Dickkopf-1 inhibits the secretion of MUC5AC induced by Mycoplasma pneumoniae P1-C in mouse lung epithelial cells / 生物工程学报

Mycoplasma pneumoniae is the most common pathogen of respiratory tract infection in children and adults. Clinical observation shows that M. pneumoniae infection can cause massive mucus secretion in the respiratory tract, which makes the breathing of patients difficult. Studies have shown that M. pneumoniae infection can cause massive secretion of mucin 5AC (MUC5AC). Adhesin P1 plays an important role in the pathogenesis of M. pneumoniae infection by mediating the adhesion of pathogens to host cells, and the C-terminal residues of P1 (P1-C) are immunogenic. This study investigated the molecular mechanism of Wnt/β-catenin signaling pathway inhibitor Dickkopf-1 (DKK1) in the secretion of MUC5AC in mouse airway epithelial cells (MAECs) induced by P1-C. Scanning electron microscope and hematoxylin-eosin staining were used to observe the effect of P1-C on mucus secretion of MAECs. Protein chip was used to detect the secretion of cytokines and analyse the enrichment of related signaling pathways induced by P1-C in MAECs. Periodic acid schiff stain (PAS) staining, Tunel staining and Masson staining were used to detect the damage of the lungs of mouse exposed to P1-C. Immunohistochemistry was used to detect the secretion of MUC5AC expression, and Western blotting was used to reveal the molecular mechanism of DKK1-regulated secretion of MUC5AC induced by P1-C protein in MACES. The results showed that P1-C induced the massive secretion of mucus and inflammatory factors in MAECs. During P1-C infection, DKK1 down-regulated janus kinase 2 (JAK2), phosphorylation signaling and transcription activator 1 (p-STAT1) and phosphorylation signaling and activator of transcription 3 (p-STAT3) expression. Overexpression of DKK1 significantly up-regulated the expression of MUC5AC repressor transcription factor fork-head box protein A2 (FOXA2). At the same time, the expression of MUC5AC induced by P1-C was inhibited significantly. It is speculated that DKK1 can effectively reduce the secretion of MUC5AC in MAECs induced by P1-C by inhibiting the JAK/STAT1-STAT3 signaling pathway and up-regulating the expression of FOXA2.

Research progress on the relationship between Fusobacterium nucleatum and periodontitis / 口腔疾病防治

@#Periodontitis is an infectious disease caused by a variety of microorganisms. Fusobacterium nucleatum is closely related to periodontitis with a high detection rate. Fusobacterium nucleatum is able to coaggregate with other microorganisms and attach and invade epithelial cells with the help of adhesins. It can also promote the occurrence and development of periodontal diseases and even systemic diseases by destroying periodontal tissues with virulence factors and metabolites and inducing a host immune response. However, at present, drugs assisting periodontal nonsurgical treatment clinically cannot target specific periodontal pathogens, such as Fusobacterium nucleatum, which may lead to problems such as dysbacteriosis or drug resistance. Therefore, studies on the pathogenic mechanism of Fusobacterium nucleatum provide new ideas for the prevention and treatment of periodontitis. The idea is to develop materials, drugs, or probiotics that target adhesins, virulence factors, and metabolites or cut off each pathogenic pathway of Fusobacterium nucleatum to inhibit its proliferation and inflammatory responses in deep periodontal pockets and achieve a balance with other oral microorganisms, and the host is beneficial for the control of periodontitis.

Gene expression of Paracoccidioides virulence factors after interaction with macrophages and fibroblasts

BACKGROUND Paracoccidioidomycosis (PCM) is a systemic mycosis with high prevalence in Latin America that is caused by thermodimorphic fungal species of the Paracoccidioides genus. OBJECTIVES In this study, we used quantitative polymerase chain reaction (qPCR) to investigate the expression of genes related to the virulence of Paracoccidioides brasiliensis (Pb18) and P. lutzii (Pb01) strains in their mycelial (M) and yeast (Y) forms after contact with alveolar macrophages (AMJ2-C11 cell line) and fibroblasts (MRC-5 cell line). METHODS The selected genes were those coding for 43 kDa glycoprotein (gp43), enolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 14-3-3 protein (30 kDa), phospholipase, and aspartyl protease. FINDINGS In the Pb18 M form, the aspartyl protease gene showed the highest expression among all genes tested, both before and after infection of host cells. In the Pb18 Y form after macrophage infection, the 14-3-3 gene showed the highest expression among all genes tested, followed by the phospholipase and gp43 genes, and their expression was 50-fold, 10-fold, and 6-fold higher, respectively, than that in the M form. After fibroblast infection with the Pb18 Y form, the 14-3-3 gene showed the highest expression, followed by the phospholipase and aspartyl protease genes, and their expression was 25-fold, 10-fold, and 10-fold higher, respectively, than that in the M form. Enolase and aspartyl protease genes were expressed upon infection of both cell lines. After macrophage infection with the Pb01 Y form, the 14-3-3 gene showed the highest expression, followed by the phospholipase and aspartyl protease genes, and their expression was 18-fold, 12.5-fold, and 6-fold higher, respectively, than that in the M form. MAIN CONCLUSIONS In conclusion, the data show that the expression of the genes analysed may be upregulated upon fungus-host interaction. Therefore, these genes may be involved in the pathogenesis of paracoccidioidomycosis.

Study on the bifidobacterial adhesion to the host tissue in ulcerative colitis and related serum inflammatory factors with Pingkui enema / 国际中医中药杂志

Objective@#This study is designed to investigate the effect and mechanism of Pingkui enema in treatment of ulcerative colitis.@*Methods@#Sixty SD rats at SPF level were randomly divided into 6 groups which were normal group, model group, and the low, medium and high dose groups of Chinese medicine Pingkui enema, and sulfasalazine (SASP) enema group. The normal group and model group were given an equal volume of saline enema after modeling. After continuous administration for 10 days, the histopathological effects were evaluated after extraction, and the serum interleukin-8 (IL-8), interleukin-13 (IL-13), tumor necrosis factor (TNF-α), rat intestinal mucosal bifidobacteria adhesion and adhesin receptor were measured by enzyme-linked immunosorbent assay (ELISA). The real-time fluorescence quantitative PCR (RT-PCR) were used to detect the intestinal bifidobacterial content.@*Results@#Compared with the model group, the content of IL-8 (153.50 ± 8.59 pg/ml, 150.84 ± 5.38 pg/ml vs. 167.13 ± 13.66 pg/ml), TNF-α (330.08 ± 10.02 pg/ml, 287.27 ± 13.89 pg/ml vs. 376.50 ± 19.06 pg/ml) in medium, high dose group of Pingkui enema significantly decreased (P<0.05), while the IL-13 (37.04 ± 3.62 pg/ml, 40.64 ± 5.98 pg/ml vs. 31.57 ± 2.95 pg/ml) significantly increased (P<0.05). The bifidobacteria adhesin (124.84 ± 9.74 ng/L, 149.93 ± 9.57 ng/L, 176.74 ± 11.01 ng/L vs. 101.11 ± 17.18 ng/L) and adhesin receptor (78.44 ± 14.03 ng/L, 99.50 ± 3.63 ng/L, 107.36 ± 5.05 ng/L vs. 60.96 ± 13.89 ng/L) in low-, medium-, high dose group of Pingkui enema significantly increased (P<0.05). The content of bifidobacteria (1 331.4 ± 242.2 copies/μl vs. 490.5 ± 106.0 copies/μl) in the middle dose Pingkui enema group significantly increased (P<0.05).@*Conclusions@#The Pingkui enema can promote the colonization of intestinal epithelial cells by promoting bifidobacteria, increase bifidobacterium adhesin and adhesin receptors, up-regulate the expression of IL-13, and down-regulate the expression of IL-8, TNF-α.

Study on the bifidobacterial adhesion to the host tissue in ulcerative colitis and related serum inflammatory factors with Pingkui enema / 国际中医中药杂志

Objective This study is designed to investigate the effect and mechanism of Pingkui enema in treatment of ulcerative colitis. Methods Sixty SD rats at SPF level were randomly divided into 6 groups which were normal group, model group, and the low, medium and high dose groups of Chinese medicine Pingkui enema, and sulfasalazine (SASP) enema group. The normal group and model group were given an equal volume of saline enema after modeling. After continuous administration for 10 days, the histopathological effects were evaluated after extraction, and the serum interleukin-8 (IL-8), interleukin-13 (IL-13), tumor necrosis factor (TNF-α), rat intestinal mucosal bifidobacteria adhesion and adhesin receptor were measured by enzyme-linked immunosorbent assay (ELISA). The real-time fluorescence quantitative PCR (RT-PCR) were used to detect the intestinal bifidobacterial content. Results Compared with the model group, the content of IL-8 (153.50 ± 8.59 pg/ml, 150.84 ± 5.38 pg/ml vs. 167.13 ± 13.66 pg/ml), TNF-α (330.08 ± 10.02 pg/ml, 287.27 ± 13.89 pg/ml vs. 376.50 ± 19.06 pg/ml) in medium, high dose group of Pingkui enema significantly decreased (P<0.05), while the IL-13 (37.04 ± 3.62 pg/ml, 40.64 ± 5.98 pg/ml vs. 31.57 ± 2.95 pg/ml) significantly increased (P<0.05). The bifidobacteria adhesin (124.84 ± 9.74 ng/L, 149.93 ± 9.57 ng/L, 176.74 ± 11.01 ng/L vs. 101.11 ± 17.18 ng/L) and adhesin receptor (78.44 ± 14.03 ng/L, 99.50 ± 3.63 ng/L, 107.36 ± 5.05 ng/L vs. 60.96 ± 13.89 ng/L) in low-, medium-, high dose group of Pingkui enema significantly increased (P<0.05). The content of bifidobacteria (1 331.4 ± 242.2 copies/μl vs. 490.5 ± 106.0 copies/μl) in the middle dose Pingkui enema group significantly increased (P<0.05). Conclusions The Pingkui enema can promote the colonization of intestinal epithelial cells by promoting bifidobacteria, increase bifidobacterium adhesin and adhesin receptors, up-regulate the expression of IL-13, and down-regulate the expression of IL-8, TNF-α.

Parenteral adjuvant potential of recombinant B subunit of Escherichia coli heat-labile enterotoxin

BACKGROUND The B subunit of Escherichia coli heat-labile enterotoxin (LTB) is a potent mucosal immune adjuvant. However, there is little information about LTB's potential as a parenteral adjuvant. OBJECTIVES We aimed at evaluating and better understanding rLTB's potential as a parenteral adjuvant using the fused R1 repeat of Mycoplasma hyopneumoniae P97 adhesin as an antigen to characterise the humoral immune response induced by this construct and comparing it to that generated when aluminium hydroxide is used as adjuvant instead. METHODS BALB/c mice were immunised intraperitoneally with either rLTBR1 or recombinant R1 adsorbed onto aluminium hydroxide. The levels of systemic anti-rR1 antibodies (total Ig, IgG1, IgG2a, and IgA) were assessed by enzyme-linked immunosorbent assay (ELISA). The ratio of IgG1 and IgG2a was used to characterise a Th1, Th2, or mixed Th1/Th2 immune response. FINDINGS Western blot confirmed rR1, either alone or fused to LTB, remained antigenic; anti-cholera toxin ELISA confirmed that LTB retained its activity when expressed in a heterologous system. Mice immunised with the rLTBR1 fusion protein produced approximately twice as much anti-rR1 immunoglobulins as mice vaccinated with rR1 adsorbed onto aluminium hydroxide. Animals vaccinated with either rLTBR1 or rR1 adsorbed onto aluminium hydroxide presented a mixed Th1/Th2 immune response. We speculate this might be a result of rR1 immune modulation rather than adjuvant modulation. Mice immunised with rLTBR1 produced approximately 1.5-fold more serum IgA than animals immunised with rR1 and aluminium hydroxide. MAIN CONCLUSIONS The results suggest that rLTB is a more powerful parenteral adjuvant than aluminium hydroxide when administered intraperitoneally as it induced higher antibody titres. Therefore, we recommend that rLTB be considered an alternative adjuvant, even if different administration routes are employed.

Evaluation of the role of Mycobacterium tuberculosis pili (MTP) as an adhesin, invasin, and cytokine inducer of epithelial cells

Abstract This study was undertaken in order to assess the involvement of Mycobacterium tuberculosis pili (MTP) as an adhesin, invasin, and cytokine inducer in the M. tuberculosis-epithelial cell interaction. A MTP-deficient strain of M. tuberculosis demonstrated a significant reduction of 69.39% (p = 0.047) and 56.20% (p = 0.033) in its ability to adhere to and invade A549 pulmonary epithelial cells, respectively, in comparison with the wild-type strain. Complementation of the MTP-deficient mutant restored its adhesion and invasion capacity back to the wild-type levels. Overall, it was found that similar concentrations of IL-1β, IL-4, IL-6, IL-8, G-CSF, IFN-γ, MCP-1, and TNF-α were induced in A549 cells infected with the MTP-proficient and MTP-deficient strains. However, at 48 h post-infection, the MTP-deficient mutant induced significantly lower levels of TNF-α than the wild-type strain (p = 0.033). Furthermore, at 72 h post-infection, the mutant induced significantly higher levels of IL-8 than the wild-type (p = 0.005). We conclude that MTP is an adhesin/invasin of epithelial cells and, while playing a role in M. tuberculosis entry, they do not appear to largely influence the epithelial cell cytokine response.

Screening and identification of the polypeptides specifically binding to the adhesion protein of Myco-plasma genitalium / 中华微生物学和免疫学杂志

Objective To screen and identify the polypeptides specifically binding to the adhesion protein of Mycoplasma genitalium(MgPa) by using the Ph. D.-12TM phage display peptide library for further understanding the biological function and the possible pathogenic mechanism of the MgPa. Methods The Ph. D.-12TM phage display peptide library was used for 3 rounds of biopanning with the purified recombinant MgPa ( rMgPa) as the given target. The phages were collected for amplification after biopanning. The single strand DNA of phage clones were extracted and purified by using the sodium iodide method for further se-quencing. ELISA, competitive binding assay and dot immunobinding assay were performed to analyze the specific binding of positive phages to rMgPa. Results A significant enrichment of phages was achieved after 3 rounds of biopanning. Eleven different phage exogenous sequences (P1-P11) were detected among the 38 phages randomly selected from the agar. Two core sequences were deduced according to the repeating times of amino acids among the 11 polypeptide sequences, which were V-H-W-D-F-R-Q-W-W-Q-P-S and D-W-S-S-W-V-H/Y-R-D-P-Q-T/S. Ten out of the 11 representative phages ( P1-P10 ) specifically combined with the rMgPa. Conclusion Two polypeptides specifically binding to rMgPa were successfully screened out, which provided the tool for further investigation on the biological function of MgPa and the pathogenic mecha-nism of Mycoplasma genitalium.

PREVALENCE OF Entamoeba histolytica/Entamoeba dispar IN THE CITY OF CAMPINA GRANDE, IN NORTHEASTERN BRAZIL / Prevalência de Entamoeba histolytica/Entamoeba dispar na cidade de Campina Grande, Nordeste do Brasil

There is a clear need to perform epidemiological studies to find the true prevalence of Entamoeba histolytica around the world. The evaluation of this prevalence has been hindered by the existence of two different species which are morphologically identical, but genetically different, namely E. histolytica, which causes amebiasis, and E. dispar, which is non-pathogenic. In Brazil, the E. dispar has been detected in communities in the Southeastern (SE) and Northeastern (NE) regions with poor sanitation. However, individuals infected with E. histolytica have been identified in other regions. There is an absence of reports on the prevalence of these parasites in the state of Paraíba, which also has areas with poor sanitary conditions where a high prevalence of the E. histolytica/E. dispar complex has been detected in children from urban slums. The present study evaluated the prevalence of E. histolytica and E. dispar in 1,195 asymptomatic children between two and 10 years of age, living in a sprawling urban slum in Campina Grande, in the state of Paraíba, in Northeastern Brazil. These children were examined and their feces samples were analyzed microscopically. A total of 553 children tested positive for the E. histolytica/E. dispar complex, and 456 of the positive samples were tested with the E. histolytica II® ELISA kit. All 456 samples were negative for the presence of the adhesin E. histolytica specific antigen. The evidence suggests that in this community E. histolytica is absent and E. dispar is the dominant species.

A prevalência mundial de Entamoeba histolytica não está bem estabelecida. Este fato deve-se à complicação derivada da existência de duas espécies morfologicamente idênticas, mas geneticamente diferentes: a E. histolytica que causa amebíases e a E. dispar descrita como não patogênica. No Brasil, em comunidades com precárias condições sanitárias e endêmicas para várias parasitoses, localizadas nas regiões Sudeste (SE) e Nordeste (NE), somente E. dispar tem sido encontrada, porém outras regiões, apresentam indivíduos infectados por E. histolytica. Na região agreste do Estado da Paraíba (NE) que apresenta as mesmas precárias condições sanitárias, não tem sido reportada prevalência específica destes parasitos, embora fosse encontrada alta prevalência do complexo E. dispar/E. histolytica em crianças em favela urbana. O presente estudo foi realizado em favela da cidade de Campina Grande, Estado da Paraíba, onde 1.195 crianças de dois a 10 anos sem sintomatologia foram examinadas. Amostras de fezes destas crianças foram analisadas microscopicamente, encontrando-se 553 positivas para o complexo E. dispar/E. histolytica. Do total de amostras positivas, 456 foram submetidas à pesquisa do antígeno especifico para E. histolytica pelo teste ELISA E. histolytica II®,obtendose resultado negativo para a presença do antígeno adesina específico de E. histolytica, em todas as amostras testadas. Os resultados sugerem que nesta comunidade não há infecção por E. histolytica, e que E. dispar é a espécie dominante na região.

Effect of bifidobacterial adhesin on nuclear factor-κB and cytokines in intestinal mucosa of stressed rats / 中华临床营养杂志

Objective To investigate the effect of bifidobacterial adhesin (BA) on nuclear factor of κB (NF-κB) and cytokines of intestinal mucosa of stressed rats.Methods Forty-eight rats were divided into stress group (n =24) and BA group (n =24) using the stochastic indicator method.After the stressed rat models were established withfettering as the stress condition,the experiment lasted 8 days.Both groups were given enteral nutrition (EN) with heat 125.4 kJ/(kg · d) and nitrogen 0.2 g/(kg · d).The BA group was fed with EN plus 5 mg/ (kg · d) bifidobacterial adhesin,and the stress group was fed with EN plus equivalent volume of normal saline [5 mg/ (kg · d)].The levels of NF-κB,interleukin-10 (IL-10),tumor necrosis factor (TNF-α),and interferon-γ (IFN-γ) were measured in both groups before modeling,after modeling,on the 3rd intervention day,and on the 8th intervention day.The changes in the morphology of intestinal mucosal were observed by transmission electron microscopy.Results (1) Expression of NF-κB:The positive expression rate of NF-κB in the intestinal mucosa was 0,79.2％,63.5％,and 66.7％ in the control group and 0,68.4％,55.7％,and 45.8％ in the BA group before modeling,after modeling,on the 3rd intervention day,and on the 8th intervention day.The expressions of NF-κB in both groups significantly increased after the modeling (both P =0.000).Even on the 3rd and 8th intervention days,the positive expression rates of NF-κB in the intestinal mucosa were still significantly higher than the pre-modeling level (both P =0.000).Compared with the levels after modeling and in the control group,the expression of NF-κB in the intestinal mucosa in the BA group on the 8th intervention day was significantly down-regulated (P =0.015,P =0.021).(2) Quantitative expressions of TNF-α and IFN-γ:Compared with the pre-modeling levels,the intestinal mncosa levels of TNF-α [stressed group:(154.63 ± 17.52) pg/g,(198.72 ±26.59) pg/g; BA group:(154.63 ±17.52) pg/g,(201.45 ±28.16) pg/g],IFN-γ [stressed group:(39.47 ±5.76) pg/g,(55.32 ±5.93) pg/g; BA group:(39.47 ± 5.76),(60.75 ± 7.68) pg/g] and the plasma levels of TNF-α [stressed group:(17.35±2.62) pg/g,(30.56±4.85) ng/L; BA group:(83.31 ±9.78) pg/g,(114.82±13.78) ng/L] and IFN-γ [stressed group:(17.35 ±2.62) pg/g,(28.73 ±4.17) ng/L; BA group:(17.35 ± 2.62) pg/g,(30.56 ± 4.85) ng/L] significantl increased (all P ＜ 0.05).On the 3rd and 8th intervention day,the intestinal mucosa levels of IFN-γ [(58.16 ± 7.38) pg/g,(56.37 ± 7.29) pg/g] and TNF-α [(215.76 ±31.54) pg/g and (211.83 ±33.61) pg/g] and plasma levels of IFN-γ [(29.35 ±4.76) ng/L,(30.25±3.67) ng/L] andTNF-α [(125.71 ±17.38) ng/L,(141.26±19.65) ng/L] in the stressed group were significantly higher than the pre-modeling levels (all P ＜ 0.05).On the 3rd and 8th intervention day,the intestinal mucosa levels of IFN-γ [(165.43 ± 24.58) pg/g,(171.57 ± 26.87) pg/g]and IFN-γ [(42.35 ±4.92) pg/g,(40.58 ±4.65) pg/g] and the plasma levels of TNF-α [(103.96 ±13.68) ng/L,(94.53±12.66) ng/L] and IFN-γ [(20.78±2.84) ng/L,(19.65±2.45) ng/L] in the BA group were significantly lower than the post-modeling levels (all P ＜ 0.05),whereas those of IL (intestinal mucosa:(62.82 ±8.34) pg/g,(75.16 ±9.65) pg/g; plasma:(43.32 ±5.28) ng/L,(55.64 ±6.87) ng/L] were significantly higher than the post-modeling levels (all P ＜ 0.05).Compared with the stressed group,the intestinal mucosa levels of TNF-α and IFN-γand plasma levels of IFN-γ and TNF-α significantly decreased while the IL-10 level significantly increased (all P ＜0.05) in the BA group.(3) Histomorphology showed that,compared that the ileal mucosal villi and crypt structure were recovered in the BA group on the 8th intervention day.Compared with the post-modeling conditions,the ileal mucosal villi and crypt structure were damaged in the stressed group,showing edema of the lamina propria,in which inflammatory cell infiltration was observed.Conclusions BA is helpful for the repair of the intestinal mucosa injury after stress by regulating the release of inflammatory mediators and cytokines of intestinal mucosa.

Observation on the immunological competence of multiple antigen peptides based on the mimic epitope of the adhesion protein of Mycoplasma genitalium / 中华微生物学和免疫学杂志

Objective To provide experimental evidence for the development of multi-epitope-baseded marker vaccines through investigating the humoral and cellular immune responses in BALB/c mice induced by the multiple antigen peptides (MAPs) with the mimic epitope.Methods Three types of MAPs in eight branched forms containing the mimic epitope of Mycoplasma genitalium adhesion protein (MgPa) were prepared using poly-lysine as the core matrix.The purity of MAPs was analyzed by reverse phase high performance liquid chromatography (RP-HPLC).The molecular weights of MAPs were characterized by Mass Spectrometry.The BALB/c mice were immunized intramuscularly for four times with single or mixed MAPs.The specific IgG antibody and the subtype of IgG antibody in serum of the immunized mice were detected by indirect ELISA.The proliferative responses of the spleen lymphocytes were detected using MTT assay.The ELISA were used to detect IFN-γ and IL-4 levels in the cultured supematant of spleen lymphocytes.Results The three types of MAPs containing the mimic epitopes were successfully prepared with high purity.They,could stimulate mice to produce specific IgG antibodies,of which,the major antibody isotype was Th1 immune response-associated IgG2a.Compared with the single MAP immunization group,the mixed-MAPs immunized mice produced more IgG,IgG1 and IgG2a antibody (P＜0.05).Furthermore,these MAPs could enhance the specific proliferation of spleen lymphocytes in immunized mice and induce the production of IFN-γ and IL-4.The levels of IFN-γand IL-4 in mixed-MAPs group were significantly higher than those of the single MAPs group (P＜0.01).Conclusion The three types of MAPs could induce strong specific cellular and humoral immune responses.The immunological competence of the mixed-MAPs was stronger than those of the single MAP.

Identificação de adesinas de Leptospira interrogans por shotgun phage display / Identification of Leptospira interrogans adhesins by shotgun phage display

Em Leptospira interrogans algumas proteínas com capacidade de ligação aos componentes de matriz extracelular foram identificadas e, em sua maioria, são fatores de virulência. Phage display é considerada uma técnica poderosa na identificação de novos ligantes, inclusive de moléculas adesinas, importantes no primeiro estágio de infecção do hospedeiro. A técnica de shotgun phage display foi utilizada visando à obtenção de ligantes à células de mamíferos. Quatro bibliotecas, por inserção de fragmentos aleatórios obtidos por sonicação do DNA de L. interrogans nos fagomídeos pG8SAET (BBT1 e BBT2) e pG3DSS (BBT5 e BBT6), foram construídas. As bibliotecas BBT1 e BBT5 contém insertos maiores e as BBT2 e BBT6 contém insertos menores, com tamanhos médios de 1500 pb e 350 pb, respectivamente. Após ensaio de panning da BBT5 contra células de mamíferos e soro fetal bovino, as sequências de clones selecionados foram analisadas quanto a orientação correta e se a fusão estava em fase com a proteína pIII. As proteínas codificadas pelos genes LIC11719, LIC10769, LIC13143 e LIC12976 foram selecionadas com estas características. Os genes que codificam a LIC12976, LIC10768, LIC10769 e LIC13418, tiveram sua conservação avaliada em diferentes sorovares da espécie patogênica L. interrogans e no sorovar Patoc da espécie de vida livre L. biflexa. As proteínas LIC12976 (selecionada pela técnica de phage display) e LIC13418 (selecionada por ferramentas de bioinformática) tiveram suas sequências amplificadas por PCR, clonadas em pGEM T easy, subclonadas em vetor de expressão pAE e expressas na fração celular correspondente ao corpúsculo de inclusão em E. coli BL21 (DE3) Star pLysS e E. coli BL21 SI, respectivamente. Após renaturação e purificação destas proteínas por cromatografia de afinidade a metal bivalente, um grupo de cinco animais BALB/c fêmeas foi imunizado. Ambas as proteínas se mostraram imunogênicas com títulos dos soros policlonais 1:256000 e 1:512000, respectivamente. Em ensaio de Western Blot os soros foram específicos no reconhecimento das proteínas recombinantes e as proteínas nativas foram verificadas em extratos de sorovares patogênicos de L. interrogans. Em ensaios de adesão, as proteínas recombinantes aderiram às células A31, LLC-PK1 e Vero e especificamente à laminina. Em ensaios de interferência em células usando laminina houve um aumento da adesão das proteínas recombinantes, o que pode ser explicado pela ligação da laminina às células e uma maior ligação das LICs estudadas. Em ensaio de localização celular usando imunofluorescência e microscopia eletrônica, foi observado que ambas as proteínas se encontram na superfície da L. interrogans. No experimento de desafio animal, a LIC12976 e a LIC13418 não se mostraram protetoras. Este trabalho contribuiu para a identificação das novas adesinas LIC13418 e LIC12976 que podem participar da virulência de leptospiras patogênicas envolvendo a primeira etapa da infecção na interação patógeno-hospedeiro

In Leptospira interrogans, proteins capable to bind to extracellular matrix components have been identified and most of them are important virulence factors. Phage display is a powerful technique to identify new ligands, including adhesin molecules that are important in the first stage of host infection. A shotgun phage display technique was used in order to obtain cell ligands. Four libraries were constructed by inserting random fragments obtained by sonication of L. interrogans DNA into phagemids pG8SAET (BBT1 and BBT2) and pG3DSS (BBT5 and BBT6). The libraries BBT1 and BBT5 contain larger inserts and BBT2 and BBT6 contain smaller inserts, with 1500 bp and 350 bp average sizes, respectively. After panning of BBT5 against mammalian cells and bovine fetal serum, the sequences of selected clones were analyzed for correct orientation and fusion with pIII protein. The proteins encoded by genes LIC11719, LIC10769, LIC13143 and LIC12976 were selected. The genes LIC12976, LIC10768, LIC10769 and LIC13418 were evaluated for their conservation in different pathogenic serovars of L. interrogans and free-living L. biflexa serovar Patoc. Proteins LIC12976 (selected by phage display technique) and also LIC13418 that was selected by bioinformatic tools, were amplified by PCR, cloned into pGEM T easy, subcloned into expression vector pAE and expressed in cellular fraction corresponding to the inclusion body in E. coli BL21 (DE3) Star pLysS and E. coli BL21 SI, respectively. After protein renaturation protocol and purification by affinity chromatography, a group of five BALB/c mice was immunized with the purified proteins. Both proteins were shown to be immunogenic with 1:256000 and 1:512000 polyclonal sera titers, respectively. In Western blot the sera were specific to recognize recombinant proteins and native proteins were detected in pathogenic L. interrogans serovars extracts. In binding assays, recombinant proteins bind to A31, LLC-PK1 and Vero cells and specifically to laminin. In interference cell assay using laminin there was an increase of recombinant protein bindings, which can be explained by the laminin binding to cells and further binding of the recombinant LICs. In cellular localization assay using immunofluorescence and electron microscopy, it was observed that both are surface proteins of L. interrogans. In the animal challenge, the LIC12976 and LIC13418 were not protective. As a whole, this work contributed to the identification of LIC12976 and LIC13418 as new adhesins and they can participate in the virulence of pathogenic Leptospira in the first stage of host pathogen interaction.

Screening and identification of antigenic mimics of the adhesion protein of Mycoplasma genitalium by phage display peptide library / 中华微生物学和免疫学杂志

ObjectiveTo screen a 12-mer phage display peptide library by the polyclonal antibody (pAb) against the recombinant adhesion protein of Mycoplasma genitalium (rMgPa) in order to obtain the antigenic mimic epitopes of MgPa.MethodsThe purified pAb was used to screen the immunodominant mimic epitopes of MgPa by a random 12-peptide phage display library.Seventy-four recombinant phage clones were randomly selected,and then DNA sequence analysis and computer-based bioinformatics analysis were performed to define the consensus amino acid residues of the mimotopes by MIMOX.The binding specificities of the selected phage-displayed peptides to the purified pAb were confirmed by ELISA,competitive ELISA and Western blot analysis.Results After four rounds of biopanning,a significant enrichment of phages was achieved,the inserts from 74 phage clones distinguished 45 peptides based on the different amino acids sequences.Amongst 45 peptides,36 peptides were ELISA positive and 23 peptides that absorbance values were higher than 1.5 showed high reactivities with pAb and effectively inhibited the binding of pAb to rMgPa.Immunoscreening via phage display peptide library revealed three different mimptopes of adhesion protein of M.genitalium,P-S-A-A/V-X-R-F/W-E/S-L-S-P,A-K-I/L-T/Q-X-T-L-X-L and K-S-L-S-R-X-D-X-I.Results of bioinformatics analysis by MIMOX demonstrated that S,A,F for cluster 1,A,K,I,T and L for cluster 2,K,S,L,R,D and I for cluster 3,may be the key consensus amino acid residues in the aligned mimotopes,respectively.ConclusionAntigenic mimics on MgPa were successfully identified and the motif P-S-A-A/V-X-R-F/W-E/S-L-S-P,A-K-I/L-T/Q-X-T-L-X-L and K-S-L-S-R-X-D-X-I may represent the immunodominant mimic epitopes of MgPa.And S,A,F K,I,T,L,R and D may be the key amino acid residues for the epitopes of MgPa.

Identificação de adesinas de Leptospira interrogans por shotgun phage display / Identification of Leptospira interrogans adhesins by shotgun phage display

Em Leptospira interrogans algumas proteínas com capacidade de ligação aos componentes de matriz extracelular foram identificadas e, em sua maioria, são fatores de virulência. Phage display é considerada uma técnica poderosa na identificação de novos ligantes, inclusive de moléculas adesinas, importantes no primeiro estágio de infecção do hospedeiro. A técnica de shotgun phage display foi utilizada visando à obtenção de ligantes à células de mamíferos. Quatro bibliotecas, por inserção de fragmentos aleatórios obtidos por sonicação do DNA de L. interrogans nos fagomídeos pG8SAET (BBT1 e BBT2) e pG3DSS (BBT5 e BBT6), foram construídas. As bibliotecas BBT1 e BBT5 contém insertos maiores e as BBT2 e BBT6 contém insertos menores, com tamanhos médios de 1500 pb e 350 pb, respectivamente. Após ensaio de panning da BBT5 contra células de mamíferos e soro fetal bovino, as sequências de clones selecionados foram analisadas quanto a orientação correta e se a fusão estava em fase com a proteína pIII. As proteínas codificadas pelos genes LIC11719, LIC10769, LIC13143 e LIC12976 foram selecionadas com estas características. Os genes que codificam a LIC12976, LIC10768, LIC10769 e LIC13418, tiveram sua conservação avaliada em diferentes sorovares da espécie patogênica L. interrogans e no sorovar Patoc da espécie de vida livre L. biflexa. As proteínas LIC12976 (selecionada pela técnica de phage display) e LIC13418 (selecionada por ferramentas de bioinformática) tiveram suas sequências amplificadas por PCR, clonadas em pGEM T easy, subclonadas em vetor de expressão pAE e expressas na fração celular correspondente ao corpúsculo de inclusão em E. coli BL21 (DE3) Star pLysS e E. coli BL21 SI, respectivamente. Após renaturação e purificação destas proteínas por cromatografia de afinidade a metal bivalente, um grupo de cinco animais BALB/c fêmeas foi imunizado. Ambas as proteínas se mostraram imunogênicas com títulos dos soros policlonais 1:256000 e 1:512000, respectivamente...

In Leptospira interrogans, proteins capable to bind to extracellular matrix components have been identified and most of them are important virulence factors. Phage display is a powerful technique to identify new ligands, including adhesin molecules that are important in the first stage of host infection. A shotgun phage display technique was used in order to obtain cell ligands. Four libraries were constructed by inserting random fragments obtained by sonication of L. interrogans DNA into phagemids pG8SAET (BBT1 and BBT2) and pG3DSS (BBT5 and BBT6). The libraries BBT1 and BBT5 contain larger inserts and BBT2 and BBT6 contain smaller inserts, with 1500 bp and 350 bp average sizes, respectively. After panning of BBT5 against mammalian cells and bovine fetal serum, the sequences of selected clones were analyzed for correct orientation and fusion with pIII protein. The proteins encoded by genes LIC11719, LIC10769, LIC13143 and LIC12976 were selected. The genes LIC12976, LIC10768, LIC10769 and LIC13418 were evaluated for their conservation in different pathogenic serovars of L. interrogans and free-living L. biflexa serovar Patoc. Proteins LIC12976 (selected by phage display technique) and also LIC13418 that was selected by bioinformatic tools, were amplified by PCR, cloned into pGEM T easy, subcloned into expression vector pAE and expressed in cellular fraction corresponding to the inclusion body in E. coli BL21 (DE3) Star pLysS and E. coli BL21 SI, respectively. After protein renaturation protocol and purification by affinity chromatography, a group of five BALB/c mice was immunized with the purified proteins. Both proteins were shown to be immunogenic with 1:256000 and 1:512000 polyclonal sera titers, respectively. In Western blot the sera were specific to recognize recombinant proteins and native proteins were detected in pathogenic L. interrogans serovars extracts...

Expression and immunological characterization of the major epitope of P1 adhesin protein of Mycoplasma pneumoniae and its clinical application research / 中华微生物学和免疫学杂志

Objective To study the application of P1 adhesin protein epitopes in diagnosis of Mycoplasma pneumoniae(Mp) infected patient. Methods The major epitope(P1-534) of P1 adhesin protein were predicted by ProPred and ANTIGENIC according to its primary structure. The high value fragment was cloned into a constructed recombinant vector. The gene was induced to express fusion protein in E. coli host strain BL21(DE3) and the fusion protein was identified by Western blot. BALB/c mice were immunized with purified protein to test its immunogenicity. Then the purified protein was used as antigen to test the serum of Mp infected patient by ELISA, and compared with the Mp whole cell antigen. Results The P1-534 protein was successfully expressed and purified. ELISA data showed that P1-534 protein could elicit high levels of IgG in immunized mice, the sensitivity and specificity of P1-534 were determined to be 85.00% and 97.67%, while the Mp whole cell antigen were 72.50% and 74.42%. Conclusion The results conformed that the recombinant epitope has certain immunogenicity,and its sensitivity and specificity are better than Mp whole cell antigen. P1-534 protein can be used as an antigen for immunodiagnosis of Mp infection.

The protective effects of bifidobacterial adhesin on ischemic reperfusion injury of intestine in rats / 中华内科杂志

ObjectiveTo investigate the protection effect of bifidobacterial adhesin for intestine ischemia/reperfusion (I/R) injury on gut barrier function in rat.MethodsSeventy-two male SD rats were randomly divided into sham operation group (n =24), I/R model group (n =24) and pretreatment group of bifidobacterial adhesin (pretreatment group, n = 24).Six rats were anatomized at 6 h, 1 d, 4 d and 7d after inducing I/R model in each group, respectively.The pathological changes of the terminal ilea and the blood levels of TNFα, IL-6, IL-10, diamine oxidase (DAO), and the activity and content of D-lactic acid were observed.ResultsThe blood levels of TNFα, IL-6, DAO and D-lactic acid in I/R model group were significantly higher than sham operation group at all time points (P ＜0.05) , while the blood level of IL-10 was no significantly change.The activity of IL-6 and DAO in pretreatment group was significantly lower than I/R model group at all time points (P ＜ 0.05), the blood level of TNFαt in pretreatment group was significantly lower than I/R model group at 1 d, the blood level of D-lactic was significantly lower than I/R model group at 4 d and 7 d (P ＜ 0.05). Intestinal pathological damages were obviously milder in pretreatment group than I/R model group at all time points (Chiu's pathological scores: 6 h, 3.22 ±0.22 vs 3.57 ±0.20;1 d,3.77 ±0.13 vs 3.90 ±0.12;4 d,2.93 ±0.23 vs 3.07 ±0.21;7 d,2.10 ±0.30 vs 2.22 ±0.17,all P ＜ 0.05).ConclusionThe pretreatment of bifidobacterial adhesin could protect the intestinal mucosa from I/R injury, and alleviate intestinal ischemic reperfusion injury.

Papel das hemaglutininas 67-72p de Corynebacterium diphtheriae na ligação a proteínas plasmáticas, superfícies celulares, invasão e indução de apoptose / Role of haemagglutinins 67-72p de Corynebacterium diphtheriae in binding to plasma proteins, cell surfaces, invasion and induction of apoptosis

Corynebacterium diphtheriae pode ser isolado tanto de quadros de difteria clássica, quanto de infecções sistêmicas, como endocardite. O fibrinogênio (Fbn) e a fibronectina (Fn) são glicoproteínas presentes na matriz extracelular de tecidos conjuntivos. A influência destas proteínas na patogênese das infecções locais e invasivas causadas por C. diphtheriae é objeto de estudo devido ao fato do bacilo diftérico poder ser encontrado em lesões nas quais o Fbn e a Fn são predominantes, incluindo a pseudomembrana diftérica e vegetações cardíacas presentes na endocardite infecciosa. São crescentes as evidências de que o C. diphtheriae pode, além de aderir, ser internalizado por células em cultura. No presente estudo, investigou-se a participação de C. diphtheriae e das proteínas de superfície 67-72p na aderência à Fn e ao Fbn de plasma humano e a eritrócitos. A aderência às células HEp-2 e internalização também foram analisadas. A participação de 67-72p nos mecanismos de morte celular foi avaliada através das colorações por Azul de Tripan e 4'6-diamidino-2-fenil indol (DAPI), pelo ensaio de redução utilizando dimetil-tiazol-difenil tetrazólio (MTT) e por citometria de fluxo. As 67-72p foram extraídas da superfície da amostra toxigênica C. diphtheriae subsp. mitis CDC-E8392 através de processos mecânicos e precipitação com sulfato de amônio saturado. Análises por SDS-PAGE e immunoblotting detectaram a presença das bandas protéicas de 67 e 72kDa nas amostras toxinogênicas e atoxinogênicas analisadas, as quais pertenciam aos biotipos fermentador e não fermentador de sacarose. C. diphtheriae foi capaz não só de formar agregados na presença de plasma de coelho, mas também de converter Fbn em fibrina independentemente da presença do gene tox. No entanto, a amostra atoxinogênica ATCC 27010 (tox-) foi menos aderente ao Fbn do que a homóloga ATCC 27012 (tox+). A interação bacteriana com eritrócitos foi inibida somente pela Fn. Ligações entre Fn e/ou Fbn com 67-72p foram ...

Corynbacterium diphtheriae have been isolated from classical diphtheria and systemic infections such as endocarditis. Fibrinogen (Fbn) and fibronectin (Fn) are high molecular-weight glycoproteins that may be found in extracellular matrix of connective tissues. Their influence in the pathogenesis of local and in invasive C. diphtheriae infection is object of interest due to the fact that diphtheria bacilli is recovered from lesions where such proteins are predominant, including pharyngeal pseudomembrane and valve heart vegetations in infectious endocarditis. There is growing evidence that C. diphtheriae may adhere to and be internalized by cells in culture. The present study investigated the participation of C. diphtheriae strains and 67-72p, a surface protein, in adherence to human plasma Fn, Fbn, erityrocytes, adherence to and internalization by HEp-2 cells. The participation of 67-72p in promoting cell death was evaluated by the Trypan blue, DAPI staining methods, methylthiazole tetrazolium (MTT) reduction assay and flow cytometry. The 67-72p was extracted from C. diphtheriae subsp. mitis CDC-E8392 toxigenic strain, by mechanical process and ammonium sulfate fractionation. SDS-PAGE and immunoblotting analysis detected the polypeptide bands of 67 and 72 kDa in all toxigenic and nontoxigenic strains from both sucrose-fermenting and non-fermanting biotypes. Diphtheria bacilli were capable to both form bacterial aggregates in rabbit plasma and to convert Fbn to fibrin independently to the presence of tox gene, albeit the ATCC 27010 (tox-) strain was less adherent to Fbn than the paental strain ATCC 27012 (tox+). Bacteria-erythrocytes interaction was inhibited only ...

Expression and purification of Tp0751 recombinant protein of Treponema pallidum and its immuno-competence analysis / 中华微生物学和免疫学杂志

Objective To express Tp0751 laminin-binding adhesion of Treponema pallidum (T. pallidum) ,and assess the immunocompetence. Methods The Tp0751 ORF without upstream non-cod-ing region was ligated into the expression vector pET-28a( + ), and expressed in E. coli R2566. Its immuno-gen was analyzed by Western blot and ELISA. Results A fusion protein with molecular weight about 26×103 was attained after expression and purification. Western blot proved that the recombinant protein can specifically react with T. palliclum IgG positive sera. Specific humoral response were elicited after introducing recombinant protein in Zealand rabbit and the specific antibody titer was above 1:10 2400 detected by indi-rect ELISA. Conclusion The expressed recombinant protein showed excellent immunoeompetence, and the results lay the foundation for the research on its function to T. paUidum infection.

Ica operon of staphylococcus epidermidis and biomaterial infection / 国际生物医学工程杂志

Staphylococcus epidermidis(SE)is the main opportunistic pathogen in nosocomial infections.It usually enter the body along with biomedical materials and adheres to the surface of biomaterials to form bacterial bi06lm.SE bi06lm bears high organized multi.cell colony structure.Bacterial biofilm iS the key reason for refractoriness of biomaterial originated infection.This paper gives an overview of the research progresses in biomaterial related infection,including SE ica operon,formation of biofilm,biofilm treatment,and SO on.Our review shows that studies of SE biofilm were COnfined to the mutated strain from laboratory and growth pattern of plankton and thus the natural clinical pathogenic procedure of SE can't be fully described.Further study should be carried out on formation of SE biofilm and treatment of infections related to the clinical application of biomaterials.

Effect of hypoxia on migration and adhesion of human pulmonary adenocarcinoma A549 cells / 肿瘤

Objective: To evaluate the effect of hypoxia on migration, invasion and adhesion to endothelial cells of human pulmonary adenocarcinoma A549 cells. Methods: Wound-healing and Transwell invasion assays were performed to study the effect of hypoxia on migration and invasion of A549 cells. Cell adhesion test was used to detect the adhesion of A549 cells to a monolayer of human umbilical vein endothelial cells (HUVECs). Immunofluorescence assay was used to evaluate the effect of hypoxia on distribution of E-cadherin, β-catenin, and F-actin. The luciferase reporter gene assay was performed to detect the transcription of hypoxia-inducible factor-1 (HIF-1) alpha. Results: Hypoxia promotes A549 cell migration, invasion, and adhesion to endothelial cells, and modulated the distribution of E-cadherin and β-catenin and rearrangement of actin cytoskeletal protein, and up-regulated HIF-1-dependent reporter gene expression in A549 cells. Conclusion: Hypoxia promoted A549 cell migration, invasion, and adhesion to endothelial cells by upregulating HIF-1-dependent gene expression, subsequently affecting the redistribution of E-cadherin and β-catenin and rearrangement of F-actin cytoskeletal protein.