The pathogenic importance of CCL21 and CCR7 in rheumatoid arthritis

Innate and adaptive immunity regulate the inflammatory and erosive phenotypes observed in rheumatoid arthritis (RA) patients. Hence, identifying novel pathways that participate in different stages of RA pathology will provide valuable insights concerning the mechanistic behavior of different joint leukocytes and the strategy to restrain their activity. Recent findings have revealed that CCL21 poses as a risk factor for RA and expression of its receptor, CCR7, on circulating monocytes is representative of the patient’s disease activity score. Expression of CCR7 was found to be the hallmark of RA synovial fluid (SF) M1 macrophages (MФs) and its levels were potentiated in response to M1 mediating factors and curtailed by M2 mediators in naïve MФs. Intriguingly, although both CCR7 ligands, CCL19 and CCL21, are elevated in RA specimens, only CCL21 was predominately responsible for CCR7’s pathological manifestation of RA. Unique subset of MФs differentiated in response to CCL21 stimulation, exhibited upregulation in Th17-polarizing monokines. Moreover, CCL21-activated monokines were capable of differentiating naïve T cells into joint Th17 cells, which also partook in RA osteoclastogenesis. Finally, to conserve chronic inflammation, SF CCL21 amplified RA neovascularization directly and indirectly by promoting RA FLS and MΦs to secrete proangiogenic factors, VEGF and IL-17. This review aims to shed light on the broad pathogenic impact of CCL21, linking immunostimulatory MФs with Th17 cells, while concurrently advancing RA bone destruction and neovascularization.     

磁性纳米粒介导沉默PI3Kγ基因调控肿瘤相关巨噬细胞抗小鼠肺癌细胞效应的研究

为了探讨超顺磁性Fe3O4纳米粒子(superparamagnetic iron oxide nanoparticles,SPIONs)介导的磷脂酰肌醇3激酶γ(phosphatidylinositol 3 kinaseγ,PI3Kγ)抑制表达调控的肿瘤相关巨噬细胞(tumor-associated macrophages,TAM)对小鼠Lewis肺癌细胞(Lewis lung carcinoma,LLC)增殖和凋亡的影响,该研究构建了能启动巨噬细胞(macrophage,MФ)特异性表达PI3Kγ催化亚基p100 siRNA的pSilencer-EGFP-SP-p110质粒,通过SPIONs负载成磁性纳米质粒复合物(SPIONs-DNA),在强磁作用下转染MФ,通过普鲁士蓝染色法检测SPIONs-DNA在细胞内的分布,Real-time PCR和Western blot检测细胞PI3Kγp110亚基的表达水平。建立M1、M2型MФ模型,将SPIONs-DNA在强磁作用下转染M2型MФ,通过Real-time PCR和Western blot鉴定细胞表型,明确M2型MФ转化为M1型的强度。采用Transwell系统建立SPIONs-DNA转染的M2型MФ与小鼠LLC细胞的共培养模型,通过锥虫蓝染色法检测LLC细胞的活细胞数并绘制细胞生长曲线,CCK-8法检测LLC细胞增殖情况,硝酸还原酶法检测共培养液上清中NO含量,流式细胞术检测LLC细胞凋亡情况。结果显示,制备的SPIONs-DNA在强磁作用下成功转染MФ并大量分布在细胞胞核周围,SPIONs-DNA转染组细胞PI3Kγp110 mRNA和蛋白表达水平显著低于空白细胞对照组(P<0.05)。建立的M1型MФ高表达iNOS(P<0.001),M2型MФ高表达ARG-1(P<0.001)。M2型MФ转染SPIONs-DNA后细胞iNOS mRNA和蛋白的表达显著增加(P<0.001),ARG-1 mRNA和蛋白的表达显著降低(P<0.01)。在共培养组中,SPIONs-DNA转染的M2型MФ组能大量分泌NO,LLC细胞生长和增殖能力显著降低(P<0.05),凋亡率显著增高(P<0.01)。结果表明,磁性纳米粒负载pSilencer-EGFP-SP-p110重组质粒能够特异性靶向抑制巨噬细胞PI3Kγp110的表达,诱导M2型MФ转化为M1型;其转染的M2型MФ可显著抑制LLC细胞的生长和增值,促进细胞凋亡,这与其大量分泌NO有关。该磁性纳米质粒复合物可诱导TAM发挥抗肿瘤作用,为研究开发有效的抗肺癌基因治疗措施奠定基础。     

LRRK2 deficiency mitigates colitis progression by favoring resolution of inflammation and restoring homeostasis of gut microbiota

Leucine rich-repeat kinase 2 (LRRK2) has been considered a susceptibility gene for ulcerative colitis (UC), and its protein abundance was enhanced in the peripheral blood mononuclear cells (PBMCs) from UC cohorts as compared to healthy volunteers. In preclinical models of colitis, Lrrk2 deficiency ameliorated dextran sodium sulfate (DSS)-induced colitis progression, whereas the processes were aggravated by R1441C mutation. While intestinal macrophages (MФs) from Lrrk2 knock-out (Lrrk2^?/?) mice exhibited a tendency to transit to alternatively activated MФs, R1441C MФs mutation facilitated the pro-inflammatory phenotype polarization, determined by RNA sequencing and qPCR. Moreover, we characterized their microbiota profiles and found that loss of Lrrk2 increased the bacterial richness and altered bacterial community structure, and this shift contributed to the alleviation of colitis development and progression. We proposed that Lrrk2 deficiency promotes M2 MФ transition and facilitates probiotics colonization, providing a protective role during colitis.     

The CD14 receptor does not mediate entry of Mycobacterium tuberculosis into human mononuclear phagocytes

Prior reports have suggested that CD14 mediates uptake of Mycobacterium tuberculosis into porcine alveolar macrophages and human fetal microglia, but the contribution of CD14 to cell entry in human macrophages has not been studied. To address this question, we used flow cytometry to quantify uptake by human monocytes and alveolar macrophages of M. tuberculosis expressing green fluorescent protein. Neutralizing anti-CD14 antibodies did not affect bacillary uptake and the efficiency of bacillary entry was similar in THP-1 cells expressing low and high levels of CD14. However, most internalized bacteria were found in CD14+ but not in CD14- monocytes because M. tuberculosis infection upregulated CD14 expression. We conclude that: (1) CD14 does not mediate cellular entry by M. tuberculosis; (2) M. tuberculosis infection upregulates CD14 expression on mononuclear phagocytes, and this may facilitate the pathogen's capacity to modulate the immune response.     

香鱼主要组织相容性复合体MHCIIB的分子克隆及其在鳗弧菌感染下的功能鉴定

主要组织相容性复合体(major histocompatibility complex classⅡ, MHC Ⅱ)在脊椎动物免疫反应中发挥重要作用。本研究从香鱼(Plecoglossus altivelis) 单核/巨噬细胞(monocytes/ macrophages,MO/MФ)转录组中获得了MHC II基因β链(PaMHCIIB) cDNA序列。PaMHCIIB由920个核苷酸组成,包含一个大的开放阅读框,编码251个氨基酸,预测分子质量为28.23 kD。氨基酸序列分析表明,PaMHCIIB具有MHC IIB的典型特征,主要包括信号肽、2个胞外区和1个保守的connecting peptide/transmembrane/cytoplasmic (CP/TM/CYT)结构域,与北极红点鲑(Salvelinus alpinus) MHC IIB同源性最高,为65.04%;系统发育分析表明,PaMHCIIB与北极红点鲑MHC IIB进化相关性最高。实时荧光定量PCR(quantitative real-time PCR, qPCR)结果显示,PaMHCIIB mRNA主要在香鱼鳃、肠和脾中表达;鳗弧菌感染(Vibrio anguillarum)后香鱼肝中PaMHCIIB mRNA在感染后12 h(hours post infection, hpi)时上调显著,在24 hpi达到峰值,为对照组的3.18倍,脾、头肾、肠和鳃中PaMHCIIB mRNA均在4 hpi时上调显著,分别在4、8、24和12 hpi时达到峰值,分别为对照组的85.18、2.06、4.21和6.81倍(P<0.05)。香鱼MO/MФ经鳗弧菌感染后,PaMHCIIB mRNA的表达水平在4 hpi时上调显著,在12 hpi时达到峰值,为对照组的3.35倍(P<0.05)。原核表达了PaMHCIIB胞外区并制备其抗体。Western 印迹分析结果表明,香鱼MO/MФ中PaMHCIIB具有N糖基化修饰,且鳗弧菌感染后其蛋白表达水平在12 hpi时显著增加,在24 hpi时达到峰值,为对照组的3.19倍(P<0.05)。抗体封闭PaMHCIIB后,香鱼MO/MФ吞噬活性被抑制,为对照组的0.28倍(P<0.05),而且鳗弧菌诱导的4个细胞因子TNF-α、IL-1β、IL-10和TGF-β表达均受到抑制,均在8 hpi时被抑制最明显,表达量分别为对照组的0.23、0.41、0.51和0.20倍(P<0.05)。本研究结果揭示PaMHCIIB可能参与香鱼MO/MФ抵抗病原菌感染的免疫防御。     

Mudskipper (<Emphasis Type="Italic">Boleophthalmus pectinirostris</Emphasis>) Hepcidin-1 and Hepcidin-2 Present Different Gene Expression Profile and Antibacterial Activity and Possess Distinct Protective Effect against <Emphasis Type="Italic">Edwardsiella tarda</Emphasis> Infection

Hepcidins are small cysteine-rich antimicrobial peptides that play an important role in fish immunity against pathogens. Most fish species have two or more hepcidin homologs that have distinct functions. This study investigated the immune functions of mudskipper (Boleophthalmus pectinirostris) hepcidin-1 (BpHep-1) and hepcidin-2 (BpHep-2) in vitro and in vivo. Upon infection with Edwardsiella tarda, the expression of BpHep-1 and BpHep-2 mRNA in immune tissues was significantly upregulated, but the expression profiles were different. Chemically synthesized BpHep-1 and BpHep-2 mature peptides exhibited selective antibacterial activity against various bacterial species, and BpHep-2 exhibited a stronger antibacterial activity and broader spectrum than BpHep-1. BpHep-1 and BpHep-2 both inhibited the growth of E. tarda in vitro, with the latter being more effective than the former. In addition, both peptides induced hydrolysis of purified bacterial genomic DNA (gDNA) or gDNA in live bacteria. In vivo, an intraperitoneal injection of 1.0 μg/g BpHep-2 significantly improved the survival rate of mudskippers against E. tarda infection compared with 0.1 μg/g BpHep-2 or 0.1 and 1.0 μg/g BpHep-1. Similarly, only BpHep-2 treatment effectively reduced the tissue bacterial load in E. tarda-infected mudskippers. Furthermore, treatment with 1.0 or 10.0 μg/ml BpHep-2 promoted the phagocytic and bactericidal activities of mudskipper monocytes/macrophages (MO/MФ). However, only the highest dose (10.0 μg/ml) of BpHep-1 enhanced phagocytosis, and BpHep-1 exerted no obvious effects on bactericidal activity. In conclusion, BpHep-2 is a stronger bactericide than BpHep-1 in mudskippers, and acts not only by directly killing bacteria but also through an immunomodulatory function on MO/MФ.     

生化反应中一类三次系统的极限环

研究生化反应中一类三次系统：dx/dt=-x-Ф1（x）＋yФ2（x）,dy/dt=a0＋Ф1（x）-yФ2（x）其中Ф1（x）=Ax^3＋ax^2＋bx＋B,Ф2（x）=cx^2＋dx＋e．较完整地解决了该系统极限环的存在性,唯一性与不存在性等问题．     

Quantification of uncultured Ruminococcus obeum-like bacteria in human fecal samples by fluorescent in situ hybridization and flow cytometry using 16S rRNA-targeted probes

A 16S rRNA-targeted probe was designed and validated in order to quantify the number of uncultured Ruminococcus obeum-like bacteria by fluorescent in situ hybridization (FISH). These bacteria have frequently been found in 16S ribosomal DNA clone libraries prepared from bacterial communities in the human intestine. Thirty-two reference strains from the human intestine, including a phylogenetically related strain and strains of some other Ruminococcus species, were used as negative controls and did not hybridize with the new probe. Microscopic and flow cytometric analyses revealed that a group of morphologically similar bacteria in feces did hybridize with this probe. Moreover, it was found that all hybridizing cells also hybridized with a probe specific for the Clostridium coccoides-Eubacterium rectale group, a group that includes the uncultured R. obeum-like bacteria. Quantification of the uncultured R. obeum-like bacteria and the C. coccoides-E. rectale group by flow cytometry and microscopy revealed that these groups comprised approximately 2.5 and 16% of the total community in fecal samples, respectively. The uncultured R. obeum-like bacteria comprise about 16% of the C. coccoides-E. rectale group. These results indicate that the uncultured R. obeum-like bacteria are numerically important in human feces. Statistical analysis revealed no significant difference between the microscopic and flow cytometric counts and the different feces sampling times, while a significant host-specific effect on the counts was observed. Our data demonstrate that the combination of FISH and flow cytometry is a useful approach for studying the ecology of uncultured bacteria in the human gastrointestinal tract.     

流式细胞术检测胞内重组耻垢分枝杆菌的活力研究

目的建立一种快速检测胞内分枝杆菌活力的方法。方法将一定量培养至对数生长期的含pMV-eis的重组耻垢分枝杆菌感染U937巨噬细胞,以含空质粒的耻垢分枝杆菌为对照,吞噬作用2 h后洗去胞外细菌,再分别培养4、12、24和48 h后收集细胞并裂解之。获得的胞内细菌用FDA荧光染料染色后用流式细胞仪检测死亡率,并与平板菌落计数法进行比较。结果流式细胞仪检测出感染12 h后重组耻垢分枝杆菌胞内死亡率较对照组均有显著下降（P〈0.05）,流式细胞仪检测法与平板菌落计数法相比差异无统计学意义（P〉0.05）。结论流式细胞术与传统的平板计数法相比具有快速、敏感、方便的特点,可用于分枝杆菌活菌快速检测。     

Overland flow delivery of faecal bacteria to a headwater pastoral stream

AIMS: To quantify and derive statistical relationships with which to predict the delivery of faecal bacteria (Escherichia coli) to a pastoral stream, by overland flow. METHODS AND RESULTS: A large-scale (1050 m2) rainfall simulator, located upon a steep (18 degrees) grazed hillside in New Zealand, was used to simulate 11 heavy rainfall events. Overland flow was generated and sampled throughout each event, before discharging to a headwater stream. The samples were subsequently analysed to determine the concentration of E. coli. Statistical analysis showed that the time elapsed since the last period of grazing was a statistically significant predictor of both the total number (load) and concentrations of E. coli in overland flow. Between 10(5) and 10(8)E. coli per m2 of hillside were delivered to the stream within overland flow during each event, and peak concentrations ranged between 10(3) and 10(7) most probable number per 100 ml. CONCLUSIONS: Under heavy rainfall on steep pastoral land, overland flow can transport substantial levels of faecal bacteria to streams. Under such conditions, it is unlikely that vegetated buffer strips will be particularly effective at attenuating bacteria within overland flow. SIGNIFICANCE AND IMPACT OF THE STUDY: This work has improved understanding of the importance of overland flow as a process contributing to the contamination of pastoral streams by faecal bacteria. In addition, the predictive relationships derived can be incorporated within catchment models.     

Ammonium availability affects the ratio of ammonia-oxidizing bacteria to ammonia-oxidizing archaea in simulated creek ecosystems

The ammonia-oxidizing microbial community colonizing clay tiles in flow channels changed in favor of ammonia-oxidizing bacteria during a 12-week incubation period even at originally high ratios of ammonia-oxidizing archaea to ammonia-oxidizing bacteria (AOB). AOB predominance was established more rapidly in flow channels incubated at 350 μM NH(4)(+) than in those incubated at 50 or 20 μM NH(4)(+). Biofilm-associated potential nitrification activity was first detected after 28 days and was positively correlated with bacterial but not archaeal amoA gene copy numbers.     

In situ abundance and carbon fixation activity of distinct anoxygenic phototrophs in the stratified seawater lake Rogoznica

Sulphide-driven anoxygenic photosynthesis is an ancient microbial metabolism that contributes significantly to inorganic carbon fixation in stratified, sulphidic water bodies. Methods commonly applied to quantify inorganic carbon fixation by anoxygenic phototrophs, however, cannot resolve the contributions of distinct microbial populations to the overall process. We implemented a straightforward workflow, consisting of radioisotope labelling and flow cytometric cell sorting based on the distinct autofluorescence of bacterial photopigments, to discriminate and quantify contributions of co-occurring anoxygenic phototrophic populations to in situ inorganic carbon fixation in environmental samples. This allowed us to assign 89.3% ± 7.6% of daytime inorganic carbon fixation by anoxygenic phototrophs in Lake Rogoznica (Croatia) to an abundant chemocline-dwelling population of green sulphur bacteria (dominated by Chlorobium phaeobacteroides), whereas the co-occurring purple sulphur bacteria (Halochromatium sp.) contributed only 1.8% ± 1.4%. Furthermore, we obtained two metagenome assembled genomes of green sulphur bacteria and one of a purple sulphur bacterium which provides the first genomic insights into the genus Halochromatium, confirming its high metabolic flexibility and physiological potential for mixo- and heterotrophic growth.     

The sodium and potassium concentration in rabbit anti-R pneumococeal sera and Its relationship to type-specific transformation

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Quantification of Uncultured Ruminococcus obeum-Like Bacteria in Human Fecal Samples by Fluorescent In Situ Hybridization and Flow Cytometry Using 16S rRNA-Targeted Probes

A 16S rRNA-targeted probe was designed and validated in order to quantify the number of uncultured Ruminococcus obeum-like bacteria by fluorescent in situ hybridization (FISH). These bacteria have frequently been found in 16S ribosomal DNA clone libraries prepared from bacterial communities in the human intestine. Thirty-two reference strains from the human intestine, including a phylogenetically related strain and strains of some other Ruminococcus species, were used as negative controls and did not hybridize with the new probe. Microscopic and flow cytometric analyses revealed that a group of morphologically similar bacteria in feces did hybridize with this probe. Moreover, it was found that all hybridizing cells also hybridized with a probe specific for the Clostridium coccoides-Eubacterium rectale group, a group that includes the uncultured R. obeum-like bacteria. Quantification of the uncultured R. obeum-like bacteria and the C. coccoides-E. rectale group by flow cytometry and microscopy revealed that these groups comprised approximately 2.5 and 16% of the total community in fecal samples, respectively. The uncultured R. obeum-like bacteria comprise about 16% of the C. coccoides-E. rectale group. These results indicate that the uncultured R. obeum-like bacteria are numerically important in human feces. Statistical analysis revealed no significant difference between the microscopic and flow cytometric counts and the different feces sampling times, while a significant host-specific effect on the counts was observed. Our data demonstrate that the combination of FISH and flow cytometry is a useful approach for studying the ecology of uncultured bacteria in the human gastrointestinal tract.     

Survival of Mycobacterium avium in drinking water biofilms as affected by water flow velocity, availability of phosphorus, and temperature

Mycobacterium avium is a potential pathogen occurring in drinking water systems. It is a slowly growing bacterium producing a thick cell wall containing mycolic acids, and it is known to resist chlorine better than many other microbes. Several studies have shown that pathogenic bacteria survive better in biofilms than in water. By using Propella biofilm reactors, we studied how factors generally influencing the growth of biofilms (flow rate, phosphorus concentration, and temperature) influence the survival of M. avium in drinking water biofilms. The growth of biofilms was followed by culture and DAPI (4',6'-diamidino-2-phenylindole) staining, and concentrations of M. avium were determined by culture and fluorescence in situ hybridization methods. The spiked M. avium survived in biofilms for the 4-week study period without a dramatic decline in concentration. The addition of phosphorus (10 microg/liter) increased the number of heterotrophic bacteria in biofilms but decreased the culturability of M. avium. The reason for this result is probably that phosphorus increased competition with other microbes. An increase in flow velocity had no effect on the survival of M. avium, although it increased the growth of biofilms. A higher temperature (20 degrees C versus 7 degrees C) increased both the number of heterotrophic bacteria and the survival of M. avium in biofilms. In conclusion, the results show that in terms of affecting the survival of slowly growing M. avium in biofilms, temperature is a more important factor than the availability of nutrients like phosphorus.     

Streptococcus pyogenes bacteria modulate membrane traffic in human neutrophils and selectively inhibit azurophilic granule fusion with phagosomes

We recently reported that the human pathogen Streptococcus pyogenes of the M1 serotype survives and replicates intracellularly after being phagocytosed by human neutrophils. These data raised the possibility that the generation of reactive oxygen metabolites by neutrophils, and the release of microbicidal molecules from their azurophilic and specific granules into phagosomes, can be modulated by S. pyogenes bacteria expressing surface-associated M and/or M-like proteins. We now demonstrate, using flow cytometry, immunofluorescence microscopy and transmission electron microscopy, that live wild-type S. pyogenes, after internalization by human neutrophils, inhibits the fusion of azurophilic granules with phagosomes. In contrast, azurophilic granule-content is efficiently delivered to phagosomes containing bacteria not expressing M and/or M-like proteins. Also, when heat-killed wild-type bacteria are used as the phagocytic prey, fusion of azurophilic granules with phagosomes is observed. The inhibition caused by live wild-type S. pyogenes is specific for azurophilic granule-phagosome fusion, because the mobilization of specific granules and the production of reactive oxygen species are induced to a similar extent by all strains tested. In conclusion, our results demonstrate that viable S. pyogenes bacteria expressing M and M-like proteins selectively prevent the fusion of azurophilic granules with phagosomes.     

Catch bond-mediated adhesion without a shear threshold: trimannose versus monomannose interactions with the FimH adhesin of Escherichia coli

The FimH protein is the adhesive subunit of Escherichia coli type 1 fimbriae. It mediates shear-dependent bacterial binding to monomannose (1M)-coated surfaces manifested by the existence of a shear threshold for binding, below which bacteria do not adhere. The 1M-specific shear-dependent binding of FimH is consistent with so-called catch bond interactions, whose lifetime is increased by tensile force. We show here that the oligosaccharide-specific interaction of FimH with another of its ligands, trimannose (3M), lacks a shear threshold for binding, since the number of bacteria binding under static conditions is higher than under any flow. However, similar to 1M, the binding strength of surface-interacting bacteria is enhanced by shear. Bacteria transition from rolling into firm stationary surface adhesion as the shear increases. The shear-enhanced bacterial binding on 3M is mediated by catch bond properties of the 1M-binding subsite within the extended oligosaccharide-binding pocket of FimH, since structural mutations in the putative force-responsive region and in the binding site affect 1M- and 3M-specific binding in an identical manner. A shear-dependent conversion of the adhesion mode is also exhibited by P-fimbriated E. coli adhering to digalactose surfaces.     

Shewanella putrefaciens adhesion and biofilm formation on food processing surfaces

Laboratory model systems were developed for studying Shewanella putrefaciens adhesion and biofilm formation under batch and flow conditions. S. putrefaciens plays a major role in food spoilage and may cause microbially induced corrosion on steel surfaces. S. putrefaciens bacteria suspended in buffer adhered readily to stainless steel surfaces. Maximum numbers of adherent bacteria per square centimeter were reached in 8 h at 25 degrees C and reflected the cell density in suspension. Numbers of adhering bacteria from a suspension containing 10(8) CFU/ml were much lower in a laminar flow system (modified Robbins device) (reaching 10(2) CFU/cm(2)) than in a batch system (reaching 10(7) CFU/cm(2)), and maximum numbers were reached after 24 h. When nutrients were supplied, S. putrefaciens grew in biofilms with layers of bacteria. The rate of biofilm formation and the thickness of the film were not dependent on the availability of carbohydrate (lactate or glucose) or on iron starvation. The number of S. putrefaciens bacteria on the surface was partly influenced by the presence of other bacteria (Pseudomonas fluorescens) which reduced the numbers of S. putrefaciens bacteria in the biofilm. Numbers of bacteria on the surface must be quantified to evaluate the influence of environmental factors on adhesion and biofilm formation. We used a combination of fluorescence microscopy (4',6'-diamidino-2-phenylindole staining and in situ hybridization, for mixed-culture studies), ultrasonic removal of bacteria from surfaces, and indirect conductometry and found this combination sufficient to quantify bacteria on surfaces.