Role of ST6GAL1 and ST6GAL2 in subversion of cellular signaling during enteroaggregative Escherichia coli infection of human intestinal epithelial cell lines.

Emerging evidence have suggested that aberrant sialylation on cell-surface carbohydrate architecture may influence host-pathogen interactions. The α2,6-sialyltransferase (ST) enzymes were found to alter the glycosylation pattern of the pathogen-infected host cell-surface proteins, which could facilitate its invasion. In this study, we assessed the role of specific α2,6-ST enzymes in the regulation of enteroaggregative E. coli (EAEC)-induced cell signaling pathways in human intestinal epithelial cells. EAEC-induced expression of α2,6-ST family genes in HCT-15 and INT-407 cell lines was assessed at mRNA level by qRT-PCR. Specific esi-RNA was used to silence the target ST-gene in each of the EAEC-infected cell type. Subsequently, the role of these enzymes in regulation of EAEC-induced cell signaling pathways was unraveled by analyzing the expression of MAPkinases (ERK1/2, p38, JNK) and transcription factors (NFκB, cJun, cFos, STAT) at mRNA and protein levels by qRT-PCR and western immunoblotting, respectively, expression of selected sialoglycoproteins by western immunoblotting along with the secretory IL-8 response using sandwich ELISA. ST6GAL-1 and ST6GAL-2 were efficiently silenced in EAEC-infected HCT-15 and INT-407 cells, respectively. Significant reduction in EAEC-induced activation of MAPKs, transcription factors, sialoglycoproteins, and IL-8 secretion was noted in ST-silenced cells in comparison to the respective control cells. We propose that ST6GAL-1 and ST6GAL-2 are quintessential for EAEC-induced stimulation of MAPK-mediated pathways, resulting in activation of transcription factors, leading to an inflammatory response in the human intestinal epithelial cells. Our study may be helpful to design better therapeutic strategies to control EAEC- infection. KEY POINTS: • EAEC induces α2,6-sialyltransferase (ST) upregulation in intestinal epithelial cells • Target STs (ST6GAL-1 & ST6GAL-2) were efficiently silenced using specific esiRNAs • Expression of MAPKs, transcription factors & IL-8 was reduced in ST silenced cells.

Heat shock protein 60 is a disease-associated sialoglycoprotein in human non-small cell lung cancer.

The diagnostic and therapeutic potential of Maackia amurensis agglutinin (MAA) have been reported in various malignancies. Earlier, we have found that MAA specifically interacted with human non-small cell lung-cancer (NSCLC) cells and induced apoptosis in these cells. The present study was designed to identify M. amurensis leukoagglutinin (MAL-I, one of the components of MAA, having the same carbohydrate specificity as MAA) interacting membrane sialoglycoprotein(s) of two subtypes of human NSCLC cell lines. Nine proteins were identified using two-dimensional (2D)-polyacrylamide gel electrophoresis (PAGE) followed by MAL-I-overlay transblotting and matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). Among these proteins, HSP60 was selected for further characterization. The sialoglycoprotein nature of membrane-HSP60 of NSCLC cell lines was confirmed by its reduced reactivity with MAL-I in Western blots in the presence of GM2 and by dual staining of the cell lines with MAL-I and HSP60-antibody. These findings were further substantiated by enzymatic analysis of membrane-HSP60 as well as in-silico evidence regarding this protein. Our observations were validated by immunohistochemical analysis of both subtypes of NSCLC tissue sections. Membrane-HSP60 was found to be involved in the inhibition of MAL-I-induced morphological alteration of NSCLC cells and also in the proliferation and migration of these cells, indicating the probable role of sialylated membrane-HSP60 in this disease.

Maackia amurensis agglutinin induces apoptosis in cultured drug resistant human non-small cell lung cancer cells.

The emergence of multi drug resistance in non-small cell lung cancer (NSCLC) patients is a major challenge towards the efficacy of chemotherapy. Thus, there is an urgent need for the newer, better clinically targeted strategies to treat this disease. Earlier studies from our laboratory revealed the apoptotic activity of Maackia amurensis agglutinin (MAA) in human NSCLC cells. In this study, the effect of MAA on drug resistant NSCLC cells was investigated. Two Paclitaxel-resistant NSCLC sub-lines (A549/PTX100 and NCI-H460/PTX100) were developed from A549 & NCI-H460 cell lines respectively. The generation of drug resistance phenotype was confirmed by the expression of cell surface MDR-1. Both the drug resistant sub-lines showed distinct morphological alterations. MAA interacted with the cell-surface protein(s) of apparent Mr ~66 kDa and induced apoptosis in both the sub-lines through intrinsic/mitochondrial pathway, involving reduction in mitochondrial trans-membrane potential, up-regulation of Bax, unaltered/decreased expression of Bcl-XL, release of mitochondrial cytochrome c into the cytosol and activation of pro-caspases (-9&-3). Our findings highlighted the potential of this plant agglutinin to serve as an apoptosis inducing agent in drug resistant NSCLC cells.

A cytosol derived factor of Group B streptococcus prevent its invasion into human epithelial cells.

Group B streptococcus (GBS) or Streptococcus agalactiae, is an opportunistic pathogen causing a wide range of infections like pneumonia, sepsis, and meningitis in newborn, pregnant women and adults. While this bacterium has adapted well to asymptomatic colonization of adult humans, it still remains a potentially devastating pathogen to susceptible infants. Advances in molecular techniques and refinement of in vitro and in vivo model systems have elucidated key elements of the pathogenic process, from initial attachment to the maternal vaginal epithelium to penetration of the newborn blood-brain barrier. Still, the formidable array of GBS virulence factors makes this bacterium at the forefront of neonatal pathogens. The involvement of bacterial components in the host-pathogen interaction of GBS pathogenesis and its related diseases is not clearly understood. In this study we demonstrated the role of a 39 kDa factor from GBS which plays an important role in the process of its invasion. We found a homogeneous 39 kDa factor from the cytosol of GBS after following a combination of sequential purification steps involving molecular sieving and ion exchange chromatography using ACTA-FPLC system. Its N-terminal sequence showed a homology with xenobiotic response element type transcriptional regulator protein, a 40 kDa protein of Streptococcus. This factor leads to inhibition of GBS invasion in HeLa and A549 cells. This protein also showed sensitivity and specific cross reactivity with the antibodies raised against it in New Zealand white rabbits by western immunoblotting. This inhibitory factor was further confirmed tolerant for its cytotoxicity. These results add a novel aspect to bacterial pathogenesis where bacteria's own intracellular protein component can act as a potential therapeutic candidate by decreasing the severity of disease thus promoting its invasion inhibition.

In vitro study of virulence potential of Acinetobacter baumannii outer membrane vesicles.

Acinetobacter baumannii has emerged as an important opportunistic pathogen mostly causing nosocomial infections. The virulence factors of this important pathogen are largely unknown. Outer membrane vesicles (OMV) are naturally secreted by many gram negative and gram positive bacteria. These vesicles contain outer membrane proteins, lipids, periplasmic proteins, DNA and RNA. Their role in intracellular and intercellular signaling, transfer of virulence factors and eliciting immune response in host cells has been established in many pathogens. In this study, we investigated OMVs from three multi-drug resistant (MDR) clinical isolates and a non-MDR reference strain of A. baumannii for virulence potential. A. baumannii OMVs showed phospholipase C, hemolytic and leukotoxic activities. We found large variations in virulence potential between OMVs of MDR clinical isolates and non-MDR reference strain. These effector molecules were concentrated in OMVs than whole cell bacterial culture and cell-free supernatant. OMV-mediated phospholipase, hemolytic and leucotoxic activities may have a key role in pathogenicity of A. baumannii infection and may be future targets for therapeutic and preventive strategies. This is, to the best of our knowledge, the first report showing virulence potential of A. baumannii OMVs.

Efficacy of intraarticular dexamethasone for postoperative analgesia after arthroscopic knee surgery.

BACKGROUND AND AIMS: In an attempt to improve the recovery and early rehabilitation after arthroscopic knee surgery, various medications have been administered via intra-articular route to prolong the duration and improve the quality of postoperative analgesia. Among the potentially effective substances, steroids like dexamethasone could be of particular interest. MATERIALS AND METHODS: Fifty patients undergoing elective knee arthroscopy were randomly assigned to one of the following groups containing 25 patients each. Group D patients received 8 mg (2 mL) of dexamethasone added to 18 mL of 0.25% levobupivacaine intra-articularly, (total volume 20 mL). Group L patients received 18 mL of 0.25% levobupivacaine and 2 mL of isotonic saline (20 mL in total) intra-articularly. Analgesic effect was evaluated by measuring pain intensity visual analogue scale score and duration of analgesia. RESULTS: A longer delay was observed between intra-articular injection of study medication and first requirement of supplementary analgesic in Group D (10.24 ± 2.8 hours) compared with Group L (5.48 ± 1.6 h). Total consumption of diclofenac sodium in first 24 h in postoperative period was significantly less in Group D. No significant side effects were noted. CONCLUSION: Dexamethasone, used as adjunct to levobupivacaine in patients undergoing arthroscopic knee surgery, improves the quality and prolongs the duration of postoperative analgesia.

Overexpression of microRNA-122 enhances in vitro hepatic differentiation of fetal liver-derived stem/progenitor cells.

MicroRNAs (miRNAs) are a versatile class of tiny non-coding RNAs involved in regulation of various biological processes. miRNA-122 (miR-122) is specifically and abundantly expressed in human liver. However, the role of miR-122 in differentiation of fetal liver stem/progenitor cells into hepatocytes remains unclear. In this study, dual positive CD34+/CD117+ expressing human fetal liver stem/progenitor cells was enriched by magnetic cell sorting and cultured in vitro. The level of miR-122 was found to be increased at specific time intervals. Interestingly, during the differentiation process of hepatocyte-like cells, the increase in expression of miR-122 was positively correlated with expression of hepatocyte-specific genes. The status of differentiation process was improved by transfection of miR-122 into enriched stem/progenitor cells. The expression level of hepatic-specific genes as well as liver-enriched transcription factors (LETFs) was significantly increased by overexpression of miR-122 in fetal liver stem/progenitor cells. Thus, the study delineated the role of hepato-specific miR-122 in differentiation of fetal liver stem/progenitor cells into hepatocyte-like cells which could be used as a therapeutic target molecule to generate abundant hepatocytes.

Potential importance of Maackia amurensis agglutinin in non-small cell lung cancer.

Maackia amurensis agglutinin is a NeuNAcα (2-3) Galß (1-4) GlcNAc/Glc-specific lectin, which was shown to have diagnostic potential in cancers of different origin. In a previous report, we demonstrated that GM3 specific IgG from bronchoalveolar lavage fluid (BALF) of non-small cell lung cancer (NSCLC) patients interacted with ∼66kDa membrane glycoprotein band of NSCLC cell lines, which was also recognised by this lectin. This observation prompted us to assess the potential of Maackia amurensis agglutinin in NSCLC. Accordingly, we examined the reactivity of this lectin with NSCLC cell lines as well as the tissue biopsies and cells obtained from fine needle aspirations of NSCLC patients. Maackia amurensis agglutinin showed strong reactivity, specifically with cells and biopsy samples of NSCLC origin. Furthermore, this lectin was found to induce apoptosis in NSCLC cells. The mechanism of this lectin-induced apoptosis involved downregulation of Bcl-XL, upregulation of Bax, release of cytochrome c and activation of procaspase-3. Collectively our results have suggested that Maackia amurensis agglutinin may have the potential to serve as a unique probe for detection of NSCLC and also as a specific apoptosis-inducing agent in NSCLC cells.

Role of TLRs in EGFR-mediated IL-8 secretion by enteroaggregative Escherichia coli-infected cultured human intestinal epithelial cells.

Enteroaggregative Escherichia coli (EAEC) is an emerging enteric pathogen associated with persistent diarrhea in travelers, immunocompromised patients and children worldwide. However, the pathogenesis of this organism is yet to be established. In this study, the role of Toll-like receptors (TLRs) was evaluated in epidermal growth factor receptor (EGFR)-mediated IL-8 secretion by EAEC-infected human small intestinal and colonic epithelial cells (INT-407 and HCT-15, respectively). We observed that EAEC-induced upregulation of TLR2, TLR4 and TLR5 transcripts in both types of cells, and the maximum level of these transcripts was seen in cells infected with EAEC-T8 (an invasive clinical isolate). All these TLRs made a significant contribution to the EAEC-T8-mediated EGFR activation in these cells. Furthermore, these TLRs were found to be associated with activation of the downstream effectors (ERK-1/2, PI3 kinase and Akt) and transcription factors (NF-κB, c-Jun, c-Fos and STAT-3) of EGFR-mediated signal transduction pathways. Moreover, the involvement of these TLRs was also noted in IL-8 secretion by both EAEC-T8-infected cell types. Our findings suggest that EAEC-induced upregulation of TLR2, TLR4 and TLR5 is important for the IL-8 response via EGFR-mediated signal transduction pathways in these cells.

Enteroaggregative Escherichia coli induced activation of epidermal growth factor receptor contributes to IL-8 secretion by cultured human intestinal epithelial cells.

Enteroaggregative Escherichia coli (EAEC) has been identified as a new enteropathogen that causes acute and chronic diarrhea in children and travelers. One defining aspect of EAEC-pathogenesis is the induction of an inflammatory response in intestinal epithelium. In this study, we have found that EAEC-induced EGFR activation in human small intestinal and colonic epithelial was attenuated in the presence of a specific inhibitor of EGFR (Tyrphostin AG1478). Further, the aggregative stacked-brick type of adherence of this organism to both the cell lines and this pathogen-induced cytoskeletal rearrangement of these cells was also reduced in the presence of Tyrphostin AG1478. Moreover, EAEC-induced activation of downstream effectors (ERK-1/2, PI3K and Akt) of EGFR mediated cell signaling pathways were found to be suppressed in the presence of EGFR inhibitor. A decrease in IL-8 response in EAEC infected both the cell types were also noted in the presence of specific inhibitors of these downstream effectors, transcription factors and Tyrphostin AG1478. We propose that EAEC-induced activation of EGFR is quintessential for stacked-brick adherence of EAEC to human intestinal epithelial cells, their cytoskeletal rearrangements and stimulation of ERK-1/2 and PI3K/Akt mediated signal transduction pathways, resulting in the activation of NF-κB, AP-1, STAT-3 and finally IL-8 secretion by these cells.

Molecular mechanism of Enteroaggregative Escherichia coli induced apoptosis in cultured human intestinal epithelial cells.

Background. Enteroaggregative Escherichia coli (EAEC) is an evolving etiological agent of acute and persistent diarrhoea worldwide. The previous study from our laboratory has reported the apoptosis-inducing activity of EAEC in human small intestinal and colonic epithelial cell lines. In the present investigation, we have explored the underlying mechanism of EAEC-induced apoptosis in human intestinal epithelial cell lines.Methods. INT-407 and HCT-15 cells were infected with EAEC-T8 and EAEC-pT8 (plasmid cured strain of EAEC-T8) separately. Cells cultured in the absence of bacteria served as a negative control in all the experiments. For the subsequent experiments, the molecular mechanism(s) of epithelial cell aposptosis was measured in EAEC infecting both the cell lines by flow cytometry, real-time PCR and Western blotting.Results and conclusions. EAEC was found to activate the intrinsic/mitochondrial apoptotic pathway in both the cell lines through upregulation of pro-apoptotic Bax and Bak, un-alteration/reduction in the level of anti-apoptotic Bcl-2 and Bcl-XL, decrease in mitochondrial transmembrane potential, accumulation of cytosolic cytochrome c leading to activation of procaspase-9 and procaspase-3, which ultimately resulted in DNA fragmentation and apoptosis. Further, an increased expression of Fas, activation of procaspase-8 and upregulation of pro-apoptotic Bid in the EAEC-infected cells indicated the involvement of extrinsic apoptotic pathway too in this process. Our finding has undoubtedly led to an increased understanding of EAEC pathogenesis, which may be helpful to develop an improved strategy to combat the infection.

Enteroaggregative Escherichia coli induced increase in intracellular calcium concentration modulates cytoskeletal F-actin rearrangement and bacterial entry in INT-407 cells.

BACKGROUND: Enteroaggregative Escherichia coli (EAEC) is an emerging enteric pathogen, associated with cases of acute and persistent diarrhoea worldwide. The pathogenesis of EAEC is yet to be understood. In intestinal epithelium, an increase in [Ca²âº](i) has been attributed due to the action of different enteric pathogens. EAEC was shown to increase [Ca²âº](i) in HEp-2 cells.The present study was undertaken to investigate the effect of EAEC induced increase in [Ca²âº](i) oncultured human intestinal epithelial cells. METHODS: INT-407 cells were infected with EAEC (T8 strain) in the absence and presence of dantrolene (inhibitor of release of Ca²âº from intracellular stores)/verapamil (L-type Ca²âº channel blocker)/BAPTAAM (Ca²âº chelator)/U73122 (PLC inhibitor)/Cytochalasin-D (inhibitor of actin polymerization). [Ca²âº](i) was estimated using Fura-2/AM. Cytoskeletal rearrangement was assessed by F-actin staining using TRITC-phalloidin. The invasiveness of EAEC-T8 to INT-407 cells was checked by electron microscopy and invasion assay. RESULTS: A significant increase in [Ca²âº](i) was observed in EAEC-T8 infected INT-407 cells, which was reduced in presence of dantrolene/verapamil/U73122. EAEC-T8 could induce cytoskeletal F-actin polymerization in INT-407 cells and was found to be invasive in nature. The cytoskeletal rearrangement as well as invasion of EAEC-T8 was attenuated in presence of U73122/dantrolene/BAPTA-AM/verapamil/cytochalasin D. CONCLUSIONS: EAEC induced increase in [Ca²âº](i) seems to play a major role in host cytoskeletal F-actin rearrangements leading to invasion of the organism. GENERAL SIGNIFICANCE: Our study undoubtedly will lead to an improved understanding of EAEC-pathogenesis.

Enteroaggregative Escherichia coli induces altered glycosylation in membrane proteins of cultured human intestinal epithelial cells.

Emerging evidences have suggested that pathogens are capable of manipulating the glycosylation pattern of host-cell glycoconjugates, which may promote their attachment to these cells. Several enteric pathogens are known to induce such altered glycosylation in intestinal epithelium thereby, facilitating the disease process. Enteroaggregative Escherichia coli (EAEC), is one of such pathogens, known to cause acute and persistent diarrhea worldwide. However, glycosylation modulation due to EAEC infection has not been explored so far. In this study, EAEC-induced glycosylation changes in membrane proteins of human small-intestinal and colonic epithelial cell lines were found as revealed by lectin-overlay transblotting using four lectins, among which Sambucus nigra agglutinin (SNA) was selected for subsequent experiments. Several differentially expressed membrane-proteins were detected on SNA-overlay transblots following 2D-PAGE and identified by MALDI-TOF/TOF mass spectrometric analysis. Among these, voltage-dependent anion-selective channel-protein 2 (VDAC2) and prohibitin 2 (PHB2), common to both the cell lines were chosen for further characterization. Reactivity of these proteins to SNA was substantiated by their presence in SNA-agarose affinity chromatography eluted fractions. The plasma membrane localization of VDAC2 and PHB2 in EAEC infected cell lines was validated by confocal microscopy. These proteins were characterized as sialoglycoproteins by SNA-overlay transblots in presence a specific SNA inhibitor i.e., 6'sialyl lactose and deglycosylation using PNGase F, O-glycosidase and neuraminidase. Membrane localization of these sialoglycoproteins was found to facilitate EAEC adherence to human intestinal epithelial cells. SIGNIFICANCE: Our findings regarding EAEC induced altered glycosylation pattern of host cell membrane proteins may help in better understanding of the disease pathogenesis.

Role of a Disease-associated ST3Gal-4 in Non-small Cell Lung Cancer.

Sialylation promotes tumorigenesis by affecting various cancer-related events, including apoptosis inhibition, cell growth, invasion, migration, metastasis, chemo-resistance, and immunomodulation in favor of tumor progression. An altered expression of sialyltransferase enzymes is responsible for synthesizing various tumor-associated sialylated structures. In the present study, our findings have revealed a significant up-regulation of ST3Gal-4 transcript in the two major subtypes of NSCLC cell lines [squamous cell carcinoma cell line (NCI-H520) and adenocarcinoma cell line (A549)]. Thus, the role of the ST3Gal-4 gene was assessed on cancer-associated signal transduction pathways in these cells in view of proliferation, invasion, and migration. ST3Gal-4 was silenced by transfection of both the cell lines with esi-ST3Gal-4-RNA, which RT-PCR and western immunoblotting confirmed. Silencing of ST3Gal-4 resulted in a decreased expression of MAL-I interacting membrane-HSP60, identified earlier as an α2,3-sialylated glycoprotein, thus pointing towards the possible role of ST3Gal-4 in its sialylation. The proliferation, invasion, and migration of both types of NSCLC cells were reduced significantly in the ST3Gal-4 silenced cells. Our findings were substantiated by the down-regulation of ß-catenin and E-cadherin, a reduced expression of activated AKT1, ERK1/2, and NF-ƙB in these cells. We propose that ST3Gal-4 may be the disease-associated sialyltransferase involved in α2,3 sialylation of the membrane proteins, including HSP60 of the NSCLC cells. This may lead to the conformational alteration of these proteins, required for the activation of E-cadherin/ß-catenin, AKT, and ERK/NF-ƙB mediated signal transduction pathways in these cells, resulting in their proliferation, invasion, and migration.

Galactose specific adhesin of enteroaggregative E. coli induces IL-8 secretion via activation of MAPK and STAT-3 in INT-407 cells.

BACKGROUND: Enteroaggregative Escherichia coli (EAEC) is one of the most common bacterial pathogens associated with the etiology of persistent diarrhea. A characteristic feature of EAEC-pathogenesis is the induction of profound inflammatory response in the intestinal epithelium. The present study was designed to investigate the underlying mechanism of inflammatory responses induced by a novel galactose specific adhesin of T7 strain of EAEC (EAEC-T7) in human intestinal epithelial cell line (INT-407). METHODS: INT-407 cells were stimulated with the adhesin in the absence and presence of anti-adhesin (IgG(AD))/d-galactose/H7/staurosporin (inhibitor of PKC)/PD098059 (inhibitor of MEK)/SB203580 (inhibitor of p38-MAPkinase)/AG490 (inhibitor of JAK (-2,-3)/STAT-3 pathway). The expression of activated Raf-1, MEK-1, ERK1/2, JNK, p38-MAPK and STAT-3 was analyzed by Western immunoblot. Release of interleukin-8 (IL-8) was measured by ELISA. RESULTS: The adhesin was found to induce activation of Raf-1, MEK-1, ERK1/2, p38-MAPK and STAT-3, which was reduced in the presence of IgG(AD)/d-galactose. The activation of Raf-1 was found to be attenuated in the presence of H7/staurosporin. The expression of phosphorylated STAT-3 was downregulated in the presence of AG490 and PD098059. Further, the adhesin induced IL-8 secretion was reduced in the presence of the inhibitors of MEK (PD098059), p38-MAPK (SB203580) and JAK (-2,-3)/STAT-3 pathway (AG490). CONCLUSIONS: We propose that STAT-3 activation is quintessential for the galactose specific adhesin induced IL-8 secretion by INT-407 cells and must occur in concert with the activation of ERK1/2. GENERAL SIGNIFICANCE: Our contribution regarding the galactose specific adhesin mediated signaling leads to an improved understanding of the EAEC-pathogenesis and may provide novel therapeutic approaches to combat EAEC infection.

Identification and characterization of non-small cell lung cancer associated sialoglycoproteins.

Aberrantly sialylated cellular glycoconjugates were found to be involved in different processes during tumorigenesis. Such alteration was also noted in case of lung cancer, an important cause of cancer-related death throughout the world. Thus, study on lung cancer associated sialoglycoproteins is of paramount relevance to have a deeper insight into the mechanism of the disease pathogenesis. In the present study, sialic acid specific lectin (Maackia amurensis agglutinin and Sambcus nigra agglutinin)-based affinity chromatography followed by 2D-PAGE and MALDI-TOF/TOF mass spectrometric analysis were done to explore the disease-associated serum proteins of squamous cell carcinoma and adenocarcinoma [the major two subtypes of NSCLC (non-small cell lung carcinoma)] patients. Among seven identified proteins, α1-antitrypsin and haptoglobin-ß were preferred for further studies. These two proteins were characterized as the disease associated serum-sialoglycoproteins of NSCLC-patients by western immunoblotting using each lectin specific inhibitor. The presence of these sialoglycoproteins was found on NSCLC cell lines (NCI-H520 & A549) by confocal microscopy. Both these proteins were also present in tissue samples of NSCLC origin and involved in proliferation, invasion and migration of NSCLC cells. Our findings suggest that α1-antitrypsin and haptoglobin-ß may be the disease-associated sialoglycoproteins in NSCLC, which seem to be involved in disease progression. SIGNIFICANCE: Our contribution regarding the identification of the NSCLC associated sialoglycoproteins may provide a new vision towards the development of clinically useful newer strategies for the treatment of this disease.

Detection of disease specific sialoglycoconjugate specific antibodies in bronchoalveolar lavage fluid of non-small cell lung cancer patients.

In this study, we report the presence of significantly higher level of GM3 specific IgG antibodies (IgG(TL)) in the bronchoalveolar lavage fluid obtained from tumor bearing lung of non-small cell lung cancer (NSCLC) patients as compared to other non-neoplastic controls. The antibodies were isolated using DEAE-cellulose anion exchange chromatography and molecular weight of the subunits of IgG(TL) was confirmed in SDS-PAGE. IgG(TL) revealed high specificity to GM3 and the IgG distribution was confined to IgG1. Furthermore, IgG(TL) showed strong reactivity with NSCLC cell lines as well as the tissue biopsies and cells obtained from fine needle aspirations of NSCLC patients. A 66 kDa membrane glycoprotein of NSCLC cell lines was found to interact specifically with IgG(TL), the intensity of which was drastically reduced in presence of GM3. Further, binding of Maackia amurensis agglutinin [specific for NeuAcalpha(2-->3)Gal unit , the same disaccharide unit also known to be present in GM3] to the 66 kDa band confirmed it to be a sialoglycoprotein in nature. IgG(TL) could not show any reactivity to alkaline borohydrate treated or periodate oxidised membrane fractions, suggesting the probable involvement of the carbohydrate moiety of the 66 kDa glycoprotein in the interaction with IgG(TL). Thus, the 66 kDa sialoglycoprotein seems to be the NSCLC specific sialoglycoconjugate. Taken together, IgG(TL) antibodies may have the potential to serve as a unique probe for detail investigation of NSCLC specific cell surface sialoglycoconjugate. Further, due to high specificity of IgG(TL) to GM3, it may be possible to develop a simple alternative diagnostic approach (GM3-ELISA) for NSCLC.

Enteroaggregative Escherichia coli infection induces IL-8 production via activation of mitogen-activated protein kinases and the transcription factors NF-kappaB and AP-1 in INT-407 cells.

Enteroaggregative Escherichia coli (EAEC) is emerging as a cause of acute and persistent diarrhea in developing countries. An important feature of EAEC pathogenesis is the induction of profound inflammatory response in the intestinal epithelium. In this article, we have shown that EAEC-induced activation of mitogen-activated protein kinases (MAPK) (ERK-1/2, JNK and p38MAPK) in cultured human intestinal epithelial cells (INT-407) leads to the induction of DNA-binding activity of NF-kappaB and AP-1, resulting in IL-8 production. Plasmid-cured EAEC could also activate the MAPK and the transcription factors leading to IL-8 secretion, but to a lesser extent than that of wild-type EAEC. Further, pretreatment of these cells with the highly specific MEK inhibitor (PD 098059), the JNK inhibitor (SP 600125), and the p38MAPK inhibitor (SB 203580) resulted in inhibition of the IL-8 secretion by EAEC (wild type as well as plasmid cured)-infected INT-407 cells. These findings demonstrate that the inflammatory response induced by EAEC may be due to the specific stimulation of MAPK signaling pathways leading to nuclear responses. To our knowledge, this is the first article regarding the detailed mechanism of IL-8 secretion from the EAEC-infected human intestinal epithelial cell line.

Normal and reconstituted high-density lipoprotein protects differentiated monocytes from oxidized low-density lipoprotein-induced apoptosis.

BACKGROUND: The progression of atherosclerosis is an ongoing struggle between cell division and cell death. Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), a novel receptor for oxidized low-density lipoprotein (ox-LDL), mediates ox-LDL-induced apoptosis of monocytes. The anti-atherogenic function of plasma high-density lipoprotein (HDL) includes the ability to inhibit apoptosis of macrophage, although the exact mechanism and consequences of apoptosis in the development and progression of this disease are still controversial. Thus, in the present study, the effect of normal HDL (nHDL) and reconstituted HDL (rHDL) on ox-LDL-induced cellular responses in differentiated monocytes in view of apoptosis and LOX-1 receptor expression was investigated. METHODS: The expression of B-cell lymphoma-2 (Bcl-2), B-cell lymphoma-extra large (Bcl-xL), caspase-3, and cytochrome c (cyt c) was assessed and substantiated in 30 hyper-LDL and control subjects. To assess the expression of LOX-1 on differentiated THP-1 cells, western blotting was carried out, followed by statistical analysis in 30 patients and control subjects. RESULTS: nHDL/rHDL inhibited the ox-LDL-induced apoptosis in the differentiated human monocytic cells, THP-1 cells, and differentiated monocytes of patient and control subjects. Enhanced expression of scavenger receptor, LOX-1, in the differentiated monocytes was also downregulated in presence of nHDL/rHDL. nHDL/rHDL could inhibit the ox-LDL-induced mitochondrial apoptotic pathway and aberrant expression of LOX-1 in patients. Double immunostaining using fluorescein isothiocyanate (FITC)-conjugated ox-LDL and LOX-1 in apoptotic cells indicates its significant role in the differentiated monocytes. CONCLUSION: It was observed that nHDL/rHDL could promote macrophage survival by preserving mitochondrial integrity from ox-LDL-induced apoptosis.