PBS Control Group Research Articles

Evaluation of protective immune responses induced by DNA vaccines encoding Echinococcus granulosus EgM123 protein in Beagle dogs.

Echinococcus granulosus, known as cystic echinococcosis, is a prominent zoonotic parasitic disease of significant global concern. The definitive hosts serves as the primary reservoir for the transmission of echinococcosis, as well as a main factor in the prevention and control of the disease. Unfortunately, there is currently no commercially available vaccine for these hosts. Nevertheless, DNA vaccines show potential as a feasible strategy for the control and management of parasitic diseases. In this study, the EgM123 antigen was selected for its well-documented immunogenic properties to develop a DNA vaccine aimed at combating E. granulosus infection in canines. The results showed a marked increase in IgG levels in the group vaccinated with pVAX1-EgM123 DNA compared to the PBS group. Additionally, the cytokines IL-1, IFN-γ, IL-4, and IL-6 were significantly upregulated in the pVAX1-EgM123 DNA vaccine group. Furthermore, in comparison to the PBS control group, the EgM123 DNA vaccine group exhibited a notable 87.85% reduction in worm burden and a 65.00% inhibition in segment development. These findings indicate that the pVAX1-EgM123 DNA vaccine shows promising immunogenicity, successfully eliciting a targeted immune response in canines. Moreover, it significantly diminishes the worm burden and hinders the progression of tapeworms in the pVAX1-EgM123 DNA vaccine group. These findings suggest that the pVAX1-EgM123 DNA vaccine holds promise as a potential candidate vaccine for combating E. granulosus infection in dogs.

Niosomes containing enciprazine hydrochloride have been shown to efficiently inhibit the proliferation and induce apoptosis in colorectal cancer cells

Colorectal cancer (CRC), currently the second most widespread cancer globally, exhibits a higher incidence in young individuals. Advancements have been made in developing anti-colorectal cancer drugs, including cytotoxic chemicals, in the past few decades. There is a need for new and innovative medications to overcome the current challenges in cancer treatment. Recent research examined the efficacy of innovative formulations in the prevention of colorectal cancer. In this study, we evaluated the efficacy of a niosome formulation loaded with Enciprazine hydrochloride (Nio-USAN). We assessed the anti-colorectal cancer characteristics of Nio-USAN by employing several techniques including CCK-8, invasion test, MTT test, flow cytometry, and cell cycle assessment. Quantitative real-time PCR was utilized to assess the transcription of genes linked to apoptosis. The F1-Nio-USAN and F2-Nio-USAN have average sizes of 200 and 500 nm, respectively. The entrapment effectiveness (EE%) of the F1-Nio-USAN and F2-Nio-USAN was measured to be 85.32 ± 0.27 % and 87.12 ± 0.35 %, respectively. The F1-Nio-USAN group exhibited the following percentages of HT-29 cell states: 43 % early apoptosis, 21 % late apoptosis, 7 % necrotic, and 29 % viable. The levels of transcription for cas8, Bid, BAX, cas9, and cas3 were significantly elevated in the treatment groups as compared to the PBS control group (P < 0.001). In addition, the treatment group exhibited significantly reduced levels of BCL2 gene transcription compared to the PBS control group (P < 0.01). These results suggest that it may be possible to improve the efficacy of USAN formulations in combating cancer by utilizing noisome encapsulation.

Oral administration of recombinant Lactococcus lactis expressing largemouth bass (Micropterus salmoides) IFNa3 protein enhances immune response against largemouth bass virus (LMBV) infection

Largemouth bass virus (LMBV) is a highly pathogenic pathogen that often causes high mortality of affected largemouth bass and significant financial losses. Type I interferon as an effective and broad spectrum tool has been successfully used for therapeutic or prophylactic treatment some viral infections. However, the implementation of immunotherapies based on interferon administration to combat LMBV infections has not been reported. And Lactic Acid Bacteria (LAB) are a powerful vehicle for expressing cytokines or immunostimulant peptides at the gastrointestinal level after oral administration. In this study, Lactococcus lactis (L. lactis) expression system with lactose as a screening marker was utilized to express the Micropterus salmoides interferon a3 (IFNa3) protein and orally administered to largemouth bass. The genetically engineered strain pNZ8149-Usp45-IFNa3-6His/L. lactis NZ3900 was successfully constructed, and its potential to elicit immune protection response by oral administration was evaluated. After orally administration, the recombinant L. lactis was detected in guts of experimental fish and remained detectable for 72 h. Additionally, IFNa3 was able to enhance the test fish's immune response, as determined by the relatively increased mRNA relative expression of immune-related genes in the liver, spleen, and kidney tissues, including IFN-γ, TNF-α, IL-1β, IL-8, IgM and IgT. Following LMBV challenge, the experiment group of pNZ8149-Usp45-IFNa3-6His/L. lactis NZ3900 exhibited a 70 % survival rate, while survival rate were 15 % in the PBS control group, 45 % in the pNZ8149/L. lactis NZ3900 group. Furthermore, the viral load in the surviving fish was significantly lower than that of the control groups. These findings suggest that oral administration of recombinant L. lactis producing IFNa3 induces largemouth bass immune responses at a systemic level to effective prevent and combat of LMBV infection.

High expression of CX3CL1/CX3CR1 at the mother-fetus interface of preeclampsia inhibits trophoblast invasion and migration

IntroductionPreeclampsia is associated with maternal inflammatory overreaction and imbalanced immunity at the mother-fetus interface. The pro-inflammatory chemokine fractalkine (CX3CL1) is recently recognized apart from imbalanced immunity. In this study, CX3CL1- CX3C chemokine receptor 1(CX3CR1) regulation of decidual macrophage function and trophoblast invasion ability in preeclampsia was initially explored. MethodsThe study comprised 60 women allocated to NP group (normotensive pregnant woman, n = 30) and sPE group (woman with severe preeclampsia, n = 30). After the delivery, the expression of CX3CL1 in placental tissues of the two groups was detected by immunohistochemical analysis. The protein level of CX3CL1 in placental tissue and CX3CR1 in decidua tissue was detected by Western Blot and the localization of CX3CR1 expression in decidua was detected by immunofluorescence. Macrophages were polarized into classically activated (M1) macrophages. M1 were treat with PBS (control group), recombinant human CX3CL1 (CX3CL1 group), recombinant human CX3CL1+ selective CX3CR1 antagonist-JMS-17-2 (CX3CL1+anti-CX3CR1 group) and recombinant human CX3CL1 + selective CX3CR1 antagonist-JMS-17-2 + VS-6063 (CX3CL1+anti-CX3CR1+ FAK inhibitor group). M1 and HTR8/SVneo cells were co-cultured as described previously to assess invasion and migration capacity by transwell assays and Wound-healing assay. ResultsIn this study, CX3CL1 expression is high in the placental tissues of severe preeclampsia (sPE) patients than in normotensive pregnancies (NP). CX3CR1 expression is high in the decidual tissues of severe preeclampsia patients and mainly expressed in macrophages of decidual tissues. CX3CL1/CX3CR1 decreased VEGF expression in M1 macrophages and reduced the invasion and migration function of HTR-8/SVneo through the FAK signaling pathway. DiscussionThese findings revealed that CX3CL1-CX3CR1 regulate the trophoblast function by FAK and provided new insights into the pathogenesis of preeclampsia.

Novel strategy of S. mutans gcrR gene over-expression plus antibacterial dimethylaminohexadecyl methacrylate suppresses biofilm acids and reduces dental caries in rats

ObjectiveStreptococcus mutans (S. mutans) is a major contributor to dental caries, with its ability to synthesize extracellular polysaccharides (EPS) and biofilms. The gcrR gene is a regulator of EPS synthesis and biofilm formation. The objectives of this study were to investigate a novel strategy of combining gcrR gene over-expression with dimethylaminohexadecyl methacrylate (DMAHDM), and to determine their in vivo efficacy in reducing caries in rats for the first time. MethodsTwo types of S. mutans were tested: Parent S. mutans; and gcrR gene over-expressed S. mutans (gcrR OE S. mutans). Bacterial minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were measured with DMAHDM and chlorhexidine (CHX). Biofilm biomass, polysaccharide, lactic acid production, live/dead staining, colony-forming units (CFUs), and metabolic activity (MTT) were evaluated. A Sprague-Dawley rat model was used with parent S. mutans and gcrR OE S. mutans colonization to determine caries-inhibition in vivo. ResultsDrug-susceptibility of gcrR OE S. mutans to DMAHDM or CHX was 2-fold higher than that of parent S. mutans. DMAHDM reduced biofilm CFU by 3–4 logs. Importantly, the combined gcrR OE S. mutans+ DMAHDM dual strategy reduced biofilm CFU by 5 logs. In the rat model, the parent S. mutans group had a higher cariogenicity in dentinal (Dm) and extensive dentinal (Dx) regions. The DMAHDM + gcrR OE group reduced the Dm and Dx caries to only 20 % and 0 %, those of parent S. mutans + PBS control group (p < 0.05). The total caries severity of gcrR OE + DMAHDM group was decreased to 51 % that of parent S. mutans control (p < 0.05). SignificanceThe strategy of combining S. mutans gcrR over-expression with antibacterial monomer reducing biofilm acids by 97 %, and reduced in vivo total caries in rats by 48 %. The gcrR over-expression + DMAHDM strategy is promising for a wide range of dental applications to inhibit caries and protect tooth structures.

NOX2 Enzyme Mimicking Nano-Networks Regulate Tumor-Associated Macrophages to Initiate Both Innate and Adaptive Immune Effects.

Macrophages, capable of both direct killing and antigen presentation, are crucial for the interplay between innate and adaptive immunity. However, strategies mainly focus on polarizing tumor-associated macrophages (TAMs) to M1 phenotype, while overlooking the inefficient antigen cross-presentation due to hyperactive hydrolytic protease within lysosomes which leads to antigen degradation. In light of the significant influence of reactive oxygen species (ROS) on TAMs' polarization and the inhibition of phagosomal proteolysis, a novel nanosystem termed OVA-Fe-GA (OFG) is engineered, drawing inspiration from the NOX2 enzyme's role. OFG integrates ovalbumin (OVA) and a network composed of Fe-gallic acid (GA), emulating the NOX2 enzyme's sequential ROS generation process ("O2 to O2 •- to H2O2/•OH"). Furthermore, it elucidates a biological mechanism that augments antigen cross-presentation by suppressing the expression of cysteine proteases. OFG restores the innate anti-tumor functionality of TAMs and significantly amplifies their antigen cross-presentation (4.5-fold compared to the PBS control group) in B16-OVA tumor-bearing mice. Notably, the infiltration and activity of intratumoral CD8+ T cells are enhanced, indicating an adaptive immune response. Moreover, OFG exhibits excellent photothermal properties, thereby fostering a system antitumor immune response. This study provides a promising strategy for initiating both innate and adaptive immunity via TAMs activation.

Salt-Sensitive Hypertension and Renal Damage are Amplified by Adoptive Transfer of CD4+ and Not CD8+ T Cells: A Role of NADPH Oxidase 2 (NOX2)

Infiltration of T cells into the kidney of the Dahl Salt Sensitive (SS) rat model of human disease accompanies the development of salt-sensitive hypertension and renal damage. Deletion of T cells on the Dahl SS genetic background (SSCD247−/−) attenuates hypertension and renal damage, and subsequent replacement of T cells by splenocyte transfer restores the disease phenotype. The present studies were performed to assess if the T cell subtype (CD4+ vs CD8+) is important in Dahl SS hypertension and interrogate the mechanism of action. In initial experiments, adoptive transfer of purified CD4+ or CD8+ T cells (~5 million) from splenocytes isolated from the Dahl SS (CD4+ SS→CD247 or CD8+ SS→CD247) or PBS vehicle (PBS→CD247) into the SSCD247−/− rat was performed on postnatal day 5. Animals were recovered and instrumented with radiotelemeters at 7 weeks of age. After a week of recovery, baseline measurements of blood pressure and albumin excretion were obtained while rats were fed a low salt (LS, 0.4% NaCl) diet. Rats were then switched to a high salt (HS, 4.0% NaCl) diet for 21 days, with continuous blood pressure measurements and urine collections every 7 days. On day 21, the rats were euthanized, and their kidneys were perfused and collected for immune cell analysis by flow cytometry. During the LS baseline period there was no significant difference observed in mean arterial pressure (MAP) between the CD4+ SS→CD247, CD8+ SS→CD247, or PBS→CD247 groups (113±1, 115±1, 114±1 mmHg; n=21, 10, 24, respectively). After three weeks of HS, rats receiving CD4+ T cells from the SS (CD4+ SS→CD247) demonstrated an amplified salt-sensitive hypertension (154±3 mmHg) and albuminuria (184±22 mg/day) compared to blood pressure (140±3 and 145±3 mmHg) and albuminuria (82±9 and 85±8 mg/day) in the CD8+ SS→CD247 or PBS→CD247 groups, respectively. Since the deletion of the p67phox subunit of NADPH oxidase 2 (NOX2) in adoptively transferred splenocytes attenuated Dahl SS hypertension and kidney damage, parallel studies examined the influence of deletion of p67phox in CD4+ (n=14) and CD8+ T cells (n=8). The adoptive transfer of either CD4+ or CD8+ T cells lacking functional NOX2 did not alter the LS or HS blood pressure or albuminuria when compared to the PBS control group. Finally, a flow cytometric analysis at the end of the experiment confirmed the absence of CD3+ T cells in the PBS→CD247 and documented the reconstitution of equal numbers of CD4+ cells in the blood and kidney of the rats receiving CD4+ cells with intact or deficient NOX2. Interestingly, no difference was observed in CD8+ T cells in the kidney and blood of rats receiving CD8+ cells with intact or deficient NOX2, but a significant number of CD4+ T cells were also observed in the SSCD247−/− rats receiving the purified CD8+ T cells. In summary, these studies demonstrate that CD4+ T cells amplify salt-sensitive hypertension and renal damage in the Dahl SS via a NOX2-dependent mechanism while CD8+ T cells do not alter disease phenotypes when compared to PBS control. GRANTS: HL161231, HL166458, Georgia Research Alliance. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Prevotella intermedia boosts OSCC progression through ISG15 upregulation: a new target for intervention

PurposePeriodontitis-associated bacteria, such as Porphyromonas gingivalis and Fusobacterium nucleatum, are closely linked to the risk of oral squamous cell carcinoma (OSCC). Emerging studies have indicated that another common periodontal pathogen, Prevotella intermedia (P. intermedia), is enriched in OSCC and could affect the occurrence and progression of OSCC. Our aim is to determine the effects of P. intermedia on the progression of OSCC and the role of antibiotics in reversing these effects.MethodsIn this study, a murine xenograft model of OSCC was established, and the mice were injected intratumorally with PBS (control group), P. intermedia (P.i group), or P. intermedia combined with an antibiotic cocktail administration (P.i + ABX group), respectively. The effects of P. intermedia and ABX administration on xenograft tumor growth, invasion, angiogenesis, and metastasis were investigated by tumor volume measurement and histopathological examination. Enzyme-linked immunosorbent assay (ELISA) was used to investigate the changes in serum cytokine levels. Immunohistochemistry (IHC) was adopted to analyze the alterations in the levels of inflammatory cytokines and infiltrated immune cells in OSCC tissues of xenograft tumors. Transcriptome sequencing and analysis were conducted to determine differential expression genes among various groups.ResultsCompared with the control treatment, P. intermedia treatment significantly promoted tumor growth, invasion, angiogenesis, and metastasis, markedly affected the levels of inflammatory cytokines, and markedly altered M2 macrophages and regulatory T cells (Tregs) infiltration in the tumor microenvironment. However, ABX administration clearly abolished these effects of P. intermedia. Transcriptome and immunohistochemical analyses revealed that P. intermedia infection increased the expression of interferon-stimulated gene 15 (ISG15). Correlation analysis indicated that the expression level of ISG15 was positively correlated with the Ki67 expression level, microvessel density, serum concentrations and tissue expression levels of inflammatory cytokines, and quantities of infiltrated M2 macrophages and Tregs. However, it is negatively correlated with the quantities of infiltrated CD4+ and CD8+ T cells.ConclusionIn conclusion, intratumoral P. intermedia infection aggravated OSCC progression, which may be achieved through upregulation of ISG15. This study sheds new light on the possible pathogenic mechanism of intratumoral P. intermedia in OSCC progression, which could be a prospective target for OSCC prevention and treatment.

Abstract 4068: In vitro masked effect, in vivo anti-tumor activity and toxicology study of KGX101, an interleukin-12 prodrug, in monotherapy or in combination with anti-PD-L1 antibody

Abstract Interleukin-12 (IL-12) has long been limited in its clinical use due to its short half-life (t1/2) and dose-related toxicity, despite its remarkable ability to activate effector functions in T cells and natural killer (NK) cells. To address these challenges, we engineered an IL-12 prodrug called KGX101. In KGX101, the C-terminus of IL-12 is fused with ADCC-impaired IgG1 Fc to extend its half-life. Meanwhile, the N-terminus is concealed with its corresponding receptor soluble domains, IL-12Rβ1 and IL-12Rβ2, using tumor-specific protease cleavable linkers. This innovative design prevents systemic immune system overactivation. Once the linker is cleaved by tumor microenvironment (TME)-specific proteases, KGX101 functions like IL-12, effectively stimulating T cells and NK cells, reshaping the TME, and displaying significant potential in cancer therapy. To confirm KGX101's masking effect, we conducted IFNγ induction efficacy tests using peripheral blood mononuclear cells (PBMCs) from three donors. KGX101 demonstrated a 300 to 2500-fold reduction in activity compared to unmasked IL-12-Fc. Importantly, the masking effect of KGX101 was nearly abolished after protease digestion, affirming its mechanism of action. To further establish KGX101's effectiveness, we conducted in vivo antitumor studies in hPBMC xenograft mouse models using human colorectal cancer HCT116 and PD-L1-overexpressed human malignant melanoma A375. Mice were subcutaneously implanted with a specific mixture of cancer cells and hPBMCs, and then intravenously injected with PBS or KGX101 at various dose levels twice a week for 4-5 cycles. Compared to the PBS control group, KGX101 demonstrated up to a 42% tumor growth inhibition (TGI) in the HCT116 model at 0.18 mg/kg and a 76% TGI in the hPD-L1-A375 model at 0.06 mg/kg. Analysis of tumor-infiltrating lymphocytes (TILs) revealed that KGX101 increased the percentage of T cells in the tumor environment in a dose-dependent manner. We also evaluated the combination of KGX101 with an anti-PD-L1 antibody in an HCT116/hPBMC xenograft mouse model, which showed limited response to anti-PD-L1 monotherapy. The TGI% for KGX101 at 0.06 mg/kg, anti-PD-L1 antibody at 1 mg/kg, and their combination were 36.56%, 6.93%, and 66.22%, respectively, demonstrating a promising antitumor synergy. In terms of pharmacokinetics, a single dose of KGX101 at 0.06 mg/kg and 0.18 mg/kg in the HCT116/hPBMC xenograft mouse model resulted in a serum half-life ranging from 52.75 to 64.87 hours, while the t1/2 in Cynomolgus monkeys was approximately 3 days. In a 4-week repeat dose toxicity study in Cynomolgus monkeys, the highest non-severely toxic dose (HNSTD) of KGX101 was determined to be 0.1 mg/kg. The results from nonclinical toxicology studies support the potential of KGX101 as a well-tolerated therapy for the First-in-Human (FIH) clinical trial. Citation Format: Shumin Yang, Nuo Li, Jing Wang, Li Ma, Yi Zhao, Hui Chen, Haixiang Wu, Weidong Jiang. In vitro masked effect, in vivo anti-tumor activity and toxicology study of KGX101, an interleukin-12 prodrug, in monotherapy or in combination with anti-PD-L1 antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4068.

Lipolysis inhibition improves the survival of fat grafts through ameliorating lipotoxicity and inflammation.

Fat grafting is a promising technique for correcting soft tissue abnormalities, but oil cyst formation and graft fibrosis frequently impede the therapeutic benefit of fat grafting. The lipolysis of released oil droplets after grafting may make the inflammation and fibrosis in the grafts worse; therefore, by regulating adipose triglyceride lipase (ATGL) via Atglistatin (ATG) and Forskolin (FSK), we investigated the impact of lipolysis on fat grafts in this study. After being removed from the mice and chopped into small pieces, the subcutaneous fat from wild-type C57BL/6J mice was placed in three different solutions for two hours: serum-free cell culture medium, culture medium+FSK (50 μM), and culture medium+ATG (100 μM). Following centrifugation to remove water and free oil droplets, 0.3 mL of the fat particles per mouse was subcutaneously injected into the back of mice. Additionally, the subcutaneous fat grafting area was immediately injected with PBS (control group), ATG (30 mg/kg), and FSK (15 mg/kg) following fat transplantation. Detailed cellular events after grafting were investigated by histological staining, real-time polymerase chain reaction, immunohistochemistry/immunofluorescent staining, and quantification. Two weeks after grafting, grafts treated with ATG showed lower expression of ATGL and decreased mRNA levels of TNFα and IL-6. In contrast, grafts treated with ATG showed elevated expression levels of IL-4 and IL-13 compared to the control grafts. In addition, fewer apoptotic cells and oil cysts were observed in ATG grafts. Meanwhile, a higher CD206+/CD68+ ratio of macrophages and more CD31+ vascular endothelial cells existed in the 2-month ATG grafts. In comparison to the control, ATG treatment improved the volume retention of grafts, and decreased graft fibrosis and oil cyst formation. By preventing oil droplet lipolysis, pharmacological suppression of ATGL shielded adipocytes from lipotoxicity following grafting. Additionally, ATG ameliorated the apoptosis and inflammation brought on by adipocyte death and oil droplet lipolysis in grafted fat. These all indicate that lipolysis inhibition improved transplanted fat survival and decreased the development of oil cysts and graft fibrosis, offering a potential postoperative pharmacological intervention for bettering fat grafting.

Transcriptome analysis provides new insights into the immune response of Ruditapes philippinarum infected with Vibrio alginolyticus

Manila clam (Ruditapes philippinarum) is a bivalve species with commercial value, but it is easily infected by pathogenic microorganisms in aquaculture, which restricts the shellfish industry. Notably, the impact of Vibrio alginolyticus on clam culture is obvious. In this study, RNA-seq was performed to analyze clam hepatopancreas tissue in 48 h (challenge group, G48h) and 96 h (challenge group, G96h) after infection with V. alginolyticus and 0 h after injection of PBS (control group, C). The results showed that a total of 1670 differentially expressed genes were detected in the G48h vs C group, and 1427 differentially expressed genes were detected in the G96h vs C group. In addition, KEGG analysis showed that DEGs were significantly enriched in pathways such as Lysosome and Mitophagy. Moreover, 15 immune related DEGs were selected for qRT-PCR analysis to verify the accuracy of RNA-seq, and the results showed that the expression level of DEGs was consistent with that of RNA-seq. Therefore, the results obtained in this study provides a preliminary understanding of the immune defense of R. philippinarum and molecular insights for genetic breeding of V. alginolyticus resistance in Manila clam.

Immunoprotective Potential of Adenylosuccinate Synthetase Protein (PurA) in Streptococcus equi ssp. zooepidemicus Infections

Background:: Streptococcus equi ssp. zooepidemicus (SEZ) is one important pathogen. There are still sporadic outbreaks in China, northern United States and the Netherlands. Adenylosuccinate synthetase PurA, a newly discovered protein in prior research, requires further assessment of its protective effectiveness. Methods:: In this study, we focused on the expression of recombinant PurA from SEZ ATCC 35246. We evaluated the immunoreactivity of this recombinant protein using convalescent minipig sera. Additionally, we conducted experiments in mice to assess its immunogenic properties. Results:: Our findings revealed that the recombinant PurA triggered a substantial antibody response in mice, resulting in an 80% protection rate against SEZ infection. Notably, mice immunized with PurA exhibited significantly reduced bacterial colonization in all organs compared to the PBS control group. Furthermore, the levels of IL-6, IL-8, IL-1β, and TNF-α in mouse serum were significantly elevated in the PurA-immunized group compared to the control group. Hyperimmune sera targeting PurA effectively eliminated SEZ in bactericidal tests. Remarkably, antibodies against PurA demonstrated a significant inhibitory effect on developing SEZ biofilm. Conclusion:: Immunization with PurA elicited robust humoral and cellular immune responses in mice. These promising results suggest the potential utility of PurA in developing SEZ vaccine immunogens, providing a valuable avenue for further research into SEZ infection prevention and control.

Soluble Angiotensin-Converting Enzyme 2 Protein Improves Survival and Lowers Viral Titers in Lethal Mouse Model of Severe Acute Respiratory Syndrome Coronavirus Type 2 Infection with the Delta Variant.

Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) utilizes angiotensin-converting enzyme 2 (ACE2) as its main receptor for cell entry. We bioengineered a soluble ACE2 protein termed ACE2 618-DDC-ABD that has increased binding to SARS-CoV-2 and prolonged duration of action. Here, we investigated the protective effect of this protein when administered intranasally to k18-hACE2 mice infected with the aggressive SARS-CoV-2 Delta variant. k18-hACE2 mice were infected with the SARS-CoV-2 Delta variant by inoculation of a lethal dose (2 × 104 PFU). ACE2 618-DDC-ABD (10 mg/kg) or PBS was administered intranasally six hours prior and 24 and 48 h post-viral inoculation. All animals in the PBS control group succumbed to the disease on day seven post-infection (0% survival), whereas, in contrast, there was only one casualty in the group that received ACE2 618-DDC-ABD (90% survival). Mice in the ACE2 618-DDC-ABD group had minimal disease as assessed using a clinical score and stable weight, and both brain and lung viral titers were markedly reduced. These findings demonstrate the efficacy of a bioengineered soluble ACE2 decoy with an extended duration of action in protecting against the aggressive Delta SARS-CoV-2 variant. Together with previous work, these findings underline the universal protective potential against current and future emerging SARS-CoV-2 variants.

Theacrine alleviates inflammation and lung injury in septic mice by mediating SIRT3

Purpose: To evaluate the effect of theacrine on sepsis progression and sepsis-induced lung injury and its mechanism of action.&#x0D; Methods: Lung injury model of lipopolysaccharide (LPS)-induced sepsis was made in the mice by injected with 3 mg/kg LPS dissolved in 50 μL PBS or only PBS (control group = 10). The remaining three groups (divided into ten mice per group) were administered theacrine (10, 20, and 40 mg/kg), by oral gavage 1 hour after LPS treatment. Hematoxylin and eosin (H&amp;E) staining was performed to confirm the effect of theacrine on lung injury. Quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA) were conducted to determine inflammatory and oxidative stress factors. TUNEL as well as immunoblot assays were carried to confirm its effect on apoptosis and mechanism of action.&#x0D; Results: Theacrine significantly improved lung injury as well as lung relief score in LPS-induced sepsis mice (p &lt; 0.01) and it significantly reversed the increased levels of inflammatory cytokines in a dose-dependent manner in LPS-induced sepsis mice (p &lt; 0.01), In addition, theacrine administration significantly reduced the increased intensity of ROS and MDA levels, and significantly improved the levels of SOD in lung tissues (p &lt; 0.01). Furthermore, it improved LPS-induced apoptosis of lung tissue cells and alleviated lung injury by significantly activating SIRT3 pathway (p &lt; 0.01).&#x0D; Conclusion: Theacrine alleviates inflammation and lung injury in septic mice by mediating SIRT3, thereby making it a potential lead in the development of drugs against sepsis-related inflammation and lung injury.

Design of an oral vaccine using Lactococcus lactis against brucellosis: an in vitro and in vivo study

Brucellosis is regarded as one of the world’s most severe zoonotic diseases. This study aimed to investigate the possibility of using recombinant Lactococcus lactis (L. lactis) as a live vector to produce recombinant Brucella abortus (B. abortus) Omp10. The gene sequences were obtained from GenBank. The proteins’ immunogenicity was assessed using Vaxijen. After confirming the cloning of the Omp10 gene in the pNZ8148 vector by enzymatic digestion and PCR, transformation into L. lactis was done. SDS-PAGE and western blot methods evaluated omp10 protein expression. Mice received oral recombinant L. lactis vaccines. IgG antibodies against Omp10 were tested using ELISA. Real-time PCR and ELISA were used to analyze cytokine responses. Survival rate and histopathological changes were evaluated after the challenge. Omp10 was chosen for its 1.5524 antigenicity score. Enzymatic digestion and PCR identified a 381-bp gene fragment. A 10 kDa band indicated the success of L. lactis transformation. Mice administered the L. lactis-pNZ8148-Omp10-Usp45 vaccination 14 days after priming showed significantly higher Omp10-specific total IgG and IgG1 (P < 0.001) than the PBS control group. The mice who received the L. lactis-pNZ8148-Omp10-Usp45 and IRBA vaccines had significantly elevated levels of IFN-γ, TNFα, IL-4, and IL-10 in samples collected on days 14 and 28 (P < 0.001). Inflammatory response, morphological damage, alveolar edema, and lymphocyte infiltration were reduced in the target group. A recombinant L. lactis expressing the Omp10 protein was constructed as an oral Lactococcus-based vaccine and compared to live attenuated vaccines for future brucellosis investigations.

DNMT1 regulates hypermethylation and silences hsa_circ_401351 in hydroquinone-induced malignant TK6 cells.

Benzene and its metabolite hydroquinone (HQ) are widely used in daily life, and long-term exposure to benzene or HQ can induce acute myeloid leukemia (AML). Circular RNAs (circRNAs) are mostly produced by reverse splicing of gene exon mRNA precursors. The modulation of circRNA expression is connected to leukemia progression; however, the molecular mechanism is still unknown. In this study, the cells were divided into four groups: PBS control group (PBS-TK6), TK6 malignantly transformed cells induced by 10.0 μmol/L HQ (HQ-TK6), and HQ-TK6 cells treated with 5 μmol/L 5-AzaC (DNA methyltransferase inhibitor) for 24 h (HQ + 5-AzaC). HQ-TK6 cells were treated with 200 nmol/L TSA (histone deacetylation inhibitor) for 24 h (HQ + TSA). qRT-PCR was used to identify the differential hsa_circ_401351 expression between the four groups. We further determined the hsa_circ_401351 promoter methylation level with methylation-specific PCR. DNMT1 and DNMT3b were knocked down by CRISPR/Cas9 to elucidate the specific molecular mechanism of hsa_circ_401351 in HQ-TK6 cells. CCK-8 and flow cytometry detected cell proliferation and apoptosis, respectively, after hsa_circ_401351 was overexpressed in HQ-TK6 cells. Compared with the PBS-TK6 group, the expression of hsa_circ_401351 was found to be lower in the HQ-TK6 group. Nevertheless, treatment with 5-AzaC or TSA increased hsa_circ_401351 expression, with the upregulation being more pronounced in the TSA group. The expression of hsa_circ_401351 in the DNMT1 knockdown group was dramatically increased by 50% compared to that in the control group, and the DNA methylation level of the hsa_circ_401351 promoter region was decreased. When hsa_circ_401351 was overexpressed, HQ-TK6 cell proliferation was significantly slowed after 48 h compared with the control group. Flow cytometry showed that cells were mainly arrested in G1 phase, and apoptosis was significantly enhanced. Similarly, qRT-PCR and Western blot data showed significant reductions in Caspase-3 mRNA and protein production, and Bcl-2 mRNA levels were also elevated. Overall, our research showed that elevated DNMT1 expression in HQ-TK6 cells increased methylation levels and decreased expression of the hsa_circ_401351 promoter region, limiting its ability to suppress HQ-TK6 cell growth and enhance apoptosis.

Avian pathogenic Escherichia coli infection causes infiltration of heterophilic granulocytes of chick tracheal by the complement and coagulation cascades pathway

BackgroundAvian pathogenic Escherichia coli (APEC) causes tracheal damage and heterophilic granulocytic infiltration and inflammation in infected chicks. In this study, we infected chick tracheal tissue with strain AE17 and produced pathological sections with proteomic sequencing. We compared the results of pathological sections from the APEC-infected group with those from the PBS control group; the pathological sections from the experimental group showed hemorrhage, fibrinization, and infiltration of heterophilic granulocytes in the tracheal tissue. In order to explore the effect on proteomics on inflammation and to further search for the caus.ResultsThe tandem mass tag-based (TMT) sequencing analysis showed 224 upregulated and 140 downregulated proteins after infection with the AE17 strain. Based on the results of KEGG in Complement and coagulation cascades, differential protein expression in the Protein export pathway was upregulated.ConclusionsWith these results, we found that chemokines produced by the Complement and coagulation cascades pathway may cause infiltration of heterophilic granulocytes involved in inflammation, as well as antimicrobial factors produced by the complement system to fight the infection together.These results suggest that APEC causes the infiltration of heterophilic granulocytes through the involvement of the complement system with serine protease inhibitors.

Transcriptomics analysis of allergen-induced inflammatory gene expression in the Four-Core Genotype mouse model.

Sex differences in allergic inflammation have been reported, but the mechanisms underlying these differences remain unknown. Contributions of both sex hormones and sex-related genes to these mechanisms have been previously suggested in clinical and animal studies. Here, Four-Core Genotypes (FCG) mouse model was used to study the inflammatory response to house dust mite (HDM) challenge and identify differentially expressed genes (DEGs) and regulatory pathways in lung tissue. Briefly, adult mice (8-10 wk old) of the FCG (XXM, XXF, XYM, XYF) were challenged intranasally with 25 μg of HDM or vehicle (PBS-control group) 5 days/wk for 5 wk (n = 3/10 group). At 72 h after the last exposure, we analyzed the eosinophils and neutrophils in the bronchoalveolar lavage (BAL) of FCG mice. We extracted lung tissue and determined DEGs using Templated Oligo-Sequencing (TempO-Seq). DEG analysis was performed using the DESeq2 package and gene enrichment analysis was done using Ingenuity Pathway Analysis. A total of 2,863 DEGs were identified in the FCG. Results revealed increased eosinophilia and neutrophilia in the HDM-treated group with the most significantly expressed genes in XYF phenotype and a predominant effect of female hormones vs. chromosomes. Regardless of the sex hormones, mice with female chromosomes had more downregulated genes in the HDM group but this was reversed in the control group. Interestingly, genes associated with inflammatory responses were overrepresented in the XXM and XYF genotypes treated with HDM. Sex hormones and chromosomes contribute to inflammatory responses to HDM challenge, with female hormones exerting a predominant effect mediated by inflammatory DEGs.NEW & NOTEWORTHY Gene expression profiling helps to provide deep insight into the global view of disease-related mechanisms and responses to therapy. Using the Four-Core Genotype mouse model, our findings revealed the influence of sex hormones and sex chromosomes in the gene expression of lungs exposed to an aeroallergen (House Dust Mite) and identified sex-specific pathways to better understand sex disparities associated with allergic airway inflammation.

Development of a Novel Solid Tumor-Bearing Humanized Animal Model for Simultaneously Evaluating the Toxicity and Efficacy of Bispecific T Cell Engager

The development of novel therapeutics for cancer immunotherapy is tightly restricted by using human samples to estimate the drug toxicity before moving to clinical trials. Currently numerous tumor-bearing animal models have been utilized to test the efficacy of anti-tumor drugs. However, the success rate remains low in the development of clinical treatment. One prominent reason is that the utilized animal models could not accurately predict the toxic response of human immune system to the drugs. Thus, establishing a predictive nonclinical animal platform that can detect potential unwanted toxicity to human patient will be critical for improving the safety of immune drug development. Previously, we have demonstrated a disseminated tumor-bearing humanized mouse model that can evaluate the toxicity and efficacy of bispecific T cell engager (BiTE). Here we aim to develop a novel tumor-bearing mouse model that can simultaneously evaluate the long-term efficacy against solid tumor in addition to cytokine level assessment. In this study, we co-engrafted disseminated and solid tumor cells to the same mice and compared with mice engrafted with disseminated tumor or solid tumor. Three donors were tested. Method: Human triple negative breast cancer MDA-MB-231/Luciferase-2A-GFP (MDA) cell line is epidermal growth factor receptor (EGFR) positive and is labeled with luciferin. The cells were implanted into PBMC humanized NSG-MHC class I/II double knock out (DKO) mice via orthotopic injection into mammary fat pad (MFP, 5x10 6 cells/mouse) and/or via intravenous (IV, 1x10 6/mouse) injection. Disseminated MDA cells normally colonize lung of mice after IV injection. Following EGFRxCD3 BiTE dosing initiation, tumor burden in MFP and lung were evaluated by a in vivo Xenogen imaging system, and solid tumor volume in MFP was measured 2 to 3 times a week by a digital caliper. To evaluate the toxicity levels in response to EGFRxCD3 BiTE treatment, the level of inflammatory cytokine levels in serum of mice were analyzed by BD cytometric bead array. Clinical observations, body weight change, and human immune cell activation were also monitored for drug toxicity assessment. Results: EGFRxCD3 BiTE treatment induced elevated human inflammatory cytokines in serum from humanized mice implanted with combined MFP and IV tumor. By comparison, the serum cytokine levels were low in mice implanted with MDA cells via MFP alone, suggesting that IV injection of tumor cells is required for cytokine production induced by EGFRxCD3 BiTE. MFP solid tumor continued to grow in the PBS control group and EGFRxCD3 BiTE treatments significantly inhibited solid tumor growth regardless the presence of IV implanted with MDA cells. Conclusion: Without disseminated tumor, MFP solid tumor-bearing mice served as a model for efficacy but did not produce inflammatory cytokines after treatment with EGFRxCD3 BiTE. Combination of solid and disseminated tumor implantation provides a new tumor inoculation approach that can be used for simultaneously evaluating the toxic cytokine release and long-term efficacy of immunotherapy drugs. This is a promising preclinical platform to define optimal approaches for clinical cancer treatment.

Protective efficacy of a novel recombinant influenza virus carrying partial human metapneumovirus F protein epitopes

ABSTRACT Human metapneumovirus (HMPV) is a leading cause of acute respiratory tract infections in infants and children. Currently, no approved HMPV vaccine is available. We developed a novel recombinant influenza virus, which carried partial HMPV F protein (HMPV-F) epitopes, utilizing reverse genetics. The novel single-stranded RNA virus, termed rFLU-HMPV/F-NA, was synthesized in the neuraminidase (NA) fragment of influenza virus A/PuertoRico/8/34 (PR8). The morphological characteristics of rFLU-HMPV/F-NA were consistent with the wild-type flu virus. The virus could passage in specific pathogen-free (SPF) chicken embryos for at least five consecutive generations with haemagglutinin (HA) titres of 28–9 or 8–9LogTCID50/mL. BALB/c mice were intranasally immunized at 21-day intervals with 104 TCID50 (low-dose group) or 106 TCID50 (high-dose group) rFLU-HMPV/F-NA, and PBS or PR8 vaccine was used for the control group. rFLU-HMPV/F-NA induced robust humoral, mucosal, and cellular immune responses in vivo in a dose-dependent manner. More importantly, wt clinical HMPV isolate challenge studies showed that rFLU-HMPV/F-NA provided significant immune protection against HMPV infection compared to the PBS or PR8 vaccine control group, as shown by improved histopathological changes and reduced viral titres in the lungs of immunized mice post-challenge. These findings demonstrate that rFLU-HMPV/F-NA has potential as a promising HMPV candidate vaccine and warrants further investigation into its control of HMPV infection.