Baicalin attenuated Mycoplasma gallisepticum-induced immune impairment in chicken bursa of fabricius through modulation of autophagy and inhibited inflammation and apoptosis.

BACKGROUND: Mycoplasma gallisepticum (MG) is the primary etiologic agent of chronic respiratory disease in poultry. However, the mechanism underlying MG-induced immune dysregulation in chicken is still elusive. Baicalin shows excellent anti-bacterial, anti-inflammatory, anti-carcinogenic and anti-viral properties. In the present study, the preventive effects of baicalin against immune impairment in chicken bursa of fabricius (BF) were studied in an MG infection model. RESULTS: Histopathological examination showed increased inflammatory cell infiltrations and fragmented nuclei in the model group. Ultrastructural analysis revealed the phenomenon of apoptosis in bursal cells, along with the deformation of mitochondrial membrane and swollen mitochondria in the model group. However, these abnormal morphological changes were partially alleviated by baicalin. Meanwhile, baicalin treatment attenuated the level of proinflammatory cytokines, and suppressed nuclear factor-kappa B expression at both protein and mRNA level. Terminal deoxynucleotidyl transferase-mediated dUTP nick endlabeling assay showed extensive apoptosis in BF in the model group. The mRNA and protein expression levels of apoptosis-related genes were upregulated in BF, while baicalin treatment significantly alleviated apoptosis in BF. In addition, alterations in mRNA and protein expression levels of autophagy-related genes and mitochondrial dynamics proteins were significantly alleviated by baicalin. Moreover, baicalin treatment significantly attenuated MG-induced decrease in CD8+ cells and reduced bacterial load in chicken BF compared to the model group. CONCLUSIONS: These results suggested that baicalin could effectively inhibit MG-induced immune impairment and alleviate inflammatory responses and apoptosis in chicken BF. © 2020 Society of Chemical Industry.

Interaction of Mycoplasma synoviae with chicken synovial sheath cells contributes to macrophage recruitment and inflammation.

Mycoplasma synoviae (MS) is an important avian pathogen causing considerable economic hardship in the poultry industry. A major inflammation caused by MS is synovitis that occurs in the synovial tendon sheath and joint synovium. However, the overall appearance of pathological changes in the tendon sheath and surrounding tissues caused by MS infection at the level of pathological tissue sections was poor. Studies on the role of MS and synovial sheath cells (SSCs) interaction in the development of synovitis have not been carried out. Through histopathological observation, our study found that a major MS-induced pathological change of the tendon sheath synovium was extensive scattered and focal inflammatory cell infiltration of the tendon sheath synovial layer. In vitro research experiments revealed that the CFU numbers of MS adherent and invading SSC, the levels of expression of various pattern recognition receptors, inflammatory cytokines, and chemokines coding genes, such as IL-1ß, IL-6, IL-8, CCL-20, RANTES, MIP-1ß, TLR7, and TLR15 in SSCs, and chemotaxis of macrophages were significantly increased when the multiplicity of infection (MOI) of MS to SSC were increased tenfold. The expression level of IL-12p40 in SSC was significantly higher when the MOIs of MS to SSC were increased by a factor of 100. The interaction between MS and SSC can activate macrophages, which was manifested by a significant increase in the expression of IL-1ß, IL-6, IL-8, CCL-20, RANTES, MIP-1ß, and CXCL-13. This study systematically demonstrated that the interaction of MS with chicken SSC contributes to the inflammatory response caused by the robust expression of related cytokines and macrophage chemotaxis. These findings are helpful in elucidating the molecular mechanism of MS-induced synovitis in chickens.

Baicalin inhibits inflammation caused by coinfection of Mycoplasma gallisepticum and Escherichia coli involving IL-17 signaling pathway.

Coinfection of Mycoplasma gallisepticum (MG) and Escherichia coli (E. coli) is frequently reported in poultry farms. Baicalin possess various pharmacological properties such as anti-inflammatory, anticancer, and antioxidant, etc. However, the protective effects of baicalin against coinfection of MG and E. coli are still elusive. In this study, baicalin (450 mg/kg) treatment was started on day 13 after infection and continued for 5 d. Histopathological examination, qRT-PCR, ELISA, and molecular docking technique were used to evaluate the effects of baicalin on MG and E. coli coinfection in chicken lung and trachea. The results showed that coinfection caused severe lesions in the lung and tracheal tissues. However, baicalin treatment partially alleviated these lesions in coinfection group. Histopathological examination showed the alveolar spaces and mucosal layer thickening was restored and cilia gradually recovered with baicalin treatment compared in coinfection group and MG-infection group. Meanwhile, IL-17 singling pathway-related genes were significantly reduced (P < 0.05) in baicalin treatment group in lung, including IL-17C, TRAF6, NF-κB, CXCL1, CXCL2, MMP1, GM-CSF, and MUC5AC. The activities of cytokines and chemokines (CXCL1, CXCL2, MMP1, GMCSF, and MUC5AC) were decreased significantly (P < 0.05) in baicalin-treated group. The molecular docking of baicalin and NF-κB showed the highest fitness score and interaction. From these results, it has been suggested that baicalin proved effective against coinfection of MG and E. coli in chicken and provided scientific basis for further dose-response and drug-target interaction studies.

Mycoplasma Infection Mediates Sensitivity of Multidrug-Resistant Cell Lines to Tiopronin: A Cautionary Tale.

We previously reported that some, but not all, multidrug-resistant cells that overexpressed various drug-resistance transporters were collaterally sensitive to tiopronin. In recent follow-up studies, we discovered that sensitivity to tiopronin in the original study was mediated by infection of the cells by a human-specific strain of mycoplasma. These results strongly support the need to constantly monitor cells for mycoplasma infection and keep stored samples of all cells that are used for in vitro studies.

Bovine Epithelial in vitro Infection Models for Mycoplasma bovis.

Mycoplasma bovis causes bovine mycoplasmosis. The major clinical manifestations are pneumonia and mastitis. Recently an increase in the severity of mastitis cases was reported in Switzerland. At the molecular level, there is limited understanding of the mechanisms of pathogenicity of M. bovis. Host-pathogen interactions were primarily studied using primary bovine blood cells. Therefore, little is known about the impact of M. bovis on other cell types present in infected tissues. Clear in vitro phenotypes linked to the virulence of M. bovis strains or tissue predilection of specific M. bovis strains have not yet been described. We adapted bovine in vitro systems to investigate infection of epithelial cells with M. bovis using a cell line (MDBK: Madin-Darby bovine kidney cells) and two primary cells (PECT: bovine embryonic turbinate cells and bMec: bovine mammary gland epithelial cells). Two strains isolated before and after the emergence of severe mastitis cases were selected. Strain JF4278 isolated from a cow with mastitis and pneumonia in 2008 and strain L22/93 isolated in 1993 were used to assess the virulence of M. bovis genotypes toward epithelial cells with particular emphasis on mammary gland cells. Our findings indicate that M. bovis is able to adhere to and invade different epithelial cell types. Higher titers of JF4278 than L22/93 were observed in co-cultures with cells. The differences in titers reached between the two strains was more prominent for bMec cells than for MDBK and PECT cells. Moreover, M. bovis strain L22/93 induced apoptosis in MDBK cells and cytotoxicity in PECT cells but not in bMec cells. Dose-dependent variations in proliferation of primary epithelial cells were observed after M. bovis infection. Nevertheless, an indisputable phenotype that could be related to the increased virulence toward mammary gland cells is not obvious.

Mycoplasma hyorhinis reduces sensitivity of human lung carcinoma cells to Nutlin-3 and promotes their malignant phenotype.

PURPOSE: MDM2 inhibitors are promising anticancer agents that induce cell cycle arrest and tumor cells death via p53 reactivation. We examined the influence of Mycoplasma hyorhinis infection on sensitivity of human lung carcinoma cells NCI-H292 to MDM2 inhibitor Nutlin-3. In order to unveil possible mechanisms underlying the revealed effect, we investigated gene expression changes and signal transduction networks activated in NCI-H292 cells in response to mycoplasma infection. METHODS: Sensitivity of NCI-Н292 cells to Nutlin-3 was estimated by resazurin-based cell viability assay. Genome-wide transcriptional profiles of NCI-H292 and NCI-Н292Myc.h cell lines were determined using Illumina Human HT-12 v3 Expression BeadChip. Search for key transcription factors and key node molecules was performed using the geneXplain platform. Ability for anchorage-independent growth was tested by soft agar colony formation assay. RESULTS: NCI-Н292Myc.h cells were shown to be 1.5- and 5.2-fold more resistant to killing by Nutlin-3 at concentrations of 15 and 30 µM than uninfected NCI-Н292 cells (P < 0.05 and P < 0.001, respectively). Transcriptome analysis revealed differential expression of multiple genes involved in cancer progression and metastasis as well as epithelial-mesenchymal transition (EMT). Moreover, we have shown experimentally that NCI-Н292Myc.h cells were more capable of growing and dividing without binding to a substrate. The most likely mechanism explaining the observed changes was found to be TLR4- and IL-1b-mediated activation of NF-κB pathway. CONCLUSIONS: Our results provide evidence that mycoplasma infection is an important factor modulating the effect of MDM2 inhibitors on cancer cells and is able to induce EMT-related changes.

Novel role of Vpmas as major adhesins of Mycoplasma agalactiae mediating differential cell adhesion and invasion of Vpma expression variants.

Mycoplasma agalactiae exhibits antigenic variation by switching the expression of multiple surface lipoproteins called Vpmas. Although implicated to have a significant influence on the pathogenicity, their exact role in pathogen-host interactions has not been investigated so far. Initial attachment to host cells is regarded as one of the most important steps for colonization but this pathogen lacks the typical mycoplasma attachment organelle. The aim of this study was to determine the role of Vpmas in adhesion of M. agalactiae to host cells. 'Phase-Locked' Mutants (PLMs) steadily expressing single well-characterized Vpma lipoproteins served as ideal tools to evaluate the role of each of the six Vpmas in cytadhesion, which was otherwise not possible due to the high-frequency switching of Vpmas in the wildtype strain PG2. Using in vitro adhesion assays with HeLa and sheep mammary epithelial (MECs) and stromal (MSCs) cells, we could demonstrate differences in the adhesion capabilities of each of the six PLMs compared to the wildtype strain. The PLMV mutant expressing VpmaV exhibited the highest adhesion rate, whereas PLMU, which expresses VpmaU showed the lowest adhesion values explaining the reduced in vivo fitness of PLMU in sheep during experimental intramammary and conjunctival infections. Furthermore, adhesion inhibition assays using Vpma-specific polyclonal antisera were performed to confirm the role of Vpmas in M. agalactiae cytadhesion. This led to a significant decrease (p<0.05) in the adhesion percentage of each PLM. Immunofluorescence staining of TX-114 phase proteins extracted from each PLM showed binding of the respective Vpma to HeLa cells and MECs proving the direct role of Vpmas in cytadhesion. Furthermore, as adhesion is a prerequisite for cell invasion, the ability of the six PLMs to invade HeLa cells was also evaluated using the gentamicin protection assay. The results showed a strong correlation between the adhesion rates and invasion frequencies of the individual PLMs. This is the first report that describes a novel function of Vpma proteins in cell adhesion and invasion. Besides the variability of these proteins causing surface antigenic variation, the newly identified phenotypes are likely to play critical roles in the pathogenicity potential of this ruminant pathogen.

[Mixed infections and inflammatory ophthalmic diseases: clinical and laboratory observations]. / Mikst-infektsii i vospalitel'naia oftal'mopatologiia: kliniko-laboratornye nabliudeniia.

In recent years, all medical specialists, including ophthalmologists, have been facing the problem of mixed infections. Recurrent inflammation in the anterior and posterior eye segments is often a result of infection by more than one variety of pathogens. MATERIAL AND METHODS: Over the period 2013-2016, 34 patients (14 men and 20 women) with different inflammatory processes in the eye who appeared DNA-positive for mycoplasmas (Mycoplasma hominis, Ureaplasma urealyticum) and/or chlamydiae (Chlamydia trachomatis) (PCR testing of tear fluid and/or urine) were followed up. All patients were examined for intensive production of herpesvirus, adenovirus, and enterovirus DNA in biological fluids. After being consulted by related specialists, all the patients started local and systemic (antibacterial and antiviral) therapy. In the end of the latter, laboratory tests were repeated. RESULTS: Among all the clinical forms, anterior segment inflammation (i.e. of conjunctiva, cornea, and the anterior vascular tract) prevailed - 76%. In most patients, mycoplasmas and/or chlamydiae formed associations with herpesviruses (n=19; 56%). Bacterial DNA alone (mycoplasma and/or chlamydia) was detected in 12 cases (35%). In 4 cases, mycoplasma and/or chlamydia DNA was detected in tear fluid, in 19 patients - in urine, and in 10 patients - in both secreta. Local and systemic causal treatment enabled resolution of the complaints and symptoms and yielded negative results of follow-up laboratory tests. CONCLUSION: More than a half of the patients demonstrated concomitant viral-bacterial infection (22 cases). The presence of bacterial/viral DNA in biological secreta, as revealed by PCR, reflects the systemic nature of the infection process and, thus, necessitates engagement of related specialists (dermatologists, urologists, gynecologists).

Neurological Manifestations of Mycoplasma pneumoniae Infection in Hospitalized Children and Their Long-Term Follow-Up.

Objective In this retrospective study, we aimed to assess frequency, types, and long-term outcome of neurological disease during acute Mycoplasma pneumoniae (M. pneumoniae) infection in pediatric patients. Materials and Methods Medical records of patients hospitalized with acute M. pneumoniae infection were reviewed. Possible risk factors were analyzed by uni- and multivariate regression. Patients with neurological symptoms were followed up by expanded disability status score (EDSS) and the cognitive problems in children and adolescents (KOPKJ) scale. Results Out of 89 patients, 22 suffered from neurological symptoms and signs. Neurological disorders were diagnosed in 11 patients: (meningo-) encephalitis (n = 6), aseptic meningitis (n = 3), transverse myelitis (n = 1), and vestibular neuritis (n = 1), 11 patients had nonspecific neurological symptoms and signs. Multivariate logistic regression identified lower respiratory tract symptoms as a negative predictor (odds ratio [OR] = 0.1, p < 0.001), a preexisting immune deficit was associated with a trend for a decreased risk (OR = 0.12, p = 0.058). Long-term follow-up after a median of 5.1 years (range, 0.6-13 years) showed ongoing neurological deficits in the EDSS in 8/18, and in the KOPKJ in 7/17. Conclusion Neurological symptoms occurred in 25% of hospitalized pediatric patients with M. pneumoniae infection. Outcome was often favorable, but significant sequels were reported by 45%.

Interactive host cells related to Mycoplasma suis α-enolase by yeast two-hybrid analysis.

Mycoplasma suis belongs to the haemotrophic mycoplasmas, which colonise the red blood cells of a wide range of vertebrates. Adhesion to red blood cells is the crucial step in the unique lifecycle of M. suis. In addition to MSG1 protein, α-enolase is the second adhesion protein of M. suis, and may be involved in the adhesion of M. suis to porcine red blood cells (RBC). To simulate the environment of the RBC, we established the cDNA library of swine peripheral blood mononuclear cells (PBMC). The yeast two-hybrid (Y2H) system was adopted to screen α-enolase interactive proteins in the PBMC line. Alignment with the NCBI database revealed four interactive proteins: beta-actin, 60S ribosomal protein L11, clusterin precursor and endonuclease/reverse transcriptase. However, the M. suis α-enolase interactive proteins in the PBMC cDNA library obtained in the current study provide valuable information about the host cell interactions of the M. suis α-enolase protein.

Synergism between upregulation of Rab7 and inhibition of autophagic degradation caused by mycoplasma facilitates intracellular mycoplasma infection.

Following fusion of a mycoplasma with a host cell membrane, the inserted components of mycoplasma may then be transported through the endocytic pathway. However, the effects of mycoplasmas on the host cell endomembrane system are largely unknown. In this study, mycoplasma­induced changes in the dynamics of endocytic and autophagic systems were investigated. Endocytosis and autophagy are two major processes involved in the survival of intracellular prokaryotic pathogens. It was found that, immediately following infection, mycoplasmas induce endocytosis in the host cell; however, in the long term the mycoplasmas suppress turnover of the components of the endocytic pathway. Immunofluorescence microscopy revealed that Rab7 and LC3­II are recruited to the intracellular mycoplasma­containing compartments. Western blot analysis and quantitative reverse transcription-polymerase chain reaction (qPCR) showed that mycoplasmas increase expression of Rab7 by upregulating transcription, but increase levels of LC3­II and p62 by post­translational regulation. Furthermore, it was demonstrated that mycoplasma infection causes inhibition of autophagic degradation of LC3­II and p62. In addition, it was found that upregulation of Rab7 and inhibition of autophagic degradation synergistically contributes to intracellular mycoplasma accumulation. In conclusion, these findings suggest that mycoplasmas may manipulate host cell endosomal and autophagic systems in order to facilitate intracellular infection.

Molecular investigation of menstrual tissue for the presence of Chlamydia trachomatis, Ureaplasma urealyticum and Mycoplasma hominis collected by women with a history of infertility.

AIM: At present, routine laboratory investigation of the infectious agents implicated in female genital infections is mainly based on culture/direct fluorescence antibody (DFA) (immunofluorescence antibody test) results of cervicovaginal secretions. In this study the use of the menstrual tissue is introduced for the molecular detection of pathogens which are implicated in female infertility. MATERIAL AND METHODS: Cervicovaginal secretions and menstrual tissue samples of 87 women (mean age 34.07 ± 5.17) experiencing infertility problems were screened for Chlamydia trachomatis, Ureaplasma urealyticum and Mycoplasma hominis presence using polymerase chain reaction (PCR, light cycler-PCR). Cervicovaginal secretions were also tested by the culture/DFA technique. The results were compared using the binomial test. RESULTS: In the overall study group, the prevalence of C. trachomatis was 25.3%, 18.3%, and 13.8%, the prevalence of U. urealyticum was 18.3%, 16.09% and 12.6% and the prevalence of M. hominis was 13.7%, 19.5% and 8.0% in the menstrual tissue, cervicovaginal secretions using PCR and cervicovaginal secretions culture/DFA, respectively. A statistically significant difference was revealed between the two methods for all three microbes and between menstrual tissue and cervicovaginal secretions PCR for chlamydia. CONCLUSIONS: The use of menstrual tissue along with the PCR method seems to be an effective and thus novel alternative for the investigation of the infectious agents lying in the genital tract. One of the main advantages of this technique compared to cervicovaginal secretions is that it is non-invasive and the sample can be collected at home, thus allowing the early detection and treatment of a condition that can otherwise lead to serious consequences, such as tubal obstruction, pelvic inflammatory disease, ectopic pregnancy, spontaneous abortions and unexplained infertility.

Detección de infección por Mycoplasma en las gestantes con riesgo de parto pretérmino / Detection of Mycoplasma infection in pregnants with a risk of preterm delivery

Introducción: el parto pretérmino es el que se produce antes de las 37 sem de gestación; la sepsis vaginal constituye uno de los factores de riesgo predisponentes para este, de ahí que continúe siendo motivo de preocupación para obstetras y neonatólogos. Objetivo: evaluar el uso del Test Mycoplasma System Plus en gestaciones con riesgo de parto pretérmino, así como algunos de sus aspectos clínicos. Métodos: se realizó un estudio retrospectivo descriptivo de enero a septiembre de 2010 en el Hospital Profesor Eusebio Hernández, la muestra estuvo constituida por 88 pacientes ingresadas en el servicio de cuidados especiales perinatales con gestaciones menores de 34 sem a las que se les realizó el test y tuvieron el parto en este centro, los datos fueron recogidos de las historias clínicas e informes de microbiología agrupados en un formulario y procesados mediante estadísticas descriptivas y de distribución de frecuencia. Resultados: el 67 porciento, de las pacientes presentaban infección moderada o severa a ureaplasma mientras que los exudados vaginales simples fueron negativos en un 71,5 porciento, el antimicrobiano más utilizado fue la eritromicina. Conclusiones: la mayoría de las pacientes después del tratamiento adecuado llegaron al término de la gestación, así como presentaban exudados vaginales simple negativos, pero con un alto índice de infección a ureaplasma urealyticum

Introduction: the pre-term labor is produced before the 37 weeks of pregnancy; the vaginal sepsis is one of the predisposing risk factors to it, being a motive of worry for obstetricians and neonatologists. Objective: to assess the use of Test Mycoplasma System Plus in pregnancies with risk of pre-term labor, as well as of its clinical features. Methods: a descriptive and retrospective study was conducted from January to September, 2010 in the Profesor Eusebio Hernández Hospital; sample included 88 patients admitted in the perinatal special care unit with pregnancies under 34 weeks and performing of test who gave birth in this institution; data were collected from the medical records and microbiology reports grouped in a form and processed by descriptive statistics and of frequency distribution. Results: the 6l7 percent of patients had a moderate or severe infection due to ureaplasma where as the vaginal exudates were negatives in the 71.5 percent, the more used antimicrobial agents was the erythromycin. Conclusions: most of patients after an appropriate treatment arrive to a term pregnancy and also had negative simple vaginal exudate but with a high rate of infection due to ureaplasma urelyticum

alpha5beta1 Integrin blockade inhibits lymphangiogenesis in airway inflammation.

The integrin alpha5beta1 has been previously implicated in tumor angiogenesis, but its role in the remodeling of both blood vessels and lymphatics during inflammation is at an early stage of understanding. We examined this issue using a selective, small-molecule inhibitor of alpha5beta1 integrin, 2-aroylamino-3-{4-[(pyridin-2-ylaminomethyl)heterocyclyl]phenyl}propionic acid (JSM8757), in a model of sustained airway inflammation in mice with Mycoplasma pulmonis infection, which is known to be accompanied by robust blood vessel remodeling and lymphangiogenesis. The inhibitor significantly decreased the proliferation of lymphatic endothelial cells in culture and the number of lymphatic sprouts and new lymphatics in airways of mice infected for 2 weeks but did not reduce remodeling of blood vessels in the same airways. In inflamed airways, alpha5 integrin immunoreactivity was present on lymphatic sprouts, but not on collecting lymphatics or blood vessels, and was not found on any lymphatics of normal airways. Macrophages, potential targets of the inhibitor, did not have alpha5 integrin immunoreactivity in inflamed airways. In addition, macrophage recruitment, assessed in infected airways by quantitative reverse transcription-polymerase chain reaction measurements of expression of the marker protein ionized calcium-binding adapter molecule 1 (Iba1), was not reduced by JSM8757. We conclude that inhibition of the alpha5beta1 integrin reduces lymphangiogenesis in inflamed airways after M. pulmonis infection because expression of the integrin is selectively increased on lymphatic sprouts and plays an essential role in lymphatic growth.

Mycoplasma pneumoniae--associated transverse myelitis and rhabdomyolysis.

Mycoplasma pneumoniae is a common cause of respiratory tract infection. Extrapulmonary manifestations of M. pneumoniae infection are also common. The present case is that of a previously healthy 4-year-old boy who displayed a novel simultaneous onset of both acute rhabdomyolysis and transverse myelitis associated with an infection of M. pneumoniae. He had no preceding symptoms or signs of respiratory tract infection. Intravenous immunoglobulin (1 g/kg per day) for 2 days was prescribed initially for the deterioration of neurologic condition. His rhabdomyolysis resolved without complication, but neurologic sequelae remained during 2 years of follow-up. Evaluation for M. pneumoniae infection is recommended in patients with idiopathic rhabdomyolysis and transverse myelitis, even if in the absence of antecedent respiratory symptoms.

Micoplasma en patología pulmonar pediátrica / Micoplasma in pediatric pulmonology patology

Mycoplasma pneumoniae es un germen patógeno frecuente del tracto respiratorio humano, especialmente en niños y adultos jóvenes. El desarrollo en los últimos años de nuevos métodos diagnósticos como el de reacción en cadena de la polimerasa (PCR), unido a métodos diagnósticos tradicionales, ha permitido ahondar en las características de la enfermedad por M. pneumoniae en pediatría.

Interaction between cigarette smoke and mycoplasma infection: a murine model.

Cigarette smoke has a major impact on health issues worldwide. Although genetics certainly is a factor in the sensitivity to cigarette smoke, other lung environmental factors, such as infection, potentially could interact with cigarette smoke to induce inflammatory changes associated with various diseases. Four groups of BALB/c mice (smoking only; smoking + M. pneumoniae infection; mycoplasma only; saline control) were studied for eight weeks to determine the interactive outcomes of inflammation and structural changes in the smoking plus mycoplasma group. This group did have significantly higher amounts of neutrophil degranulation in the outer airway wall area (smooth muscle to alveolar attachments) (p = 0.03) and mRNA expression of matrix metalloproteinase-9 (p= 0.045). Although there was not a significant difference in alveolar tissue elastin between the groups, the smoking plus mycoplasma group had a level approximately 20% below the other groups. Even in this relatively short duration study, it appears that an infectious process can interact with cigarette smoke to produce a destructive type of inflammatory response (activated neutrophils and metalloproteinase-9) seen in the outer airway wall area.

Central nervous system manifestations of Mycoplasma pneumoniae infections.

Mycoplasma pneumoniae infection is associated with several manifestations from the central nervous system (CNS) such as encephalitis, aseptic meningitis, acute transverse myelitis, stroke, and polyradiculopathy. In the current paper epidemiologic, clinical, laboratory and treatment data on these manifestations are reviewed. The M. pneumoniae induced immune dysregulation and its contributing role in the pathogenesis of neurological insult is discussed. The recent introduction in clinical practice of newer molecular diagnostic techniques has helped in establishing a firmer association between M. pneumoniae infection and CNS disease especially encephalitis. Clinicians should be aware of the potential association between M. pneumoniae infection and several CNS manifestations. The role of various anti-microbial or immunomodulating therapies in treating such manifestations should be further explored.

Identification of antigens by monoclonal antibody PD4 and its expression in Escherichia coli.

AIM: To clone and express the antigen of monoclonal antibody (MAb) PD4 for further investigation of its function. METHODS: MGC803 cDNA expression library was constructed and screened with PD4 as probes to clone the antigen. After failed in the library screening, immunoprecipitation and SDS-polyacrylamide gel electrophoresis were applied to purify the antigen for sequence analysis. The antigen coming from Mycoplasma hyorhinis (M. hyorhinis) was further confirmed with Western blot analysis by infecting M. hyorhinis -free HeLa cells and eliminating the M. hyorhinis from MGC803 cells. The full p37 gene was cloned by PCR and expressed successfully in Escherichia coli after site-directed mutations. Immunofluorescence assay was used to demonstrate if p37 protein could directly bind to gastric tumor cell AGS. RESULTS: The cDNA library constructed with MGC803 cells was screened by MAb PD4 as probes. Unfortunately, the positive clones identified with MAb PD4 were also reacted with unrelated antibodies. Then, immunoprecipitation was performed and the purified antigen was identified to be a membrane protein of Mycoplasma hyorhinis (M. hyorhinis) by sequencing of N-terminal amino acid residues. The membrane protein was intensively verified with Western blot by eliminating M. hyorhinis from MGC803 cells and by infecting M. hyorhinis-free HeLa cells. The full p37 gene was cloned and expressed successfully in Escherichia coli after site-directed mutations. Immunofluorescence demonstrated that p37 protein could directly bind to gastric tumor cell AGS. CONCLUSION: The antigen recognized by MAb PD4 is from M. hyorhinis, which suggests the actions involved in MAb PD4 is possibly mediated by p37 protein or M. hyorhinis. As p37 protein can bind directly to tumor cells, the pathogenic role of p37 involved in tumorigenesis justifies further investigation.