Towards Complete Tumor Resection: Novel Dual-Modality Probes for Improved Image-Guided Surgery of GRPR-Expressing Prostate Cancer

Nuclear and optical dual-modality probes can be of great assistance in prostate cancer localization, providing the means for both preoperative nuclear imaging and intraoperative surgical guidance. We developed a series of probes based on the backbone of the established GRPR-targeting radiotracer NeoB. The inverse electron demand of the Diels–Alder reaction was used to integrate the sulfo-cyanine 5 dye. Indium-111 radiolabeling, stability studies and a competition binding assay were carried out. Pilot biodistribution and imaging studies were performed in PC-3 tumor-bearing mice, using the best two dual-labeled probes. The dual-modality probes were radiolabeled with a high yield (> 92%), were proven to be hydrophilic and demonstrated high stability in mouse serum (> 94% intact labeled ligand at 4 h). The binding affinity for the GRPR was in the nanomolar range (21.9–118.7 nM). SPECT/CT images at 2 h p.i. clearly visualized the tumor xenograft and biodistribution studies, after scanning confirmed the high tumor uptake (8.47 ± 0.46%ID/g and 6.90 ± 0.81%ID/g for probe [111In]In-12 and [111In]In-15, respectively). Receptor specificity was illustrated with blocking studies, and co-localization of the radioactive and fluorescent signal was verified by ex vivo fluorescent imaging. Although optimal tumor-to-blood and tumor-to-kidney ratios might not yet have been reached due to the prolonged blood circulation, our probes are promising candidates for the preoperative and intraoperative visualization of GRPR-positive prostate cancer.

Optimization of SARS-CoV-2 Spike Protein Expression in the Silkworm and Induction of Efficient Protective Immunity by Inoculation With Alum Adjuvants

The newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing a spread of coronavirus disease 2019 (COVID-19) globally. In order to end the COVID-19 pandemic, an effective vaccine against SARS-CoV-2 must be produced at low cost and disseminated worldwide. The spike (S) protein of coronaviruses plays a pivotal role in the infection to host cells. Therefore, targeting the S protein is one of the most rational approaches in developing vaccines and therapeutic agents. In this study, we optimized the expression of secreted trimerized S protein of SARS-CoV-2 using a silkworm-baculovirus expression vector system and evaluated its immunogenicity in mice. The results showed that the S protein forming the trimeric structure was the most stable when the chicken cartilage matrix protein was used as the trimeric motif and could be purified in large amounts from the serum of silkworm larvae. The purified S protein efficiently induced antigen-specific antibodies in mouse serum without adjuvant, but its ability to induce neutralizing antibodies was low. After examining several adjuvants, the use of Alum adjuvant was the most effective in inducing strong neutralizing antibody induction. We also examined the adjuvant effect of paramylon from Euglena gracilis when administered with the S protein. Our results highlight the effectiveness and suitable construct design of the S protein produced in silkworms for the subunit vaccine development against SARS-CoV-2.

The downregulation of type I IFN signaling in G-MDSCs under tumor conditions promotes their development towards an immunosuppressive phenotype

Tumors modify myeloid cell differentiation and induce an immunosuppressive microenvironment. Granulocytic myeloid-derived suppressor cells (G-MDSCs), the main subgroup of myeloid-derived suppressor cells (MDSCs), are immature myeloid cells (IMCs) with immunosuppressive activity and exist in tumor-bearing hosts. The reason why these cells diverge from a normal differentiation pathway and are shaped into immunosuppressive cells remains unclear. Here, we reported that the increase of granulocyte colony-stimulating factor (G-CSF) in mouse serum with tumor progression encouraged G-MDSCs to obtain immunosuppressive traits in peripheral blood through the PI3K-Akt/mTOR pathway. Importantly, we found that downregulation of type I interferon (IFN-I) signaling in G-MDSCs was a prerequisite for their immunosuppressive effects. Suppressor of cytokine signaling (SOCS1), the action of which is dependent on IFN-I signaling, inhibited the activation of the PI3K-Akt/mTOR pathway by directly interacting with Akt, indicating that the differentiation of immunosuppressive G-MDSCs involves a transition from immune activation to immune tolerance. Our study suggests that increasing IFN-I signaling in G-MDSCs may be a strategy for reprograming immunosuppressive myelopoiesis and slowing tumor progression.

Complement-Mediated Selective Tumor Cell Lysis Enabled by Bi-Functional RNA Aptamers

Unlike microbes that infect the human body, cancer cells are descended from normal cells and are not easily recognizable as “foreign” by the immune system of the host. However, if the malignant cells can be specifically earmarked for attack by a synthetic “designator”, the powerful effector mechanisms of the immune response can be conscripted to treat cancer. To implement this strategy, we have been developing aptamer-derived molecular adaptors to invoke synthetic immune responses against cancer cells. Here we describe multi-valent aptamers that simultaneously bind target molecules on the surface of cancer cells and an activated complement protein, which would tag the target molecules and their associated cells as “foreign” and trigger multiple effector mechanisms. Increased deposition of the complement proteins on the surface of cancer cells via aptamer binding to membrane targets could induce the formation of the membrane attack complex or cytotoxic degranulation by phagocytes and natural killer cells, thereby causing irreversible destruction of the targeted cells. Specifically, we designed and constructed a bi-functional aptamer linking EGFR and C3b/iC3b, and used it in a cell-based assay to cause lysis of MDA-MB-231 and BT-20 breast cancer cells, with either human or mouse serum as the source of complement factors.

Exosome-Mediated eCIRP Release From Macrophages to Induce Inflammation in Sepsis

Extracellular cold-inducible RNA-binding protein (eCIRP) is an important damage-associated molecular pattern (DAMP). Despite our understanding of the potentially harmful effects of eCIRP in sepsis, how eCIRP is released from cells remains elusive. Exosomes are endosome-derived extracellular vesicles, which carry proteins, lipids, and nucleic acids to facilitate intercellular communication and several extracellular functions. We hypothesized that eCIRP is released via exosomes to induce inflammation in sepsis. Exosomes isolated from the supernatants of LPS-treated macrophage culture and serum of endotoxemia and polymicrobial sepsis mice showed high purity, as revealed by their unique median sizes ranging between 70 and 126 nm in diameter. eCIRP levels of the exosomes were significantly increased after LPS treatment in the supernatants of macrophage culture, mouse serum, and cecal ligation and puncture (CLP)-induced sepsis mouse serum. Protease protection assay demonstrated the majority of eCIRP was present on the surface of exosomes. Treatment of WT macrophages and mice with exosomes isolated from LPS-treated WT mice serum increased TNFα and IL-6 production. However, treatment with CIRP−/- mice serum exosomes significantly decreased these levels compared with WT exosome-treated conditions. CIRP−/- mice serum exosomes significantly decreased neutrophil migration in vitro compared with WT exosomes. Treatment of mice with serum exosomes isolated from CIRP−/- mice significantly reduced neutrophil infiltration into the peritoneal cavity. Our data suggest that eCIRP can be released via exosomes to induce cytokine production and neutrophil migration. Thus, exosomal eCIRP could be a potential target to inhibit inflammation.

Serum Amyloid A promotes Acetaminophen-induced liver injury by damaging sinusoidal endothelial cell and exacerbating platelet aggregation in liver

BackgroundAcetaminophen (APAP) is the most commonly used non-prescription antipyretic and analgesic drugs. Overuse of APAP can cause hepatotoxicity. Liver sinusoidal endothelial cells (LSECs) damage is an important early event in APAP-induced liver injury. Serum amyloid A (SAA) is an acute phase protein that mainly produced by hepatocytes, and promotes endothelial dysfunction via a pro-inflammatory and pro-thrombotic effect in atherosclerosis and renal disease. However, the role of SAA in APAP-induced liver injury remains unclear.MethodsIn this study, we used neutralizing antibody (anti-SAA) or antagonistic small peptide derived from sequence of human SAA1/2 (SAA-pep) to block the functional activity of Saa1/2 in mouse serum. Immunohistochemistry staining, Evans blue and platelet adhesion assays were performed to examine the liver damage, the integrity of sinusoidal endothelium and platelets accumulation in APAP-induce liver injury.ResultsOur study showed that in the early stage of APAP-induced acute liver injury in mice, the intrahepatic and serum Saa1/2 levels were significantly increased within 24 hours, and then gradually reduced to normal level from 3 days. Neutralization of Saa1/2 by antibodies or peptides effectively prevented the destruction of hepatic sinusoids, reduced the intrahepatic hemorrhage and platelet accumulation in liver, as well as increased the survival rate of mice treated with lethal dose of APAP. In vitro experiments showed that Saa1/2 aggravated LSECs death induced by APAP. Moreover, Saa1/2 promoted platelets adhesion on LSECs via Tlr2/Vcam-1 axis.ConclusionOur findings suggest that Saa1/2 promotes APAP-induced liver injury by damaged LSECs and exacerbated platelets aggregation. This study provides a potential target for intervention of acute liver injury/failure caused by hepatotoxic drugs such as APAP.

Silk Sericin Activates Mild Immune Response and Increases Antibody Production

To clarify whether nanoparticles of silk sericin (SS) and silk fibroin (SF) can induce inflammation and immune responses, we analyzed splenocyte proliferation, apoptosis and cytokine release to identify the effects of SS and SF on mouse splenocytes in vitro. We implanted mice with SS and SF through intraperitoneal, intramuscular, and subcutaneous routes to evaluate the innate and adaptive immune response to SS and SF in vivo. Cytokines in the serum and spleen were analyzed by Luminex and antibody array. Antigen-specific antibodies were evaluated by enzyme-linked immunosorbent assay (ELISA) at week 1 and 5 after implantation. Distinct cell populations in the spleen and bone marrow were analyzed by flow cytometry. SS suppressed the proliferation of splenocytes and CD11b+CD27− NK cells, induced splenocyte apoptosis, and increased interleukin-1 β (IL-1 β) and tumor necrosis factor-α (TNF-α) in the culture supernatant. SF suppressed splenocyte proliferation, induced splenocyte apoptosis, and increased the titer of TNF-α in culture supernatants. At both week 1 and 5 after implantation with SS, mouse serum interleukin-1 α (IL-1 α) and keratinocyte chemoattractant (KC) were decreased, SS-specific antibody was increased, the proportion of bone marrow CD4+ T cells was increased, and the proportion of splenic neutrophils was decreased. At week 5 after subcutaneous implantation with SF, mouse serum IL-1α, and splenic IL-6, TIMP-1, IL-4, MCP-1, IFN-γ, TCA-3, TNF-α, and IL-17 were decreased. SS was able to induce a mild immune response, as evidenced by CD4+ T cell activation, splenocyte apoptosis, and antigen-specific antibody secretion. Comparatively, SF had low immunogenicity and anti-inflammatory properties.

A host-directed macrocyclic peptide therapeutic for MDR gram negative bacterial infections

The emergence of infections by carbapenem resistant Enterobacteriaceae (CRE) pathogens has created an urgent public health threat, as carbapenems are among the drugs of last resort for infections caused by a growing fraction of multi-drug resistant (MDR) bacteria. There is global consensus that new preventive and therapeutic strategies are urgently needed to combat the growing problem of MDR bacterial infections. Here, we report on the efficacy of a novel macrocyclic peptide, minimized theta-defensin (MTD)-12813 in CRE sepsis. MTD12813 is a theta-defensin inspired cyclic peptide that is highly effective against CRE pathogens K. pneumoniae and E. coli in vivo. In mouse septicemia models, single dose administration of MTD12813 significantly enhanced survival by promoting rapid host-mediated bacterial clearance and by modulating pathologic cytokine responses, restoring immune homeostasis, and preventing lethal septic shock. The peptide lacks direct antibacterial activity in the presence of mouse serum or in peritoneal fluid, further evidence for its indirect antibacterial mode of action. MTD12813 is highly stable in biological matrices, resistant to bacterial proteases, and nontoxic to mice at dose levels 100 times the therapeutic dose level, properties which support further development of the peptide as a first in class anti-infective therapeutic.

Fortified Fermented Rice-Acid Can Regulate the Gut Microbiota in Mice and Improve the Antioxidant Capacity

The study aimed to explore the effects of fortified fermented rice-acid on the antioxidant capacity of mouse serum and the gut microbiota. Hair characteristics, body mass index, intestinal villus height, intestinal crypt depth, serum antioxidant capacity, and gut microbiota of mice were first measured and the correlation between the antioxidant capacity of mouse serum and the gut microbiota was then explored. The mice in the lactic acid bacteria group (L-group), the mixed bacteria group (LY-group), and the rice soup group (R-group) kept their weight well and had better digestion. The mice in the L-group had the better hair quality (dense), but the hair quality in the R-group and the yeast group (Y-group) was relatively poor (sparse). In addition, the inoculation of Lactobacillus paracasei H4-11 (L. paracasei H4-11) and Kluyveromyces marxianus L1-1 (K. marxianus L1-1) increased the villus height/crypt depth of the mice (3.043 ± 0.406) compared to the non-inoculation group (R-group) (2.258 ± 0.248). The inoculation of L. paracasei H4-11 and K. marxianus L1-1 in fermented rice-acid enhanced the blood antioxidant capacity of mouse serum (glutathione 29.503 ± 6.604 umol/L, malonaldehyde 0.687 ± 0.125 mmol/L, catalase 15.644 ± 4.618 U/mL, superoxide dismutase 2.292 ± 0.201 U/mL). In the gut microbiota of L-group and LY-group, beneficial microorganisms (Lactobacillus and Blautia) increased, but harmful microorganisms (Candidatus Arthromitus and Erysipelotrichales) decreased. L. paracasei H4-11 and K. marxianus L1-1 might have a certain synergistic effect on the improvement in antibacterial function since they reduced harmful microorganisms in the gut microbiota of mice. The study provides the basis for the development of fortified fermented rice-acid products for regulating the gut microbiota and improving the antioxidant capacity.

Lactobacillus fermentum ZS40 Ameliorates Inflammation in Mice With Ulcerative Colitis Induced by Dextran Sulfate Sodium

Ulcerative colitis is an inflammatory disease of the intestine caused by many reasons, and it may even develop into colon cancer. Probiotics are normal bacteria that exist in the human body and have been proven to regulate the balance of intestinal flora and alleviate inflammation. The current study aimed to study the effect of Lactobacillus fermentum ZS40 (ZS40) on dextran sulfate sodium (DSS)-induced ulcerative colitis mice. The length and weight of the colon were measured, and the histopathological morphological changes of colon tissue were observed to evaluate the effects of ZS40 on colitis. Biochemical kits, ELISA kits, real-time quantitative PCR (RT-qPCR), and western blot were also used to detect the effects of ZS40 on serum and colon tissue related oxidative indicators and pro-inflammatory and anti-inflammatory cytokines. We found that ZS40 could reduce colonic inflammatory cell infiltration and goblet cell necrosis, increase total superoxide dismutase and catalase in mouse serum, and reduce myeloperoxidase and malondialdehyde levels. ZS40 could down-regulate the level of proinflammatory cytokines and up-regulate the level of anti-inflammatory cytokines. More importantly, ZS40 down-regulated the relative expression of nuclear factor-κB p65 (NF-κBp65), IL-6, and TNF-α mRNA and protein, up-regulated the relative expression of inhibitor kapa B alpha (IκB-α). By regulating the NF-κB and MAPK pathways to down-regulated the relative expression of p38 and JNK1/2 mRNA and p38, p-p38, JNK1/2, and p-JNK1/2 proteins. Our study suggested that ZS40 may serve as a potential therapeutical strategy for ulcerative colitis.