Cuprous oxide-demethyleneberberine nanospheres for single near-infrared light-triggered photoresponsive-enhanced enzymatic synergistic antibacterial therapy.

In this work, novel cuprous oxide-demethyleneberberine (Cu2O-DMB) nanomaterials are successfully synthesized for photoresponsive-enhanced enzymatic synergistic antibacterial therapy under near-infrared (NIR) irradiation (808 nm). Cu2O-DMB has a spherical morphology with a smaller nanosize and positive ζ potential, can trap bacteria through electrostatic interactions resulting in a targeting function. Cu2O-DMB nanospheres show both oxidase-like and peroxidase-like activities, and serve as a self-cascade platform, which can deplete high concentrations of GSH to produce O2˙- and H2O2, then H2O2 is transformed into ˙OH, without introducing exogenous H2O2. At the same time, Cu2O-DMB nanospheres become photoresponsive, producing 1O2 and having an efficient photothermal conversion effect upon NIR irradiation. The proposed mechanism is that the generated ROS (O2˙-, ˙OH and 1O2) and hyperthermia can have synergetic effects for killing bacteria. Moreover, hyperthermia is not only beneficial for destroying bacteria, but also effectively enhances the efficiency of ˙OH production and accelerates GSH oxidation. Upon NIR irradiation, Cu2O-DMB nanospheres exhibit excellent antibacterial ability against methicillin-resistant Staphylococcus aureus (MRSA) and ampicillin-resistant Escherichia coli (AREC) with low cytotoxicity and bare bacterial resistance, destroy the bacterial membrane causing an efflux of proteins and disrupt the bacterial biofilm formation. Animal experiments show that the Cu2O-DMB + NIR group can efficiently treat MRSA infection and promote wound healing. These results suggest that Cu2O-DMB nanospheres are effective materials for combating bacterial infections highly efficiently and to aid the development of photoresponsive enzymatic synergistic antibacterial therapy.

Extranodal natural killer/T-cell lymphoma (nasal type) presenting as a perianal abscess: A case report.

BACKGROUND: Extranodal natural killer (NK) T-cell lymphoma (ENKTL), nasal type is a rare subtype of extranodal non-Hodgkin lymphoma characterized by vascular damage and necrosis. The lesions usually present in the nasal cavity and adjacent tissues, however, the disease originates from the gastrointestinal or genitourinary tract in 25% of cases. Since rectal involvement in ENKTL is rare, rectal symptoms in the course of ENKTL are often misdiagnosed and considered to be related to benign diseases such as rectal fistula or perianal abscess. CASE SUMMARY: We report the case of a 24-year-old Han Chinese female who initially presented with a perianal abscess that was subsequently diagnosed as nasal type ENKTL. Due to typical perianal pain, perianal abscess was diagnosed and surgical incision and drainage were performed. After recurrent, severe anal hemorrhages leading to hypovolemic shock and multiple surgeries, a diagnosis of ENKTL was made. The patient's condition gradually deteriorated, and she died shortly after initiation of chemotherapy. CONCLUSION: Systemic and neoplastic diseases should be included in the differential diagnosis of any potentially benign perianal abscess complicated with recurrent hemorrhages.

[A novel treatment regimen for acute liver failure based on a combination of mesenchymal stem cells transplantation and IL-lRa-loaded chitosan nanoparticles].

OBJECTIVE: To evaluate whether a combination therapy using allogeneic mesenchymal stem cell (MSC) transplantation and interleukin-1 receptor antagonist (IL-1Ra) chitosan nanoparticles is more robust than MSC transplantation alone for treating acute liver failure and to investigate the mechanisms of the improved therapeutic effect using a swine model system. METHODS: IL-1Ra-loaded nanoparticles were made of lactosylated chitosan-FITC using the electrostatic spray method and analyzed by enzyme-linked immunosorbent assay. The active live targeting of these nanopaticles were investigated by fluorescence microscope and flow cytometer(FCM). The combined therapy was given and the Detailed Steps as follows: Chinese experimental mini swine were given D-galactosamine to build models of acute liver failure. Thirty-four pigs were randomly divided into five groups. In the control group(A),the normal saline was injected into liver via portal veins after 24 h; in IL-1Ra group(B), IL-1Ra was injected via ear veins 6 h before normal saline; In the MSCs transplantation group (C), 8 * 107 MSCs were injected into liver via portal veins after 24 h; IL-1Ra together with MSCs transplantation group(D) and nanopaticles group(E) as follows: on the one hand, 8 * 107 MSCs were injected into liver via portal veins after 24 h, on the other hand, rhIL-1Ra in group C or IL-1Ra chitosan nanopaticles in group D was injected via ear veins respectively at 6 h before. Liver function, serum inflammation and pathological changes were measured. The fate of MSCs was also observed. RESULTS: The profiles in vitro shown that there was a steady-state release after a fast linear release; The live targeting of the lactosylated chitosan-based nanoparticles was achieved by ligand-receptor specificity; The biochemical assay, the serum inflammation lever and pathological changes of the nanopaticles group were all greatly different from the other groups, the hepatocytes grow rate was significantly improved after 1 week; The liver engraftment was very low in group C and D with significantly higher numbers found in nanopaticles group, but the differentiation of MSCs after 2 weeks relatively rare; western blot showed that there was more HGF and VEGF secreted in nanopaticles group. CONCLUSION: IL-lRa-loaded lactosylated chitosan-based nanopaticles have significant liver targeting abilities and slow release characteristics in PBS solution. The combined therapy showed great synergistic effects through suppression of liver inflammation and paracrine.

Administration of IL-1Ra chitosan nanoparticles enhances the therapeutic efficacy of mesenchymal stem cell transplantation in acute liver failure.

BACKGROUND AND AIMS: To investigate the synergistic effect of IL-1Ra administration and stem cell transplantation in swine suffering from acute liver failure (ALF), to elucidate the mechanism of IL-1Ra activity and to demonstrate mesenchymal stem cell (MSC) transplantation as a potential treatment for ALF. METHODS: Thirty-five Chinese experimental mini-swine were divided into five groups randomly. Group A (n = 7) is the control group and all swine were injected with saline via portal veins. Group B (n = 7) received IL-1Ra via ear veins 6 h before receiving saline. Group C (n = 7) received MSC transplantation and all swine were injected with 8 × 107 MSCs via portal veins. Group D (n = 7) swine were treated with a combination of IL-1Ra and MSC transplantation E (n = 7) also received a combined treatment of both IL-1Ra and bone marrow (BM-MSC) transplantation, except that the IL-1Ra was in the form of chitosan nanoparticles. Liver function, level of cytokines and liver pathological changes were measured in the following 4 weeks. RESULTS: IL-1Ra chitosan nanoparticles exhibited controlled-release ability in PBS. Swine in Group E showed a significant improvement in inflammation environment, liver function and hepatocyte proliferation. Levels of hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) in Group E were elevated compared to other groups. CONCLUSIONS: IL-1Ra chitosan nanoparticles showed significant liver targeting ability and controlled-release characteristics. Combined therapy with IL-1Ra chitosan nanoparticles and MCS transplantation exhibits great synergistic effects through paracrine function and suppression of inflammation.

Effect evaluation of interleukin-1 receptor antagonist nanoparticles for mesenchymal stem cell transplantation.

AIM: To study the efficacy of marrow mesenchymal stem cells (MSCs) transplantation combined with interleukin-1 receptor antagonist (IL-1Ra) for acute liver failure (ALF). METHODS: Chinese experimental miniature swine were randomly divided into four groups (n = 7), and all animals were given D-galactosamine (D-gal) to induce ALF. Group A animals were then injected with 40 mL saline via the portal vein 24 h after D-gal induction; Group B animals were injected with 2 mg/kg IL-1Ra via the ear vein 18 h, 2 d and 4 d after D-gal induction; Group C received approximately 1 × 10(8) green fluorescence protein (GFP)-labeled MSCs (GFP-MSCs) suspended in 40 mL normal saline via the portal vein 24 h after D-gal induction; Group D animals were injected with 2 mg/kg IL-1Ra via the ear vein 18 h after D-gal induction, MSCs transplantation was then carried out at 24 h after D-gal induction, and finally 2 mg/kg IL-1Ra was injected via the ear vein 1 d and 3 d after surgery as before. Liver function, serum inflammatory parameters and pathological changes were measured and the fate of MSCs was determined. RESULTS: The optimal efficiency of transfection (97%) was achieved at an multiplicity of infection of 80, as observed by fluorescence microscopy and flow cytometry (FCM). Over 90% of GFP-MSCs were identified as CD44+ CD90+ CD45- MSCs by FCM, which indicated that most GFP-MSCs retained MSCs characteristics. Biochemical assays, the levels of serum inflammatory parameters and histological results in Group D all showed a significant improvement in liver injury compared with the other groups (P < 0.05). The number of GFP-MSCs in Group D was also greater than that in Group B, and the long-term cell proliferation rate was also better in Group D than in the other groups. CONCLUSION: MSCs transplantation is useful in ALF, IL-1Ra plays an important role in alleviating the inflammatory condition, and combination therapy with MSCs transplantation and IL-1Ra is a promising treatment for ALF.

Evaluation of a novel hybrid bioartificial liver based on a multi-layer flat-plate bioreactor.

AIM: To evaluate the efficacy and safety of a hybrid bioartificial liver (HBAL) system in the treatment of acute liver failure. METHODS: Canine models with acute liver failure were introduced with intravenous administration of D-galactosamine. The animals were divided into: the HBAL treatment group (n = 8), in which the canines received a 3-h treatment of HBAL; the bioartificial liver (BAL) treatment group (n = 8), in which the canines received a 3-h treatment of BAL; the non-bioartificial liver (NBAL) treatment group (n = 8), in which the canines received a 3-h treatment of NBAL; the control group (n = 8), in which the canines received no additional treatment. Biochemical parameters and survival time were determined. Levels of xenoantibodies, RNA of porcine endogenous retrovirus (PERV) and reverse transcriptase (RT) activity in the plasma were detected. RESULTS: Biochemical parameters were significantly decreased in all treatment groups. The TBIL level in the HBAL group was lower than that in other groups (2.19 ± 0.55 µmol/L vs 24.2 ± 6.45 µmol/L, 12.47 ± 3.62 µmol/L, 3.77 ± 1.83 µmol/L, P < 0.05). The prothrombin time (PT) in the BAL and HBAL groups was significantly shorter than the NBAL and control groups (18.47 ± 4.41 s, 15.5 ± 1.56 s vs 28.67 ± 5.71 s, 21.71 ± 3.4 s, P < 0.05), and the PT in the HBAL group was shortest of all the groups. The albumin in the BAL and HBAL groups significantly increased and a significantly higher level was observed in the HBAL group compared with the BAL group (27.7 ± 1.7 g/L vs 25.24 ± 1.93 g/L). In the HBAL group, the ammonia levels significantly decreased from 54.37 ± 6.86 to 37.75 ± 6.09 after treatment (P < 0.05); there were significant difference in ammonia levels between other the groups (P < 0.05). The levels of antibodies were similar before and after treatment. The PERV RNA and the RT activity in the canine plasma were all negative. CONCLUSION: The HBAL showed great efficiency and safety in the treatment of acute liver failure.

Microbiological safety of a novel bio-artificial liver support system based on porcine hepatocytes: a experimental study.

BACKGROUND: Our institute has developed a novel bio-artificial liver (BAL) support system, based on a multi-layer radial-flow bioreactor carrying porcine hepatocytes and mesenchymal stem cells. It has been shown that porcine hepatocytes are capable of carrying infectious porcine endogenous retroviruses (PERVs) into human cells, thus the microbiological safety of any such system must be confirmed before clinical trials can be performed. In this study, we focused on assessing the status of PERV infection in beagles treated with the novel BAL. METHODS: Five normal beagles were treated with the novel BAL for 6 hours. The study was conducted for 6 months, during which plasma was collected from the BAL and whole blood from the beagles at regular intervals. DNA and RNA in both the collected peripheral blood mononuclear cells (PBMCs) and plasma samples were extracted for conventional PCR and reverse transcriptase (RT)-PCR with PERV-specific primers and the porcine-specific primer Sus scrofa cytochrome B. Meanwhile, the RT activity and the in vitro infectivity of the plasma were measured. RESULTS: Positive PERV RNA and RT activity were detected only in the plasma samples taken from the third circuit of the BAL system. All other samples including PBMCs and other plasma samples were negative for PERV RNA, PERV DNA, and RT activity. In the in vitro infection experiment, no infection was found in HEK293 cells treated with plasma. CONCLUSIONS: No infective PERV was detected in the experimental animals, thus the novel BAL had a reliable microbiological safety profile.

Factors influencing the transfer of porcine endogenous retroviruses across the membrane in bioartificial livers.

OBJECTIVES: to investigate the factors influencing the transfer of porcine endogenous retroviruses (PERVs) across the membrane in a new bioartificial liver (BAL). METHODS: A new BAL containing 2 circuits was constructed using plasma component separators with membrane pore sizes of 10 nm, 20 nm, 30 nm, and 35 nm, or a plasma filter with a membrane pore size of 500 nm. Cocultured cells of porcine hepatocytes and mesenchymal stem cells or single porcine hepatocytes were incubated in the bioreactors, and the BAL worked for 72 hours, with supernatant samples in internal and external circuits collected every 12 hours. PERV RNA, reverse transcriptase (RT) activity, and in vitro infectivity of the supernatant were detected. RESULTS: With the plasma filters, the results of PERV detection were the same in both circuits. With plasma component separators, PERV RNA was found in the external circuits, but no positive RT activity or HEK293 cell infection was found. The time at which the PERV RNA was first detected varied with the pore size of membrane; the larger the membrane pore size was, the earlier the RNA was detected. The PERV RNA level in the external circuits was reduced significantly compared with that in the internal circuits at any detecting time. CONCLUSIONS: The plasma component separators with membrane pore size =35 nm could significantly reduce the passage of infectious PERVs. And the membrane pore size, the treatment duration, and the viral level in the internal circuit were potential factors influencing the transfer of PERVs across the membrane in a BAL. In addition, a low risk of PERV transmission from porcine hepatocytes to human cells was found.

[New evidence of porcine endogenous retrovirus transmission with new bio-artificial liver system: a experimental study].

OBJECTIVE: To investigate the potential transmissibility of porcine endogenous retrovirus (PERV) from a newly-developed porcine hepatocyte bioartificial liver (BAL) system prior to human clinical trial by using a live canine model. METHODS: Five normal beagles were treated with the new BAL support system for six hours. Samples of plasma from the BAL system and whole blood from the beagles were collected at regular intervals over the six month study period. DNA and RNA were isolated from both the peripheral blood mononuclear cells (PBMCs) and plasma for evaluation by polymerase chain reaction (PCR) and reverse transcription (RT)-PCR, respectively, to detect PERV and the Sus scrofa cytochrome B normalization standard. In addition, RT activity and the in vitro infectivity of the plasma were detected in HEK293 cells. RESULTS: All five beagles remained in stable physical health throughout the treatment and survived until the end of the study. PERV RNA-positivity and RT activity were only detected in the plasma samples from the 3rd BAL treatment cycle. All other samples, including PBMCs and plasma, were negative for PERV RNA, PERV DNA, and RT activity. In addition, none of the sera samples showed in vitro infectivity. CONCLUSION: Application of our BAL system does not lead to PERV transmission.

Influence of chitosan nanofiber scaffold on porcine endogenous retroviral expression and infectivity in pig hepatocytes.

AIM: To investigate the influence of chitosan nanofiber scaffold on the production and infectivity of porcine endogenous retrovirus (PERV) expressed by porcine hepatocytes. METHODS: Freshly isolated porcine hepatocytes were cultured with or without chitosan nanofiber scaffold (defined as Nano group and Hep group) for 7 d. The daily collection of culture medium was used to detect reverse transcriptase (RT) activity with RT activity assay kits and PERV RNA by reverse transcription-polymerase chain reaction (PCR) and real time PCR with the PERV specific primers. And Western blotting was performed with the lysates of daily retrieved cells to determine the PERV protein gag p30. Besides, the in-vitro infectivity of the supernatant was tested by incubating the human embryo kidney 293 (HEK293) cells. RESULTS: The similar changing trends between two groups were observed in real time PCR, RT activity assay and Western blotting. Two peaks of PERV expression at 10H and Day 2 were found and followed by a regular decline. No significant difference was found between two groups except the significantly high level of PERV RNA at Day 6 and PERV protein at Day 5 in Nano group than that in Hep group. And in the in-vitro infection experiment, no HEK293 cell was infected by the supernatant. CONCLUSION: Chitosan nanofiber scaffold might prolong the PERV secreting time in pig hepatocytes but would not obviously influence its productive amount and infectivity, so it could be applied in the bioartificial liver without the increased risk of the virus transmission.

[The efficacy research of multi-layer flat-plate bioartificial liver on acute liver failure].

OBJECTIVE: To evaluate the efficacy of newly developed multi-layer flat-plate bioartificial liver in treatment of canines with acute liver failure. METHODS: Porcine hepatocytes and bone marrow mesenchymal stem cells were cocultured in newly developed multi-layer flat-plate bioreactor. Acute liver failure in canine models was induced by D-galactosamine administration.Sixteen canine models were divided into two groups: treatment group (n = 8) and control group (n = 8). Biochemical parameters were determined for 7 days after treatment and liver specimens were collected for histological analysis. RESULTS: Hepatic encephalopathy and general conditions were significantly improved in the treatment group, but no changes in the control group. Alanine aminotransferase was significantly decreased from (1512 ± 183) U/L to (86 ± 25) U/L in the treatment group, aspartate aminotransferase was significantly decreased from (1472 ± 365) U/L to (46 ± 11) U/L, lactate dehydrogenase was significantly decreased from (463 ± 76) U/L to (312 ± 84) U/L, total bilirubin was significantly decreased from (28.8 ± 6.2) µmol/L to (12.5 ± 3.6) µmol/L, ammonia was significantly decreased from (56 ± 15) µmol/L to (34 ± 10) µmol/L, and prothrombin time were significantly decreased in the treatment group but increased in the control group, albumin was improved in the treatment group but decreased in the control group. There were 5 canines survived in the treatment group but only 3 in the control group. But there was no difference on survival rates between the two group (P = 0.294). CONCLUSION: The application of newly developed multi-layer flat-plate bioartificial liver system was effective in the treatment of canines with acute liver failure.