Platelets directly regulate DNA damage and division of Staphylococcus aureus.

Platelets (PLTs) are classically used in the clinical setting to maintain hemostasis. Recent evidence supports important roles for PLTs in host inflammatory and immune responses, and PLT-rich plasma has been demonstrated to inhibit the growth of bacteria in vitro and in vivo; however, few studies have examined whether PLTs can inhibit bacterial growth directly, and related mechanisms have not been elucidated further. Accordingly, in this study, we evaluated the effects of PLTs on bacterial growth. We washed and purified PLTs from peripheral blood, then confirmed that PLTs significantly inhibited the growth of Staphylococcus aureus when cocultured in vitro. Moreover, PLTs damaged DNA and blocked cell division in S. aureus. During coculture, PLT-derived TGF-ß1 was dramatically down-regulated compared with that in PLT culture alone, and the addition of TGF-ß1 to the coculture system promoted the inhibition of PLTs on S. aureus. Analysis of a murine S. aureus infection model demonstrated that the depletion of PLTs exacerbated the severity of infection, whereas the transfusion of PLTs alleviated this infection. Our observations demonstrate that PLTs could directly inhibit the growth of S. aureus by damaging DNA and blockage cell division, and that PLT-derived TGF-ß1 may play an important role in this machinery.-Xu, J., Yi, J., Zhang, H., Feng, F., Gu, S., Weng, L., Zhang, J., Chen, Y., An, N., Liu, Z., An, Q., Yin, W., Hu, X. Platelets directly regulate DNA damage and division of Staphylococcus aureus.

Evaluation of the advantages of platelet concentrates stored at 4°C versus 22°C.

BACKGROUND: Platelet (PLT) storage at cold temperatures (4°C) can reduce bacterial contamination and lower the risk of transfusion-related complications. We compared the effects of 22 and 4°C storage conditions for PLTs to further explore the efficiency of hemostasis in acute bleeding and extended PLT shelf life. STUDY DESIGN AND METHODS: Manually prepared PLTs (PLT concentrates in plasma, not PLT additive solution) were stored at 4 and 22°C. The PLT counts, scanning electronic microscope observations, blood gas indices, biochemical indices, PLT aggregative function, and surface CD62P expression were monitored and compared between the groups. RESULTS: There was no obvious change in PLT counts between Day 21 at 4°C and Day 5 at 22°C. PLTs stored at 4°C for 10 to 14 days were dramatically activated, had rough surfaces, and showed a significant degree of long pseudopodia formation. The pH of the PLTs on Day 5 was lower at 22°C than at 4°C, while the lactate dehydrogenase and lactic acid levels in the former group were significantly higher (p < 0.005). The maximum aggregation rates induced by collagen and arachidonic acid in the PLTs stored at 4°C for 5 days remained higher than 80%, while the rates induced by four inducers in the PLTs stored at 22°C were less than 5%. PLTs stored at 4°C for 10 to 14 days showed higher surface expression of PAC-1 and CD62P. CONCLUSION: PLT counts, cellular morphologies, PLT membranes, cytoplasmic structures, aggregation rates, and hemostatic PLT function stored at 4°C for 10 to 14 days were better than those stored at 22°C for 5 days.

Supernatant of platelet-Klebsiella pneumoniae coculture induces apoptosis-like death in Klebsiella pneumoniae.

Multidrug-resistant Klebsiella pneumoniae strains, especially carbapenem-resistant K. pneumoniae, have become a rapidly emerging crisis worldwide, greatly limiting current therapeutic options and posing new challenges to infection management. Therefore, it is imperative to develop novel and effective biological agents for the treatment of multidrug-resistant K. pneumoniae infections. Platelets play an important role in the development of inflammation and immune responses. The main component responsible for platelet antibacterial activity lies in the supernatant stimulated by gram-positive bacteria. However, little research has been conducted on the interaction of gram-negative bacteria with platelets. Therefore, we aimed to explore the bacteriostatic effect of the supernatant derived from platelet-K. pneumoniae coculture and the mechanism underlying this effect to further assess the potential of platelet-bacterial coculture supernatant. We conducted this study on the gram-negative bacteria K. pneumoniae and CRKP and detected turbidity changes in K. pneumoniae and CRKP cultures when grown with platelet-K. pneumoniae coculture supernatant added to the culture medium. We found that platelet-K. pneumoniae coculture supernatant significantly inhibited the growth of K. pneumoniae and CRKP in vitro. Furthermore, transfusion of platelet-K. pneumoniae coculture supernatant alleviated the symptoms of K. pneumoniae and CRKP infection in a murine model. Additionally, we observed apoptosis-like changes, such as phosphatidylserine exposure, chromosome condensation, DNA fragmentation, and overproduction of reactive oxygen species in K. pneumoniae following treatment with the supernatant. Our study demonstrates that the platelet-K. pneumoniae coculture supernatant can inhibit K. pneumoniae growth by inducing an apoptosis-like death, which is important for the antibacterial strategies development in the future.IMPORTANCEWith the widespread use of antibiotics, bacterial resistance is increasing, and a variety of multi-drug resistant Gram-negative bacteria have emerged, which brings great challenges to the treatment of infections caused by Gram-negative bacteria. Therefore, finding new strategies to inhibit Gram-negative bacteria and even multi-drug- resistant Gram-negative bacteria is crucial for treating infections caused by Gram-negative bacteria, improving the abuse of antibiotics, and maintaining the balance between bacteria and antibiotics. K. pneumoniae is a common clinical pathogen, and drug-resistant CRKP is increasingly difficult to cure, which brings great clinical challenges. In this study, we found that the platelet-K. pneumoniae coculture supernatant can inhibit K. pneumoniae growth by inducing an apoptosis-like death. This finding has inspired the development of future antimicrobial strategies, which are expected to improve the clinical treatment of Gram-negative bacteria and control the development of multidrug-resistant strains.

[Laboratory testing strategies for human immunodeficiency virus (HIV) in blood donors].

Objective To propose the blood detection strategies for human immunodeficiency virus (HIV) among blood donors, and provide reference for the detection, early diagnosis and transmission blocking of HIV. Methods A total of 117 987 blood samples from blood donors were screened using the third- and fourth-generation ELISA HIV detection reagents. Western blot analysis was used to verify the reactive results of the third-generation reagent alone, or both the third-generation and fourth-generation reagents. HIV nucleic acid test was carried out for those with negative test results of the third- and fourth-generation reagents. For those with positive results of the fourth-generation reagent only, nucleic acid test followed by a confirmatory test by Western blot analysis was carried out. Results 117 987 blood samples from blood donors were tested by different reagents. Among them, 55 were tested positive by both the third- and fourth-generation HIV detection reagents at the same time, accounting for 0.047% and 54 cases were confirmed HIV-positive by Western blot analysis, and 1 case was indeterminate, then turned positive during follow-up testing. 26 cases were positive by the third-generation reagent test alone, among which 24 cases were negative and 2 were indeterminate by Western blot analysis. The band types were p24 and gp160 respectively detected by Western blot analysis, and were confirmed to be HIV negative in follow-up testing. 31 cases were positive by the fourth-generation HIV reagent alone, among which 29 were negative by nucleic acid test, and 2 were positive according to the nucleic acid test.Western blot analysis was used to verify that the two cases were negative. However, after 2~4 weeks, the results turned positive when the blood sample was retested by Western blot analysis during the follow-up of these two cases. All the specimens that were tested negative by both the third- and fourth-generation HIV reagents were validated negative by HIV nucleic acid test. Conclusion A combined strategy with both third- and fourth-generation HIV detection reagents can play a complementary role in blood screening among blood donors. The application of complementary tests, such as nucleic acid test and Western blot analysis, can further improve the safety of blood supply, thus contributing to the early diagnosis, prevention, transmission and treatment of blood donors potentially infected by HIV.

Rational design of dual-functional surfaces on polypropylene with antifouling and antibacterial performances via a micropatterning strategy.

The hydrophobicity and inertness of the polypropylene (PP) material surface usually lead to serious biofouling and bacterial infections, which hamper its potential application as a biomedical polymer. Many strategies have been developed to improve its antifouling or antibacterial properties, yet designing a surface to achieve both antifouling and antibacterial performances simultaneously remains a challenge. Herein, we construct a dual-function micropatterned PP surface with antifouling and antibacterial properties through plasma activation, photomask technology and ultraviolet light-induced graft polymerization. Based on the antifouling agent poly(2-methacryloyloxyethyl phosphate choline) (PMPC) and the antibacterial agent quaternized poly(N,N-dimethylamino)ethyl methacrylate (QPDMAEMA), two different micropatterning structures have been successfully prepared: PP-PMPC-QPDMAEMA in which QPDMAEMA is the micropattern and PMPC is the coating polymer, and PP-QPDMAEMA-PMPC in which PMPC is the micropattern and QPDMAEMA is the coating polymer. The composition, elemental distribution and surface morphology of PP-PMPC-QPDMAEMA and PP-QPDMAEMA-PMPC have been thoroughly characterized by Fourier transform infrared (FT-IR) spectroscopy, X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM), respectively. Compared with pristine PP, the two types of micropatterned PP films exhibit good surface hydrophilicity as characterized by water contact angle measurements. The results of anti-protein adsorption, platelet adhesion and antibacterial evaluation showed that PP-PMPC-QPDMAEMA and PP-QPDMAEMA-PMPC had good anti-protein adsorption properties, especially for lysozyme (Lyz). They can effectively prevent platelet adhesion, and the anti-platelet adhesion performance of PP-QPDMAEMA-PMPC is slightly better than that of the PP-PMPC-QPDMAEMA sample. The sterilization rate of S. aureus and E. coli is as high as 95% for the two types of micropatterned PP films. Due to the rational design of micropatterns on the PP surface, the two classes of dual-functional PP materials realize both the resistance of protein and platelet adhesion, and the killing of bacteria at the same time. We anticipate that this work could provide a design strategy for the construction of multifunctional biomedical polymer materials.

Platelets Inhibit Methicillin-Resistant Staphylococcus aureus by Inducing Hydroxyl Radical-Mediated Apoptosis-Like Cell Death.

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most common drug-resistant bacteria and poses a significant threat to human health. Due to the emergence of multidrug resistance, limited drugs are available for the treatment of MRSA infections. In recent years, platelets have been reported to play important roles in inflammation and immune responses, in addition to their functions in blood hemostasis and clotting. We and other researchers have previously reported that platelets can inhibit Staphylococcus aureus growth. However, it remained unclear whether platelets have the same antibacterial effect on drug-resistant strains. In this study, we hypothesized that platelets may also inhibit the growth of MRSA; the results confirmed that platelets significantly inhibited the growth of MRSA in vitro. In a murine model of MRSA infection, we found that a platelet transfusion alleviated the symptoms of MRSA infection; in contrast, depletion of platelets aggravated infective symptoms. Moreover, we observed an overproduction of hydroxyl radicals in MRSA following platelet treatment, which induced apoptosis-like death of MRSA. Our findings demonstrate that platelets can inhibit MRSA growth by promoting the overproduction of hydroxyl radicals and inducing apoptosis-like death. IMPORTANCE The widespread use of antibiotics has led to the emergence of drug-resistant bacteria, particularly multidrug-resistant bacteria. MRSA is the most common drug-resistant bacterium that causes suppurative infections in humans. As only a limited number of drugs are available to treat the infections caused by drug-resistant pathogens, it is imperative to develop novel and effective biological agents for treating MRSA infections. This is the first study to show that platelets can inhibit MRSA growth in vitro and in vivo. Our results revealed that platelets enhanced the production of hydroxyl radicals in MRSA, which induced a series of apoptosis hallmarks in MRSA, including DNA fragmentation, chromosome condensation, phosphatidylserine exposure, membrane potential depolarization, and increased intracellular caspase activity. These findings may further our understanding of platelet function.

[High expression of P-selectin (CD62P) after platelet activation in vitro inhibits bacterial proliferation and induces apoptosis-like changes].

Objective To compare the expression level of P-selectin (CD62P) on platelets surface under the stimulation of Staphylococcus aureus (SA) and Escherichia coli (E.coli), explore the inhibitory effects of platelets on the their proliferation, and further investigate the molecular mechanism by which platelets inhibit the proliferation of bacteria. Methods 106 CFU/mL SA and E.coli were co-cultured with 2×1011/L purified platelets, and the A600 values of the two groups were detected; The CD62P of platelets was detected by flow cytometry after platelets co-cultured with SA and E.coli for 2 hours and 4 hours. The platelet factor 4 (PF4) released by platelets was detected by ELISA; After co-cultured with SA and E.coli for 12 hours, the proliferation, phosphatidylserine (PS) eversion and cell membrane potential of SA and E.coli were analyzed by flow cytometry. Results After platelets co-cultured with SA and E.coli for 6 hours, the turbidity of SA decreased significantly and the turbidity of E.coli showed a slight decrease. Compared with the control group, the counts of bacterial plates decreased after two kinds of bacteria co-cultured with platelets. After co-cultured with SA and E.coli for 2 hours and 4 hours, the CD62P levels of platelets increased. In particular, the CD62P level of platelets co-cultured with SA was significantly higher than that of platelets co-cultured with E.coli. The release of intracellular protein PF4 of platelet increased significantly after bacteria stimulation. The proliferation rate of SA and E.coli decreased after co-cultured with platelets, and SA and E.coli exhibited PS eversion and depolarization of cell membrane potential. Conclusion High expression of CD62P inhibits the proliferation and induces apoptotic changes of SA and E.coli after platelets activation in vitro, and the inhibitory effect of platelets on SA was better than that of E.coli.

Caspase-3/NLRP3 signaling in the mesenchymal stromal niche regulates myeloid-biased hematopoiesis.

BACKGROUND: The fate of hematopoietic stem cells (HSCs) is determined by a complex regulatory network that includes both intrinsic and extrinsic signals. In the past decades, many intrinsic key molecules of HSCs have been shown to control hematopoiesis homeostasis. Non-hematopoietic niche cells also contribute to the self-renewal, quiescence, and differentiation of HSCs. Mesenchymal stromal cells (MSCs) have been identified as important components of the niche. However, the regulatory role of MSCs in hematopoiesis has not been fully understood. METHODS: Caspase-3 and NLRP3 gene knockout mice were generated respectively, and hematopoietic development was evaluated in the peripheral circulation and bone marrow by flow cytometry, colony formation assay, and bone marrow transplantation. Bone-associated MSCs (BA-MSCs) were then isolated from gene knockout mice, and the effect of Caspase-3/NLRP3 deficient BA-MSCs on hematopoiesis regulation was explored in vivo and ex vivo. RESULTS: We report that Caspase-3 deficient mice exhibit increased myelopoiesis and an aberrant HSC pool. Ablation of Caspase-3 in BA-MSCs regulates myeloid lineage expansion by altering the expression of hematopoietic retention cytokines, including SCF and CXCL12. Interestingly, NLRP3 gene knockout mice share phenotypic similarities with Caspase-3 deficient mice. Additionally, we found that NLRP3 may play a role in myeloid development by affecting the cell cycle and apoptosis of hematopoietic progenitors. CONCLUSIONS: Our data demonstrate that the Caspase-3/NLRP3 signaling functions as an important regulator in physiological hematopoiesis, which provides new insights regarding niche signals that influence hematopoiesis regulation in the bone marrow.

NLRP3 inflammasome of renal tubular epithelial cells induces kidney injury in acute hemolytic transfusion reactions.

BACKGROUND: Blood transfusion, a common basic supporting therapy, can lead to acute hemolytic transfusion reaction (AHTR). AHTR poses a great risk to patients through kidney function damage in a short time. Previous reports found that heme from destroyed red blood cells impaired kidney function, and NLR family pyrin domain containing 3 (NLRP3) inflammasome was augmented in case of kidney injury. However, the detailed mechanism regarding whether NLRP3 inflammasome is involved in kidney function injury in AHTR is not fully understood yet. METHODS: Hemolysis models were established by vein injection with human blood plasma or mouse heme from destroyed red blood cells. The injured renal tubular epithelial cells (RTECs) were evaluated by tubular damage markers staining in hemolysis models and in primary RTECs in vitro. The activation of NLRP3 inflammasome in RTECs by hemes was investigated by Western blot, ELISA, scanning electron microscopy, immunofluorescent staining, flow cytometry, and hemolysis models. NLRP3 gene knockout mice were employed to confirm these observations in vitro and in vivo. The binding between a novel inhibitor (66PR) and NLRP3 was affirmed by molecule docking and co-immunoprecipitation. The rescue of 66PR on kidney function impairment was explored in murine hemolysis models. RESULTS: We found that heme could activate NLRP3 inflammasome in RTECs to induce kidney function injury. NLRP3 gene knockout could prevent the damage of RTECs caused by hemes and recover kidney function in AHTR. Moreover, NLRP3 inflammasome chemical inhibitor, 66PR, could bind to NLRP3 protein and inhibit inflammasome activation in RTECs, which consequently relieved the injury of RTECs caused by hemes, and alleviated kidney function damage in the AHTR model. CONCLUSIONS: Hemes could activate NLRP3 inflammasome in RTECs, and a novel NLRP3 inflammasome inhibitor named 66PR relieved kidney function damage in AHTR. Our findings provided a new possible strategy to treat kidney function failure in AHTR.

Rational Design of PMPC/PDMC/PEGDA Hydrogel Micropatterns onto Polylactic Acid with Enhanced Biological Activity.

Polylactic acid (PLA) is one of the biodegradable materials that has been used in the areas of surgical healing lines, cancer treatment, and wound healing. However, the application of PLA is still rather limited due to its high hydrophobicity and poor antibacterial activity. In order to enhance the antifouling and antibacterial performances of PLA, here we modified the surface of PLA with various sizes of hydrogel micropatterns in negative or positive mode using plasma treatment, the photomask technique, and UV-graft polymerization. The hydrogel micropatterns consist of poly(ethylene glycol) diacrylate (PEGDA), poly(2-methacryloyloxyethylphosphorylcholine) (PMPC), and poly(methacryloyloxyethyltrimethylammonium chloride) (PDMC). Compared to PLA, the patterned PLA (PLA-PMPC/PDMC/PEGDA) shows obviously enhanced antifouling and antibacterial activities. For PLA-PMPC/PDMC/PEGDA with either positive or negative micropatterns, the antifouling and antibacterial properties are gradually increasing with decreasing the size of micropatterns. Compared with PLA-PMPC/PDMC/PEGDA bearing positive and negative micropatterns in the same size, the PLA-PMPC/PDMC/PEGDA with negative micropatterns exhibits slightly better biological activity and the PLA-PMPC/PDMC/PEGDA with 3 µm negative hydrogel micropatterns shows the best hydrophilicity, antifouling, and antibacterial properties. Combining the in vitro hemolysis assay, cytotoxicity, water absorption test, and degradation test results, it is suggested that the fabrication of hydrogel micropatterns onto the PLA surface could significantly improve biological activities of PLA. We expect that this work would provide a new strategy to potentially develop PLA as a promising wound dressing.

[Macrophage and platelets inhibit proliferation of methicillin-resistant Staphylococcus aureus in vitro].

Objective To investigate the effect of platelet and macrophage on the proliferation of methicillin-resistant Staphylococcus aureus (MRSA) in vitro, to explore the mechanisms of the inhibition effect, and to further reveal platelet function. Methods LB culture system with 4×108 CFU/mL MRSA inoculation concentration was established in vitro. Leukocyte-free platelets with a number of 200×109/L (platelet group) and macrophages with a number of 1×109/L (macrophage group) were added, respectively. The system without platelets and macrophages was used as negative control (control group). The absorbance of bacterial solution of the two groups was measured for reflect the proliferation of MRSA at different time points. After 2 hours culture, the bacterial liquid was collected for plating after double dilution, and the concentration of bacterial liquid was obtained through calculation after colony counting. Flow cytometry was also performed to verify the phagocytosis of macrophages and the capture ability of platelets. Results Both macrophages and platelets showed suppressive effect on the proliferation of MRSA compared to control group, while the effect of platelets was more significant. Conclusion Platelets have stronger inhibitory ability on MRSA proliferation in vitro than macrophages.

Mesenchymal stromal cells can be applied to red blood cells storage as a kind of cellular additive.

During storage in blood banks, red blood cells (RBCs) undergo the mechanical and metabolic damage, which may lead to the diminished capacity to deliver oxygen. At high altitude regions, the above-mentioned damage may get worse. Thus, more attention should be paid to preserve RBCs when these components need transfer from plain to plateau regions. Recently, we found that mesenchymal stromal cells (MSCs) could rescue from anemia, and MSCs have been demonstrated in hematopoietic stem cells (HSCs) transplantation to reconstitute hematopoiesis in vivo by us. Considering the functions and advantages of MSCs mentioned above, we are trying to find out whether they are helpful to RBCs in storage duration at high altitudes. In the present study, we first found that mice MSCs could be preserved in citrate phosphate dextrose adenine-1 (CPDA-1) at 4 ± 2°C for 14 days, and still maintained great viability, even at plateau region. Thus, we attempted to use MSCs as an available supplement to decrease RBCs lesion during storage. We found that MSCs were helpful to support RBCs to maintain biochemical parameters and kept RBCs function well on relieving anemia in an acute hemolytic murine model. Therefore, our investigation developed a method to get a better storage of RBCs through adding MSCs, which may be applied in RBCs storage as a kind of cellular additive into preservation solution.