Venous thromboembolism in IBD: Increased risk for women in CD?

Although coagulation disturbances have been described in inflammatory bowel disease (IBD), it remains unclear how common venous thromboembolism (VTE) is in IBD, and what factors influence VTE frequency. We evaluated VTE in Crohn's disease (CD) and ulcerative colitis (UC) at LSUHSC-S, a southern US medical center with an approximately equal White: African-American (AA) (1.12:1) patient base. This retrospective study evaluated VTE as a co-morbidity in IBD as a function of age, gender and race based on ICD-10 coding (2011-2015.) Results. Of 276 IBD diagnostic records, 213 were for CD (77.17%) and 63 for UC (22.8%). 52% of the CD patients were white, 42% were AA, and 6% were other. 42% of CD patients were male, with 58% were female. 6.1% (13 patients) of the 213 CD patients had a VTE. Of these 13 CD patients, 9 had active disease and 4 were in remission. 9 of 13 were female and 4 were male, with 5 white patients and 4 A A patients. 63 patients were diagnosed with UC, 3.38-fold fewer cases than CD. 25 UC patients were white, 25 were AA and 13 were in other ethnic groups. Of 63 UC cases, 2 UC patients had a VTE, both with active disease. At our institution, VTE appears to be 3x more frequently associated with CD than UC and was more common in white female patients. The recognition of VTE risk in CD, particularly in women, may be an important observation which may guide therapy and limit potentially life-threatening consequences.

Differential cytokine responses in human and mouse lymphatic endothelial cells to cytokines in vitro.

BACKGROUND: Inflammatory cytokines dysregulate microvascular function, yet how cytokines affect lymphatic endothelial cells (LEC) are unclear. METHODS AND RESULTS: We examined effects of TNF-α, IL-1 beta, and IFN-gamma on LEC proliferation, endothelial cell adhesion molecule (ECAM) expression, capillary formation, and barrier changes in murine (SV-LEC) and human LECs (HMEC-1a). RESULTS: All cytokines induced ICAM-1, VCAM-1, MAdCAM-1, and E-selectin in SV-LECs; TNF-α, IL-1 beta; and IFN-gamma induced ECAMs (but not MAdCAM-1) in HMEC-1a. IL-1 beta increased, while IFN-gamma and TNF-α reduced SV-LEC proliferation. While TNF-α induced, IFN-gamma decreased, and IL-1 beta did not show any effect on HMEC-1a proliferation. TNF-α, IL-1 beta, and IFN-gamma each reduced capillary formation in SV-LEC and in HMEC-1a. TNF-α and IL-1 beta reduced barrier in SV-LEC and HMEC-1a; IFN-gamma did not affect SV-LEC barrier, but enhanced HMEC-1a barrier. Inflammatory cytokines alter LEC growth, activation and barrier function in vitro and may disturb lymphatic clearance increasing tissue edema in vivo. CONCLUSION: Therapies that maintain or restore lymphatic function (including cytokines blockade), may represent important strategies for limiting inflammation.

Gastrointestinal lymphatics in health and disease.

Lymphatics perform essential transport and immune regulatory functions to maintain homeostasis in the gastrointestinal (GI) system. Although blood and lymphatic vessels function as parallel and integrated systems, our understanding of lymphatic structure, regulation and functioning lags far behind that of the blood vascular system. This chapter reviews lymphatic flow, differences in lymphangiogenic and hemangiogenic factors, lymphatic fate determinants and structural features, and examines how altered molecular signaling influences lymphatic function in organs of the GI system. Innate errors in lymphatic development frequently disturb GI functioning and physiology. Expansion of lymphatics, a prominent feature of GI inflammation, may also play an important role in tissue restitution following injury. Destruction or dysregulation of lymphatics, following injury, surgery or chronic inflammation also exacerbates GI disease activity. Understanding the physiological roles played by GI lymphatics is essential to elucidating their underlying contributions to forms of congenital and acquired forms of GI pathology, and will provide novel approaches for therapy.

The regulation of integrin-linked kinase in human platelets: evidence for involvement in the regulation of integrin alpha 2 beta 1.

BACKGROUND: Activation of the platelet integrin alpha 2 beta 1 is closely regulated due to the high thrombogenicity of its ligand. As a beta 1 interacting kinase, ILK represents a candidate intracellular regulator of alpha 2 beta 1 in human platelets. OBJECTIVES: We investigated the regulation of ILK in human platelets and the role of ILK in regulating alpha 2 beta 1 activation in HEL cells, a megakaryocytic cell line. METHODS: An in-vitro kinase assay was used to determine the effect of platelet agonists on ILK kinase activity together with the contribution of PI3K and PKC on ILK activation. Interaction of ILK with beta 1-integrin subunits was investigated by coimmunoprecipitation and the role of ILK in regulating alpha 2 beta 1 function assessed by overexpression studies in HEL cells. RESULTS: We report that collagen and thrombin modulate ILK kinase activity in human platelets in an aggregation-independent manner. Furthermore, ILK activity is dually regulated by PI3K and PKC in thrombin-stimulated platelets and regulated by PI3K in collagen-stimulated cells. ILK associates with the beta 1-integrin subunits immunoprecipitated from platelet cell lysates, an association which increased upon collagen stimulation. Overexpression of ILK in HEL cells enhanced alpha 2 beta 1-mediated adhesion whereas overexpression of kinase-dead ILK reduced adhesion, indicating a role for this kinase in the positive regulation of alpha 2 beta 1. CONCLUSIONS: Our findings that ILK regulates alpha 2 beta 1 in HEL cells, is activated in platelets and associates with beta 1-integrins, raise the possibility that it may play a key role in adhesion events upon agonist stimulation of platelets.

Quercetin inhibits collagen-stimulated platelet activation through inhibition of multiple components of the glycoprotein VI signaling pathway.

BACKGROUND: The regulation of platelet function by pharmacological agents that modulate platelet signaling has proven a successful approach to the prevention of thrombosis. A variety of molecules present in the diet have been shown to inhibit platelet activation, including the antioxidant quercetin. OBJECTIVES: In this report we investigate the molecular mechanisms through which quercetin inhibits collagen-stimulated platelet aggregation. METHODS: The effect of quercetin on platelet aggregation, intracellular calcium release, whole cell tyrosine phosphorylation and intracellular signaling events including tyrosine phosphorylation and kinase activity of proteins involved in the collagen-stimulated glycoprotein (GP) signaling pathway were investigated. RESULTS: We report that quercetin inhibits collagen-stimulated whole cell protein tyrosine phosphorylation and intracellular mobilization of calcium, in a concentration-dependent manner. Quercetin was also found to inhibit various events in signaling generated by the collagen receptor GPVI. This includes collagen-stimulated tyrosine phosphorylation of the Fc receptor gamma-chain, Syk, LAT and phospholipase Cgamma2. Inhibition of phosphorylation of the Fc receptor gamma-chain suggests that quercetin inhibits early signaling events following stimulation of platelets with collagen. The activity of the kinases that phosphorylate the Fc receptor gamma-chain, Fyn and Lyn, as well as the tyrosine kinase Syk and phosphoinositide 3-kinase was also inhibited by quercetin in a concentration-dependent manner, both in whole cells and in isolation. CONCLUSIONS: The present results provide a molecular basis for the inhibition by quercetin of collagen-stimulated platelet activation, through inhibition of multiple components of the GPVI signaling pathway, and may begin to explain the proposed health benefits of high quercetin intake.

Radionuclides in soil and water near a low-level disposal site and potential ecological and human health impacts.

Material Disposal Area G is the primary low-level radioactive waste disposal site at Los Alamos National Laboratory, New Mexico, and is adjacent to Pueblo of San Ildefonso lands. Pueblo residents and Los Alamos scientists are concerned about radiological doses resulting from uptake of Area G radionuclides by mule deer (Odocoileus hemionus) and Rocky Mountain elk (Cervus elaphus), then consumption of deer and elk meat by humans. Tissue samples were collected from deer and elk accidentally killed near Area G and were analyzed for 3H, 90Sr, total U, 238Pu, 239,240Pu, 241Am, and 137Cs. These data were used to estimate human doses based on meat consumption of 23 kg y(-1). Human doses were also modeled using RESRAD, and dose rates to deer and elk were estimated with a screening model. Dose estimates to humans from tissue consumption were 2.9 x 10(-3) mSv y(-1) and 1.6 x 10(-3) mSv y(-1) from deer and elk, respectively, and RESRAD dose estimates were of the same order of magnitude. Estimated dose rates to deer and elk were 2.1 x 10(-4) mGy d(-1) and 4.7 x 10(-4) mGy d(-1), respectively. All estimated doses were significantly less than established exposure limits or guidelines.

New insights into the metal center of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase.

Metal binding properties for a series of metal-substituted forms of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, DAHPS(Tyr), have been followed by UV-vis and EPR spectroscopy. The results show that there are two metal species present at pH = 7.0 and these are coordinated in a distorted metal binding site with a mixed nitrogen and oxygen donor atom coordination set. There is no spectroscopic evidence for strong M-S interactions in this system at any pH. Metal saturation occurs at a substoichiometric ratio of 0.8-0.85 metal/monomer, and the binding trends mirror previously published enzyme activity profiles. There is a conformational change for CuDAHPS under basic conditions, and equivalent protein handling for apoDAHPS leads to apparent loss of metal binding ability. Addition of the substrate PEP does not alter the UV-vis spectra, but there are small changes in the EPR spectra of CuDAHPS(Tyr). Further addition of the substrate analogue A5P has no effect on either spectra. Taken together, these results serve to link previous studies on enzyme activity with the recently determined X-ray crystal structure for DAHPS(Phe) and represent the first detailed spectroscopic characterization of the metal binding properties of DAHPS(Tyr).

Anaerobic acquisition of [4FE 4S] clusters by the inactive FNR(C20S) variant and restoration of activity by second-site amino acid substitutions.

The FNR protein of Escherichia coli controls the transcription of target genes in response to anoxia. The anaerobic incorporation of oxygen-sensitive [4Fe 4S] clusters promotes dimerization, which in turn enhances DNA binding. Four potential iron ligands (C20, C23, C29 and C122) are essential for normal FNR activity in vivo. Three FNR variants (C20S, C23G and C29G) retained the ability to incorporate oxygen-sensitive [4Fe 4S] clusters and to bind target DNA with essentially unimpaired affinity, suggesting that their failure to function normally in vivo resides at a later stage in the signal transduction pathway. The C122 variant failed to assemble iron-sulphur clusters and to bind DNA. Second-site substitutions that partially restore activity to FNR(C20S) were generated by error-prone polymerase chain reaction and were located in the dimer interface, in the activating regions (AR1, 2 or 3) or close to C122. Substitutions at E47, R48, E123, I124, E127 or T128 allowed the extent of the FNR AR2 surface to be defined. Only one revertant, FNR(C20S Y69F G149S), specifically corrected the C20S defect. It was concluded that [4Fe 4S] cluster acquisition, dimerization and DNA binding are not sufficient to confer transcription regulatory activity on FNR: the iron-sulphur cluster must also be correctly liganded in order to establish effective activating contacts between FNR and RNA polymerase.

Biochemical and spectroscopic characterization of Escherichia coli aconitases (AcnA and AcnB).

Escherichia coli contains two major aconitases (Acns), AcnA and AcnB. They are distantly related monomeric Fe-S proteins that contain different arrangements of four structural domains. On the basis of the differential expression of the acnA and acnB genes, AcnA has been designated as an aerobic-stationary-phase enzyme that is specifically induced by iron and oxidative stress, whereas AcnB functions as the major citric-acid-cycle enzyme during exponential growth. The biochemical and kinetic properties of the purified enzymes have now shown that AcnA is more stable than AcnB, has a higher affinity for citrate, and operates optimally over a wider pH range, consistent with its role as a maintenance or survival enzyme during nutritional or oxidative stress. In contrast, the better performance at high substrate concentrations and greater instability of AcnB indicate that AcnB is specifically adapted to function as the main catabolic enzyme and, by inactivation, to rapidly modulate energy metabolism in response to oxidative or pH stress, either directly or indirectly by regulating post-transcriptional gene expression. EPR and magnetic-CD spectroscopy showed that the iron-sulphur clusters of the bacterial Acns (and their binding sites) strongly resemble those of the mammalian enzymes. The EPR and MCD spectra of the oxidized inactive form of AcnB confirmed the presence of a [3Fe-4S](1+) (S=1/2) cluster. Comparisons showed that the EPR spectrum of AcnB more closely resembled that of mammalian mitochondrial Acn (m-Acn), whereas the spectrum of AcnA more closely resembled that of the cytoplasmic enzyme (c-Acn). The MCD spectra revealed spectroscopic signatures similar to that of m-Acn. Reconstitution of the active [4Fe-4S](2+) forms followed by one-electron reduction gave rise to EPR spectra that are almost identical with those reported for the mammalian enzymes.

Reversible and irreversible hemichrome generation by the oxygenation of nitrosylmyoglobin.

The repeated oxygenation/reduction/nitrosylation of nitrosylmyoglobin produces low-spin ferric heme hemichromes which have been characterized by electron spin resonance spectroscopy. The predominant myoglobin hemichrome is a chemically reversible dihistidyl complex identified by the g values 1.53, 2.21, and 2.97. Also present is a low-spin ferric hydroxide derivative which is represented by the g values 1.83, 2.18, and 2.59. The formation of these species goes undetected by UV-vis spectroscopy, but the oxygenation of myoglobin to metmyoglobin is correlated with complete conversion of nitric oxide to nitrate which is released following a clear induction period. These results are interpreted in terms of the intermediates generated during the MbNO oxygenation reaction.

The value of screening for Down's syndrome in a socioeconomically deprived area with a high ethnic population.

OBJECTIVE: To assess the utility of biochemical antenatal screening for Down's syndrome in a socioeconomically deprived area with a high proportion of Asian women from the Indian Subcontinent. DESIGN: Audit of Down's syndrome biochemical screening service over a four-year period. SETTING: Teaching hospital and community antenatal clinic in inner city Birmingham. POPULATION: Women booked between October 1992 and December 1996. METHODS: Blood for screening was collected between 14 and 21 weeks gestation, alpha-fetoprotein and intact human chorionic gonadotrophin were measured in serum and the risk of Down's syndrome was calculated. MAIN OUTCOME MEASURES: Uptakes of screening and amniocentesis, screen positive rate, odds of being affected given a positive result, miscarriages associated with amniocentesis offered following a high risk result, detection rate, number of Down's cases prevented and a cost analysis. Outcome measures were compared between Asians and Caucasians. RESULTS: Overall 11,974 women (71%) accepted serum screening. The screen positive rate was 8.3% in Asians and 5.0% in Caucasians. The uptake of amniocentesis in women following a high risk result was 54% overall (35% Asian, 67% Caucasian). Nineteen cases of Down's syndrome were identified, of which 13 occurred in women who opted for biochemical screening. The detection rate of the biochemical screening programme was 85% (11/13). Of these 11 cases, six (none of whom were Asian) elected to have an amniocentesis, of whom four thereafter had a termination. CONCLUSION: In this study the public health benefits of screening for Down's syndrome in a socioeconomically deprived area with a high Asian population, were small.

FNR is a direct oxygen sensor having a biphasic response curve.

FNR is a transcription regulator that controls the expression of target genes in response to anoxia. Anaerobiosis is accompanied by the acquisition of two [4Fe-4S]2+ clusters per FNR dimer and the ability to bind DNA site-specifically. Oxidation of the [4Fe-4S]2+ form of FNR by O2 produced a non-DNA-binding, transcriptionally inactive form which also contains an iron-sulfur cluster, recently identified by Mossbauer spectroscopy as a [2Fe-2S] cluster (Khoroshilova et al., 1997, PNAS. 94, 6078). Complete conversion needed at least 2.5-3.0 molecules of O2 per [4Fe-4S]2+ cluster. Using sub-stoicheiometric amounts of air-saturated buffer, stable equilibria were established in which the [4Fe-4S]2+ and [2Fe-2S]2+ forms co-exist and no EPR detectable free ferric ions were released. In contrast, a 20-fold molar excess K3Fe(CN)6 was required to oxidise the [4Fe-4S]2+ cluster and in this case, ferric ions were released. FNR is therefore a sensitive O2 sensor.

Determination of proliferation index with MIB-1 in advanced ovarian cancer using quantitative image analysis.

A new monoclonal antibody, MIB-1, reacts with the same epitope recognized by Ki-67. The authors investigated the feasibility of using image analysis to quantitate the MIB-1 staining (proliferation index [PI]) of epithelial ovarian cancers. The PI was determined in 50 advanced-stage primary ovarian cancers. Paraffin sections were immunostained with the MIB-1 monoclonal antibody, and the PI was calculated using a CAS 200 image analyzer. Among 36 stage III ovarian carcinomas, the median PI was 15.1%, compared with 18.9% in 14 stage IV cancers (P = .47). Based on exploratory methods, a cutoff point of 7% best dichotomized these patients into two prognostic groups. Of 39 patients whose cancers had a high MIB-1 expression (> or = 7%), the median survival was 16.5 months, which differed significantly (P = .01) from the median survival of 33.2 months observed in the 11 patients whose tumors demonstrated low MIB-1 expression (< 7%). Using MIB-1 as a binary variable, a strong correlation was found between overexpression of c-erbB-2 (2+ and 3+) and MIB-1 > or = 7% (P = .001). No relationship was found between PI and histologic grade. Further studies are warranted to investigate the relationship between MIB-1, PI expression, and other known clinicopathologic and genetic features of early- and late-stage ovarian cancer.

Assessment of c-erbB-2 amplification by immunohistochemistry in paraffin-embedded breast cancer.

The c-erbB-2 (HER-2/neu) proto-oncogenes is important in oncogenesis and for determination of prognosis in a number of human malignancies. DNA (Southern) hybridization and immunoblotting (Western) techniques are most commonly utilized for determining amplification status and protein expression of this proto-oncogene, respectively. These extraction techniques are often time-consuming, costly, and subject to variability depending on the histological characteristics of the tumor. Paraffin-immunohistochemistry (P-IHC), on the other hand, is time and cost-effective. In addition, this technique may offer enhanced sensitivity and specificity over extraction techniques due to the in situ nature of analysis. In data presented here, 67 cases of human mammary carcinoma were concomitantly assessed for c-erbB-2 gene copy number and oncoprotein expression by dilutional DNA hybridization and P-IHC, respectively. In 64 (95.5%) of 67 cases, high level expression was associated with gene amplification, whereas no detectable expression was associated with a normal diploid gene copy number. In two of the three discrepant cases, P-IHC predicted amplification not corroborated by Southern analysis. In these cases, tumor mass was limited by the intraductal component of the lesion or by an abundance of stromal elements within the specimen. We conclude that P-IHC offers a favorable alternative to Southern analysis in the assessment of c-erbB-2 gene copy number of this oncoprotein in human mammary carcinoma. Furthermore, immunohistochemistry may prove superior to either extraction technique in specimens with limited tumor mass, such as biopsy materials, stroma-rich tumors, or early stage lesions such as intraductal carcinoma.

Determination of proliferation index in advanced ovarian cancer using quantitative image analysis.

It has been shown that the monoclonal antibody Ki-67 reacts with a nuclear antigen that is expressed only by proliferating cells. The feasibility of using image analysis to quantitate Ki-67 staining (proliferation index [PI]) of epithelial ovarian cancers was investigated. The PI was determined in 50 advanced-stage primary ovarian cancers. Frozen sections were immunostained with the Ki-67 monoclonal antibody, and the PI was calculated using static image analysis. Among 35 stage III ovarian carcinomas, the median PI was 8.9%, compared with 17.7% in 15 stage IV cancers (P = 0.06). There was no relationship between PI and histologic grade. The median survival time of 32 patients whose cancers had a high Ki-67 expression (> or = 7.5%) was 16.8 months, which differed significantly (P < 0.01) from the median survival of 31.5 months observed in patients whose tumors demonstrated low Ki-67 expression (< 7.5%). Quantitative image analysis of Ki-67-stained fresh-frozen ovarian cancers may provide useful prognostic information. Further studies are warranted to investigate the relationship between Ki-67 expression and other known clinicopathologic and genetic features of ovarian cancer.

DNA ploidy of basal cell carcinoma determined by image cytometry of fresh smears.

Image analysis of nuclear DNA content (DNA ploidy) was performed on smears of basal cell carcinoma (BCC) obtained during Mohs microscopically-controlled surgery from 51 tumors. DNA ploidy was compared with histologic growth pattern and the contour of the invading edge. There was a statistically significantly increased frequency of DNA aneuploidy in smears from BCC exhibiting partial or total diffuse (infiltrative and superficial multicentric) growth patterns (80%; 32 of 40) as compared to solely circumscribed growth patterns (0%; 0 of 11) (p < 0.001).

p53 overexpression in formalin-fixed, paraffin-embedded tissue detected by immunohistochemistry.

Mutation and overexpression of the p53 gene have been noted in a wide range of human cancers and are thought to play a role in malignant transformation. Previously, immunohistochemical detection of p53 has been possible only in fresh-frozen tissues. We examined p53 expression in paraffin-embedded tissues from 50 epithelial ovarian cancers and 25 primary breast cancers with a modified immunohistochemical (IHC) technique developed in this laboratory, using monoclonal antibody (MAb) PAb1801. The 75 cases were selected from a group of patients in whom the expression levels had already been assessed in a fresh-tissue IHC assay. An identical staining reactivity was observed in both formalin-fixed, paraffin-embedded tissue and fresh-frozen tissue in 48 of 50 (96%) epithelial ovarian cancers and in 23 of 25 (92%) primary breast cancers. Immunodetection of p53 in paraffin-embedded tissue blocks will be a useful alternative to the standard fresh-tissue assay and can accurately reflect the level of p53 expression in human tumors.

Pragmatic factors influencing the detection of Neisseria gonorrhoeae.

The effects of the suboptimal temperature caused by delayed incubation or prolonged transport on the recovery of Neisseria gonorrhoeae were studied in 2 separate patient populations. The individual components of the John E. Martin Biological Environmental Chamber were used to define more precisely other variables that might influence the incidence of recovery. In a study of 1500 urethral and endocervical specimens processed in parallel for N gonorrhoeae, results quantitatively and qualitatively superior were achieved when an immediate source of CO2 and a zip-lock bag were used. The zip-lock bag appears to be a significant factor in the enhanced demonstration of N gonorrhoeae. Failure to incubate specimens before transportation decreased the numeric representation of N gonorrhoeae in 50 sets of triple cultures studied in parallel. Most low-inoculum cultures subjected to prolonged transportation without prior incubation will not demonstrate the presence of N gonorrhoeae. Even if preincubated before transportation, the cultures demonstrate a significant reduction in the number of colony-forming units per plate when subjected to normal room temperature during the course of transportation.

Urinary tract infection caused by Staphylococcus saprophyticus.

This study was designed to determine whether Staphylococcus saprophyticus was an important cause of urinary tract infection (UTI), as has been reported by European, but not by American, investigators, S. saprophyticus was the second most common cause of UTI in young (mean age, 20 years), sexually active female outpatients without known preexisting kidney disease or preceding manipulation of the urinary tract. Most cases presented as acute cystitis, but frank pyelonephritis and UTI in pregnant females were observed. The organism was rarely found as a contaminant in urine cultures. When present in the mucocutaneous flora of the anal-urogenital area, the organism was significantly associated with UTI by the same organism. These results show that S. saprophyticus should be accepted as an important urinary tract pathogen of young female patients in the United States. A simple adequate laboratory identification may be based on resistance to novobiocin (disk diffusion test), absence of hemolysis and coagulase, and intense pigment production (65% of strains yellow, 35% white).