Klebsiella pneumoniae secreted protein-containing antigens in the system of adaptive immunity

The study was aimed at the evaluation of the antigenic properties of K. pneumoniae secreted protein-containing antigens with a molecular weightt of 21 and 34-35 kD, obtained from supernatant culture fluid. As confirmed by the method of flow cytofluorimetry, the protein-containing fractions belonged to the secreted components of the microbial cell. The fraction with a molecular weight of 34-35 kD possessed high antigenic activity and contributed to the formation of specific antibodies after the immunization of mice. At the same time none of the protein fractions lead to an increase in the level of autoantibodies in mouse blood sera to organ-unspecific and organ-specific antigens. As revealed by the method of solid-phase, in 6 (27.3%) from 22 patients of patients with rhizomelic spondylitis had an increased level of IgG to K. pneumoniae cell-wall antigens with a molecular weight of 34-35 kD. An increase in the level of IgG to the secreted protein-containing fraction with a molecular weight of 34-35 kD was detected only in one patient (4.5%) (p<0.05).     

Transport of 4-hydroxyphenylacetic acid in Klebsiella pneumoniae.

Klebsiella pneumoniae M5a1 has been shown to possess an inducible transport system for 4-hydroxyphenylacetate (4-HPA). This transport system has a Kt of 16.3 microM and a maximal velocity of 31.2 nmol/min (milligrams dry weight). The transport system has been inhibited by inhibitors of energy metabolism with a concomitant decrease in cellular ATP concentrations, and the 4-HPA binding activity has been detected in the crude shock extracts. All these observations indicate that 4-HPA uptake is an active transport which involves a periplasmic binding protein and it seems to be energized by phosphate bond energy.     

Protective effect of the aerosol immunization of mice with the polycomponent vaccine Immunovac VP-4 after the challenge of the animals with Klebsiella pneumoniae virulent strain

Used four schemes of the administration of the preparation with different time of the exposition of the animals in an aerosol chamber were tested with their subsequent intraperitoneal challenge with K. pneumoniae virulent strain K16. Irrespective of the number of immunization courses, the administration of the preparation made at intervals of 1 day, or daily, did not ensure any protective effect, but only led to an insignificant increase in their survival time in comparison with nonimmunized animals. After intervals between immunizations were increased to 3 days the protective effect of aerosol immumization was obtained (the survival rate was 65-80 % and considerably differed from that of the controls). The protective effect of aerosol immunization thus obtained was comparable with the effectiveness immunization made in a single subcutaneous injection. Aerosol immunization resulted in low antibody titers to the antigens contained in the vaccine, while after a single subcutaneous injection high antibody titers to Klebsiella and Proteus antigens were detected. The antigen-stimulated blast transformation of spleen lymphocytes in mice subjected to aerosol immunizations in 5 exposures was high. After subcutaneous immunization significant changes in such characteristics were detected on day 15. The data thus obtained were indicative of good prospects in the development Immunovac VP-4 as the medicinal form intended for use in aerosols.     

Playing with cellular and humoral immunity

An interesting and easy game for the teaching of immunology was developed by the authors. Previously distributed figure cards are used by students that have to ask questions of each other. Most students considered the game to be interesting (97.7%) and declared that it helped them to understand the subject. The authors emphasise the importance of improving the quality of teaching using visual memory, fun and competition.     

Analysis of 2,3-butanediol production with Klebsiella pneumoniae using sugar mixture under different oxygen supply conditions

以筛选的肺炎克雷伯氏菌(Klebsiella pneumoniae UV-86)为对象,考察供氧条件分别对菌体生长、葡萄糖和木糖双底物利用和产物合成的影响.研究发现生物量随氧供应量增加而增加.不同供氧条件对菌体消耗葡萄糖过程的影响较小,而代谢木糖的能力随氧供应量的增大而增强.微氧条件下2,3-丁二醇的生物合成能力最强,2,3-丁二醇产量在1.5 vvm下达到最高为30.1 g/L,是好氧时的2.5倍,最大体积产率为0.485 g/(L·h).不同条件下两底物产物分布有所区别,木糖代谢中乙酸生产增强.因此根据不同阶段代谢特点选择适合的供氧策略可以提高过程产量和产率.     

氧供应量对Klebsiella pneumoniae利用混合糖生物合成2,3-丁二醇过程的影响分析

以筛选的肺炎克雷伯氏茵（Klebsiella pneumoniaeUV-86）为对象,考察供氧条件分别对茵体生长、葡萄糖和木糖双底物利用和产物合成的影响。研究发现生物量随氧供应量增加而增加。不同供氧条件对茵体消耗葡萄糖过程的影响较小,而代谢木糖的能力随氧供应量的增大而增强。微氧条件下2,3-丁二醇的生物合成能力最强,2,3-丁二醇产量在1．5wm下达到最高为30．1g／L,是好氧时的2．5倍,最大体积产率为0．485g／（L·h）。不同条件下两底物产物分布有所区别,木糖代谢中乙酸生产增强。因此根据不同阶段代谢特点选择适合的供氧策略可以提高过程产量和产率。     

Hemolytic activity of Klebsiella pneumoniae and Klebsiella oxytoca

We demonstrated that Klebsiella pneumoniae and Klebsiella oxytoca possess a selective haemolytic activity on rabbit erythrocytes. Thirty one Klebsiella strains (18 strains of K. pneumoniae and 13 strains of K. oxytoca) were isolated from hospitalized patients. The liquid (Trypcase-soy broth--TSB) and solid (Trypcase-soy agar--TSA) medium, containing the red cells were used for the tests. All the screened strains showed a haemolytic effect on rabbit erythrocytes, provided that the supernatants of the cultures were preincubated with beta-mercaptoethanol or calcium chloride. There was no human and sheep erythrocyte lysis.     

Intranasal immunization with recombinant vesicular stomatitis virus expressing murine cytomegalovirus glycoprotein B induces humoral and cellular immunity

Cytomegalovirus is a leading cause of morbidity and mortality among neonatal and immunocompromised patients. The use of vaccine prophylaxis continues to be an effective approach to reducing viral infections and their associated diseases. Murine cytomegalovirus (mCMV) has proven to be a valuable animal model in determining the efficacy of newly developed vaccine strategies in vivo. Live recombinant vesicular stomatitis viruses (rVSV) have successfully been used as vaccine vectors for several viruses to induce strong humoral and cellular immunity. We tested the ability of intranasal immunization with an rVSV expressing the major envelope protein of mCMV, glycoprotein B (gB), to protect against challenge with mCMV in a mouse model. rVSV-gB-infected cells showed strong cytoplasmic and cell surface expression of gB, and neutralizing antibodies to gB were present in mice after a single intranasal vaccination of VSV-gB. After challenge with mCMV, recovery of live virus and viral DNA was significantly reduced in immunized mice. In addition, primed splenocytes produced a CD8+ IFNgamma response to gB. The ability to induce an immune response to a gene product through mucosal vaccination with rVSV-gB represents a potentially effective approach to limiting CMV-induced disease.     

Three immunity types of klebicins which use the cloacin DF13 receptor of Klebsiella pneumoniae

We have investigated a group of bacteriocinogenic strains used in the epidemiological investigation of Klebsiella infections. Transfer of plasmids from these strains to laboratory strains allowed the identification of three klebicins which use the cloacin DF13 receptor in Klebsiella, but are of three distinct immunity types. These klebicins use the ferric-aerobactin receptor determined by ColV-K30 in Escherichia coli, which is also used by cloacin DF13. We propose to call them group A klebicins, of immunity types A1, A2 and A3. On the basis of immunity, cloacin DF13 also belongs to the klebicin A1 group.     

Sublingual immunization with Japanese encephalitis virus vaccine effectively induces immunity through both cellular and humoral immune responses in mice

The Japanese encephalitis virus (JEV) is the leading cause of viral encephalitis. Although there are four classes of vaccines against JEV, all of them are administered by s.c or i.m injection. Here, the effectiveness of sublingual (s.l.) administration of a JEV live‐attenuated vaccine or recombinant modified vaccinia virus Ankara (MVA) vaccine, including JEV prM/E, was investigated. The mice were immunized three times i.m. or s.c. One week after the final immunization by both s.l. and i.m. routes, the titers of IgG1 induced by the recombinant MVA vaccine were higher than those induced by the live‐attenuated vaccine, whereas the titers of IgG2a induced by the live‐attenuated vaccine were higher than those induced by the recombinant MVA vaccine. However, both vaccines induced neutralizing antibodies when given by either s.l. or i.m. routes, indicating that both vaccines induce appropriate Th1 and Th2 cell responses through the s.l. and i.m. routes. Moreover, both vaccines protected against induction of proinflammatory cytokines and focal spleen white pulp hyperplasia after viral challenge. Virus‐specific IFN‐γ⁺ CD4⁺ and CD8⁺ T cells appeared to increase in mice immunized via both s.l. and i.m. routes. Interestingly, virus‐specific IL‐17⁺ CD4⁺ T cells increased significantly only in the mice immunized via the s.l. route; however, the increased IL‐17 did not affect pathogenicity after viral challenge. These results suggest that s.l. immunization may be as useful as i.m. injection for induction of protective immune responses against JEV by both live‐attenuated and recombinant MVA vaccines.     

Catabolism of 3- and 4-hydroxyphenylacetic acid by Klebsiella pneumoniae

Klebsiella pneumoniae catabolizes both 4-hydroxyphenylacetic acid and 3-hydroxyphenylacetic acid via meta-cleavage of 3,4-dihydroxyphenylacetic acid, ultimately yielding pyruvate and succinate. The organism can synthesize two hydroxylases catalysing 3,4-dihydroxyphenylacetic acid formation, which differ in substrate specificity, cofactor requirement, kinetics and regulation. Five enzymes sequentially involved in the catabolism of 3,4-dihydroxyphenylacetic acid are encoded on a 7 kbp fragment of the K. pneumoniae chromosome that has been isolated in a recombinant plasmid.     

脑梗死患者的细胞免疫与体液免疫的变化规律研究

目的探讨脑梗死患者的细胞免疫功能与体液免疫功能的变化规律。 方法2016年5月~ 2017年5月期间广东省佛山市禅城区中心医院收治的脑梗死患者120例设为观察组,观察组患者按照脑梗死的面积分为轻症组（大脑中动脉区域脑梗,脑梗范围的直径≤5 cm者,60例）和中重症组（大脑中动脉区域脑梗,脑梗范围的直径> 5 cm者,60例）;选择120例健康体检者设为对照组。采用流式细胞仪和速率散射免疫透射比浊法测定各组血免疫球蛋白IgA、IgM、IgG、补体C3、C4水平来反应体液免疫功能;用试剂盒、外周血中T淋巴细胞亚群CD3⁺、CD4⁺、CD8⁺、NK细胞、B细胞和CD4⁺/CD8⁺百分率水平的测定;对抗心磷脂抗体（ACA）浓度进行测定;通过ACA浓度反映的颈动脉粥样硬化（CAS）中生化指标的变化分析脑梗死患者的细胞免疫功能与体液免疫功能的关联。多组间比较采用单因素方差分析;患者性别比例采用χ²分析;通过直线相关分析细胞免疫与体液免疫的相关性。 结果脑梗死中重症组T淋巴细胞含量低于脑梗死轻症组和对照组,差异具有统计学意义（P < 0.05）,CD8⁺细胞百分率（27.91%±2.97%）T淋巴细胞含量高于脑梗死轻症组和对照组,CD4⁺/CD8⁺比值（1.98±0.03）水平高于脑梗死轻症组和对照组（P < 0.05）;与脑梗死轻症组和对照组比较,脑梗死中重症组NK细胞（12.29%±1.58%）和B细胞（12.76%±2.00%）均下降（P < 0.05）。与脑梗死轻症组和对照组比较,脑梗死中重症组血清中血球免疫球蛋白IgG（10.60±1.06）IU/ml水平降低（P < 0.05）;血球免疫球蛋白IgA（4.01±0.35）IU/ml、补体C3（2.13±0.04）IU/ml和补体C4（0.39±0.01）IU/ml的水平升高（P < 0.05）,而IgM（2.60±0.05）IU/ml无明显变化,差异无统计学意义（P > 0.05）。与脑梗死轻症组（14.11±2.09）PLU/ml和对照组（7.82±1.15）PLU/ml比较,脑梗死中重症组（16.88±2.50）PLU/ml血清ACA浓度提高,差异具有统计学意义（t = 9.153,P < 0.01）。与脑梗死轻症组和对照组比较,脑梗死中重症组总胆固醇[（1.75±0.03）mmol/L]和甘油三酯[（2.42±0.33）mmol/L]含量增加;高密度脂蛋白胆固醇[（0.41±0.03）mmol/L]含量降低;与对照组比较,脑梗死中重症组低密度脂蛋白胆固醇[（1.55±0.21）mmol/L]含量升高（P < 0.05）。通过细胞免疫参数与体液免疫参数之间的相关性分析显示呈负相关,且相关性曲线陡峭（P < 0.05）。 结论脑梗死患者的细胞免疫与体液免疫的变化规律可指导脑梗死治疗和预后。     

Ineffective humoral immunity in the elderly

As individuals age, dysfunction of the immune system leads to an increased incidence of infectious disease. Due to the complex network of cellular interactions and the multi-factorial process of aging, numerous impairments in humoral immunity have been reported. Advances in technology have allowed scientists to begin to identify the molecular mechanisms behind the age-associated decline.     

Epidermal langerhans cells are dispensable for humoral and cell-mediated immunity elicited by gene gun immunization

Gene gun immunization, i.e., bombardment of skin with DNA-coated particles, is an efficient method for the administration of DNA vaccines. Direct transfection of APC or cross-presentation of exogenous Ag acquired from transfected nonimmune cells enables MHC-I-restricted activation of CD8(+) T cells. Additionally, MHC-II-restricted presentation of exogenous Ag activates CD4(+) Th cells. Being the principal APC in the epidermis, Langerhans cells (LC) seem ideal candidates to accomplish these functions. However, the dependence on LC of gene gun-induced immune reactions has not yet been demonstrated directly. This was primarily hampered by difficulties to discriminate the contributions of LC from those of other dermal dendritic cells. To address this problem, we have used Langerin-diphtheria toxin receptor knockin mice that allow for selective inducible ablation of LC. LC deficiency, even over the entire duration of experiments, did not affect any of the gene gun-induced immune functions examined, including proliferation of CD4(+) and CD8(+) T cells, IFN-gamma secretion by spleen cells, Ab production, CTL activity, and development of protective antitumor immunity. Together, our data show that gene gun immunization is capable of inducing humoral and cell-mediated immune reactions independently of LC.     

Hydrophobic and hemagglutinating properties of Klebsiella pneumoniae and Klebsiella oxytoca

The aim of this study was to estimated hydrophobic and hemnagglutinating properties of Klebsiella pneumoniae and Klebsiella oxytoca rods. The hydrophobcity was evaluated according to the method of Rosenberg et al. The hemagglutinating properties were estimated by method of Blanco et al. Forty seven hydrophobic Klebsiella strains (30 K. pneumoniae strains and 17 K. oxytoca strains) were detected. Hemagglutinating properties were observed in 65 Klebsiella strains (45 K. pneumoniae strains and 20 K. oxytoca strains). Hemagglutination of sheep tannined erythrocytes the most frequently was observed. Inhibition of hemagglutination of erythrocytes by K. pneumoniae strains was most frequently observed in presence of D-glucose and D-mannose and by K. oxytoca strains in presence of D-glucose.     

Hydrolytic and haemolytic activity of Klebsiella pneumoniae and Klebsiella oxytoca

Hydrolytic enzymes and haemolysins are important extracellular substances produced by many bacteria. We investigated 57 K. pneumoniae strains and 40 K. oxytoca strains isolated from clinical materials. We estimated the ability to produce: proteases hydrolyzing milk powder, caseinase, gelatinase, elastase, lecithinase, lipases, DNase and haemolysins on human, sheep and horse erythrocytes on TSA medium with or without 5% Egg Yolk. We detected that K. oxytoca strains produced proteases hydrolyzing milk powder (37.5%), caseinase (15.0%) and gelatinase (17.5%) more frequently than K. pneumoniae strains (respectively 21.0%, 5.3%, 8.9%). None of the analysed Klebsiella spp. strains produced elastase. Only K. pneumoniae strains produced lecithinase (5.3%). Lipases hydrolyzing Tween were produced from 3.5% (for Tween 60 and 80) to 7.0% (for Tween 20). Among K. oxytoca strains only one (2.5%) hydrolyzing Tween 20. DNase was produced by 38.6% of K. pneumoniae strains and by 27.5% K. oxytoca strains. Haemolytic properties on human erythrocytes were detected in 5.3% K. pneumoniae strains on TSA medium and 29,8% on medium with Egg Yolk. In K. oxytoca strains haemolytic properties on human erythrocytes were detected only on medium with Egg Yolk (12.5%). Haemolytic properties on sheep erythrocytes were detected respectively in 21.0% and 22.8% K. pneumoniae strains and in 7.5% K. oxytoca strains on each medium. Haemolytic properties on horse erythrocytes were detected respectively in 33.4% and 52.6% K. pneumoniae strains and in 15.0% and 20.0% K. oxytoca strains.     

Induction of humoral immunity in response to immunization with recombinant Mycobacterium bovis BCG expressing the S1 subunit of Bordetella pertussis toxin

Two recombinant Mycobacterium bovis BCG (rBCG) vaccine strains were developed for the expression of cytoplasmically located S1 subunit of pertussis toxin, with expression driven by the hsp60 promoter of M. bovis (rBCG/pPB10) or the pAN promoter of Mycobacterium paratuberculosis (rBCG/pPB12). Both strains showed stable expression of equivalent levels of recombinant S1 in vitro and induced long-term (up to 8 months) humoral immune responses in BALB/c mice, although these responses differed quantitatively and qualitatively. Specifically, rBCG/pPB12 induced markedly higher levels of IgG1 than did rBCG/pPB10, and mice immunized with the former strain developed specific long-term memory to S1, as indicated by the production of high levels of S1-specific IgG in response to a sublethal challenge with pertussis toxin 15 months after initial immunization. When considered in combination with previous studies, our data encourage further evaluation of rBCG as a potential means of developing a low-cost whooping cough vaccine based on defined antigens.