Metabolomics Method in Understanding and Sensitizing Carbapenem-Resistant Acinetobacter baumannii to Meropenem.

Carbapenem-resistant Acinetobacter baumannii (CRAB) strains are prevalent worldwide and represent a major threat to public health. However, treatment options for infections caused by CRAB are very limited as they are resistant to most of the commonly used antibiotics. Consequently, understanding the mechanisms underlying carbapenem resistance and restoring bacterial susceptibility to carbapenems hold immense importance. The present study used gas chromatography-mass spectrometry (GC-MS)-based metabolomics to investigate the metabolic mechanisms of antibiotic resistance in clinically isolated CRAB. Inactivation of the pyruvate cycle and purine metabolism is the most typical characteristic of CRAB. The CRAB exhibited a reduction in the activity of enzymes involved in the pyruvate cycle, proton motive force, and ATP levels. This decline in central carbon metabolism resulted in a decrease in the metabolic flux of the α-ketoglutarate-glutamate-glutamine pathway toward purine metabolism, ultimately leading to a decline in adenine nucleotide interconversion. Exogenous adenosine monophosphate (AMP) and adenosine triphosphate (ATP) enhance the killing efficacy of Meropenem against CRAB. The combination of ATP and Meropenem also has a synergistic effect on eliminating CRAB persisters and the biofilm, as well as protecting mice against peritonitis-sepsis. This study presents a novel therapeutic modality to treat infections caused by CRAB based on the metabolism reprogramming strategy.

Ampicillin-controlled glucose metabolism manipulates the transition from tolerance to resistance in bacteria.

The mechanism(s) of how bacteria acquire tolerance and then resistance to antibiotics remains poorly understood. Here, we show that glucose abundance decreases progressively as ampicillin-sensitive strains acquire resistance to ampicillin. The mechanism involves that ampicillin initiates this event via targeting pts promoter and pyruvate dehydrogenase (PDH) to promote glucose transport and inhibit glycolysis, respectively. Thus, glucose fluxes into pentose phosphate pathway to generate reactive oxygen species (ROS) causing genetic mutations. Meanwhile, PDH activity is gradually restored due to the competitive binding of accumulated pyruvate and ampicillin, which lowers glucose level, and activates cyclic adenosine monophosphate (cAMP)/cAMP receptor protein (CRP) complex. cAMP/CRP negatively regulates glucose transport and ROS but enhances DNA repair, leading to ampicillin resistance. Glucose and Mn2+ delay the acquisition, providing an effective approach to control the resistance. The same effect is also determined in the intracellular pathogen Edwardsiella tarda. Thus, glucose metabolism represents a promising target to stop/delay the transition of tolerance to resistance.

Untargeted serum metabolomics reveals potential biomarkers and metabolic pathways associated with esophageal cancer.

Metabolomics has been reported as an efficient tool to screen biomarkers that are related to esophageal cancer. However, the metabolic biomarkers identifying malignant degrees and therapeutic efficacy are still largely unknown in the disease. Here, GC-MS-based metabolomics was used to understand metabolic alteration in 137 serum specimens from patients with esophageal cancer, which is approximately two- to fivefold as many plasma specimens as the previous reports. The elevated amino acid metabolism is in sharp contrast to the reduced carbohydrate as a characteristic feature of esophageal cancer. Comparative metabolomics showed that most metabolic differences were determined between the early stage (0-II) and the late stage (III and IV) among the 0-IV stages of esophageal cancer and between patients who received treatment and those who did not receive treatment. Glycine, serine, and threonine metabolism and glycine were identified as the potentially overlapped metabolic pathway and metabolite, respectively, in both disease progress and treatment effect. Glycine, fructose, ornithine, and threonine can be a potential array for the evaluation of disease prognosis and therapy in esophageal cancer. These results highlight the means of identifying previously unknown biomarkers related to esophageal cancer by a metabolomics approach.

Succinate and inosine coordinate innate immune response to bacterial infection.

Macrophages restrict bacterial infection partly by stimulating phagocytosis and partly by stimulating release of cytokines and complement components. Here, we treat macrophages with LPS and a bacterial pathogen, and demonstrate that expression of cytokine IL-1ß and bacterial phagocytosis increase to a transient peak 8 to 12 h post-treatment, while expression of complement component 3 (C3) continues to rise for 24 h post-treatment. Metabolomic analysis suggests a correlation between the cellular concentrations of succinate and IL-1ß and of inosine and C3. This may involve a regulatory feedback mechanism, whereby succinate stimulates and inosine inhibits HIF-1α through their competitive interactions with prolyl hydroxylase. Furthermore, increased level of inosine in LPS-stimulated macrophages is linked to accumulation of adenosine monophosphate and that exogenous inosine improves the survival of bacterial pathogen-infected mice and tilapia. The implications of these data suggests potential therapeutic tools to prevent, manage or treat bacterial infections.

Nitrite Promotes ROS Production to Potentiate Cefoperazone-Sulbactam-Mediated Elimination to Lab-Evolved and Clinical-Evolved Pseudomonas aeruginosa.

Cefoperazone-sulbactam (SCF)-resistant Pseudomonas aeruginosa poses a big challenge in the use of SCF to treat infection caused by the pathogen. We have recently shown exogenous nitrite-enabled killing of naturally and artificially evolved Pseudomonas aeruginosa strains (AP-RCLIN-EVO and AP-RLAB-EVO, respectively) by SCF. However, the underlying mechanism is unknown. Here, reprogramming metabolomics was adopted to investigate how nitrite enhanced the SCF-mediated killing efficacy. Nitrite-reprogrammed metabolome displayed an activated pyruvate cycle (P cycle), which was confirmed by elevated activity of pyruvate dehydrogenase (PDH), α-ketoglutarate dehydrogenase, succinate dehydrogenase, and malate dehydrogenase. The activated P cycle provided NADH for the electron transport chain and thereby increased reactive oxygen species (ROS), which potentiated SCF to kill AP-RCLIN-EVO and AP-RLAB-EVO. The nitrite-enabled killing of AP-RCLIN-EVO and AP-RLAB-EVO by SCF was inhibited by PDH inhibitor furfural and ROS scavenger N-Acetyl-L-cysteine but promoted by ROS promoter Fe3+. SCF alone could not induce ROS, but SCF-mediated killing efficacy was enhanced by ROS. In addition, the present study demonstrated that nitrite repressed antioxidants, which were partly responsible for the elevated ROS. These results reveal a nitrite-reprogrammed metabolome mechanism by which AP-RCLIN-EVO and AP-RLAB-EVO sensitivity to SCF is elevated. IMPORTANCE Antibiotic-resistant Pseudomonas aeruginosa has become a real concern in hospital-acquired infections, especially in critically ill and immunocompromised patients. Understanding antibiotic resistance mechanisms and developing novel control measures are highly appreciated. We have recently shown that a reduced nitrite-dependent NO biosynthesis contributes to cefoperazone-sulbactam (SCF) resistance, which is reverted by exogenous nitrite, in both naturally and artificially evolved P. aeruginosa strains (AP-RCLIN-EVO and AP-RLAB-EVO, respectively). However, the mechanism is unknown. The present study reports that the nitrite-enabled killing of AP-RCLIN-EVO and AP-RLAB-EVO by SCF is attributed to the promoted production of reactive oxygen species (ROS). Nitrite activates the pyruvate cycle to generate NADH for the electron transport chain, which in turn promotes ROS generation. Nitrite-potentiated SCF-mediated killing is decreased by pyruvate dehydrogenase inhibitor furfural and ROS scavenger N-Acetyl-L-cysteine but increased by ROS promoter Fe3+. Furthermore, SCF-mediated killing is promoted by H2O2 in a dose-dependent manner. In addition, the combination of nitrite and H2O2 greatly enhances SCF-mediated killing. These results not only disclose a nitrite-ROS-potentiated SCF-mediated killing, but also show SCF-mediated killing is dependent upon ROS.

Inactivation of Nitrite-Dependent Nitric Oxide Biosynthesis Is Responsible for Overlapped Antibiotic Resistance between Naturally and Artificially Evolved Pseudomonas aeruginosa.

Metabolic flexibility of Pseudomonas aeruginosa could lead to new strategies to combat bacterial infection. The present study used gas chromatography-mass spectrometry (GC-MS)-based metabolomics to investigate global metabolism in naturally and artificially evolved strains with cefoperazone-sulbactam (SCF) resistance (AP-RCLIN-EVO and AP-RLAB-EVO, respectively) from the same parent strain (AP-RCLIN). Inactivation of the pyruvate cycle and nitric oxide (NO) biosynthesis was identified as characteristic features of SCF resistance in both evolved strains. Nitrite-dependent NO biosynthesis instead of an arginine-dependent NO pathway is responsible for the reduced NO, which is attributed to lower nitrite and electrons from the oxidation of NADH to NAD+ provided by the pyruvate cycle. Exogenous fumarate, NADH, nitrate, and nitrite promoted the NO level and thereby potentiated SCF-mediated killing. Unexpectedly, fumarate caused the elevation of nitrite, while nitrite/nitrate resulted in the increase of Cyt bc1 complex (providing electrons). These interesting findings indicate that the nitrite-dependent NO biosynthesis and the pyruvate cycle are mutual to promote NO metabolism. In addition, the NO-potentiated sensitivity to SCF was validated by NO donor sodium nitroprusside. These results reveal an endogenous NO-mediated SCF resistance and develop its reversion by metabolites in P. aeruginosa. IMPORTANCE Infections with Pseudomonas aeruginosa have become a real concern among hospital-acquired infections, especially in cystic fibrosis patients and immunocompromised individuals. Control of the pathogen is challenging due to antibiotic resistance. Since bacterial metabolic state impacts sensitivity and resistance to antibiotics, exploring and disclosing bacterial metabolic mechanisms can be used to develop a metabolome-reprogramming approach to elevate bacterial sensitivity to antibiotics. Therefore, GC-MS-based metabolomics is used to explore the similarities and differences of metabolomes between naturally and artificially evolved cefoperazone-sulbactam (SCF)-resistant P. aeruginosa (AP-RCLIN-EVO and AP-RLAB-EVO, respectively) from the same parent strain (AP-RCLIN). It identifies the depressed nitrite-dependent nitric oxide (NO) biosynthesis as the most overlapping characteristic feature between AP-RCLIN-EVO and AP-RLAB-EVO. This is because the pyruvate cycle fluctuates, thereby generating fewer NADH and providing fewer electrons for nitrite-dependent NO biosynthesis than the control. Interestingly, exogenous fumarate, NADH, nitrate, and nitrite as well as NO donor sodium nitroprusside promote NO generation to elevate sensitivity to SCF. These results highlight the way to understand metabolic mechanisms of antibiotic resistance and explore metabolic modulation to combat the bacterial pathogen.

Na+-NQR Confers Aminoglycoside Resistance via the Regulation of l-Alanine Metabolism.

Sodium-translocating NADH:quinone oxidoreductase (Na+-NQR) functions as a unique redox-driven sodium pump, generating membrane potential, which is related to aminoglycoside antibiotic resistance. However, whether it modulates other metabolisms to confer antibiotic resistance is unknown. The present study showed that loss of nqrA or nqrF led to differential metabolomes with elevated resistance to aminoglycoside antibiotics. Decreased alanine, aspartate, and glutamate metabolism and depressed abundance of alanine were characterized as the most impacted pathway and crucial biomarker, respectively. Further data showed that higher viability was detected in ΔnqrA and ΔnqrF mutant strains than their parent strain ATCC 33787 in the presence of gentamicin but recovered by exogenous l-alanine. It proceeds by the following events. The loss of nqrA or nqrF led to the decrease of membrane potential, ATPase activity, and then ATP and cyclic AMP (cAMP), which reduced the cAMP/CRP (cAMP receptor protein) complex. The reduced cAMP/CRP complex promoted l-alanine catabolism and inhibited l-alanine anabolism, causing reduced levels of alanine. Reduced alanine affected the expression of antiporter families Atp and Mnh genes. Our results suggest a novel mechanism by which the Na+-NQR system regulates antibiotic resistance via l-alanine metabolism in a cAMP/CRP complex-dependent manner.IMPORTANCE The Na+-NQR complex functions as a unique redox-driven sodium pump, generating membrane potential directly. However, whether it mediates generation of membrane potential indirectly is unknown. The present study shows that the Na+-NQR complex impacts membrane potential through other antiporter families Atp and Mnh. It proceeds by ATP and then cAMP/CRP regulon, which inhibits l-alanine catabolism and promotes l-alanine anabolism. When the Na+-NQR complex is reduced as in antibiotic-resistant bacteria, l-alanine is depressed, which is related to the antibiotic resistance phenotypes. However, exogenous l-alanine reverts the phenotype and promotes antibiotic-mediated killing. These findings suggest a novel mechanism by which the Na+-NQR system regulates antibiotic resistance via l-alanine metabolism in a cAMP/CRP complex-dependent manner.

The depressed P cycle contributes to the acquisition of ampicillin resistance in Edwardsiella piscicida.

Antibiotic-resistant bacteria are an increasingly serious threat to human health and aquaculture. To further explore bacterial antibiotic resistance mechanism, iTRAQ is used to identify a differential proteome in ampicillin-resistant LTB4 (LTB4-RAMP), a strain of Edwardsiella piscicida. A total of 102 differentially proteins with 50 upregulation and 52 downregulation are identified. Since many of these changes are related to metabolism, interactive pathways explorer(iPath) is used to understand a global differentially metabolic response in LTB4-RAMP. This analysis identifies a global depressed metabolic modulation as the most characteristic feature of LTB4-RAMP. Lower membrane potential and ATP in LTB4-RAMP than control support that the central carbon metabolism and energy metabolism are reduced. Since the pyruvate cycle (the P cycle) plays a key role in the central carbon metabolism and energy metabolism, further investigation focuses on the P cycle and shows that expression of genes and activity of enzymes in the P cycle are decreased in LTB4-RAMP. These results support the conclusion that the depressed P cycle contributes to the acquisition of ampicillin resistance in E.piscicida. These findings indicate that the combination of proteomics and iPath analysis can provide a global metabolic profile, which helps us better understand the correlation between ampicillin resistance and cellular metabolism. SIGNIFICANCE: The present study uses iTRAQ to explore ampicillin resistance mechanism in Edwardsiella piscicida and finds many of these differential abundances of proteins are related to metabolism. IPath further identifies a global depressed metabolic modulation and characterizes the reduced pyruvate cycle as the most characteristic feature of the ampicillin-resistant E. piscicida, which is supported by reduced expression of genes and activity of enzymes in the pyruvate cycle. Consisitently, lower membrane potential and ATP are detetced. These results reveal the metabolic mechanism of ampicillin resistance and provide a solid proof to revert the resistance by reprogramming metabolomics.

Glycine, serine and threonine metabolism confounds efficacy of complement-mediated killing.

Serum resistance is a poorly understood but common trait of some difficult-to-treat pathogenic strains of bacteria. Here, we report that glycine, serine and threonine catabolic pathway is down-regulated in serum-resistant Escherichia coli, whereas exogenous glycine reverts the serum resistance and effectively potentiates serum to eliminate clinically-relevant bacterial pathogens in vitro and in vivo. We find that exogenous glycine increases the formation of membrane attack complex on bacterial membrane through two previously unrecognized regulations: 1) glycine negatively and positively regulates metabolic flux to purine biosynthesis and Krebs cycle, respectively. 2) α-Ketoglutarate inhibits adenosine triphosphate synthase, which in together promote the formation of cAMP/CRP regulon to increase the expression of complement-binding proteins HtrE, NfrA, and YhcD. The results could lead to effective strategies for managing the infection with serum-resistant bacteria, an especially valuable approach for treating individuals with weak acquired immunity but a normal complement system.

Metabolic Mechanism for l-Leucine-Induced Metabolome To Eliminate Streptococcus iniae.

Crucial metabolites that modulate hosts' metabolome to eliminate bacterial pathogens have been documented, but the metabolic mechanisms are largely unknown. The present study explores the metabolic mechanism for l-leucine-induced metabolome to eliminate Streptococcus iniae in tilapia. GC-MS-based metabolomics was used to investigate the tilapia liver metabolic profile in the presence of exogenous l-leucine. Thirty-seven metabolites of differential abundance were determined, and 11 metabolic pathways were enriched. Pattern recognition analysis identified serine and proline as crucial metabolites, which are the two metabolites identified in survived tilapias during S. iniae infection, suggesting that the two metabolites play crucial roles in l-leucine-induced elimination of the pathogen by the host. Exogenous l-serine reduces the mortality of tilapias infected by S. iniae, providing a robust proof supporting the conclusion. Furthermore, exogenous l-serine elevates expression of genes IL-1ß and IL-8 in tilapia spleen, but not TNFα, CXCR4 and Mx, suggesting that the metabolite promotes a phagocytosis role of macrophages, which is consistent with the finding that l-leucine promotes macrophages to kill both Gram-positive and Gram-negative bacterial pathogens. Therefore, the ability of phagocytosis enhanced by exogenous l-leucine is partly attributed to elevation of l-serine. These results demonstrate a metabolic mechanism by which exogenous l-leucine modulates tilapias' metabolome to enhance innate immunity and eliminate pathogens.

GC-MS-Based Metabolome and Metabolite Regulation in Serum-Resistant Streptococcus agalactiae.

Streptococcus agalactiae causes severe systemic infections in human and fish. In the present study, we established a pathogen-plasma interaction model by which we explored how S. agalactiae evaded serum-mediated killing. We found that S. agalactiae grew faster in the presence of yellow grouper plasma than in the absence of the plasma, indicating S. agalactiae evolved a way of evading the fish immune system. To determine the events underlying this phenotype, we applied GC-MS-based metabolomics approaches to identify differential metabolomes between S. agalactiae cultured with and without yellow grouper plasma. Through bioinformatics analysis, decreased malic acid and increased adenosine were identified as the most crucial metabolites that distinguish the two groups. Meanwhile, they presented with decreased TCA cycle and elevated purine metabolism, respectively. Finally, exogenous malic acid and adenosine were used to reprogram the plasma-resistant metabolome, leading to elevated and decreased susceptibility to the plasma, respectively. Therefore, our findings reveal for the first time that S. agalactiae utilizes a metabolic trick to respond to plasma killing as a result of serum resistance, which may be reverted or enhanced by exogenous malic acid and adenosine, respectively, suggesting that the metabolic trick can be regulated by metabolites.

Tumor-associated antigen CAPERα and microvessel density in hepatocellular carcinoma.

PURPOSE: CAPERα, a tumor-associated antigen, was identified from a cDNA clone with autoantibody from a patient with hepatocellular carcinoma (HCC). It has been implicated, by way of alternative splicing of VEGF pre-mRNA, in the regulation of microvessel formation in Ewing's sarcoma. In this study, we looked for possible association of alterations in CAPERα with microvessel density in HCC. METHODS: Enzyme-linked immunosorbent assay using recombinant CAPERα as antigen were used to detect antibody against CAPERα. Immunohistochemistry (IHC) on liver sections was performed to analyze expression profiles of CAPERα, VEGF and CD34 in HCC and control tissues and was further used to assess the correlation of expression among CAPERα, VEGF and CD34 in HCC development. RESULTS: Autoantibody to CAPERα was highest in HCC (22/76, 28.9%), not detected in prostate cancer (0/79) and at 3.4% (3/88) in breast cancer. In immunohistochemical analysis of grades II and III HCC tissues, significantly decreased immunostaining for CAPERα was observed and this correlated directly with decreased immunostaining for VEGF (R=0.534, P=0.0003). Using CD34 immunostaining for detecting newly formed microvessels, strong staining was observed in grades II and III HCC. Normal liver sections, all of which have high expression of CAPERα were totally negative for CD34 immunostaining. A significant inverse correlation was seen between CAPERα and CD34 immunostaining (R=-0.481, P=0.0012). CONCLUSIONS: Decreased expression of CAPERα appears to be correlated with appearance of microvessels. It would be of interest to elucidate the cause of altered CAPERα since new formation of microvessels is important in progression of HCC.

Myo-inositol improves the host's ability to eliminate balofloxacin-resistant Escherichia coli.

Antibiotic-resistant mechanisms are associated with fitness costs. However, why antibiotic-resistant bacteria usually show increasing adaptation to hosts is largely unknown, especially from the host's perspective. The present study reveals the host's varied response to balofloxacin-resistant Escherichia coli (BLFX-R) using an integrated proteome and metabolome approach and identifies myo-inositol and phagocytosis-related proteins as crucial biomarkers. Originally, macrophages have an optimal attractive preference to BLFX-S due to more polarization of BLFX-S than BLFX-R, which renders faster elimination to BLFX-S than BLFX-R. The slower elimination to BLFX-R may be reversed by exogenous myo-inositol. Primarily, myo-inositol depolarizes macrophages, elevating adherence to both BLFX-S and BLFX-R. Since the altered adherence is equal to both strains, the myo-inositol-treated macrophages are free of the barrier to BLFX-R and thereby promote phagocytosis of BLFX-R. This work provides a novel strategy based on metabolic modulation for eliminating antibiotic-resistant bacteria with a high degree of host adaptation.

Identification of vaccine candidates from differentially expressed outer membrane proteins of Vibrio alginolyticus in response to NaCl and iron limitation.

Vibrio alginolyticus is the etiological agent that causes great losses in aquacultures and clinical emanating cases in humans. Identification of highly efficient vaccine candidates to control V. alginolyticus infection has been highly concerned since vaccines offer a powerful approach to provide efficient protection from bacterial infections. In the present study, we firstly investigated the altered outer membrane proteins (OM proteins) of V. alginolyticus in response to NaCl concentrations and iron limitation using Western blotting, and then identified the protective activity of these altered OM proteins by bacterial challenge post immunization. Ten OM proteins were differentially expressed in response to the osmolarity changing or/and iron limitation, in which VA2212, OmpV, VPA1186, OmpU, VPA1644, VA1061, VA1631 and VPA0860 were markedly altered in response to osmolarity, and VPA1186, OmpU, OmpV, VA0449, VPA0860, VPA1435 and VA1631 were determined to be iron-limited responsive proteins. Out of the ten OM proteins, VA1061, OmpU, VPA1435 and VPA0860 could be effective vaccine candidates against infection by V. alginolyticus in vivo. Further results indicated that VA1061 and VPA0860 were dominant antigens and could stimulate hosts to produce stronger antibody response than other two in live or inactivated whole-cell vaccines. These results not only expand knowledge on osmolarity-, iron-responsive proteins, but also provide a valuable strategy for identify protective proteins suitable for use in vaccine development.

Autoantibodies to tumor-associated antigens combined with abnormal alpha-fetoprotein enhance immunodiagnosis of hepatocellular carcinoma.

The identification and characterization of tumor-associated antigens (TAAs) and their use in antigen mini-arrays for cancer immunodiagnosis has been of interest recently as an approach to cancer detection. In this study, autoantibodies in sera from a patient with HCC were used as probes to immunoscreen a HepG2 cDNA expression library for the identification of TAAs involved in malignant liver transformation. Recombinant proteins from two genes identified in this manner, Sui1 and RalA were expressed, purified and used as antigens in immunoassays to detect the presence of antibodies in sera from 77 patients with HCC, 30 with chronic hepatitis (CH), 30 with liver cirrhosis (LC) and 82 normal human sera (NHS). The prevalence of antibody to Sui1 and RalA in HCC were 11.7% (9/77) and 19.5% (15/77), respectively, which were significantly higher than prevalence in liver cirrhosis (3.3% and 3.3%), chronic hepatitis (0% and 0%) and normal human sera (0% and 0%). When Sui1 and RalA were added to a panel of eight other TAAs used in a previous study, the final cumulative prevalence of anti-TAA antibodies in HCC to the 10 TAA array was raised to 66.2% (51/77). The specificity for HCC compared with LC, CH and NHS, was 66.7%, 80.0%, and 87.8%, respectively. When anti-TAA was added to abnormal serum AFP as combined diagnostic markers, it raised the diagnostic sensitivity from 66.2% to 88.7%. AFP and anti-TAA were independent markers and the simultaneous use of these two markers significantly resulted in the increased sensitivity of HCC detection.

Downregulation of Tsx and OmpW and upregulation of OmpX are required for iron homeostasis in Escherichia coli.

Upregulation of outer membrane (OM) proteins was systematically investigated in response to poor iron availability in the host and natural environments, but downregulation of OM proteins was ill-defined in this response. We utilized proteomic methodologies to characterize altered OM proteins in the sarcosine-insoluble fraction of Escherichia coli K12 cultured in LB medium with iron limitation. Notably, three novel proteins, Tsx, OmpW, and OmpX, related to iron homeostasis were identified; Tsx and OmpW were downregulated, and OmpX was upregulated. These alterations were functionally validated with the use of gene overexpression and deletion methods. Of the two downregulated proteins, Tsx was more sensitive to an iron-deficient environment than OmpW. In addition, the significantly negative correlation between Tsx with OmpW was achieved when overexpressed strains were used. These findings strongly indicate that the downregulation of Tsx and OmpW and the upregulation of OmpX are required for iron homeostasis in E. coli.

Detection of autoantibodies to multiple tumor-associated antigens in the immunodiagnosis of ovarian cancer.

Ovarian cancer is one of the most common cancers in women. Its early stages may be asymptomatic, and as a result diagnosis frequently occurrs at an advanced, often incurable, stage. The high mortality and low survival rates associated with ovarian cancer can in part be attributed to the lack of diagnostic methods allowing for early detection, yet a methodology to identify patients with early-stage ovarian cancer remains to be established. In order to investigate the frequency of antibodies against a panel of multiple carefully-selected tumor-associated antigens (TAAs) in sera from patients with ovarian cancer, and to determine the possibility and usefulness of such a panel of TAAs in the immunodiagnosis of ovarian cancer, sera from 32 ovarian cancer patients and 82 normal individuals were tested using an enzyme-linked immunosorbent assay (ELISA) for the presence of autoantibodies to a panel of 13 TAAs. ELISA results were also confirmed by immunoblotting analysis. The sensitivity and specificity of the multiple anti-TAA antibodies in the detection of ovarian cancer was 62.5 and 85.4%, respectively. With the successive addition of TAAs to a total of 7 antigens (survivin, p53, p16, cyclin B1, cyclin D1, cyclin A and cyclin E), there was a stepwise increase in sensitivity of up to 62.5%, and in specificity of 90.2%. With the addition of more antigens to the panel, no further increase in sensitivity was detected. This study further supports our previous hypothesis that a combination of antibodies might acquire higher sensitivity for the diagnosis of cancer, and also indicates that, in the selection of ovarian cancer-associated TAAs, some may be specific to ovarian cancer while others may not be. This emphasizes the importance of a comprehensive analysis of antibody response to selected TAAs in various disease conditions, such as ovarian cancer, in benign ovarian diseases, and in normal individuals, before conclusions can be drawn regarding their contribution to ovarian cancer.

Autoantibodies to Ca2+ binding protein Calnuc is a potential marker in colon cancer detection.

Calnuc is a calcium (Ca2+) binding protein found in both Golgi and cytoplasm, and it may play a role in G protein- and Ca2+-regulated signal transduction events. This study was designed to investigate the possibility of whether Calnuc protein might be a tumor-associated antigen (TAA) that induces autoantibody response in human cancers, and to evaluate the feasibility of the Calnuc antigen-antibody system as a marker in cancer detection. Purified full-length recombinant Calnuc protein was used as an antigen in enzyme-linked immunoassay and Western blotting for the detection of autoantibodies in cancers. Sera from 447 patients with 9 different types of cancer were analyzed. Although the frequency of autoantibody to Calnuc was found to be 4.7% in total groups of cancer, it was not significantly different to that of normal individuals (1.2%). However, the frequency of autoantibody to Calnuc in colon cancer (11.5%) was significantly higher than that in normal individuals (1.2%). The expression analysis of Calnuc in multiple colon cancer tissues by immunohistochemistry on tissue array further confirmed the high specificity of Calnuc in colon cancer. Of 69 colon cancer tissue specimens examined, 41 tissues (59.4%) overexpressed Calnuc, while normal colon tissues did not show any expression of Calnuc. The subcellular distribution analysis of Calnuc examined by subcellular fractionation and immunofluorescence indicates that Calnuc is a membrane associated protein and mostly distributed in Golgi, which is consistent with previous reports. With adding Calnuc into a TAA array (including p53, c-myc, cyclin B1, cyclin D1), the cumulative frequency of antibody to multiple TAAs in colon cancer was raised to 65.4% which is significantly higher than the cumulative frequency in normal individuals (6.1%). This indicates that a mini-array of multiple TAAs which includes Calnuc might provide a novel non-invasive approach to enhance antibody detection for colon cancer diagnosis.

Antibody detection using tumor-associated antigen mini-array in immunodiagnosing human hepatocellular carcinoma.

BACKGROUND/AIMS: Previous studies have demonstrated that during transition from chronic liver disease to hepatocellular carcinoma (HCC), autoantibodies can appear which are not detected in the prior pre-malignant conditions. These antibody responses may be stimulated by cellular proteins involved in carcinogenesis. This study determines the prevalence of antibodies to a selected panel of eight tumor-associated antigens (TAAs) in sera from patients with chronic hepatitis, liver cirrhosis and HCC, and considers the possibility and usefulness of antibodies to such a panel of TAAs in differentiating between these conditions. The panel of eight TAAs includes Imp1, p62, Koc, p53, c-myc, cyclin B1, survivin and p16 full-length recombinant proteins. METHODS: Enzyme-linked immunosorbent assay (ELISA) was used to detect antibodies against eight selected TAAs in 30 sera from chronic hepatitis, 30 from liver cirrhosis, and 142 from HCC. Positive results were also confirmed by slot blot, Western blotting and immunoprecipitation assay. RESULTS: Antibody frequency to any individual TAA in HCC varied from 9.9% to 21.8%. With the successive addition of TAAs to a final total of eight antigens, there was a stepwise increase of positive antibody reactions reaching a frequency of 59.8% with whole cohort of HCC patients. This was significantly higher than the frequency of antibodies in chronic hepatitis (20%), liver cirrhosis (30%) and normal individuals (12.2%). CONCLUSIONS: This study demonstrates that malignant transition to HCC is associated with increased autoantibody responses to certain cellular proteins which might have a role in tumorigenesis, and shows that a mini-array of eight TAAs enhanced antibody detection for diagnosis of HCC. More studies in patients with HCC and precursor conditions such as chronic hepatitis, alcoholic hepatitis and liver cirrhosis using enlarged TAA mini-array panels might further improve the sensitivity and specificity of this mode of cancer immunodiagnosis. Its additional usefulness might be in the early detection of cancer in some patients with predisposing conditions.

Humoral immune response to p16, a cyclin-dependent kinase inhibitor in human malignancies.

The p16 protein is a cyclin-dependent kinase (CDK) inhibitor, which plays an important role in the regulation of the cell cycle by inactivating the cyclin-dependent kinase (CDK) that phosphorylates the retinoblastoma (Rb) protein. Overexpression of p16 protein has been found in many types of human malignancy. Autoantibody response to p16 in cancer has not been reported. This study determined the extent and frequency of autoantibodies to p16 in diverse malignancies. p16 recombinant protein was expressed in E. Coli BL21 (DE3) cells, and purified using GST fusion protein purification system. In further studies, p16 recombinant proteins were used as antigens in enzyme-linked immunoassay (ELISA) and Western blotting. Sera from 479 cancer patients and 82 normal individuals were analyzed. Autoantibodies to p16 were found in 11.7% in cancer, with significant difference from the normal individuals (p<0.05). The results in this study also showed that the frequency of antibodies to p16 is relatively higher in nasopharyngeal cancer (28.6%), breast cancer (17.1%) and hepatocellular carcinoma (HCC, 21.4%). Of the 56 ELISA positive sera with the anti-p16 antibodies, 85.7% (48/56) had positive reactions in Western blotting. The antigen-antibody absorption experiment was also performed to confirm the specificity of the anti-p16 antibody. In order to increase the frequency of antibody detection in cancer, a combination of three tumor-associated antigens (TAAs) p16, p53 and c-myc were used. Increased frequencies at p<0.01 were found for antibodies to p16 in breast, esophageal, and nasopharyngeal cancer as well as HCC. For antibodies to c-myc, increased frequencies at p<0.01 were found in breast, cervical, colorectal and lung cancer. For antibodies to p53, increased frequencies at p<0.01 were only found in breast cancer. With the successive addition of three TAAs, there was a stepwise increase of positive anti-body reaction up to 44% in breast cancer and 43% in nasopharyngeal cancer. In summary, the results in this study suggest that the combination of antibodies might acquire higher sensitivity for early cancer diagnosis. It is conceivable that auto-antibody profiles involving different panels or arrays of TAAs might be developed in the future and the results could be useful for cancer diagnosis.