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Objective:To explore the influence of short-time pasteurization (62.5±0.5℃ for 5 s) on the main bioactive components and immune cells in human breast milk.Methods:Fresh breast milk was collected from 53 women whose premature infants were admitted to the neonatal intensive care unit of the Children's Hospital of Fudan University from May 2020 to October 2020. Each sample (20 ml) was divided into unsterilized, Holder pasteurized (62.5 ℃ for 30 min), or short-time pasteurized groups. The concentration of secretory immunoglobulin A (sIgA), lactoferrin (LTF), lysozyme (LZM), and insulin-like growth factor binding protein-3 (IGFBP-3) in breastmilk whey were detected by enzyme-linked immunosorbent assay and the number of viable immune cells (leukocytes, monocytes, T cells, and B cells) in breastmilk by flow cytometry.Results:(1) A total of 87 breast milk samples were collected. The levels of sIgA, LTF, and LZM were the highest in the unsterilized group, followed by the short-time and Holder pasteurized group [0.42 mg/ml (0.33-0.65 mg/ml) vs 0.40 mg/ml (0.28-0.62 mg/ml) and 0.25 mg/ml (0.17-0.37 mg/ml); (3.57±1.06) vs (3.53±1.11) and (0.85±0.58) mg/ml; 128.60 μg/ml (77.18-203.00 μg/ml) vs 121.70 μg/ml (68.66-188.20 μg/ml) and 83.40 μg/ml (47.40-151.40 μg/ml); all P<0.05]. There was no significant difference in the level of IGFBP-3 among the groups. The median retention rates of sIgA, LTF, and LZM in the Holder pasteurized group were all lower than those in the short-time pasteurized group [55.87% (46.01%-71.41%) vs 96.93% (83.03%-115.90%); 21.72% (12.54%-29.42%) vs 97.88% (88.98%-104.30%); 69.26% (49.42%-89.08%) vs 93.80% (74.85%-110.20%); all P<0.05]. No significant difference in the level of preserved IGFBP-3 was observed between the three groups ( P>0.05). (2) The number of viable leukocytes, monocytes, T cells, and B cells in the Holder pasteurized group were lower than those in the unsterilized group [leukocytes: 185.50 (87.00-356.50) vs 1 271.00 (540.50-2 283.00); monocytes: 12.00 (6.00-16.75) vs 266.00 (137.30-518.80); T cells: 1.00 (0.00-2.00) vs 47.50 (28.50-116.00); B cells: 1.00 (0.00-1.75) vs 21.00(10.00-41.50); all P<0.05]. The percentage of viable leukocyte to the total leukocyte and the viable monocytes, T cells, and B cells to the viable leukocytes were lower in the Holder pasteurized group than those in the unsterilized group [24.80%(16.00%-36.80%) vs 74.20%(63.55%-86.45%); 5.91%(4.09%-8.77%) vs 21.90%(17.40%-29.30%); 0.31%(0.00%-1.31%) vs 4.00%(2.69%-6.43%); 0.30%(0.00%-0.86%) vs 1.27%(0.57%-2.85%); all P<0.05]. A similar trend was observed between short-time pasteurization and unsterilized groups (all P<0.05). (3) The percentages of viable monocytes, T cells, and B cells in their subsets were lower in both Holder and short-time pasteurized groups than those in the unsterilized group [2.94%(1.33%-7.14%) vs 9.72%(5.77%-16.00%) and 52.60%(31.35%-68.75%); 0.00%(0.00%-1.61%) vs 0.49%(0.00%-2.53%) and 28.10%(10.55%-57.00%); 0.00%(0.00%-0.83%) vs 0.24%(0.00%-2.47%) and 13.80%(3.27%-41.00%); all P<0.05].The number and percentage of viable leukocytes in total leukocytes and viable monocytes in total monocytes [leukocytes: 279.50(116.80-548.50), 32.20%(20.70%-45.75%); monocytes: 32.00(21.00- 83.75),15.60%(11.10%-19.15%)] were higher than those in the pasteurized group (all P>0.05). The short-time pasteurized group was noted only for a higher percentage of the viable monocytes to viable leukocytes than the Holder pasteurized group (all P<0.05). Conclusions:Compared with the Holder pasteurization, sIgA, LTF, LZM level, and monocyte activity in breast milk can be better preserved by short-time pasteurization.
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Objective@#To study the antiseptic effect of compound lysostaphin disinfectant and its preventive effect on infection of artificial dermis after graft on full-thickness skin defect wound in rats.@*Methods@#(1) Each one standard strain of Klebsiella pneumoniae, Acinetobacter baumannii, and Staphylococcus aureus were selected. Each 20 clinical strains of Klebsiella pneumoniae, Acinetobacter baumannii, and Staphylococcus aureus were collected from those isolated from wound exudates of burn patients hospitalized in our wards from January 2014 to December 2016 according to the random number table. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of compound lysostaphin disinfectant to above-mentioned strains were detected. The experiment was repeated 3 times. Compared with the corresponding standard strain, the clinical strain with higher MIC and/or MBC was considered as having decreased sensitivity to the disinfectant. The percentage of strains of each of the three kinds of bacteria with decreased sensitivity was calculated. (2) Artificial dermis pieces were soaked in compound lysostaphin disinfectant for 5 min, 1 h, 2 h, and 4 h, respectively, with 21 pieces at each time point. After standing for 0 (immediately), 12, 24, 36, 48, 60, 72 h (with 3 pieces at each time point), respectively, the diameters of their inhibition zones to standard strains of Klebsiella pneumoniae, Acinetobacter baumannii, and Staphylococcus aureus were measured. The experiment was repeated 3 times. The shortest soaking time corresponding to the longest standing time, after which the disinfectant-soaked artificial dermis could form an effective inhibition zone (with diameter more than 7 mm), was the sufficient soaking time of the disinfectant to the artificial dermis. (3) Forty Sprague-Dawley rats were divided into post injury day (PID) 3, 7, 14, and 21 sampling groups according to the random number table, with 10 rats in each group. A full-thickness skin defect wound with a diameter of 20 mm was made on both sides of the spine on the back of each rat. Immediately after injury, the artificial dermis without any treatment was grafted on the wound on left side of the spine (hereinafter referred to as control wound), while the sufficiently soaked artificial dermis with compound lysostaphin disinfectant was grafted on the wound on right side of the spine (hereinafter referred to as disinfectant wound). On PID 3, 7, 14, and 21, the gross condition of wounds of all the surviving rats was observed, and the new infection rates of control wounds and disinfectant wounds were calculated. Then, the rats in the sampling group with corresponding time were killed, and the full-thickness wound tissue containing artificial dermis was collected for quantitative analysis of bacteria. Bacteria content of the uninfected control wounds and that of the uninfected disinfectant wounds were compared. Data were processed with chi-square test and Wilcoxon rank sum test.@*Results@#(1) The MIC of compound lysostaphin disinfectant to standard strains of Staphylococcus aureus, Klebsiella pneumoniae, and Acinetobacter baumannii were 1/32, 1/32, and 1/512 of the original concentration of the disinfectant, respectively, and the MBC were 1/32, 1/16, and 1/512 of the original concentration of the disinfectant, respectively. The percentages of clinical strains of Klebsiella pneumoniae, Acinetobacter baumannii and Staphylococcus aureus with decreased sensitivity to compound lysostaphin disinfectant were 15% (3/20), 20% (4/20), and 10% (2/20), respectively. (2) After being soaked in compound lysostaphin disinfectant for 2 and 4 h, the longest standing time, after which the artificial dermis could form an effective inhibition zone against Klebsiella pneumoniae, Acinetobacter baumannii, and Staphylococcus aureus, were 24, 36, and 48 h respectively, longer than 12, 24, and 24 h of soaking for 5 min and 24, 24, and 36 h of soaking for 1 h. The sufficient soaking time of compound lysostaphin disinfectant to artificial dermis was 2 h. (3) On PID 3, no infection symptom was observed in all the wounds, and so both the new infection rate of control wounds and that of disinfectant wounds were 0. The artificial dermis was transparent but not well connected with the wound. On PID 7, the new infection rate of control wounds was 20.00% (6/30), which was obviously higher than 3.33% (1/30) of disinfectant wounds, χ2=4.043, P<0.05. On the infected wound, a large amount of purulent exudates were observed, and the artificial dermis was not connected with the wound and degraded partially. On the uninfected wound, artificial dermis was transparent and had a partial connection with the wound. On PID 14 and 21, no new infected wound was observed, and so both the new infection rate of control wounds and that of disinfectant wounds were 0. There was no obvious improvement on the infected wounds. The collagen layers of artificial dermis in the uninfected wound established a good connection with the wound and were separating from the silica gel layer gradually. Infection occurred in 2, 3, 1 control wound (s) in PID 7, 14, and 21 sampling groups, respectively, and in 1 disinfectant wound in PID 14 sampling group. The bacteria content of the infected wounds tissue was 0.79×106 to 7.22×109 colony-forming unit (CFU)/g. The bacteria content of uninfected control wounds tissue in PID 3, 7, and 14 sampling groups were (3.43±1.88)×102, (2.37±0.43)×103, and (8.40±1.03)×103 CFU/g, respectively, which were significantly higher than (0.33±0.12)×102, (0.43±0.17)×103, (2.16±0.52)×103 CFU/g of uninfected disinfectant wounds tissue (Z=-3.780, -3.554, -3.334, P<0.05). The bacteria content of uninfected control wounds tissue and that of uninfected disinfectant wounds tissue in PID 21 sampling group were similar (Z=-0.490, P>0.05).@*Conclusions@#Compound lysostaphin disinfectant has quite strong antibacterial ability against Klebsiella pneumoniae, Acinetobacter baumannii, and Staphylococcus aureus. Clinical strains of the three kinds of bacteria were highly sensitive to compound lysostaphin disinfectant. Saturation of absorption of compound lysostaphin disinfectant achieves in artificial dermis after 2 hours′ soaking. After 24, 36, and 48 hours′ standing, the soaked artificial dermis still has the antibacterial effect on Klebsiella pneumoniae, Acinetobacter baumannii, and Staphylococcus aureus, respectively. The infection rate and the bacteria content of full-thickness skin defect wound in rats are all decreased when grafted with soaked artificial dermis.
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Objective To study the mechanism of platelet aggregation induced by Streptococcus suis serotype 2 muramidase-released protein (MRP) and to provide scientific proof and theoretical basis for clinical treatment of patients with Streptococcus suis infection. Methods Nickel column affinity chromatogra-phy was used to purify recombinant proteins of MRP-N and MRP-C. Platelet aggregometer, thromboelastog-raphy (TEG) and scanning electron microscope were used to observe the platelet aggregation induced by MRP. Results Streptococcus suis 2 wild type strain,but not the mutant strain ΔMRP,could induce platelet aggregation. It was MRP-N but not MRP-C that induced platelet aggregation. GPRP,an inhibitor of β2inte-grin receptor,could significantly inhibit the platelet aggregation induced by MRP. Conclusion Streptococ-cus suis 2 MRP induces platelet aggregation through β2integrin receptor pathway.
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Objective:To evaluate the synergistic antibacterial effects of lysozyme with ethylenediami-netetraacetic acid disodium salt (EDTA-2Na) on Enterococcus faecalis (E.faecalis) and Porphyromonas endodontalis ( P.endodontalis) .Methods:E.faecalis and P.endodontalis were cultured and adjusted to 108 CFU/mL.Then 0.3, 0.5, 1, 2, 5, 10, 50, 100, 150 and 300 g/L of lysozyme were prepared with deionized water;and the lysozyme solutions were mixed with 0.5, 1.0, 2.0 g/L of EDTA-2Na, re-spectively.The bacteria and lysosome with/without EDTA-2Na interacted for 15 min, then water-soluble tetrazolium (WST) working solution was added and the activity of the bacteria was calculated by mea-suring optical densities at 450 nm and 630 nm with microplate spectrophotometer .Results:Regarding the pure lysozyme from 0.5 g/L to 150 g/L, more E.faecalis and P.endodontalis were inhibited when the concentration of lysozyme was higher , especially for E.faecalis.There was synergistic effect of lysozyme with EDTA-2Na on antibacterial activity , which was related to the concentration of lysozyme .On E.fae-calis, the antibacterial activity of lysozyme with EDTA-2Na was 1.2-3.7 folds than the pure lysozyme when the concentration of lysozyme was 0.5-50 g/L (P0.05). Conclusion: For E.faecalis and P.endodontalis, a low concentration of lysozyme with EDTA-2Na showed significant synergistic antibacterial activity , while a high concentration of lysozyme with EDTA-2 Na did not .
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Desde que la lisozima fue descubierta por Alexander Fleming en 1922, muchos son los trabajos que se han lleva-do a cabo para describir las distintas actividades biológicas de esta proteína como son su actividad antibacteria-na, antiviral, antinflamatoria, analgésica, antitumoral y antioxidante. Su actividad antibacteriana frente a bacterias Gram-positivas es la actividad más ampliamente estudiada. Muchas investigaciones se han realizado para ampliar el espectro antibacteriano y poder atacar bacterias Gram-negativas. Se han llevado a cabo modificacio-nes térmicas, químicas, enzimáticas, mutaciones genéticas y efectos sinérgicos con otros compuestos, y se consiguió exitosamente ampliar el espectro antibacteriano, proponiendo en todos los casos que dicha actividad es independiente de su a ctividad enzimática natural. En este trabajo destacamos todas las propiedades estructura-les de la lisozima y sus principales actividades biológicas y las investigaciones que se han llevado a cabo para potenciar dichas actividades.
Since ly sozyme was discovered by Alexander Fleming in1922, many papers have been published on the different biological activities of this protein, such as its antibacterial, antiviral, anti-inflammatory, analgesic, antitumor andantioxidant properties. Its antibacterial activity againstGram-positive bacteria is the most widely studied. Much research has been done to widen the antibacterial spectrumand to attack the Gram-negative bacteria. Thermal, chemical and enzymatic modifications, as well as genetic mutations and synergistic effects, with other compounds have been made. The antibacterial spectrum was success fully widened in all cases, suggesting that this activity is independent of its natural enzymatic activity. In this paper we describe t he structural properties of lysozyme and its main biological activities, and also the research that has been carried out to maximize these activitie.
Desde que a lisozima foi descoberta por AlexandreFleming em 1922, muitos são os trabalhos que foram realizados para descrever as diferentes atividades biológicas desta proteína, como são sua atividade bacteriana, antiviral, anti-inflamatória, analgésica, antitumoral e antioxidante. Sua atividade antibacteriana frente a bactérias Gram-positivas é a atividade mais amplamente estudada. Muitas pesquisas foram realizadas para ampliar o espectroanti bacteriano e poder atacar bactérias Gram-negativas.Foram realizadas modificações térmicas, químicas,enzimáticas, mutações genéticas e efeitos sinérgicos com outros compostos, e obteve-se com sucesso ampliar o espectro antibacteriano, propondo em todos os casos que tal atividade é independente da sua atividade enzimática natural.Neste trabalho destacamos todas as propriedades estruturais da lisozima e suas principais atividades biológicas e as pesquisas que foram realizadas para potenciar tais atividades.
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Humanos , Clara de Ovo , Hipersensibilidade , MuramidaseRESUMO
Objective To investigate the influences of lysozyme on the mRNA expressions of MMP-1,-12 and lysyl oxidase(LOX)in cultured fibroblasts in vitro.Methods Primarily cultured fibroblasts isolated from human skin were treated with three concentrations(0.1×10~(-8),1×10~(-7)mol/L)of lysozyme followed by another 24-hour cuhure.Subsequently,total RNA was extracted from the fibroblasts and subjected to RT-PCR for the detection of MMP-1,-12 and LOX mRNA.Results There was a significant difference in the mRNA expressions of MMP-1,-12 and LOX among the fibroblasts treated with the three concentrations of lysozyme (F=6.98,4.44,5.24,respectively,all P<0.05).SNK-q test showed that untreated fibroblasts differed signifi-cantly from those treated with lysozyme of 1×10~(-7) mol/L in the mRNA expression of MMP-1 and MMP-12 (P<0.05),and from those treated with iysozyme of 1×10~(-7) mol/L.and 1×10~(-8)mol/L in the mRNA expres-sion of LOX(both P<0.05),whereas no significant difference was ohserved between fibroblasts treated with lysozyme of 1×10~(-8) mol/L and untreated fibrohlasts or those with lysozyme of 1 x 10~(-7)mol/L in the mRNA expression of MMP-1 and MMP-12.or between fibroblasts treated with lysozyme of 1 x 10~(-8)mol/L and those with that of 1×10~(-7) mol/L in the expression of LOX (all P>0.05).Conclusions Lysozyme upregulates the mRNA expression of MMP-1 and MMP-12 but downregulates the mRNA expression of LOX in cultured fibro-blasts in vitro.
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The mrp gene of SS2 human strain Habb was truncated and cloned into a prokaryotic expression vector pGEX4T-2,and a fusion-expressed protein MRP-GST of 61 000 was obtained in E.coli.The GST was cut from MRP-GST with thrombin protease to gain the purified MRP of 35 000 which showed a strong reaction to the SS2 positive sera in Western blotting.BALB/c mice were immunitied intraperitoneally with purified MRP protein.Murine myeloma cells were fused with the splenocytes of the immunized mice after the third immunization.An indirect ELISA coated with purified MRP was used to screen hybridomas for production of specific antibody.The six McAb recognized MRP specially.According to the results of 71 standard strains (48 SS and 34 SS2)by Sandwich ELISA,the coincidence rate was 97.2%,but for 34 SS standard serotype was 100 %.The ELISA method has a potential value for clinical and epidemiological applicationgs.
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BACKGROUND AND OBJECTIVES: Airway mucosa is protected by several complex of defense mechanisms, namely mucociliary clearance, immunoglobulins, cellular components, and antibacterial secretary enzymes. In particular, lysozymes, also known as muramidase, are important components of innate immunity against pathogens at mucosal surfaces. Trachea mucosa is mainly protected by mucociliary clearance, and recently, lysozymes are also known to be in the trachea tissues. The purpose of this study is to identify the distribution and expression of lysozymes in the trachea of normal rats. MATERIALS AND METHOD: Tra-cheas were collected from male Wistar-kyoto rats. The expression level and distribution of mRNA of lysozyme were analyzed by RT-PCR and immunohistochemistry. Western blot assessed and confirmed the expression of lysozyme. RESULTS: The expression of mRNA of lysozyme in trachea was observed. Immunohistochemical analysis demonstrated that lysozyme was expressed in the epithelium and the submucosal serous gland. Western blot was detected at the molecular weight of 14 kDa. CONCLUSION: We identified the distribution and expression of lysozymes in the trachea of normal rats. The result suggests that innate immunity such as that played by lysozymes is an important component of defense mechanisms along with mucociliary clearance.