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PubMed1994

[Changes in fibrinolysis-associated parameters in HELLP syndrome].

Various parameters associated with fibrinolysis were examined in plasma and placenta tissue extract in 18 patients with HELLP syndrome (haemolysis, elevated liver enzymes, and low platelet count). A significant increase in plasma concentrations of tPA and PAl-1 was observed in the HELLP patients in comparison to 18 pregnant women of the control group (equal duration of pregnancy). In contrast, there was no difference in plasma concentrations of uPA, uPA-receptor and D-dimer. In placenta tissue extract, significant differences were found just as rarely for uPA, uPA-receptor and PAl-1 as for tPA and D-dimer. Our findings indicate, that endothelial damage with release of tPA may be involved in the pathophysiological pathway of HELLP syndrome. Increased plasma levels of PAl-1 may reflect deficient fibrinolysis resulting in impairment in microcirculation. Clinical relevance of tPA and PAl-1 plasma concentration as possible predictor of hypertensive pregnancy complications will have to be studied further.

Adult↗
PubMed1987

Characterization of the 31P NMR spectrum of the schistosome vector Biomphalaria glabrata and of the changes following infection by Schistosoma mansoni.

The 31P nuclear magnetic resonance (NMR) spectrum of the digestive gland-gonad complex (DGG) of the schistosome vector Biomphalaria glabrata was characterized and the effects of infection by Schistosoma mansoni noted. The in vivo spectrum was comprised of 11 peaks, 5 downfield and 6 upfield of an external 85% phosphoric acid standard. Based on a variety of analytical procedures, the upfield peaks from the standard were demonstrated to be composed of carbamoyl phosphate + a mixture of 3 phosphatides and sphingomyelin, the gamma + beta phosphorus resonances of nucleotide triphosphate (NTP) and nucleotide diphosphate (NDP), respectively, the alpha phosphorus resonances of NTP + NDP, NAD(H) + the phosphorus resonance of uridine phosphate from uridine diphosphoglucose (UDPG), the phosphorus resonance of glucose phosphate from UDPG and, last, the beta phosphorus resonance of NTP. The downfield peaks were assigned as glycerophosphoryl choline, intracellular inorganic phosphate (Pi), sugar phosphates + phosphoryl choline, aminoethyl phosphonate (AEP), and ceramide AEP. T1 values for the in vivo NMR components were determined by inversion recovery. Infection produced distinct alterations in the levels of nonnucleotide components of the in vivo 31P NMR spectrum and the spectra of tissue extracts. Specifically, the levels of phosphonate, phospholipids, and carbamoyl phosphate were markedly reduced, and the relative level of Pi was increased. The potential significance of these changes to the parasite-host relationship was discussed. In contrast, starvation resulted in a decreased level of phosphonate only. The pH of the intact DGG was estimated by titrating the inorganic phosphate component of tissue extracts. The mean pH was 6.9 for both control and infected material.

Animals↗
PubMed1997

FGF2 suppresses neuronogenesis of a cell line derived from rat olfactory epithelium.

Neurogenesis continues throughout adulthood in the mammalian olfactory epithelium (OE), and both neurons as well as nonneuronal cells are reconstituted following experimental injury. Underlying the capacity of the OE to replenish its mature elements is a population of progenitor basal cells. Although the precise lineage relationships among progenitor and mature cell types are incompletely understood, the population of globose basal cells (GBCs) contains immediate precursors to neurons as well as amplifying progenitors, and retroviral lineage analyses suggest that multipotential GBCs are activated following direct injury to the OE. To assess the controls on the process of epithelial regeneration, we have characterized a cell line derived from rat OE and studied the effects of serum and tissue extracts, fibroblast growth factor-2 (FGF2) and transforming growth factor-alpha (TGF alpha) on the cells. Using a panel of cell type-specific markers whose patterns of labeling in the OE are well defined, including recently developed markers for GBCs, we characterized the phenotype of the cell line under differing culture conditions. In complete medium, which contains serum and tissue extracts, the cell line displayed characteristics of GBCs that are prominent during regeneration. Serum and extract withdrawal induced the cells to differentiate into neurons. In contrast, FGF2 prevented neuronal differentiation and maintained a GBC phenotype. TGF alpha had a mitogenic or differentiative effect that was context dependent. Finally, we demonstrate here that FGF2 is contained in mature olfactory neurons and sustentacular cells in vivo, suggesting a physiologic role for this growth factor in OE cell regulation.

Animals↗
PubMed1990

[Radionuclide study on the change of duct-ligated parotid gland].

Regressive changes of duct-ligated parotid glands in rabbits were examined by salivary gland scintigraphy with 99mTc O4-, subtraction sialography and soft X-ray radiography. Scintigram images were obtained with a pinhole collimator improved for small animal scintigraphy. Subtraction images were taken photographically. Contrast media were injected into extracted tissue for soft X-ray radiography. Radiological observations were carried out 3, 7, 14, 21, 28, 35 and 42 days after injection, and in the control rabbit. The images obtained by these three modalities were compared and discussed. Salivary gland scintigrams and subtraction sialograms clearly demonstrated the outlines of duct ligated parotid glands. Soft X-ray radiograms obtained from the extracted tissue showed the fine structure of the parotid glands. All of these images showed regressive changes in the duct-ligated parotid glands.

Animals↗
PubMed2002

Trace element speciation in largemouth bass ( Micropterus salmoides) from a fly ash settling basin by liquid chromatography-ICP-MS.

Analytical techniques used to examine the chemical speciation of multiple trace elements are important for the investigation of biological systems. Size exclusion chromatography (SEC) coupled to ICP-MS was used to investigate the speciation of Se, As, Cu, Cd and Zn in tissue extracts from a largemouth bass (Micropterus salmoides) collected from a coal fly ash basin and results were compared to a largemouth bass collected at a reference site. Using a Biosil SEC column, with an effective separation range of 100-7 KDa, Cu, Zn and Cd were shown to be bound to metallothionein (MT) in the liver, gill and, to a lesser extent, gonad tissue extract. In liver, muscle and gill of the ash basin bass, Se was predominantly present as low molecular weight species. Only in the gonad extract was the major fraction of Se associated with high molecular weight species. For the liver and gill extracts, further SEC-ICP-MS on a column with an effective separation range of 7000-500 Da was performed, but Se species still eluted near the total volume of the column suggesting a low molecular weight organic or inorganic species. Ion chromatography (IC)-ICP-MS using an AS7 column and HNO(3) gradient elution indicated that the Se and As species in the liver and gill extracts had similar retention times but these retention times did not correspond to retention times for As(III), As(V), dimethylarsenate, arsenobetaine, Se(IV), Se(VI), seleno-methionine, or seleno-cystine.

Animals↗
PubMed2000

Elevated humoral immune response to heat shock protein 60 (hsp60) family in periodontitis patients.

The presence of antibodies to the 60-kD human and Porphyromonas gingivalis GroEL hsp60 in the sera and inflamed gingival tissues of periodontitis patients was examined. In order to obtain the antigens, recombinant plasmids carrying human hsp60 and P. gingivalis GroEL genes were constructed and expressed as histidine-tagged recombinant proteins. Immunoreactivities of these proteins were confirmed by MoAbs specific to mammalian hsp60 and cross-reactive with both mammalian and bacterial hsp60. Western blot analysis clearly demonstrated that the number of periodontitis patients showing a positive response to P. gingivalis GroEL was higher than the number of periodontally healthy subjects. Furthermore, anti-P. gingivalis GroEL antibody was detected in all samples of gingival tissue extracts. For human hsp60, a higher frequency of seropositivity was found in the periodontitis patients than in the healthy subjects. In addition, the periodontitis patients demonstrated stronger reactivity compared with the healthy subjects. Quantitative analysis of serum antibodies by ELISA also demonstrated that the levels of antibodies in the sera of patients were significantly higher than those of control subjects. In the gingival tissue extracts, seven out of 10 patients demonstrated a positive response to human hsp60 and tso of these demonstrated strong positivity. Affinity-purified serum antibodies to human hsp60 and P. gingivalis GroEL from selected patients reacted with P. gingivalis GroEL and human hsp60, respectively, suggesting cross-reactivity of antibodies. These results suggest that molecular mimicry between GroEL of the periodontopathic bacterium P. gingivalis and autologous human hsp60 may play some role in immune mechanisms in periodontitis.

Adult↗
PubMed2002

[Effects of intrasplenic transplantation of IL-18 gene-modified fetal hepatocytes on mouse immune function]

OBJECTIVE: To investigate the effects of IL-18 gene-modified fetal hepatocytes (AdmIL-18/MNL.CL2) intrasplenic transplantation on mouse immune function. METHODS: Forty mice were evenly divided into 4 groups of 10. Each group received an intrasplenic transplantation one of the following: AdmIL-18/BNL.CL2, Ad-LacZ/BNL.CL2 (virus control), BNL.CL2 (cell control) and PBS (blank control). After two weeks, the mice were sacrificed. Serum cytokine levels, Mpsi and splenic cell culture supernatant and liver tissue extracts supernatants were measured using ELISA. Hepatic cytokines mRNA expression were determined by RT-PCR. THe cytotoxicity of peritoneal Mpsi and NK activity of spienocytes were detected by LDH release assay. The proliferation of splenic lymphocytes was determined by MTT assay. RESULTS: The IL-18, IL-2,IFN-gamma, TNF-alpha levels of serum, Mpsi and splenocyte culture supernatant, liver tissue extracts supernatants in mice transplanted with AdmIL-18/BNL.CL2 were higher and the IL-4, IL-10 levels were lower compared to their levels in other 3 groups. The highest IL-18, IL-2, IFN-gamma, TNF-alpha and the lowest IL-4, IL-10 mRNA expressions in the liver were observed in mice transplanted with AdmIL-18/BNL.CL2. The mice transplanted with AdmIL-18/BNL.Cl2 showed significantly increases cytotoxicity of Mpsi, NK activity and splenic cell proliferation compared with the other 3 groups. CONCLUSION: AdmIL-18 can be effectively transfected into mice fetal heptocytes which subsequently IL-18. Intransplenic transplantation of IL-18 gene-modified fetal hepatocytes may augment mouse immune function and provide an useful basis for targeted gene therapy of liver disease.

Journal Article↗
PubMed2005

Confirmation of peak assignments in capillary electrophoresis using immunoprecipitation. Application to D-aspartate measurements in neurons.

Capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection is a powerful tool for analysis of samples ranging from tissue extracts to single cells. However, accurate peak identification in electropherograms is challenging when complex biological samples are analyzed, as often matching a migration time between an analyte and corresponding standard may be insufficient to confirm the peak's identity. A method which combines single-step immunoprecipitation and CE-LIF analysis for investigation of the chiral amino acids in single cells and small tissue samples is demonstrated. D-Aspartate (D-Asp) has been reported in the central nervous system of the invertebrate neurobiological model Aplysia californica. In order to confirm the identity of D-Asp signal in the complex electropherograms of nerve tissue extracts and individual neurons, anti-D-Asp serum, preincubated with L-Asp conjugate, is added to the sample. This selectively binds the free D-Asp, creating an antibody-antigen complex with a migration time similar to that of antibody alone, but not that of D-Asp. The complete disappearance of the putative D-Asp peak confirms its identity and validates that there are no other detectable analytes co-migrating with D-Asp in the electropherogram.

Animals↗
PubMed1991

Kinetics of plasma fibronectin: increased lung tissue incorporation after postoperative bacteremia.

Fibronectin is an adhesive glycoprotein that may influence the permeability of the vascular barrier. We compared the plasma disappearance of purified human fibronectin (hFn) and its incorporation into lung tissue in control nonbacteremic and bacteremic sheep after surgery to determine the influence of postoperative bacteremia on the plasma clearance and lung deposition of hFn. Lymph fistulas were surgically prepared 48 h before either saline or bacterial challenge to allow for collection of timed samples of plasma, lung lymph, and peripheral lymph. On the day of the study, the sheep were anesthetized and given either 5 X 10(8) Pseudomonas aeruginosa in saline or saline alone. In parallel, they were infused intravenously with 100 mg of hFn, and the hFn concentration in plasma, lymph, and tissue extracts was subsequently determined by enzyme-linked immunosorbent assay over an 8-h experimental interval. Localization of endogenous sheep fibronectin (sFn) and the injected hFn in serial lung tissue samples was determined by dual-label immunofluorescence. In nonbacteremic control sheep, plasma hFn levels declined with an initial rapid slope (t1/2 = 0.53 +/- 0.19 min), followed by a second, gradual slope (t1/2 = 21.7 +/- 2.5 h), whereas the concentration of hFn in lung lymph and peripheral lymph rose exponentially with time. In bacteremic sheep, the early plasma disappearance of hFn was similar, but the second phase of plasma clearance was faster (t1/2 = 7.4 +/- 0.3 h). Lung tissue from control and bacteremic sheep contained the same level of extractable hFn. However, tissue extraction followed by immunofluorescence microscopy indicated that lung tissue from bacteremic sheep contained more nonextractable hFn than lung tissue from nonbacteremic sheep. Thus postoperative bacteremia that elicits acute lung vascular injury will increase the plasma disappearance of hFn and its incorporation into the lung tissue.

Animals↗
PubMed1994

Retention of a mutagen, 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), in the liver of mice infected with Schistosoma japonicum.

Regarding the mechanism underlying the suspected enhancement of hepatic cancers among Schistosoma japonicum-infected humans, we hypothesized that mutagen exposures in the livers of patients may be enhanced due to the parasitic infection. To explore this possibility, we have done a model experiment using mice and a carcinogenic mutagen, 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2). Mice infected with Schistosoma japonicum were intravenously administered Trp-P-2, and the mutagenic activities of the mouse serum and of the liver tissue extracts, which were observable during the 6-h period after the administration, were investigated. The level of serum indirect mutagenicity, which probably reflected the amount of unmetabolized Trp-P-2, was higher in the infected animals than in uninfected control animals. Direct mutagenicity in the serum, on the other hand, was higher in the control animals than in the infected mice. Furthermore, the liver tissue extracts from infected mice showed higher indirect-mutagenicity than those from the controls. These data suggest that the infection results in a decreased metabolism and an increased retention of Trp-P-2 in the liver. Consistent with this phenomenon, pigments in the liver formed by the schistosome infection were found to be an efficient adsorbent for Trp-P-2. Thus, the possibility exists that these pigments, which contain hematin as a major constituent, may function as a reservoir for the mutagen, thereby prolonging the exposure period of the liver to the mutagen.

Adsorption↗
PubMed1993

Epidermal growth factor and transforming growth factor alpha concentrations in BPH and cancer of the prostate: their relationships with tissue androgen levels.

We measured immunoreactive EGF and TGF alpha in prostate tissue extracts obtained from 19 patients with benign prostatic hyperplasia (BPH) and 19 with cancer of the prostate (CaP). Whilst both BPH and CaP expressed EGF (BPH = 195.61 +/- 19.94 ng g-1 protein; CaP = 235.60 +/- 24.45 ng g-1 protein) and TGF alpha (BPH = 92.57 +/- 7.60 ng g-1 protein; CaP = 100.73 +/- 15.47 ng g-1 protein) in equal concentrations, the levels of EGF in any tissue extract were on average twice those of TGF alpha. Furthermore analysis of the individual growth factor data revealed a direct correlation between EGF and TGF alpha in both BPH (r = 0.72, P < 0.001) and CaP (r = 0.69, P < 0.001). When the tumours were classified according to their Gleason score, a slight but significant increase in growth factor concentrations was noted as the tumour became less differentiated. We also measured the concentrations of testosterone and dihydrotestosterone (DHT) in prostate extracts with a view of elucidating the relationship between androgen and growth factors in this gland. There was a small positive correlation only between testosterone and EGF (r = 0.62, P < 0.05) and testosterone and TGF alpha (r = 0.61, P < 0.05) in CaP. The absence of any similar correlation in BPH where DHT becomes the predominant hormone may suggest an indirect role for testosterone in the regulation of growth factor production.

Androgens↗
PubMed1989

Enzymatic cycling assay for phenylpyruvate.

Enzymatic cycling assays for the determination of L-phenylalanine and phenylpyruvate in deproteinized tissue extracts are described. Assay 1 couples glutamine transaminase K with L-phenylalanine dehydrogenase. Assay 2 combines phenylalanine dehydrogenase, L-amino acid oxidase, and catalase. In both assays, tyrosine and some other amino acids (or their alpha-keto acid analogs) can replace phenylalanine (or phenylpyruvate) to a small extent. Thus, if phenylalanine is to be measured a correction must be made for the nonspecificity of the reaction. By removing phenylalanine on a cation-exchange column it was possible to measure phenylpyruvate in tissue extracts. Concentrations of phenylpyruvate (mumol/kg) in normal rat liver, kidney, and brain were 2.1 +/- 1.1 (n = 8), 1.8 +/- 0.4 (n = 4), and 3.3 +/- 0.6 (n = 4), respectively.

Amino Acid Oxidoreductases↗
PubMed1998

Direct ELISA method for the specific determination of prothymosin alpha in human specimens.

An enzyme linked immunosorbent assay, specific for prothymosin alpha (ProT alpha) was developed using an antibody against the synthetic C-terminal peptide ProT alpha[101-109] and isolated bovine ProT alpha for the preparation of standard solutions and immunoplates. Due to the antibody used, the ELISA developed was capable of fully discriminating between ProT alpha, the naturally occuring and partially homologous peptide parathymosin alpha (ParaT alpha) and the peptide thymosin alpha1 (T alpha1), whose sequence is identical to the [1-28] sequence of ProT alpha, and its in vivo occurrence is under question. Moreover, due to its improved sensitivity, the ELISA was capable of directly determining ProT alpha concentration in human serum and tissue extracts, without any pretreatment of the samples. ProT alpha levels were directly measured in sera obtained from 48 apparently healthy individuals and 27 patients with diagnosed breast cancer and found to range from 0.67 to 2.34 microg/ml (mean value 1.27 +/- 0.49 microg/ml) and from 0.47 to 1.74 microg/ml (mean value 1.02 +/- 0.29 microg/ml), respectively. ProT alpha levels were also measured in four breast tumor and adjacent normal breast tissue extracts and found to be elevated in the tumor extracts.

Adolescent↗
PubMed1977

A neutrophil chemotactic factor and its inhibitor found in DNCB-induced skin inflammatory lesions.

Neutrophil chemotactic factor(s) and their inhibitors were explored in the acute inflammatory skin lesions induced by the application of 4% DNCB solution with acetone in guinea pigs. Skin biopsies were taken periodically and tissue extracts were made from the biopsy specimens. Neutrophil chemotactic activity found in such extracts reached a peak at 12 to 24 hr after the induction of the inflammation, when the lesions were found to be infiltrated predominantly with neutrophils. After 24 hr, the activity gradually diminished. Physicochemical and antigenic characterization studies on the chemotactic substance indicated that the material was most likely the cleavage product of C3. On the other hand, inhibitors against the neutrophil chemotactic factor were found in the tissue extracts which were obtained from the lesions at a later stage (48 to 96 hr after the induction of the inflammatory reaction). These inhibitors blocked not only the complement-derived chemotactic activity but also that obtained from bacterial culture filtrates. They were heat labile and showed striking heterogeneity in size on Sephadex gel filtration.

Animals↗
PubMed1996

Up-regulation of urokinase-type plasminogen activator in squamous cell carcinoma of human larynx.

The expression of urokinase-type plasminogen activator (uPA) was investigated in squamous cell carcinoma of the human larynx. For this purpose, tissue extracts from 25 matched samples of normal mucosa and neoplastic larynx were compared for the levels of uPA activity as evaluated by a chromogenic PA assay and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) zymography. Also, uPA antigen was quantified by enzyme-linked immunosorbent assay (ELISA) in 19 cases. The results demonstrate a significant increase in the levels of uPA activity and protein in tumour tissue extracts, more pronounced in tumours with lymph node metastases. Immunohistochemistry performed on 70 biopsies showed that uPA positivity is present both in neoplastic cells and in fibroblast-like cells and macrophages. However, depending on the histological grading and invasive capacity of the tumour, a pronounced intra- and intertumoral heterogeneity in uPA staining was observed. In situ hybridisation confirmed the presence of uPA mRNA in both tumour and stromal cells. The present study provides experimental evidence for a role of uPA in the invasive growth of human laryngeal carcinoma.

Antigens, Neoplasm↗
PubMed1991

Tissue levels of bone resorptive cytokines in periodontal disease.

The levels of 3 bone resorptive cytokines, interleukin 1 alpha (IL-1 alpha), IL-1 beta, and tumor necrosis factor alpha (TNF alpha), were assessed in tissues from sites of periodontal disease. As determined by ELISA of tissue extracts, IL-1 beta and TNF alpha were detected in all diseased sites, whereas IL-1 alpha was present in 8/22 sites, IL-1 beta was present in highest concentration (mean +/- SEM: 11,695 +/- 2,888 pg/ml; 672 pM), followed by TNF alpha (434 +/- 135 pg/ml; 26 pM), and IL-1 alpha (342 +/- 160 pg/ml; 20 pM). The levels of all 3 mediators were significantly lower in clinically healthy tissues. There was a highly significant correlation between levels of IL-1 beta and TNF alpha (rs = 0.61, P less than 0.001), suggesting coordinated expression of these 2 mediators. The numbers of cells containing each mediator was also determined by indirect immunofluorescence on frozen tissue sections. Consistent with findings from tissue extracts, IL-1 beta-containing cells were present in approximately 5-fold higher numbers than TNF alpha-containing cells, and 40-fold higher numbers than IL-1-alpha-containing cells. Taken together with previous findings, these results indicate that IL-1 beta is likely to be an important mediator in the pathogenesis of periodontal disease.

Adult↗
PubMed1987

A direct determination of taurine in perchloric acid-deproteinized biological samples.

A high-performance liquid chromatographic method was developed to estimate the amount of taurine in tissue extracts and body fluids. Shodex Ionpak C-811, an adsorption-distribution type column, was chosen and combined with 3 mM perchloric acid as the mobile phase. The eluted taurine was derivatized with o-phthaladehyde-2-mercaptoethanol reagent and measured at an absorbance of 350 nm instead of fluorescent intensity. Using this method, it was possible to isolate and estimate the amount of taurine from other coexisting substances or structural analogs such as hypotaurine, cysteamine, or cysteinesulfinic acid. For rapid routine assays of taurine in many biological samples, it is advisable that the tissue extracts be pretreated with cation-exchange resin to exclude other interfering substances including O-phosphoserine. The linear relationship between the concentration of taurine and absorbance achieved over the concentration range 0.1-10.0 nmol and the recoveries obtained were 98-101% by the internal standard method. The specificity, reproducibility, and sensitivity of this method for taurine were good enough to be used for biological applications. Examples of the tissue levels of taurine in various organs of the rat, as determined by this new method, are also presented.

Animals↗
PubMed2005

Stroke-induced migration of human umbilical cord blood cells: time course and cytokines.

The therapeutic window for treatment of individuals after stroke is narrow, regardless of the treatment regime; extension of this window would provide a major therapeutic advance. In prior reports, we demonstrated significant improvements in the behavioral defects of rats that received human umbilical cord blood (HUCB) cells 24 h after a middle cerebral arterial occlusion. These effects paralleled the recruitment of these cells to the site of tissue damage. While the administration of HUCB cells 24 h after stroke was effective, the optimal time to administer these cells after stroke has not been established. Here, we investigated the migration of HUCB cells to ischemic tissue extracts. After ischemic assault, brain tissue was homogenized, and the supernatants were assayed for their ability to attract HUCB mononuclear cells as well as for levels of several cytokines. We demonstrate increased migratory activity of HUCB cells toward the extracts harvested at 24-72 h after stroke. The extracts possessed increased levels of certain cytokines and chemokines, suggesting their participation in HUCB cell migration. The results from this study are promising in that the current 3-h therapeutic window for the treatment of stroke victims, using approved anticoagulant treatment, may be extended with the use of HUCB cell therapy 24-72 h post stroke. Last, the chemokines present in the supernatant provide a sound starting point to start examining the mechanisms responsible for the in vivo migration of HUCB cells after the induction of stroke.

Animals↗