Nontypeable Haemophilus influenzae Lipooligosaccharide Expresses a Terminal Ketodeoxyoctanoate In Vivo, Which Can Be Used as a Target for Bactericidal Antibody.

Nontypeable Haemophilus influenzae (NTHi) is an important pathogen in individuals of all ages. The lipooligosaccharide (LOS) of NTHi has evolved a complex structure that can be attributed to a multiplicity of glycosyltransferases, the random switching of glycosyltransferase gene expression via phase variation, and the complex structure of its core region with multiple glycoform branch points. This article adds to that complexity by describing a multifunctional enzyme (LsgB) which optimally functions when the species is grown on a solid surface and which can add either a ketodeoxyoctanoate (KDO) or an N-acetylneuramic acid (Neu5Ac) moiety to a terminal N-acetyllactosamine structure of LOS. Our studies show that expression of lsgB is reduced four- to sixfold when NTHi is grown in broth. The substrate that the enzyme utilizes is dependent upon the concentration of free Neu5Ac (between 1 and 10 µg/ml) in the environment. In environments in which Neu5Ac is below that level, the enzyme utilizes endogenous CMP-KDO as the substrate. Our studies show that during in vivo growth in an NTHi biofilm, the KDO moiety is expressed by the organism. Monoclonal antibody 6E4, which binds KDO, is bactericidal for NTHi strains that express the KDO epitope at high levels. In a survey of 33 NTHi strains isolated from healthy and diseased individuals, the antibody was bactericidal (>90% kill) for 12 strains (36%). These studies open up the possibility of using a KDO-based glycoconjugate vaccine as part of a multicomponent vaccine against NTHi.IMPORTANCE Nontypeable Haemophilus influenzae is an important pathogen in middle ear infections in children, sinusitis in adults, and acute bronchitis in individuals with chronic obstructive lung disease. The organism is very well adapted to the human host environment, and this has hindered successful development of an effective vaccine. In this article, we describe a mechanism by which the bacteria decorates its surface lipooligosaccharide with a sugar unique to Gram-negative bacteria, ketodeoxyoctanoate (KDO). This sugar decoration is present during active infection and we have shown that an antibody directed against this sugar can result in killing of the organism. These data demonstrate that the lipooligosaccharide ketodeoxyoctanoate epitope may be a novel NTHi-specific candidate vaccine antigen.

Lentiviral Vectors Pseudotyped with Filoviral Glycoproteins.

Pseudotyping lentivirus-based vectors is a strategy used to study conferred vector tropism and mechanisms of envelope glycoprotein function. Lentiviruses and filoviruses both assemble at the plasma membrane and have homotrimeric structural envelope glycoproteins that mediate both receptor binding and fusion. Such similarities help foster efficient pseudotyping. Importantly, filovirus glycoprotein pseudotyping of lentiviral vectors allows investigators to study virus entry at substantially less restrictive levels of biosafety containment than that required for wild-type filovirus work (biosafety level-2 vs. biosafety level-4, respectively). Standard lentiviral vector production involves transient transfection of viral component expression plasmids into producer cells, supernatant collection, and centrifuge concentration. Because the envelope glycoprotein expression plasmid is provided in trans, wild type or variant filoviral glycoproteins from marburgvirus or ebolavirus species may be used for pseudotyping and compared side-by-side. In this chapter we discuss the manufacture of pseudotyped lentiviral vector with an emphasis on small-scale laboratory grade production.

Comparative Analyses of the Lipooligosaccharides from Nontypeable Haemophilus influenzae and Haemophilus haemolyticus Show Differences in Sialic Acid and Phosphorylcholine Modifications.

Haemophilus haemolyticus and nontypeable Haemophilus influenzae (NTHi) are closely related upper airway commensal bacteria that are difficult to distinguish phenotypically. NTHi causes upper and lower airway tract infections in individuals with compromised airways, while H. haemolyticus rarely causes such infections. The lipooligosaccharide (LOS) is an outer membrane component of both species and plays a role in NTHi pathogenesis. In this study, comparative analyses of the LOS structures and corresponding biosynthesis genes were performed. Mass spectrometric and immunochemical analyses showed that NTHi LOS contained terminal sialic acid more frequently and to a higher extent than H. haemolyticus LOS did. Genomic analyses of 10 strains demonstrated that H. haemolyticus lacked the sialyltransferase genes lic3A and lic3B (9/10) and siaA (10/10), but all strains contained the sialic acid uptake genes siaP and siaT (10/10). However, isothermal titration calorimetry analyses of SiaP from two H. haemolyticus strains showed a 3.4- to 7.3-fold lower affinity for sialic acid compared to that of NTHi SiaP. Additionally, mass spectrometric and immunochemical analyses showed that the LOS from H. haemolyticus contained phosphorylcholine (ChoP) less frequently than the LOS from NTHi strains. These differences observed in the levels of sialic acid and ChoP incorporation in the LOS structures from H. haemolyticus and NTHi may explain some of the differences in their propensities to cause disease.

ATP11B mediates platinum resistance in ovarian cancer.

Platinum compounds display clinical activity against a wide variety of solid tumors; however, resistance to these agents is a major limitation in cancer therapy. Reduced platinum uptake and increased platinum export are examples of resistance mechanisms that limit the extent of DNA damage. Here, we report the discovery and characterization of the role of ATP11B, a P-type ATPase membrane protein, in cisplatin resistance. We found that ATP11B expression was correlated with higher tumor grade in human ovarian cancer samples and with cisplatin resistance in human ovarian cancer cell lines. ATP11B gene silencing restored the sensitivity of ovarian cancer cell lines to cisplatin in vitro. Combined therapy of cisplatin and ATP11B-targeted siRNA significantly decreased cancer growth in mice bearing ovarian tumors derived from cisplatin-sensitive and -resistant cells. In vitro mechanistic studies on cellular platinum content and cisplatin efflux kinetics indicated that ATP11B enhances the export of cisplatin from cells. The colocalization of ATP11B with fluorescent cisplatin and with vesicular trafficking proteins, such as syntaxin-6 (STX6) and vesicular-associated membrane protein 4 (VAMP4), strongly suggests that ATP11B contributes to secretory vesicular transport of cisplatin from Golgi to plasma membrane. In conclusion, inhibition of ATP11B expression could serve as a therapeutic strategy to overcome cisplatin resistance.

Epigenetic regulation of maspin expression in the human placenta.

Maspin, a tumour suppressor gene, is differentially expressed in the human placenta. Decreased expression of maspin in the first trimester corresponds with the period of maximum trophoblast invasion, suggesting a role in cell invasion and motility. Although methylation of CpG islands regulates maspin expression in cancer cells, the mechanism of maspin regulation in the human placenta is unknown. Our objectives were to determine the role of epigenetic alterations in the regulation of maspin expression in the placenta. Placental samples obtained from 7 to 40 weeks' gestation were used for bisulphite sequencing and chromatin immunoprecipitation (ChIP) PCR. There was no significant change in the methylation indices in the promoter region of maspin throughout gestation. The levels of histone modifications associated with transcriptionally active chromatin were significantly different in placental tissues from second and third trimester relative to those from first trimester. Addition of trichostatin A (TSA) to placental explants increased the maspin mRNA expression (8- to 20-fold), whereas addition of 5-aza-cytidine (5-AzaC) had no effect on maspin expression. Our data suggest that maspin expression in the human placenta is regulated by changes in histone tail modifications. This is the first report of selective histone modifications associated with differential placental gene expression in human gestation.

Genomic instability is associated with lack of telomerase activation in ovarian cancer.

INTRODUCTION: Malignant cells are capable of an unlimited number of cell divisions, either through production of telomerase, or through the alternate lengthening of telomere (ALT) mechanism. Yeast cells with genomic instability have been shown to survive in the absence of telomerase by increased recombination events. We hypothesized that ovarian cancers with high microsatellite instability (MSI-H) are more likely to lack telomerase activation. METHODS: We examined 104 invasive ovarian cancers for MSI with six microsatellite markers (BAT25, BAT26, D5S346, D2S123, D17S250 and NME1). Telomerase activity was determined with ELISA, and its subunits human telomerase reverse transcriptase (hTERT) and human telomerase RNA (hTR) by RT-PCR. Statistical analysis was performed with Chi-square and p<0.05 was considered significant. RESULTS: Telomerase activity was detected in 79 samples (76%). The hTERT subunit was detected in 85% of samples, and hTR was found in all ovarian cancers. Presence of hTERT was positively associated with telomerase activity (p=0.001). High MSI (MSI-H), defined as two or more positive markers, was detected in 15% of ovarian cancers; low MSI (MSI-L), defined as having only one positive marker, was found in 13%; the remaining 72% were microsatellite stable (MSI-S). Telomerase activity was detected in 83% of MSI-S and 79% of MSI-L tumors, but only 40% of MSI-H tumors (p=0.002). Interestingly, hTERT was similar in all three groups (range 73-84%, p=0.59), demonstrating that the presence of hTERT transcript was not the only determinant of telomerase activity in MSI-H tumors. CONCLUSIONS: Ovarian cancers with high MSI are more likely to propagate without the need to produce telomerase.

Biological significance of focal adhesion kinase in ovarian cancer: role in migration and invasion.

Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that is activated by integrin clustering. There are limited data regarding the functional role of FAK in ovarian cancer migration and invasion. In the current study, FAK expression was evaluated in ovarian cell lines (nontransformed and cancer), 12 benign ovarian samples, and in 79 invasive epithelial ovarian cancers. All three ovarian cancer cell lines overexpressed FAK compared to the nontransformed cells. The dominant-negative construct called FAK-related nonkinase (FRNK) was introduced into two ovarian cancer cell lines (SKOV3 and 222). FRNK promoted FAK dephosphorylation without changing total FAK levels in these cell lines. Furthermore, FRNK decreased the in vitro invasive ability of ovarian cancer cells by 56 to 85% and decreased migration by 52 to 68%. FRNK-transfected cells also displayed poor cell spreading. Immunohistochemical analysis revealed that the surface epithelium from all benign ovarian samples had weak FAK expression. In contrast, 68% of invasive ovarian cancers overexpressed FAK. FAK overexpression was significantly associated with high tumor stage, high tumor grade, positive lymph nodes and presence of distant metastasis (all P values <0.05). FAK overexpression was also associated with shorter overall survival (P = 0.008). Multivariate analysis revealed that FAK overexpression and residual disease >1 cm were independent predictors of poor survival. These data indicate that FAK is overexpressed in most invasive ovarian cancers and plays a functionally significant role in ovarian cancer migration and invasion. Thus, FAK may be an important therapeutic target in ovarian carcinoma.

Functional role of matrix metalloproteinases in ovarian tumor cell plasticity.

OBJECTIVE: We previously demonstrated that aggressive ovarian cancer cells are able to display in vitro vasculogenic mimicry, which is reflected by their ability to form vasculogenic-like networks in 3-dimensional cultures and to express vascular cell-associated markers. The goal of this study was to examine the functional role of specific matrix metalloproteinases in the formation of vasculogenic-like networks and extracellular matrix remodeling in vitro. We also investigated the clinical relevance of matrix metalloproteinase-2 and -9 and membrane type 1-matrix metalloproteinase in human ovarian cancers with evidence of tumor cell-lined vasculature. STUDY DESIGN: Ovarian cancer cells (A2780-PAR, SKOV3, and EG) were seeded onto separate 3-dimensional cultures that contained either Matrigel or type I collagen, in the absence of endothelial cells or fibroblasts. These cultures were treated with either chemically modified tetracycline-3 (general matrix metalloproteinase inhibitor), recombinant tissue inhibitor of metalloproteinase-1 or -2, or function-blocking antibodies to matrix metalloproteinase-2 or -9 or membrane type 1-matrix metalloproteinase. In addition, 78 invasive epithelial ovarian cancers were evaluated for expression of matrix metalloproteinase-2 and -9 and membrane type 1-matrix metalloproteinase and correlated with various clinical parameters. RESULTS: The aggressive ovarian cancer cells (SKOV3 and EG) were able to form in vitro vasculogenic-like networks and contract 3-dimensional collagen I gels, whereas the poorly aggressive A2780-PAR cell line did not. Chemically modified tetracycline-3 completely blocked the network formation. Blocking antibodies to matrix metalloproteinase-2 and membrane type 1-matrix metalloproteinase inhibited the formation of the vasculogenic-like networks and collagen gel contraction, but the antibody to matrix metalloproteinase-9 had no effect on network formation and minimal effect on gel contraction. Treatment of 3-dimensional cultures with tissue inhibitor of metalloproteinase-2 retarded the network formation and only small, partially developed structures were noted that did not form network connections. Tissue inhibitor of metalloproteinase-1 had no appreciable effect on the extent or efficiency of network formation. Human invasive ovarian cancers with evidence of tumor cell-lined vasculature were significantly more likely to have strong epithelial and stromal matrix metalloproteinase-2 and -9 and membrane type 1-matrix metalloproteinase expression (all probability values were <.05). CONCLUSION: Matrix metalloproteinase-2 and membrane type 1-matrix metalloproteinase appear to play a key role in the development of vasculogenic-like networks and matrix remodeling by aggressive ovarian cancer cells. Human ovarian cancers with matrix metalloproteinase overexpression are more likely to have tumor cell-lined vasculature. These results may offer new insights for consideration in ovarian cancer treatment strategies.

Stress-related mediators stimulate vascular endothelial growth factor secretion by two ovarian cancer cell lines.

PURPOSE: Stress has long been believed to influence carcinogenesis, but little is known about physiological mechanisms that may underlie these effects. We have recently observed lower levels of vascular endothelial growth factor (VEGF) in ovarian cancer patients with greater social support, whereas higher VEGF was found in patients with greater distress. The goal of this study was to examine possible mechanisms underlying these relationships. EXPERIMENTAL DESIGN: The effects of stress-related mediators including norepinephrine (NE), epinephrine, isoproterenol (a nonspecific beta-adrenergic agonist), and cortisol on the production of VEGF by the ovarian cell lines SKOV3 and EG were investigated. RESULTS: NE and isoproterenol significantly enhanced VEGF production by SKOV3 cells, and all three of the adrenergic agonists enhanced VEGF production by EG cells. These effects were blocked by the beta antagonist propranolol, supporting a role for beta-adrenergic receptors in these effects. Reverse transcriptase-PCR studies indicated constitutive expression of beta-1 and beta-2 adrenergic receptors on both cell lines. Effects of cortisol on VEGF production varied according to the specific cell line and dose, with stimulating effects on SKOV3 at pharmacologic doses (1000 nM) and on EG at physiological stress level doses (10 nM), and inhibitory effects on EG at pharmacologic doses. Although priming with cortisol blunted NE-induced VEGF production from both cell lines at 3 h, significant increases in VEGF were still seen. Priming with cortisol enhanced isoproterenol-induced VEGF production from SKOV3. CONCLUSION: These findings provide the first experimental evidence of a pathway by which biobehavioral stress mediators could directly contribute to the progression of ovarian tumors.

p53 null mutations are associated with a telomerase negative phenotype in ovarian carcinoma.

Telomerase activation and p53 dysfunction are important events in the development and progression of most cancers including ovarian cancer. However, many cancer cell lines and human tumors have been shown to lack telomerase, and maintain telomerase through the ALT (alternative lengthening of telomeres). It is not known whether specific types of p53 mutations are correlated with telomerase activity in human tumors. Invasive ovarian cancers (109) were analyzed for telomerase by ELISA and its subunits human telomerase RNA (hTR), and human telomerase reverse transcriptase (hTERT) by RT-PCR. p53 protein was analyzed in the same samples using immunohistochemistry, and p53 exons 2-11 were analyzed using SSCP and sequence analysis. Telomerase activity was detected in 80 (74%) of 109 tumors. The subunit hTR was consistently present in all ovarian cancer samples, and hTERT was expressed in 96 (88%) tumors. Thirteen (16%) tumors were negative for hTERT and none of these expressed telomerase. Fifty-seven (52%) tumors stained positive for p53, and there was no correlation with telomerase activity based on p53 staining (p = 0.08). Eighty-two (75%) tumors were found to have a p53 mutation, and 40 (36%) tumors contained a null mutation. Only 14% of the tumors with wild type or missense p53 were negative for telomerase activity. In contrast, 19 (48%) of 40 tumors with a p53 null mutation were negative for telomerase activity (p <0.001). There was no difference in the incidence of telomerase positivity between critical site versus non-critical site missense p53 mutations. Seventy-five percent of the tumors with a p53 mutation in the central region were telomerase positive. In contrast, only 47% of the tumors with a mutation in either the amino- or carboxy-terminus were telomerase positive (p = 0.03). Ovarian cancers with a p53 null mutation are more likely to lack telomerase activity. This may have implications for therapeutic approaches based on telomerase.

Preoperative serum vascular endothelial growth factor levels: significance in ovarian cancer.

PURPOSE: To assess the clinical relevance of serum vascular endothelial growth factor (VEGF) levels in distinguishing patients with ovarian cancer from those with benign adnexal masses. EXPERIMENTAL DESIGN: Preoperative serum VEGF levels were assessed in 101 women with invasive epithelial ovarian cancer, 16 with low malignant potential (LMP) ovarian tumors, and 34 women with benign ovarian tumors. VEGF levels were determined using an ELISA (R&D Systems, Minneapolis, MN). RESULTS: Ovarian cancer patients had a mean preoperative VEGF level of 549 pg/ml (median 379 pg/ml), which was significantly higher than those with benign adnexal masses (mean 228 pg/ml, median 155 pg/ml; P < 0.001) and LMP tumors (mean 200 pg/ml, median 129 pg/ml; P < 0.001). There were no significant differences in VEGF levels between individuals with benign masses and LMP tumors. The ability of VEGF to differentiate malignancy from benign masses at a cutoff VEGF level of 246 pg/ml gave a sensitivity of 74%, a specificity of 71%, a positive predictive value of 88%, and a negative predictive value of 48%. VEGF levels were also significantly higher in patients with stage I ovarian cancer compared with those with benign disease or LMP tumors. Among patients with ovarian cancer, there were no significant differences in VEGF levels based on age, stage, grade, or level of cytoreduction. The presence of ascites was associated with a significantly higher VEGF level (mean 667 pg/ml, median 445 pg/ml versus mean 317 pg/ml, median 293 pg/ml; P < 0.001). Various preoperative VEGF levels were assessed as a predictor of survival, and a VEGF level >380 pg/ml was associated with a hazard ratio of 2.13 (P = 0.009) by univariate analysis. In multivariate analysis of age, stage, cytoreduction, preoperative CA-125, grade, ascites, and VEGF levels above 380 pg/ml, only VEGF levels >380 pg/ml (hazard ratio 2.33; P = 0.02) and advanced stage (hazard ratio 9.03; P = 0.004) were significant. CONCLUSIONS: Preoperative VEGF levels may be useful in differentiating benign adnexal masses from malignancy. Preoperative VEGF levels >380 pg/ml are an independent risk factor for death because of disease.

The paradoxical expression of maspin in ovarian carcinoma.

Maspin is a noninhibitory member of the serpin family that is down-regulated in breast carcinoma but overexpressed in pancreatic carcinoma. There are no published data regarding the role of maspin in ovarian carcinoma, which is the focus of the present study. Ovarian cell lines (normal and cancer) and tumors (80 invasive, 14 benign, and 10 low malignant potential) were evaluated for maspin expression and localization. Normal ovarian surface epithelial cells had low levels of maspin. Two of four ovarian cancer cell lines (OVCAR3 and SKOV3) expressed maspin, whereas the cell line EG had weak expression, and 222 had no detectable maspin. Subcellular fractionation studies revealed that the two maspin-positive ovarian cancer cell lines contained maspin in both the nuclear and cytosolic compartments. Wild-type maspin was transfected into the aggressive ovarian cancer cell lines SKOV3 and 222. The in vitro invasive activity of the maspin-transfected cell lines was 44-68% lower than respective controls. The histopathology analysis revealed that among the ovarian tumors examined, 57 (71%) were ranked positive for maspin. Thirty (37%) of the invasive tumors overexpressed maspin. Invasive cancers were more likely to have predominantly cytoplasmic staining compared with benign and low-malignant-potential tumors. Maspin overexpression was significantly associated with a high tumor grade (P = 0.004), the presence of ascites (P = 0.02), a lower likelihood of optimal surgical cytoreduction (P = 0.04), and a shorter duration of overall survival (median survival, 6.33 versus 2.67 years; P = 0.003). The Cox proportional hazards multivariate model revealed that maspin overexpression and high stage were independent predictors of survival. Thus, maspin was found to be overexpressed in a substantial proportion of ovarian tumors, which may serve as an adverse prognostic factor; however, its localization may provide new clues as to its activity and function. These paradoxical results may offer new insights regarding the role of maspin in ovarian cancer progression that may also impact diagnosis and treatment strategies.

The clinical significance of tumor cell-lined vasculature in ovarian carcinoma: implications for anti-vasculogenic therapy.

Vasculogenic mimicry reflects the plasticity of aggressive tumor cells that express vascular cell markers and line tumor vasculature; such has been demonstrated in aggressive ovarian carcinoma. This study measured the clinical significance of tumor cell-lined vasculature in ovarian carcinomas (n=77), which was detected in 23 (29.8%) tumors. The data show that tumor cell-lined vasculature was associated with aggressive tumor features and with shorter overall survival (p<0.001). Cox proportional hazards model revealed that tumor cell-lined vasculature (p=0.002) was independently associated with poor survival. This is the first study demonstrating the clinical implications of tumor cell-lined vasculature in ovarian carcinoma.