Intraperitoneal Injection In Mice Research Articles

Comparison of C-type Nanoantibody Produced in Different Expression Systems Implying Potential Clinical Applications.

In the pharmaceutical industry, the Chinese hamster ovary cell, a type of mammalian cell, is extensively employed for the production of conventional full-length monoclonal antibodies. Nanobody is one of the most attractive directions for the development of next-generation antibody drugs. However, a suitable expression system for its manufacture has not yet been comprehensively evaluated. Previously, we proposed that the immunoglobulin constant CH2 domain could be a promising scaffold for developing C-type nanoantibodies (C-Nabs) as candidate therapeutics. Here, we used an antiviral C-Nab, which we identified previously (under review), as a model for investigation. We expressed C-Nabs without a tag in different systems, including a bacterium (C-Nabbac), yeast (C-Nabyeast), and mammalian cell (C-Nabmam). After purification, the binding and neutralizing activities of C-Nabs from different expression systems are similar. Their secondary structures are rich in β-strand. The melting temperatures of C-Nabbac (71.5 °C) and C-Nabmam (70.2 °C) are similar, which are slightly higher than that of C-Nabyeast (65.6 °C), while C-Nabyeast and C-Nabmam are more resistant to urea-induced unfolding than C-Nabbac. C-Nabyeast and C-Nabmam demonstrate higher resistance to aggregation compared to C-Nabbac. C-Nabyeast exhibits greater resistance to enzyme digestion compared to C-Nabbac and C-Nabmam. Notably, when administered via intraperitoneal injection in mice, C-Nabyeast shows superior pharmacokinetics. Overall, after comparing C-Nab proteins from various expression systems, we determined that yeast is the most suitable host for producing C-Nabs. This finding is beneficial for the production of nanobodies as potential drug candidates.

Effect of GB1107, a novel galectin-3 inhibitor on pro-fibrotic signalling in the liver

Background and PurposeGalectin-3 (Gal-3) is a pro-fibrotic β-galactoside binding lectin highly expressed in fibrotic liver and implicated in hepatic fibrosis. GB1107 is a novel orally active Gal-3 small molecule inhibitor that has high affinity for Gal-3 >1000-fold selectively over other galectins. The aim of this study was to characterise GB1107 and galectin-3 in vitro and in vivo in the context of fibrosis signalling and liver disease. Experimental ApproachLiver fibrosis was induced by administration of CCl4 twice weekly by intraperitoneal injection in mice for 8 weeks. GB1107 was orally administered once daily (10 mg/kg) for the last 4 weeks of CCl4 treatment. Fibrosis was assessed by picrosirius red staining of FFPE sections. Liver enzymes, Gal-3 and downstream biomarkers were assessed in liver and plasma. Paired-end sequencing was performed on the NextSeq 2000 platform. Pathway enrichment analysis was performed to determine enrichment of differentially expressed genes (DEGs) within Reactome pathways and Gene Ontology (GO) terms. Key ResultsGB1107 significantly reduced plasma transaminases and liver Gal-3 and reduced liver fibrosis. RNAseq analysis of whole liver showed that 1,659 DEGs were identified with CCl4 treatment compared to control. Pathways enriched in up-regulated genes in the CCl4 group included those related to the extracellular matrix, collagen biosynthesis and assembly, cell cycle and the immune system. Comparing GB1107 treatment with CCl4 control 1147 DEGs were identified. GB1107 effectively reversed the majority of the CCl4 induced gene changes. Conclusions and ImplicationsGB1107 attenuated liver fibrosis and highlights Gal-3 as a therapeutic target for hepatic fibrosis.

Exploratory research on the effective chemical basis of tanreqing injection for treating acute lung injury: In vivo, in vitro and in silico

Ethnopharmacological relevanceSepsis-induced acute lung injury (ALI) presents with significant morbidity and mortality in clinical settings. Tanreqing Injection (TRQI) has been clinically recommended for the treatment of ALI; however, the specific active chemical constituents remain unidentified. Aim of the studyThis study aimed to elucidate the potential pharmacologically active components and the underlying mechanisms of TRQI in the treatment of sepsis-induced ALI. Materials and methodsHigh-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) techniques were employed to identify the effective chemical constituents of TRQI. Additionally, an in vitro study was conducted using Raw264.7 macrophage cells stimulated with lipopolysaccharide (LPS) to evaluate the inhibitory effects of TRQI. An acute lung injury model produced by LPS was intraperitoneal injection in mice to assess the ALI-inhibitory effect of TRQI. The lung's pathological characteristics were examined using hematoxylin and eosin staining. Enzyme-linked immunosorbent assay (ELISA) and QPCR were performed to confirm the pharmaceutical effect. Network pharmacology was employed for mechanistic exploration, incorporating GO, and PPI analyses of targets. Src inhibitor and JNK agonist used to investigate the dependence of associated signaling pathways. ResultsCombining pharmacokinetic characteristics, lung first-pass effect and anti-inflammatory effects, the main components of TRQI for treating sepsis induced ALI were narrowed down to seven compounds: chlorogenic acid, scutellarin, wogonoside, oroxyloside, oroxylin A and baicalein. Network pharmacology indicated that Src/JNK signaling pathway, may be the main regulatory pathway for treatment of actue lung injury. Next by using Src inhibitor, Src inhibition partly diminished the protective effects of TRQI in LPS-injected mice. Pretreatment with JNK agonist anisomycin abolished the protective effects of lung injury in vivo. ConclusionsTRQI is injected, the seven compounds could be presented in vivo, which can improve ALI by inhibiting Src-JNK signaling.

Helicobacter pylori infection promotes liver injury through an exosome-mediated mechanism

Helicobacter pylori infection has been thought to be associated with liver diseases, although the exact mechanisms remain elusive. This study identified H. pylori-induced liver inflammation and tissue damage in infected mice and examined the exosome-mediated mechanism underlying H. pylori infection's impact on liver injury. Exosomes were isolated from H. pylori-infected gastric epithelial GES-1 cells (Hp-GES-EVs), and the crucial virulence factor CagA was identified within these exosomes. Fluorescent labeling demonstrated that Hp-GES-EVs can be absorbed by liver cells. Treatment with Hp-GES-EVs enhanced the proliferation, migration, and invasion of Hep G2 and Hep 3B cells. Additionally, exposure to Hp-GES-EVs activated NF-κB and PI3K/AKT signaling pathways, which provides a reasonable explanation for the liver inflammation and neoplastic traits. Using a mouse model established via tail vein injection of Hp-GES-EVs, exosome-driven liver injury was evidenced by slight hepatocellular erosion around the central hepatic vein and elevated serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and IL-6. Administering the exosome inhibitor GW4869 via intraperitoneal injection in mice resulted in a reduction of liver damage caused by H. pylori infection. These findings illuminate the exosome-mediated pathogenesis of H. pylori-induced liver injury and offer valuable insights into the extra-gastrointestinal manifestations of H. pylori infection.

Effects of morphine on conditioned place preference and pain are independent of uptake‑2.

Morphine changes neurotransmitter release, including norepinephrine, dopamine, and serotonin. Decynium‑22 (D22) inhibits an alternative neurotransmitter removal pathway, namely uptake‑2. Uptake‑2 includes plasma membrane monoamine transporter (PMAT) and organic cation transporters that have a low affinity, but high capacity for uptake of various monoamines such as norepinephrine, dopamine, and serotonin. This study was done to assess the effect of uptake‑2 inhibition on morphine‑induced conditioned place preference (CPP) and analgesia. In this study, the effects of morphine and/or D22 on CPP were evaluated following intraperitoneal injection in mice. Afterward, changes in motor activity were evaluated by the open field test. Using the tail‑flick model, the effects of D22 and/or morphine were evaluated on the pain threshold. The results showed that 20 mg/kg of morphine induced a place preference response. D22, at the dose of 0.03 mg/kg, caused place avoidance, while at the dose of 0.3 mg/kg, it produced a notable place preference response. Co‑administration of D22 and morphine showed that morphine reversed the CPP aversion induced by D22 at the lowest dose. Motor activity did not alter. In the tail‑flick test, morphine, at the dose of 3 mg/kg but not 1 mg/kg, increased the pain threshold. D22 induced significant analgesic responses. Co‑administration of D22 and morphine caused considerable analgesic effects. The findings revealed that D22 induced both conditioned aversion and preference depending on the dose while morphine induced CPP. Both drugs produced analgesia.

Evaluation of the toxicological effects of Neltuma alpataco (Prosopis alpataco) pod alkaloid extract

The pods of Neltuma spp. have shown potential as a source of protein and energy in livestock. However, prolonged consumption of some of these species can lead to neurological symptoms in ruminants. This study aimed to determine the alkaloid content, as well as the in vitro and in vivo effects of an alkaloid-enriched extract (AEE) from N. alpataco pods. High performance liquid chromatography-high resolution mass spectrometry (HPLC-HRMS) identified juliprosine and juliprosopine as primary alkaloids, with juliprosine being most abundant. AEE from N. alpataco demonstrated dose-dependent cytotoxicity on glioma cells after 48 h, with a 50% cytotoxic concentration (CC50) of 24.69 μg/mL. However, the release of LDH was observed only at the highest tested concentration, indicating cellular damage. Further examination through phase-contrast microscopy and dual acridine orange/ethidium bromide fluorescence staining revealed morphological changes consistent with an apoptotic mechanism of cell death, ultimately leading to secondary necrosis. Finally, the LD50 after intraperitoneal injection in mice was determined to be 12.98 mg/kg. Taken together, these findings demonstrated for the first time the in vivo and in vitro toxicity of the AEE from N. alpataco pods.

Extracellular vesicles from pristane-treated CD38-deficient mice express an anti-inflammatory neutrophil protein signature, which reflects the mild lupus severity elicited in these mice.

In CD38-deficient ( Cd38-/- ) mice intraperitoneal injection of pristane induces a lupus-like disease, which is milder than that induced in WT mice, showing significant differences in the inflammatory and autoimmune processes triggered by pristane. Extracellular vesicles (EV) are present in all body fluids. Shed by cells, their molecular make-up reflects that of their cell of origin and/or tissue pathological situation. The aim of this study was to analyze the protein composition, protein abundance, and functional clustering of EV released by peritoneal exudate cells (PECs) in the pristane experimental lupus model, to identify predictive or diagnostic biomarkers that might discriminate the autoimmune process in lupus from inflammatory reactions and/or normal physiological processes. In this study, thanks to an extensive proteomic analysis and powerful bioinformatics software, distinct EV subtypes were identified in the peritoneal exudates of pristane-treated mice: 1) small EV enriched in the tetraspanin CD63 and CD9, which are likely of exosomal origin; 2) small EV enriched in CD47 and CD9, which are also enriched in plasma-membrane, membrane-associated proteins, with an ectosomal origin; 3) small EV enriched in keratins, ECM proteins, complement/coagulation proteins, fibrin clot formation proteins, and endopetidase inhibitor proteins. This enrichment may have an inflammation-mediated mesothelial-to-mesenchymal transition origin, representing a protein corona on the surface of peritoneal exudate EV; 4) HDL-enriched lipoprotein particles. Quantitative proteomic analysis allowed us to identify an anti-inflammatory, Annexin A1-enriched pro-resolving, neutrophil protein signature, which was more prominent in EV from pristane-treated Cd38-/- mice, and quantitative differences in the protein cargo of the ECM-enriched EV from Cd38-/- vs WT mice. These differences are likely to be related with the distinct inflammatory outcome shown by Cd38-/- vs WT mice in response to pristane treatment. Our results demonstrate the power of a hypothesis-free and data-driven approach to transform the heterogeneity of the peritoneal exudate EV from pristane-treated mice in valuable information about the relative proportion of different EV in a given sample and to identify potential protein markers specific for the different small EV subtypes, in particular those proteins defining EV involved in the resolution phase of chronic inflammation.

The histopathological changes induced by intra peritoneal administration of cerium oxide on mice brain tissue

The use of nanoparticles (NPs) and nanoparticle- based materials raises many concerns about their impact on health related to its many applications in diverse fields, including petroleum refining, polishing agents, and coatings, as well as the toxicity of these NPs in biological/ physiological environment is a major concern. The study conducted at college of nursing – university of Basrah –Iraq,and it aimed to investigated the effect of cerium oxide nanoparticles on mice brain tissues after 30 days of intra-peritoneal I/P injection of cerium oxide nanoparticles . White laboratory mice of Mus musculus L. strain BALB/C that were supplied from (Drug Control Center - College of Science, Thi -Qar University). The mice were placed under controlled conditions of 20-25 degrees and a 12-hour light cycle. And 12 hours of darkness in plastic cages of standard sizes (30×12×11) cm, the first group injected laboratory mice with 1 ml of normal saline physiological solution as a control group. The second treatment group. Laboratory mice were injected with 1 ml of cerium dioxide at a concentration of 3.75 µl of the raw material to 996.25 µl of physiological solution. By one injection between one day and another for a period of 30 days to investigate the effects of I/P administration of cerium oxide Nano- particles on mice brain tissue after 30 day. Mice were killed and brain isolated to prepare for the histological examination which showed that cerium oxide injected for 30 day induce pathological alteration in brain tissue especially on white matter , neurofibers and glial cells. Brain of mice show degeneration of white matter, ,necrosis of glial cell aggregation of glial cell , The histopathological examination investigate that intra-peritoneal injection in mice for thirty day induce histopathological adverse changes in mice brain tissue.

Design, synthesis, and evaluation of a novel series of mono-indolylbenzoquinones derivatives for the potential treatment of breast cancer

Breast cancer is one of the most common cancers in the world, and pro-apototic drugs activating the apoptotic pathway are a strategy for anticancer therapy. To explore new antineoplastic agents, a series of novel mono-indolylbenzoquinone derivatives have been designed and synthesized. Compared with the lead bis-indolylbenzoquinones, most of the novel mono-indolylbenzoquinone derivatives have significantly increased their activity against A549, HeLa, and especially, MDA-MB-231 cell lines. Among them, 10d has the lowest IC50 value of 70 nM on MDA-MB-231 cells. Moreover, its oral toxicity is extremely low with an LD50 value of 374 mg/kg and no obvious liver and kidney damage to mice. 10d down-regulated Bcl-2, up-regulated Bax, and increased the release of cytochrome C, caspase3 and 9. 10d also up-regulated the expression of p53, catalase, and HTRA2/Omi. Therefore, 10d may exert its anticancer activity by activating apoptotic pathway and p53 expression. In vivo, 10d suppressed breast cancer 4T1 tumor growth with 36% inhibition ratio of tumor by intraperitoneal injection in mice. Furthermore, a cross-linked cyanoacrylate (CA)-based local sustained-release drug delivery systems (LSRDDSs) improved 10d anticancer activity to 49.8% inhibition of tumor growth. Taken together, 10d could be a promising drug candidate for clinical development to treat metastatic breast cancer.

Solubilization of luteolin in PVP40 solid dispersion improves inflammation-induced insulin resistance in mice

Our previous studies have confirmed that luteolin (LU) has a good therapeutic effect on obesity and its complications. However, due to its poor water solubility, the bioavailability is low with limited clinical application. Therefore, the water-soluble solid dispersions (SD) of luteolin were prepared with polyvinylpyrrolidone (PVP) (K10, K40 & K90) by solvent evaporation. The polyvinylpyrrolidone K40 (PVP40) was selected as the ideal carrier to formulate polyvinylpyrrolidone K40-luteolin solid dispersion (PVP40-LU SD), thereby the solubility of luteolin increased about 250 times compared to the pure luteolin, without changing its physical stability and activity. The crystallinity of luteolin was reduced after the formation of solid dispersion, and no strong drug-polymer interactions were observed. This prepared water-soluble luteolin inhibits the polarization of inflammatory macrophages by decreasing the expression of pro-inflammatory cytokine genes interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in vitro. Moreover, it can improve glucose tolerance and insulin sensitivity quickly after intraperitoneal injection in mice.

The Significance of PAX8-PPARγ Expression in Thyroid Cancer and the Application of a PAX8-PPARγ-Targeted Ultrasound Contrast Agent in the Early Diagnosis of Thyroid Cancer.

Objective To investigate the significance of PAX8-PPARγ expression in thyroid cancer and the application of a PAX8-PPARγ-targeted ultrasound contrast agent in the early diagnosis of thyroid cancer. Methods In this study, the expression of PAX8-PPARγ in thyroid cancer tissues, paracancer groups, and normal thyroid tissues was detected by western and immunohistochemical techniques; the effects of PAX8-PPARγ expression inhibition on thyroid cancer cell growth, clonogenic ability, and antiapoptosis were examined. The terminal carboxylactic acid/hydroxyacetic acid copolymer (PLGA-COOH) nanoparticles were prepared by the double emulsification solvent volatilization method. The in vitro cytotoxicity of the targeted contrast agent was detected by MTS and other methods; LD50 was used to evaluate its short-term in vivo toxicity after intraperitoneal injection in mice. Results PAX8-PPARγ expression was significantly increased in thyroid cancer tissues, and the expression level of PAX8-PPARγ was closely correlated with TNM staging and lymph node metastasis (P < 0.05). In addition, PAX8-PPARγ was also expressed at high levels in thyroid cancer cell lines relative to normal thyroid cells. MTS experiments showed that the PAX8-PPARγ-targeted ultrasound nanocontrast agent had no significant toxic side effects on thyroid cells; countess observed that the contrast agent had no effect on cell survival and mortality; the LD50 assay showed that the targeted contrast agent had a wide safety range. Western blot showed the expression of caspase-3, BAX, and Bcl-2 in thyroid cancer cells, indicating that the nanocontrast agent has a good biosafety. In vitro targeting experiments showed that there were more nanospheres aggregated around the cells in the targeted contrast group. In vivo targeting imaging of nude mice revealed that the ultrasound signal was significantly enhanced in the targeted group compared with the nontargeted group after 20 min of LIFU irradiation. Conclusion PAX8-PPARγ overexpression in thyroid cancer cell lines and thyroid cancer tissues promoted the proliferation and antiapoptotic ability of thyroid cancer cells and promoted the tumorigenic ability in nude mice in vivo. We successfully prepared a PAX8-PPARγ-targeted ultrasound nanocontrast agent, which has regular morphology, uniform size, and high stability, and its liquid-gas phase change can be promoted at lower temperature. Therefore, this contrast agent can achieve US-targeted imaging and temperature phase transition function, and may have enhanced ultrasound imaging potential.

VALIDATION OF HPTM-001, A HUMANIZED CANDIDATE THERAPEUTIC ANTIBODY FOR PROMOTING MUCOSAL WOUND HEALING IN IBD

Abstract A hallmark of the clinical course of patients with Inflammatory Bowel Disease (IBD) is poorly healing erosions and ulcers in the intestinal mucosa. Despite the adverse clinical consequences of non-healing mucosal wounds in IBD, current front-line therapies that selectively target mucosal wound healing are not available. Recent studies revealed an association between colonic inflammation and aberrant glycosylation of epithelial CD44v6. Under conditions of inflammation, epithelial CD44v6 was shown to be decorated with the terminal glycan sialyl Lewis A. Importantly, targeting sialylated Lewis glycans on CD44v6 with a murine antibody GM35 was shown to promote mucosal wound healing in cell lines and in biopsy based wounding assays as well as dextran sodium sulfate (DSS) induced murine colitis models. We sequenced CDRs from GM35 and produced a humanized antibody. Eight candidate human IgG1 clones were produced and screened. hPTM-001.4772 was selected from the eight candidates based on glycan affinity and selectivity compared to GM35. In vitro wound healing studies revealed that PTM-001.4772 was as effective as GM35 in promoting repair of scratch wounds with human intestinal epithelial cells. Furthermore, intraperitoneal injection of mice with hPTM-001.4772 during induction of DSS colitis resulted in reduced weight loss compared to control IgG. These results suggest that hPTM-001.4772 is well-positioned as a unique potential candidate therapeutic for IBD that can be used to selectively promote healing of mucosal wounds and ulcers.

173. HSV-2 Isolates from Neonates with Different Clinical Outcomes Exhibit Different in Vitro and in Vivo phenotypes

BackgroundHerpes simplex virus (HSV) infection of the neonatal brain causes severe meningoencephalitis and permanent neurologic deficits. However, infants infected with HSV at the time of birth follow different clinical courses. Some infants develop only external infection of the skin, eyes, or mouth (SEM disease), while others develop invasive infection of the central nervous system resulting in encephalitis (CNS disease). The factors that explain this clinical divergence are not well understood. While adults can be predisposed to HSV CNS infection by an innate immune defect, no such host susceptibility has been identified in neonates. Therefore, we have taken a novel approach to determine whether variations in the HSV genome contribute to infection of the neonatal brain. We recently defined the viral genetic diversity among HSV-2 isolates cultured from neonates with a range of clinical presentations. Isolates collected from neonates with CNS disease contained several unique amino acid variations in HSV proteins known to contribute to cell-to-cell spread and neurovirulence in mouse models.MethodsTo understand the relevance of these findings to neonatal CNS disease, we evaluated CNS disease- and SEM disease-associated neonatal HSV-2 isolates in neurologically-relevant in vitro and in vivo models.ResultsWe found that HSV-2 isolates from neonates with CNS disease, as compared to those collected from neonates with SEM disease, displayed enhanced spread in human neuronally-differentiated SH-SY5Y or LUHMES cells and enhanced retrograde transport in rat neurons cultured in modified Campenot chambers. CNS disease-associated isolates also resulted in increased hind limb paralysis and zosteriform disease in a mouse flank scratch infection model, and increased death in a mouse direct intracerebral injection model of encephalitis. Notably, CNS disease and SEM disease-associated isolates resulted in equivalent outcomes following mouse intraperitoneal injection, suggesting similar systemic virulence.ConclusionThese data suggest that virus-mediated differences in neuronal spread and transport may contribute to neurovirulence in neonatal HSV disease.Disclosures All Authors: No reported disclosures

Toxoplasma gondii Infection in Cattle Egret (Bubulcus ibis): First Report from Shebin El-Kom, Menoufia Governorate, Egypt.

Toxoplasma gondii (T. gondii) is capable of infecting a broad range of intermediate hosts. Cattle egret (Bubulcus ibis) is a species of herons that is common in Egypt. This work aimed to study the prevalence of T. gondii in cattle egret which is an efficient tool of investigating environmental contamination with T. gondii. Serum, heart and brain tissues of 51 cattle egrets were collected from Shebin El-Kom, Menoufia, Egypt and tested using Modified Agglutination Test (MAT) and mice bioassay. There was a detection rate of 13.7% (7/51) in these birds using MAT. By intraperitoneal injection of mice with heart and brain tissues digest of MAT positive birds, the parasite was isolated from two T. gondii sero-positive birds (28.6%). The mice bioassay was confirmed by MAT, Hematoxylin and Eosin (HAND E) staining of the brain of the infected mice, also by Polymerase Chain Reaction (PCR) and sequencing. This is the first report of T. gondii infection in cattle egret from Egypt and more studies are needed on Egyptian wildlife to understand the sylvatic life cycle of the parasite.

Toxicity and Immunogenicity of a Tardigrade Cytosolic Abundant Heat Soluble Protein in Mice.

Tardigrades are microscopic animals well-known for their stress tolerance, including the ability to survive desiccation. This survival requires cytosolic abundant heat soluble (CAHS) proteins. CAHS D protects enzymes from desiccation- and lyophilization-induced inactivation in vitro and has the potential to stabilize protein-based therapeutics, including vaccines. Here, we investigate whether purified recombinant CAHS D causes hemolysis or a toxic or immunogenic response following intraperitoneal injection in mice. CAHS D did not cause hemolysis, and all mice survived the 28-day monitoring period. The mice gained weight normally and developed anti-CAHS D antibodies but did not show upregulation of the inflammatory cytokines interleukin-6 and tumor necrosis factor alpha. In summary, CAHS D is not toxic and does not promote an inflammatory immune response in mice under the conditions used here, suggesting the reasonability of further study for use as stabilizers of protein-based therapeutics.

Aldophosphamide-thiazolidine (NSC-613060) an oxazaphosphorine cytostatic that crosses the blood brain barrier

The pharmacologically active metabolite of cyclophosphamide is aldophosphamide. With cysteine, aldophosphamide forms stable aldophosphamide-thiazolidine which under physiological pH and temperature conditions hydrolyzes to aldophosphamide and cysteine. Aldophosphamide-thiazolidine was synthesized and tested for its ability as a cytostatic. The LD50 after a single intraperitoneal injection in mice was determined to be 2162 mg/kg, but after intravenous bolus administration of 500 mg/kg or in chronic toxicity tests with daily intraperitoneal injections, neurological side effects were observed. Antitumor activity was determined in therapy experiments in CD2F1 mice bearing subcutaneously transplanted P388 mouse leukemia cells. Administration of 100 mg/kg (less than 5% LD50) days 1-5 after tumor transplantation yielded an ILS of 100%. Organ distribution studies showed that aldophosphamide-thiazolidine is evenly distributed in all tissues examined, including brain tissue. The possibilities to increase the antitumor activity of aldophosphamide-thiazolidine by modulating the alkylating function are discussed.

PI3K-Akt-mTOR inhibition by GNE-477 inhibits renal cell carcinoma cell growth in vitro and in vivo.

Sustained activation of PI3K-Akt-mTOR cascade is important for renal cell carcinoma (RCC) cell progression. GNE-477 is a novel and efficacious PI3K-mTOR dual inhibitor. The current study tested its anti-RCC cell activity. In the primary cultured human RCC cells, GNE-477 potently inhibited cell growth, viability and proliferation, as well as cell cycle progression, migration and invasion. Furthermore, it induced robust apoptosis activation in primary RCC cells, but being non-cytotoxic to HK-2 epithelial cells and primary human renal epithelial cells. In the primary RCC cells GNE-477 inactivated PI3K-Akt-mTOR cascade by blocking phosphorylation of p85, Akt1, p70S6K1 and S6. Restoring Akt-mTOR activation by a constitutively-active Akt1 reversed GNE-477-induced anti-RCC cell activity. In nude mice intraperitoneal injection of GNE-477 potently suppressed RCC xenograft tumor growth. Collectively, targeting PI3K-Akt-mTOR cascade by GNE-477 inhibits RCC cell growth in vitro and in vivo.

Biochemical and immunological characterization of haemolysin produced by &lt;i&gt;Pseudomonas aeruginosa&lt;/i&gt; PAO1 isolated from burn wounds

Background: Infection of burn wounds by multidrug-resistant (MDR) Pseudomonas aeruginosa (P. aeruginosa) is a leading cause of morbidity and mortality and remains one of the most challenging concerns for the burns unit. The aim of this study is purify and characterize the haemolysin produced by multidrug resistant P. aeruginosa PAO1 isolated from burn wounds. Methods: Isolation and identification of P. aeruginosa from burns was done by standard bacteriological methods. P. aeruginosa PAO1 was identified by PCR amplification and sequencing of the 16S rRNA gene. The haemolysin of P. aeruginosa PAO1 was purified by 70% ammonium sulphate precipitation followed by gel filtration on Sephadex G-100, and separation by SDS-Poly Acrylamide Gel Electrophoresis. In vivo toxicity of the purified haemolysin was determined by intraperitoneal injection of Swiss albino mice, and in vitro toxin-antitoxin neutralization test was performed as previously described. Results: The pure haemolysin had a molecular weight of 37 kDa, with maximum activity at 25°C for 30 minutes and stable within pH range of 4-9 (maximum activity at pH 7). The haemolysin was activated by Ca2+, Fe3+ and Cu2+. Intraperitoneal injection of mice with 0.5ml of haemolysin (128 HU/ml) caused 100% mortality while 0.5 and 0.1 ml of haemolytic titer (64 HU/ml) of the heated haemolysin (toxoid) caused 50% and 0% mortality respectively. In vitro toxin-antitoxin neutralization test revealed that anti-haemolysin antitoxin was present in the serum of the mice that were previously vaccinated with heated toxin. Conclusion: This study concluded that haemolysin can be a potential vaccine component for prevention of haemolysis caused by multidrug resistant P. aeruginosa in burn patients.Keywords: haemolysin, Pseudomonas aeruginosa, multidrug resistant organism

Circ-CEP85L suppresses the proliferation and invasion of gastric cancer by regulating NFKBIA expression via miR-942-5p.

The expression pattern and role of circular RNAs (circRNAs) in the pathogenesis of gastric cancer (GC) and their underlying mechanisms remain unresolved. In this study, we identified differentially expressed circRNAs by a circRNA microarray and verified the results by quantitative reverse transcription-polymerase chain reaction using 117 clinical samples. Cell Counting Kit-8, wound healing, Transwell, and tumorsphere formation assays were conducted to assess the effects of circ-CEP85L on cell proliferation and invasion in vitro. Mouse intraperitoneal injection models were used to assess the functions of circ-CEP85L in vivo. Luciferase reporter assays, fluorescence in situ hybridization, and rescue experiments were performed to elucidate the underlying mechanism of circ-CEP85L. We found that circ-CEP85L, which has not been studied in GC, was significantly downregulated in GC tissues and that decreased circ-CEP85L expression correlated significantly with a worse prognosis. The knockdown of circ-CEP85L promoted the proliferation and invasion of GC cells, which was reversed by overexpression of circ-CEP85L. Furthermore, inhibition of circ-CEP85L promoted tumor growth in vivo. Mechanistically, circ-CEP85L was confirmed to be a direct target of miR-942-5p. In addition, rescue experiments indicated that circ-CEP85L is able to inhibit the proliferation and invasion of GC cells by sponging miR-942-5p. Finally, western blot assays verified that the downregulation of miR-942-5p efficiently reversed the inhibition of NFKBIA induced by circ-CEP85L overexpression. Therefore, we conclude that circ-CEP85L promotes NFKBIA expression by acting as a sponge of miR-942-5p; thus, inhibiting GC proliferation and invasion. circ-CEP85L is a potential target in the treatment of GC.

Washed microbiota transplantation vs. manual fecal microbiota transplantation: clinical findings, animal studies and in vitro screening

Fecal microbiota transplantation (FMT) by manual preparation has been applied to treat diseases for thousands of years. However, this method still endures safety risks and challenges the psychological endurance and acceptance of doctors, patients and donors. Population evidence showed the washed microbiota preparation with microfiltration based on an automatic purification system followed by repeated centrifugation plus suspension for three times significantly reduced FMT-related adverse events. This washing preparation makes delivering a precise dose of the enriched microbiota feasible, instead of using the weight of stool. Intraperitoneal injection in mice with the fecal microbiota supernatant obtained after repeated centrifugation plus suspension for three times induced less toxic reaction than that by the first centrifugation following the microfiltration. The toxic reactions that include death, the change in the level of peripheral white blood cells, and the proliferation of germinal center in secondary lymphoid follicles in spleen were noted. The metagenomic next-generation sequencing (NGS) indicated the increasing types and amount of viruses could be washed out during the washing process. Metabolomics analysis indicated metabolites with pro-inflammatory effects in the fecal microbiota supernatant such as leukotriene B4, corticosterone, and prostaglandin G2 could be removed by repeated washing. Near-infrared absorption spectroscopy could be served as a rapid detection method to control the quality of the washing-process. In conclusion, this study for the first time provides evidence linking clinical findings and animal experiments to support that washed microbiota transplantation (WMT) is safer, more precise and more quality-controllable than the crude FMT by manual.