDT) for oligometastatic prostate cancer (PC). METHODS: In this multi-institutional study, clinical data of 176 PC patients with 353 lesions receiving MDT between 2014 and 2019 were retrospectively evaluated. All patients had biopsy proven PC with ≤5 metastases detected with 68Ga-PSMA-PET/CT. MDT was delivered with conventional fractionation or stereotactic body radiotherapy (SBRT) techniques. CTCAE v4.0 was used for acute and RTOG/EORTC Late Radiation Morbidity Scoring Schema was used for late toxicity evaluation. RESULTS: At the time of MDT, 59 patients (33.5%) had synchronous and 117 patients (66.5%) had metachronous metastases. Median number of metastases was one and the MDT technique was SBRT in 73.3% patients. The 2­year overall survival (OS) and progression-free survival (PFS) rates were 87.6% and 63.1%, respectively. With a median follow-up of 22.9 months, 9 patients had local recurrence at the irradiated site. The 2­year local control rate at the treated oligometastatic site per patient was 93.2%. In multivariate analysis, an increased number of oligometastases and untreated primary PC were negative predictors for OS; advanced clinical tumor stage, untreated primary PC, BED3 value of ≤108 Gy, and MDT with conventional fractionation were negative predictors for PFS. No patient experienced grade ≥3 acute toxicity, but one patient had a late grade 3 toxicity of compression fracture after spinal SBRT. CONCLUSION: 68Ga-PSMA-PET/CT-based MDT is an efficient and safe treatment for oligometastatic PC patients. Proper patient selection might improve treatment outcomes.

Entecavir-resistant hepatitis B virus decreases surface antigenicity: A full genome and functional characterization.

BACKGROUND AND AIM: Since polymerase and surface genes overlap in hepatitis B virus (HBV), an antiviral-induced mutation in the polymerase gene may alter the surface antigenicity in patients with chronic hepatitis B (CHB), but this possibility has not been clearly confirmed. This study aimed to determine the drug susceptibility and surface antigenicity of the patient-derived mutants. PATIENTS AND METHODS: Full-length HBV genomes isolated from four entecavir-resistant CHB patients were cloned and sequenced. Around 10 clones of full-length HBV obtained from each patient were analysed and registered in the NCBI GenBank. Representative clones were further characterized by in vitro drug susceptibility and surface antigenicity assays. RESULTS: The rtL180M + rtM204V mutations were common among all the clones analysed. Additionally, the ETV resistance mutations rtT184A/L, rtS202G and rtM250V were found among three patients. Most of the ETV-resistant mutants had amino acid alterations within the known epitopes recognized by T- and B-cells in the HBV surface and core antigens. The in vitro drug susceptibility assay showed that all tested clones were resistant to ETV treatment. However, they were all susceptible to ADV and TDF. More importantly, the rtI169T mutation in the RT domain, led to the sF161L mutation in the overlapping S gene, which decreased in surface antigenicity. CONCLUSIONS: The ETV resistance mutations can affect the antigenicity of the HBsAg proteins due to changes in the overlapping sequence of this surface antigen. Thus, the apparent decline or disappearance of HBsAg needs to be interpreted cautiously in patients with previous or current antiviral resistance mutations.

Copper, PET/CT and prostate cancer: a systematic review of the literature.

Copper is an essential element that plays an important role in both cancer development and growth. Indeed, high levels of copper have been found in prostate cancer (PCa), and this finding have paved the way for the use of this element as a target for positron emission tomography (PET) imaging. Copper64 (64Cu) can be used alone, as 64CuCl2, and also as a precursor for the in-vitro radio-labelling of specific carriers for PET imaging in PCa, (e.g. associated to prostate-specific membrane antigen: PSMA). The use of 64Cu-PSMA can yield late acquisitions in which PET images are characterized by a higher target-to-background ratio. At the same time, the shorter positron range of 64Cu provides high spatial resolution, which leads to better detection of small lesions. In this context, the aim of this review was to systematically review studies evaluating the identification of PCa in humans by means of 64CuCl2 and other PET tracers radio-labelled with 64Cu.

Milk fat globule-epidermal growth factor-factor VIII attenuates sepsis-induced acute kidney injury.

BACKGROUND: Acute kidney injury (AKI) is most commonly caused by sepsis in critically ill patients, and it is associated with high morbidity and mortality. The pathophysiology of sepsis-induced AKI is generally accepted to include direct inflammatory injury, endothelial cell dysfunction, and apoptosis. Milk fat globule-epidermal growth factor-factor VIII (MFG-E8) is a secretory glycoprotein with a known role in the enhancement of apoptotic cell clearance and regulation of inflammation. We hypothesize that administration of recombinant mouse MFG-E8 (rmMFG-E8) can protect mice from kidney injuries caused by sepsis. METHODS: Sepsis was induced in 8-wk-old male C57BL/6 mice by cecal ligation and puncture (CLP). rmMFG-E8 or phosphate-buffered saline (vehicle) was injected intravenously at a dosage of 20 µg/kg body weight at time of CLP (n = 5-8 mice per group). After 20 h, serum and renal tissue were harvested for various analyses. The renal injury markers blood urea nitrogen (BUN) and creatinine were determined by enzymatic and chemical reactions, respectively. The gene expression analysis was carried out by real-time quantitative polymerase chain reaction. RESULTS: At 20 h after CLP, serum levels of BUN and creatinine were both significantly increased in the vehicle group compared with the sham group, whereas the mice treated with rmMFG-E8 had a significant reduction in BUN and creatinine levels by 28% and 24.1%, respectively (BUN: 197.7 ± 23.6 versus 142.3 ± 20.7 mg/dL; creatinine: 0.83 ± 0.12 versus 0.63 ± 0.06 mg/dL; P < 0.05). Expressions of novel biomarkers of renal tissue injury neutrophil gelatinase-associated lipocalin and kidney injury molecule-1 were also significantly downregulated by 58.2% and 95%, respectively, after treatment with rmMFG-E8. Proinflammatory cytokine interleukin-6 and tumor necrosis factor-α messenger RNA (mRNA) were significantly reduced by 50.8% and 50.3%, respectively, in rmMFG-E8-treated mice compared with vehicle-treated mice. The mRNA levels of the chemokines keratinocyte chemoattractant and macrophage inhibitory protein-2 were reduced by 85.1% and 78%, respectively, in mice treated with rmMFG-E8 compared with the vehicle mice. In addition, the expression of intercellular cell adhesion molecule-1 and platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) mRNA was downregulated by 35.6% and 77.8%, respectively, in rmMFG-E8-treated mice compared with the vehicle animals (P < 0.05). CONCLUSIONS: Treatment with rmMFG-E8 reduces renal tissue injury induced by sepsis through inhibiting the production of proinflammatory cytokines and chemokine, as well as through the activation of endothelial cells. Thus, MFG-E8 may have a therapeutic potential for treating AKI induced by sepsis.

Milk fat globule-epidermal growth factor-factor VIII-derived peptide MSP68 is a cytoskeletal immunomodulator of neutrophils that inhibits Rac1.

BACKGROUND: Prolonged neutrophil infiltration leads to exaggerated inflammation and tissue damage during sepsis. Neutrophil migration requires rearrangement of their cytoskeleton. Milk fat globule-epidermal growth factor-factor VIII-derived short peptide 68 (MSP68) has recently been shown to be beneficial in sepsis-induced tissue injury and mortality. We hypothesize that MSP68 inhibits neutrophil migration by modulating small GTPase Rac1-dependent cytoskeletal rearrangements. METHODS: Bone marrow-derived neutrophils (BMDNs) or whole lung digest isolated neutrophils were isolated from 8 to 10 wk old C57BL/6 mice by Percoll density gradient centrifugation. The purity of BMDN was verified by flow cytometry with CD11b/Gr-1 staining. Neutrophils were stimulated with N-formylmethionine-leucine-phenylalanine (f-MLP) (10 nM) in the presence or absence of MSP68 at 10 nM or cecal ligation and puncture (CLP) was used to induce sepsis, and MSP68 was administered at 1 mg/kg intravenously. Cytoskeletal organization was assessed by phalloidin staining, followed by analysis using fluorescence microscopy. Activity of the Rac1 GTPase in f-MLP or CLP-activated BMDN in the presence or absence of MSP68 was assessed by GTPase enzyme-linked immunosorbent assay. Mitogen-activated protein (MAP) kinase activity was determined by western blot densitometry. RESULTS: BMDN treatment with f-MLP increased cytoskeletal remodeling as revealed by the localization of filamentous actin to the periphery of the neutrophil. By contrast, cells pretreated with MSP68 had considerably reduced filamentous actin polymerization. Cytoskeletal spreading is associated with the activation of the small GTPase Rac1. We found BMDN-treated with f-MLP or that were exposed to sepsis by CLP had increased Rac1 signaling, whereas the cells pretreated with MSP68 had significantly reduced Rac1 activation (P < 0.05). MAP kinases related to cell migration including pp38 and pERK were upregulated by treatment with f-MLP. Upregulation of these MAP kinases was also significantly reduced after pretreatment with MSP68 (P < 0.05). CONCLUSIONS: MSP68 downregulates actin cytoskeleton-dependent, Rac1-MAP kinase-mediated neutrophil motility. Thus, MSP68 is a novel therapeutic candidate for regulating inflammation and tissue damage caused by excessive neutrophil migration in sepsis.

Milk fat globule epidermal growth factor 8 inhibits periodontitis in non-human primates and its gingival crevicular fluid levels can differentiate periodontal health from disease in humans.

AIM: We have previously shown that the secreted glycoprotein milk fat globule epidermal growth factor 8 (MFG-E8) has anti-inflammatory and anti-osteoclastogenic properties. Our objective was to investigate the potential of MFG-E8 as a diagnostic or therapeutic agent in periodontitis. MATERIALS AND METHODS: Periodontitis was induced in non-human primates (NHPs) by placing ligatures around posterior teeth on both halves of the mandible for a split-mouth design: one side was treated with MFG-E8-Fc and the other with Fc control. Disease was assessed by clinical periodontal examinations, radiographic analysis of bone loss, and analysis of cytokine mRNA expression in gingival biopsy samples. Gingival crevicular fluid (GCF) was collected from human healthy volunteers or subjects with gingivitis, chronic moderate periodontitis, or chronic severe periodontitis. Additionally, GCF was collected from a subset of severe periodontitis patients following scaling and root planing (SRP) and after pocket reduction surgery. GCF was analysed to quantify MFG-E8 and periodontitis-relevant cytokines using multiplex assays. RESULTS: In NHPs, sites treated with MFG-E8-Fc exhibited significantly less ligature-induced periodontal inflammation and bone loss than Fc control-treated sites. In humans, the GCF levels of MFG-E8 were significantly higher in health than in periodontitis, whereas the reverse was true for the proinflammatory cytokines tested. Consistently, MFG-E8 was elevated in GCF after both non-surgical (SRP) and surgical periodontal treatment of periodontitis patients. CONCLUSION: MFG-E8 is, in principle, a novel therapeutic agent and biomarker of periodontitis.

CD200R1 agonist attenuates glial activation, inflammatory reactions, and hypersensitivity immediately after its intrathecal application in a rat neuropathic pain model.

BACKGROUND: Interaction of CD200 with its receptor CD200R has an immunoregulatory role and attenuates various types of neuroinflammatory diseases. METHODS: Immunofluorescence staining, western blot analysis, and RT-PCR were used to investigate the modulatory effects of CD200 fusion protein (CD200Fc) on activation of microglia and astrocytes as well as synthesis of pro- (TNF, IL-1ß, IL-6) and anti-inflammatory (IL-4, IL-10) cytokines in the L4-L5 spinal cord segments in relation to behavioral signs of neuropathic pain after unilateral sterile chronic constriction injury (sCCI) of the sciatic nerve. Withdrawal thresholds for mechanical hypersensitivity and latencies for thermal hypersensitivity were measured in hind paws 1 day before operation; 1, 3, and 7 days after sCCI operation; and then 5 and 24 h after intrathecal application of artificial cerebrospinal fluid or CD200Fc. RESULTS: Seven days from sCCI operation and 5 h from intrathecal application, CD200Fc reduced mechanical and thermal hypersensitivity when compared with control animals. Simultaneously, CD200Fc attenuated activation of glial cells and decreased proinflammatory and increased anti-inflammatory cytokine messenger RNA (mRNA) levels. Administration of CD200Fc also diminished elevation of CD200 and CD200R proteins as a concomitant reaction of the modulatory system to increased neuroinflammatory reactions after nerve injury. The anti-inflammatory effect of CD200Fc dropped at 24 h after intrathecal application. CONCLUSIONS: Intrathecal administration of the CD200R1 agonist CD200Fc induces very rapid suppression of neuroinflammatory reactions associated with glial activation and neuropathic pain development. This may constitute a promising and novel therapeutic approach for the treatment of neuropathic pain.

Systemic Radioligand Therapy with (177)Lu Labeled Prostate Specific Membrane Antigen Ligand for Imaging and Therapy in Patients with Metastatic Castration Resistant Prostate Cancer.

PURPOSE: We report our initial clinical experience with ß -emitting (177)Lu-PSMA-I&T ((177)Lu labeled prostate specific membrane antigen ligand for imaging and therapy) for systemic treatment of metastatic castration resistant prostate cancer. MATERIALS AND METHODS: Patients with metastatic castration resistant prostate cancer who experienced treatment failure with chemotherapy and novel androgen receptor targeted therapy were treated for 8 weeks with up to 4 cycles of (177)Lu-PSMA-I&T. We report safety data, the antitumor response with prostate specific antigen decreases and the radiographic tumor response as well as the clinical outcome with changes in ECOG (Eastern Cooperative Oncology Group) performance status and pain severity. RESULTS: The first 3 patients were treated with a lower activity of 3.7 GBq in cycle 1. Due to a favorable safety profile the activity was increased to 7.4 GBq in 19 subsequent patients who completed a total of 40 cycles. With the higher activity no grade 3/4 toxicities were observed. The main nonhematological and hematological grade 1/2 toxicities were dry mouth in 7 patients (37%), anemia in 6 (32%) and thrombopenia in 5 (25%). The proportion of patients who achieved a maximum prostate specific antigen decrease of 30% or greater, 50% or greater and 90% or greater was 56%, 33% and 11%, respectively. Combined assessment of bone and soft tissue metastases showed complete remission in 5% of patients, stable disease in 63% and progressive disease in 32%. ECOG performance status improved or was stable in 74% of patients. Of men with bone pain 58% achieved complete resolution or reduced pain. CONCLUSIONS: Radioligand therapy with (177)Lu-PSMA-I&T appears to be safe and active in heavily pretreated patients with metastatic castration resistant prostate cancer.

Recombinant human MFG-E8 ameliorates colon damage in DSS- and TNBS-induced colitis in mice.

Inflammatory bowel disease (IBD) is characterized by chronic inflammation of the digestive system and typically requires lifelong medical care. Recombinant human MFG-E8 (rhMFG-E8) is a 364-amino acid protein, which promotes apoptotic cell clearance and reduces inflammation. This study investigates the therapeutic effect of rhMFG-E8 on two well-established mouse models of IBD. Acute mucosal injury leading to colitis was caused by exposing C57BL/6 mice to 4% dextran sodium sulfate (DSS) in the drinking water over 7 days, and BALB/c mice to a single intrarectal dose of 2.75 mg of 2,4,6-trinitrobenzene sulfonic acid (TNBS). Upon clinical onset of colitis (day 2 in the DSS model and day 1 in the TNBS model), mice were treated with daily subcutaneous injections of rhMFG-E8 (60 or 120 µg/kg/day) or vehicle (saline) for 6 days. Treatment with rhMFG-E8 significantly attenuated colitis in both models in a dose-dependent way. Treatment of DSS-induced colitis with rhMFG-E8 (120 µg/kg/day) decreased weight loss by 59%, the colitis severity score by 71%, and colon shrinkage by 49% when compared with vehicle. Similarly, treatment of TNBS-induced colitis with rhMFG-E8 (120 µg/kg/day) decreased weight loss by 97%, the colitis severity score by 82%, and colon shrinkage by 62% when compared with vehicle. In both models, the colons of animals receiving rhMFG-E8 showed marked reduction in neutrophil infiltration, cytokine and chemokine expression, and apoptotic cell counts. In conclusion, rhMFG-E8 ameliorates DSS- and TNBS-induced colitis, suggesting that it has the potential to become a novel therapeutic agent for IBD.

Milk fat globule epidermal growth factor-factor 8-derived peptide attenuates organ injury and improves survival in sepsis.

INTRODUCTION: Sepsis involves overwhelming inflammatory responses with subsequent immune-suppression that can lead to multiple organ dysfunction and ultimately death. Milk fat globule epidermal growth factor-factor 8 (MFG-E8) is a secretory protein found to have multiple biological activities against autoimmune and inflammatory diseases. MFG-E8 contains an Arg-Gly-Asp (RGD) motif involved in cell-cell and cell-matrix interactions. In sepsis, excessive neutrophils migration through endothelial cells and matrix to sites of inflammation results in organ damage. We hypothesized that MFG-E8-derived short peptides (MSP) flanking its RGD motif could provide protection against organ injury in sepsis. METHODS: The differentiated human neutrophil-like HL-60 cells (dHL60) were incubated with a series of peptides flanking the RGD motif of human MFG-E8 for a cell adhesion assay to fibronectin or human pulmonary artery endothelial cells (PAECs). For the induction of sepsis, male C57BL/6 mice (20-25 g) were subjected to cecal ligation and puncture (CLP). Peptide MSP68 (1 mg/kg body weight) or normal saline (vehicle) was injected intravenously at 2 h after CLP. Blood and tissue samples were collected at 20 h after CLP for various measurements. RESULTS: After screening, peptide MSP68 (VRGDV) had the highest inhibition of dHL-60 cell adhesion to fibronectin by 55.8 % and to PAEC by 67.7 %. MSP68 treatment significantly decreased plasma levels of organ injury marker AST by 37.1 % and the proinflammatory cytokines IL-6 and TNF-α by 61.9 % and 22.1 %, respectively after CLP. MSP68 improved the integrity of microscopic architectures, decreased IL-6 levels in the lungs by 85.1 %, and reduced apoptosis. MSP68 treatment also significantly reduced the total number of neutrophil infiltration by 61.9 % and 48.3 % as well as MPO activity by 40.8 % and 47.3 % in the lungs and liver, respectively, after CLP. Moreover, the number of bacteria translocated to mesenteric lymph nodes was decreased by 57 % with MSP68 treatment. Finally, the 10-day survival rate was increased from 26 % in the vehicle group to 58 % in the MSP68-treated group. CONCLUSIONS: MSP68 effectively inhibits excessive neutrophils infiltrating to organs, leading to moderate attenuation of organ injury and significantly improved survival in septic mice. Thus, MSP68 may be a potential therapeutic agent for treating sepsis.