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1.
J Perinatol ; 37(11): 1204-1209, 2017 11.
Article in English | MEDLINE | ID: mdl-28837136

ABSTRACT

OBJECTIVE: The significance of cord blood neutropenia as a screening tool for early-onset sepsis (EOS) is unclear. The objectives were to define reference values for cord blood neutrophil count and to determine the sensitivity and positive likelihood ratio of cord neutropenia for the detection of EOS. STUDY DESIGN: This retrospective observational cohort study included all mother-infant pairs with deliveries between 2009 and 2014 for whom cord neutrophil counts were routinely done. EOS cases were identified by interrogation of electronic charts. Maternal and perinatal factors were assessed to determine reference values of cord neutrophil. The diagnostic value of neutropenia for detecting EOS was assessed. A nested case-control design was used to measure the value of neutropenia in the detection of EOS in comparison with other risk factors. RESULTS: A total of 8,590 mother-infant pairs were included. We identified 84 sepsis cases. The neutrophil count was strongly associated with gestational age. Neutropenia adjusted for gestational age was strongly associated with EOS and had good specificity but poor sensitivity. The addition of neutropenia to other EOS risk factors increased sensitivity without decreasing specificity. CONCLUSION: Cord blood neutropenia was significantly associated with EOS and the addition of cord neutropenia to current EOS risk factors increased the detection rate of EOS.


Subject(s)
Fetal Blood , Neutropenia/blood , Sepsis/diagnosis , Case-Control Studies , Female , Gestational Age , Humans , Infant, Newborn , Inflammation Mediators/blood , Male , Predictive Value of Tests , Pregnancy , Retrospective Studies , Risk Factors , Sepsis/epidemiology
2.
J Physiol Anthropol ; 35: 9, 2016 Mar 15.
Article in English | MEDLINE | ID: mdl-26980095

ABSTRACT

Notwithstanding its effects on the classical visual system allowing image formation, light acts upon several non-image-forming (NIF) functions including body temperature, hormonal secretions, sleep-wake cycle, alertness, and cognitive performance. Studies have shown that NIF functions are maximally sensitive to blue wavelengths (460-480 nm), in comparison to longer light wavelengths. Higher blue light sensitivity has been reported for melatonin suppression, pupillary constriction, vigilance, and performance improvement but also for modulation of cognitive brain functions. Studies investigating acute stimulating effects of light on brain activity during the execution of cognitive tasks have suggested that brain activations progress from subcortical regions involved in alertness, such as the thalamus, the hypothalamus, and the brainstem, before reaching cortical regions associated with the ongoing task. In the course of aging, lower blue light sensitivity of some NIF functions has been reported. Here, we first describe neural pathways underlying effects of light on NIF functions and we discuss eye and cerebral mechanisms associated with aging which may affect NIF light sensitivity. Thereafter, we report results of investigations on pupillary constriction and cognitive brain sensitivity to light in the course of aging. Whereas the impact of light on cognitive brain responses appears to decrease substantially, pupillary constriction seems to remain more intact over the lifespan. Altogether, these results demonstrate that aging research should take into account the diversity of the pathways underlying the effects of light on specific NIF functions which may explain their differences in light sensitivity.


Subject(s)
Aging/radiation effects , Brain/radiation effects , Cognition/radiation effects , Light , Vision, Ocular/radiation effects , Aging/physiology , Brain/physiology , Cognition/physiology , Humans , Vision, Ocular/physiology
3.
J Mol Biol ; 294(2): 321-32, 1999 Nov 26.
Article in English | MEDLINE | ID: mdl-10610761

ABSTRACT

We have recently presented evidence that inhibitory R-loops form during transcription in topA null mutants when the nascent RNA anneals with the template DNA strand behind the moving RNA polymerase. This was supported by the results of in vitro transcription assays and by in vivo studies in which R-loop formation was shown to be inhibited by coupled transcription-translation. The results presented here support this model and further demonstrate the link between R-loop formation and growth inhibition of topA null mutants. First, we show that RNase H activity is essential in the absence of DNA topoisomerase I. This was observed even if the growth of the topA null mutant is compensated for by naturally selected mutations, that also reduce global supercoiling below the wild-type level. Second, we show that R-loop-dependent hypernegative supercoiling increases as the temperature decreases and correlates with growth inhibition of topA null mutants. In fact, RNase H overproduction is shown to be detrimental to cell growth at 21 degrees C. Presumably, several mRNAs are being sequestered in R-loops and their degradation by RNase H significantly impedes protein synthesis. We propose that a reduced transcription velocity at low temperatures favors the annealing of the nascent RNA with the template strand behind the moving RNA polymerase, in agreement with the results of previous studies. Finally, based on the currently available data on R-loop formation, we present a model that explains the sensitivity of topA null mutants to various environmental changes that are often accompanied by transient inhibition of translation.


Subject(s)
DNA Topoisomerases, Type I/genetics , DNA, Bacterial/chemistry , Escherichia coli/genetics , RNA, Bacterial/chemistry , Cell Division/genetics , DNA Topoisomerases, Type I/metabolism , DNA, Bacterial/genetics , Escherichia coli/cytology , Mutation , Nucleic Acid Conformation , Ribonuclease H/genetics , Ribonuclease H/metabolism , Temperature
4.
J Biol Chem ; 274(23): 16654-8, 1999 Jun 04.
Article in English | MEDLINE | ID: mdl-10347233

ABSTRACT

It has been suggested that the essential function of DNA topoisomerase I in Escherichia coli is to prevent chromosomal DNA from reaching an unacceptably high level of global negative supercoiling. However, other in vivo studies have shown that DNA topoisomerase I is very effective in removing local negative supercoiling generated during transcription elongation. To determine whether topoisomerase I is essential for controlling global or local DNA supercoiling, we have prepared a set of topA null mutant strains in combination with different plasmid DNAs. Although we found a correlation between the severity of the growth defect with both transcription-induced and global supercoiling, near to complete growth inhibition correlated only with transcription-induced supercoiling. This result strongly suggests that the major function of DNA topoisomerase I is to relax local negative supercoiling generated during transcription elongation.


Subject(s)
DNA Topoisomerases, Type I/metabolism , Escherichia coli/enzymology , Nucleic Acid Conformation , Transcription, Genetic , Antiporters/genetics , Antiporters/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , DNA, Bacterial/chemistry , DNA, Bacterial/metabolism , Escherichia coli/genetics , Escherichia coli/growth & development , Promoter Regions, Genetic , Tetracycline Resistance/genetics
5.
J Biol Chem ; 274(23): 16659-64, 1999 Jun 04.
Article in English | MEDLINE | ID: mdl-10347234

ABSTRACT

It has recently been shown that RNase H overproduction can partially compensate for the growth defect due to the absence of DNA topoisomerase I in Escherichia coli (Drolet, M., Phoenix, P., Menzel, R., Massé, E., Liu, L. F., and Crouch, R. J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 3526-3530). This result has suggested a model in which inhibitory R-loops occur during transcription in topA mutants. Results presented in this report further support this notion and demonstrate that transcription-induced supercoiling is involved in R-loop formation. First, we show that stable R-loop formation during in vitro transcription with E. coli RNA polymerase only occurs in the presence of DNA gyrase. Second, extensive R-loop formation in vivo, revealed by the production of RNase H-sensitive hypernegatively supercoiled plasmid DNAs, is observed under conditions where topA mutants fail to grow. Furthermore, we have demonstrated that the coupling of transcription and translation in bacteria is an efficient way of preventing R-loop formation.


Subject(s)
DNA Topoisomerases, Type I/metabolism , Escherichia coli/enzymology , Transcription, Genetic , Antiporters/genetics , Bacterial Proteins/genetics , DNA Topoisomerases, Type II/metabolism , DNA, Bacterial/metabolism , DNA-Directed RNA Polymerases/metabolism , Escherichia coli/drug effects , Escherichia coli/genetics , Escherichia coli/growth & development , Protein Synthesis Inhibitors/pharmacology , Ribonuclease H/metabolism , Ribosomes/metabolism , Spectinomycin/pharmacology , Tetracycline Resistance/genetics
6.
Hum Gene Ther ; 9(7): 1069-81, 1998 May 01.
Article in English | MEDLINE | ID: mdl-9607418

ABSTRACT

We have used our previously described ex vivo mesothelial cell (MC)-mediated gene therapy strategy (Gene Ther. 2:393-401, 1995) to modify the functional properties of the rat parietal peritoneal mesothelium in vivo by expression of a membrane-bound recombinant protein on the MC surface. Rat primary MCs were stably transfected (using strontium phosphate DNA coprecipitation) with a plasmid containing the gene for rat thrombomodulin (TM), a transmembrane glycoprotein that functions as an essential cofactor for the physiological activation of the anticoagulant protein C by the enzyme thrombin. As demonstrated by immunohistochemistry and by direct equilibrium binding with radiolabeled thrombin, genetically modified MCs expressed high levels of TM antigen on their surface in vitro. As judged by a thrombin-dependent protein C activation assay, such MC membrane-bound TM was biologically active. Once reseeded on the denuded parietal peritoneal surface of syngeneic recipients, these TM-transfected MCs continued to express TM antigen in vivo for at least 90 days. Moreover, the recombinant TM expressed on the reconstituted parietal mesothelium retained its ability to activate protein C in a thrombin-dependent manner. Our data indicate that MC-mediated expression of TM can be used to augment the anticoagulant properties of the parietal peritoneal surface. In general, our results suggest that ex vivo MC-mediated gene therapy can be used to deliver other therapeutic transmembrane proteins to the MC surface to enhance the functional repertoire of the parietal mesothelium in vivo.


Subject(s)
Anti-Inflammatory Agents , Anticoagulants , Epithelial Cells/metabolism , Gene Transfer Techniques , Peritoneal Cavity/cytology , Thrombomodulin/genetics , Animals , Anti-Inflammatory Agents/metabolism , Anticoagulants/metabolism , Blotting, Northern , Cattle , Female , Gene Expression , Genetic Vectors , Plasmids , Precipitin Tests , Rabbits , Rats , Rats, Inbred F344 , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Thrombomodulin/metabolism , Transfection , Tumor Necrosis Factor-alpha/pharmacology
7.
Nephrol Dial Transplant ; 13(6): 1420-9, 1998 Jun.
Article in English | MEDLINE | ID: mdl-9641171

ABSTRACT

BACKGROUND: The efficacy of peritoneal dialysis and its success as a long-term treatment depends on the preservation of the integrity of the peritoneal membrane. With increasing time on dialysis, the membrane may become compromised resulting in decreased dialysing capacity. We have pursued an innovative strategy, i.e. genetic modification of the mesothelial cell to change the properties of the membrane to potentially improve its dialysing capacity and longevity, and have demonstrated the feasibility of this approach in a rat model of ex vivo gene transfer. The potential to regulate transgene expression in this model is examined here. METHODS: Rat peritoneal mesothelial cells (MCs) were stably modified to express human growth hormone (hGH) under control of the heavy metal ion and glucocorticoid-regulatable murine metallothionein-1 promoter. The effect of zinc and the synthetic glucocorticoid dexamethasone on hGH expression was analysed in MC clones maintained in continuous passage or stationary phase, and in our rat model of ex vivo gene transfer. RESULTS: Exposure of these clones to zinc and dexamethasone, either singly or in combination, resulted in significant (i.e. 2-200-fold) increases in hGH production. Zinc-induced modulation of hGH production was demonstrated in cells in continuous passage and stationary culture. Regulation was also demonstrated after ex vivo gene transfer by both the intraperitoneal administration of zinc ions or the systemic administration of dexamethasone. CONCLUSIONS: Our results demonstrate the modulation of transgene expression in MCs in vitro and in vivo, and suggest the potential for the regulation of gene expression in a genetically modified mesothelium that may ultimately be used for the delivery of therapeutic proteins to maintain peritoneal membrane viability in the peritoneal dialysis patient.


Subject(s)
Gene Expression Regulation , Gene Transfer Techniques , Peritoneum/cytology , Peritoneum/metabolism , Animals , Animals, Genetically Modified , Base Sequence , Clone Cells , DNA Primers/genetics , Dexamethasone/pharmacology , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Gene Expression Regulation/drug effects , Glucocorticoids/pharmacology , Human Growth Hormone/biosynthesis , Human Growth Hormone/genetics , Humans , Mice , Peritoneal Dialysis , Peritoneum/drug effects , Polymerase Chain Reaction , Promoter Regions, Genetic/drug effects , Rats , Zinc/pharmacology
8.
Can J Anaesth ; 44(8): 816-9, 1997 Aug.
Article in English | MEDLINE | ID: mdl-9260008

ABSTRACT

PURPOSE: One of the factors that can affect the distribution of local anaesthetic solutions in the subarachnoid space is the direction of the spinal needle through which injections are made. This study investigated the effect of the direction of the aperture of the Whitacre needle on the spread of hyperbaric bupivacaine in parturients undergoing elective caesarean section. METHODS: Forty healthy term parturients scheduled for caesarean delivery under spinal anaesthesia with 12 mg hyperbaric bupivacaine + 0.2 mg morphine were randomly assigned to one of two groups: needle orifice cephalad (I) or caudad (II). Spinal blocks were administered in the sitting position with patients being positioned supine immediately after. A blinded observer assessed the dermatome level of analgesia to ice every minute for the first 10 minutes, every three minutes for the following 35 min, then every 15 min until the sensory level regressed to T10. RESULTS: There was no difference between the groups regarding the maximal number of segments blocked cephalad to T11 (11.4 +/- 3.4: group I and 12.0 +/- 3.4: group II), time to highest cephalad spread of sensory block (22 +/- 10: group I and 19 +/- 10 min: group II), or time to regression to T10 (164 +/- 26: group I and 153 +/- 24 min: group II). The maximum decrease in blood pressure (33.9 +/- 9.6: group I and 36.8 +/- 11.8 mmHg: group II) and dosage of ephedrine administered (14.7 +/- 10.7: group I and 16.2 +/- 11.0 mg: group II) did not differ. CONCLUSION: The direction of the aperture of the Whitacre needle does not influence the spread of hyperbaric bupivacaine in the term parturient.


Subject(s)
Anesthesia, Obstetrical , Anesthesia, Spinal , Anesthetics, Local/administration & dosage , Bupivacaine/administration & dosage , Adult , Bupivacaine/pharmacokinetics , Double-Blind Method , Female , Humans , Injections , Pregnancy
9.
J Biol Chem ; 272(19): 12816-23, 1997 May 09.
Article in English | MEDLINE | ID: mdl-9139742

ABSTRACT

Recent in vivo and in vitro studies have suggested an important role for DNA topoisomerases in regulating R-loop formation during transcription in Escherichia coli. In the present report we present genetic and biochemical evidence strongly suggesting that R-loop formation can occur during transcription of a portion of the rrnB operon and that it is regulated by DNA topoisomerase activity. We found that a multicopy plasmid (pBR322) carrying an heavily transcribed portion of the rrnB operon cannot be transformed in topA mutants unless RNase H is overproduced. Transcription of the 567-base pair HindIII fragment from the rrnB operon allows the extraction of large amount of R-looped plasmid DNAs from a topA mutant, in a manner that depends on the intracellular level of RNase H activity. When DNA gyrase is sufficiently active, hypernegatively supercoiled plasmid DNA is produced if the same DNA fragment is transcribed in a topA mutant. The formation of such topoisomers most likely reflect the presence of extensive R-loops since it is sensitive to the intracellular level of RNase H activity. Finally, the formation of R-looped plasmid DNAs in an in vitro transcription system using phage RNA polymerases is also detected when the 567-base pair HindIII fragment is transcribed on a negatively supercoiled DNA template.


Subject(s)
DNA Topoisomerases, Type I/metabolism , Nucleic Acid Conformation , Transcription, Genetic , rRNA Operon , DNA, Superhelical/metabolism , Deoxyribonuclease HindIII/metabolism , Escherichia coli , Plasmids/metabolism , Ribonuclease H/metabolism
10.
J Biol Chem ; 272(3): 1473-9, 1997 Jan 17.
Article in English | MEDLINE | ID: mdl-8999816

ABSTRACT

We have recently found that stable R-loop formation occurs in vivo and in vitro when a portion of the Escherichia coli rrnB operon is transcribed preferentially in its physiological orientation. Our results also suggested that the formation of such structures was more frequent in topA mutants and was sensitive to the template DNA supercoiling level. In the present report we investigated in greater detail the involvement of DNA topoisomerases in this process. By using an in vitro transcription system with phage RNA polymerases, we found that hypernegative supercoiling of plasmid DNAs in the presence of DNA gyrase is totally abolished by RNase H, suggesting that extensive R-looping occurs during transcription in the presence of DNA gyrase. When RNase A is present, significant hypernegative supercoiling occurs only when the 567-base pair rrnB HindIII fragment is transcribed in its physiological orientation. This result suggests that more stable R-loops are being produced in this orientation. Our results also suggest that DNA gyrase can participate in the process of R-loop elongation. The strong transcription-induced relaxing activity of E. coli DNA topoisomerase I is shown to efficiently counteract the effect of DNA gyrase and thus inhibit extensive R-looping. In addition, we found that an R-looped plasmid DNA is a better substrate for relaxation by E. coli DNA topoisomerase I as compared with a non-R-looped substrate.


Subject(s)
DNA Topoisomerases, Type I/metabolism , rRNA Operon/genetics , DNA Topoisomerases, Type II/metabolism , DNA, Recombinant/chemistry , DNA, Recombinant/genetics , DNA, Superhelical/chemistry , DNA, Superhelical/genetics , DNA-Directed RNA Polymerases/metabolism , Deoxyribonuclease HindIII , Escherichia coli/enzymology , Escherichia coli/genetics , Nucleic Acid Conformation , Plasmids , Substrate Specificity , Transcription, Genetic , Viral Proteins
12.
Gene Ther ; 2(6): 393-401, 1995 Aug.
Article in English | MEDLINE | ID: mdl-7584114

ABSTRACT

We have developed a model system in the rat to test the feasibility of recombinant protein expression by genetically modified peritoneal mesothelial cells following autologous peritoneal implantation. Rat primary peritoneal mesothelial cells, isolated from parietal peritoneum by enzymatic digestion, were stably transduced (using a Moloney murine leukemia virus (MoMLV)-derived retroviral vector, BAG, expressing the Escherichia coli lacZ gene) to mark the cells with a reporter protein (beta-galactosidase, beta-gal). Such transduced mesothelial cells, tagged with DiO, a fluorescent lipophilic dye used for long-term tracing of transplanted cells, were then reseeded on the denuded peritoneal surface of syngeneic recipients. DiO-labeled, BAG-transduced mesothelial cells were observed to repopulate the denuded areas and remain attached there for > 90 days. Moreover, these genetically modified mesothelial cells continued to express the reporter gene product in vivo (ie beta-gal activity was present for at least 1 month). Our results demonstrate the feasibility of ex vivo gene therapy using peritoneal mesothelial cells.


Subject(s)
Genetic Therapy/methods , Genetic Vectors , Moloney murine leukemia virus , Recombinant Proteins/biosynthesis , Transfection/methods , Animals , Carbocyanines , Cell Line , Cells, Cultured , Epithelium/metabolism , Epithelium/transplantation , Escherichia coli/genetics , Female , Fluorescent Dyes , Genes, Bacterial , Peritoneum , Rats , Rats, Inbred F344 , Transplantation, Homologous , beta-Galactosidase/biosynthesis , beta-Galactosidase/genetics
13.
Gene Ther ; 2(6): 402-10, 1995 Aug.
Article in English | MEDLINE | ID: mdl-7584115

ABSTRACT

To evaluate the ability of genetically modified peritoneal mesothelial cells to deliver recombinant proteins to the systemic circulation, we used our previously described mesothelial cell-based ex vivo gene therapy strategy. Rat primary peritoneal mesothelial cells, isolated from parietal peritoneum by enzymatic digestion, were stably transfected (using strontium phosphate DNA co-precipitation) with the plasmid pSVTKgh to express a secreted reporter gene product, human growth hormone (hgh). Such hgh-secreting mesothelial cells were reseeded on the denuded peritoneal surface of syngeneic recipients and delivery of the reporter gene product to the systemic circulation was monitored by analysis of serum samples for the presence of hgh at various times after mesothelial cell implantation. Polymerase chain reaction (PCR) analysis demonstrated that the hgh-transfected mesothelial cells repopulated the denuded areas and remained attached there for at least 12 weeks. Moreover, these genetically modified mesothelial cells continued to express the reporter gene product in vivo and secreted hgh in sufficient quantity to be detected in the systemic circulation (ie statistically significant amounts of hgh could be measured in the serum of cyclosporine A-treated rats for at least 2 months; Mann-Whitney test, P < 0.05). Our results demonstrate the successful, sustained, systemic delivery of a recombinant protein by genetically modified peritoneal mesothelial cells following their reattachment to the peritoneal surface, and suggest the potential of ex vivo mesothelial cell-mediated gene therapy for the treatment of inherited or acquired disorders requiring delivery of therapeutic proteins to the circulation.


Subject(s)
Genetic Therapy/methods , Growth Hormone/biosynthesis , Recombinant Proteins/biosynthesis , Transfection , Animals , Base Sequence , DNA Primers , Epithelial Cells , Epithelium/metabolism , Epithelium/transplantation , Female , Growth Hormone/blood , Humans , Kanamycin Kinase , Molecular Sequence Data , Peritoneum , Phosphotransferases (Alcohol Group Acceptor)/biosynthesis , Polymerase Chain Reaction/methods , Rats , Rats, Inbred F344 , Recombinant Proteins/blood
14.
Proc Natl Acad Sci U S A ; 92(8): 3526-30, 1995 Apr 11.
Article in English | MEDLINE | ID: mdl-7536935

ABSTRACT

Previous biochemical studies have suggested a role for bacterial DNA topoisomerase (TOPO) I in the suppression of R-loop formation during transcription. In this report, we present several pieces of genetic evidence to support a model in which R-loop formation is dynamically regulated during transcription by activities of multiple DNA TOPOs and RNase H. In addition, our results suggest that events leading to the serious growth problems in the absence of DNA TOPO I are linked to R-loop formation. We show that the overexpression of RNase H, an enzyme that degrades the RNA moiety of an R loop, can partially compensate for the absence of DNA TOPO I. We also note that a defect in DNA gyrase can correct several phenotypes associated with a mutation in the rnhA gene, which encodes the major RNase H activity. In addition, we found that a combination of topA and rnhA mutations is lethal.


Subject(s)
DNA Topoisomerases, Type I/deficiency , Escherichia coli/growth & development , Nucleic Acid Conformation , Ribonuclease H/biosynthesis , Transcription, Genetic , Cold Temperature , DNA, Bacterial/metabolism , Escherichia coli/genetics , Genetic Complementation Test , Nucleic Acid Heteroduplexes/metabolism , Phenotype , RNA, Bacterial/metabolism , Ribonuclease H/genetics
15.
Cancer Res ; 55(2): 369-75, 1995 Jan 15.
Article in English | MEDLINE | ID: mdl-7812970

ABSTRACT

In the immediately preceding paper, we demonstrated that the microvasculature supplying peritoneal lining tissues of mice bearing either of two transplantable ascites carcinomas was hyperpermeable to circulating macromolecules. Solid tumors have been shown to exhibit similar levels of microvascular hyperpermeability, leading to extravasation of plasma proteins, including fibrinogen which clots on extravasation to form an extravascular fibrin gel. To determine whether similar extravasation and clotting of plasma fibrinogen occurred in ascites tumors, we used 125I-labeled fibrinogen (125I-F) as a tracer to measure inflow of fibrinogen into the peritoneal cavities, and influx and accumulation of fibrinogen/fibrin in the peritoneal lining tissues (peritoneal wall, mesentery, and diaphragm) of mice bearing syngeneic TA3/St or MOT ascites tumors. The percentage of circulating 125I-F that extravasated into the peritoneal cavity was increased from 10- to 50-fold in mice bearing either ascites tumor. Influx into the peritoneal walls of ascites tumor-bearing mice was 3-7 times that of control mice and became maximal on day 8 (TA3/St) and day 15 (MOT). Accumulation of 125I-F in ascites fluid and peritoneal lining tissues was also increased substantially in mice bearing these ascites tumors, reaching maximal values on days 7-8 (TA3/St) and 19-29 (MOT) at levels 2- to 3-fold (peritoneal wall) and 33- to 148-fold (ascites fluid) above control levels. Significant amounts of the 125I-F that accumulated in the peritoneal lining tissues of ascites tumor-bearing animals were insoluble in 3 M urea, consistent with clotting of 125I-F to cross-linked fibrin. Autoradiographs of SDS-PAGE gels performed on extracts of peritoneal lining tissues of both ascites tumors revealed the characteristic signature of cross-linked fibrin, i.e., gamma-gamma dimers and alpha-polymers. Fibrin was also identified in peritoneal lining tissues of both ascites tumors by immunohistochemistry. Taken together, these data indicate that fibrinogen, like other circulating macromolecules, extravasates into the peritoneal cavity and peritoneal lining tissues of ascites tumor-bearing mice and does so with kinetics similar to those of other macromolecular tracers we have studied. Moreover, a portion of the fibrinogen that extravasated into peritoneal lining tissues clotted to form a cross-linked fibrin meshwork which trapped tumor cells and favored their attachment to the peritoneal surface. By analogy with solid tumors, such fibrin deposits may also be expected to have a role in initiating angiogenesis and the generation of mature tumor stroma.


Subject(s)
Ascites/etiology , Capillary Permeability , Fibrinogen/metabolism , Peritoneal Cavity/blood supply , Peritoneum/blood supply , Animals , Ascites/metabolism , Female , Fibrin/analysis , Iodine Radioisotopes/pharmacokinetics , Male , Mammary Neoplasms, Animal/metabolism , Mice , Mice, Inbred C3H , Muscle, Skeletal/metabolism , Ovarian Neoplasms/metabolism , Peritoneum/chemistry , Peritoneum/metabolism
16.
Cancer Res ; 55(2): 360-8, 1995 Jan 15.
Article in English | MEDLINE | ID: mdl-7812969

ABSTRACT

Previous studies have shown that accumulation of tumor ascites fluid results in large part from increased permeability of peritoneal lining vessels (Nagy et al., Cancer Res., 49: 5449-5458, 1989; Nagy et al., Cancer Res., 53: 2631-2643, 1993). However, the specific microvessels rendered hyperpermeable have not been identified nor has the basis of peritoneal vascular hyperpermeability been established. To address these questions, TA3/St and MOT carcinomas, well-characterized transplantable murine tumors that grow in both solid and ascites form, were studied as model systems. Ascites tumor cells of either type were injected i.p. into syngeneic A/Jax and C3Heb/FeJ mice, and ascites fluid and plasma were collected at intervals thereafter up to 8 and 28 days, respectively. Beginning several days after tumor cell injection, small blood vessels located in tissues lining the peritoneal cavity (mesentery, peritoneal wall, and diaphragm) became hyperpermeable to several macromolecular tracers (125I-human serum albumin, FITC-dextran, colloidal carbon, and Monastral Blue B). Increased microvascular permeability correlated with the appearance in ascites fluid of vascular permeability factor (VPF), a tumor cell-secreted mediator that potently enhances vascular permeability to circulating macromolecules. VPF was measured in peritoneal fluid by both a functional bioassay and a sensitive immunofluorometric assay. The VPF concentration, total peritoneal VPF, ascites fluid volume, tumor cell number, and hyperpermeability of peritoneal lining microvessels were found to increase in parallel over time. The close correlation of peritoneal fluid VPF concentration with the development of hyperpermeable peritoneal microvessels in these two well-defined ascites tumors suggests that VPF secretion by tumor cells is responsible, in whole or in part, for initiating and maintaining the ascites pattern of tumor growth.


Subject(s)
Ascitic Fluid/etiology , Capillary Permeability , Endothelial Growth Factors/analysis , Lymphokines/analysis , Peritoneal Cavity/blood supply , Animals , Ascitic Fluid/metabolism , Carbohydrate Sequence , Carbon , Cell Division , Endothelial Growth Factors/chemistry , Endothelial Growth Factors/metabolism , Female , Iodine Radioisotopes , Lymphokines/chemistry , Lymphokines/metabolism , Male , Mammary Neoplasms, Animal/blood supply , Mammary Neoplasms, Animal/pathology , Mice , Molecular Sequence Data , Ovarian Neoplasms/blood supply , Ovarian Neoplasms/pathology , Tumor Cells, Cultured , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors
17.
Cancer Res ; 53(12): 2912-8, 1993 Jun 15.
Article in English | MEDLINE | ID: mdl-8504432

ABSTRACT

Vascular permeability factor (VPF), also known as vascular endothelial growth factor, is a dimeric M(r) 34,000-42,000 glycoprotein that possesses potent vascular permeability-enhancing and endothelial cell-specific mitogenic activities. It is synthesized by many rodent and human tumor cells and also by some normal cells. Recently we developed a sensitive and specific time-resolved immunofluorometric assay for quantifying VPF in biological fluids. We here report findings with this assay in guinea pigs and patients with both malignant and nonmalignant effusions. Line 1 and line 10 tumor cells were injected into the peritoneal cavities of syngeneic strain 2 guinea pigs, and ascitic fluid, plasma, and urine were collected at various intervals. Within 2 to 4 days, we observed a time-dependent, parallel increase in VPF, ascitic fluid volume, and tumor cell numbers in animals bearing either tumor line; in contrast, VPF was not detected in plasma or urine, even in animals with extensive tumor burdens. However, low levels of VPF were detected in the inflammatory ascites induced by i.p. oil injection. In human studies, high levels of VPF (> 10 pM) were measured in 21 of 32 effusions with cytology-documented malignant cells and in only seven of 35 effusions without cytological evidence of malignancy. Thus, VPF levels in human effusions provided a diagnostic test for malignancy with a sensitivity of 66% and a specificity of 80% (perhaps as high as 97% in that six of the seven cytology-negative patients with VPF levels > 10 pM had cancer as determined by other criteria). As in the animal tumor models, VPF was not detected in serum or urine obtained from patients with or without malignant ascites. Many nonmalignant effusions contained measurable VPF but, on average, in significantly smaller amounts than were found in malignant effusions. VPF levels in such fluids correlated strongly (p = 0.59, P < 0.001) with monocyte and macrophage content. Taken together, these data relate ascitic fluid accumulation to VPF concentration in a well-defined animal tumor system and demonstrate, for the first time, the presence of VPF in human malignant effusions. It is likely that VPF expression by tumor and mononuclear cells contributes to the plasma exudation and fluid accumulation associated with malignant and certain inflammatory effusions. The VPF assay may prove useful for cancer diagnosis as a supplement to cytology, especially in tumors that grow in the pleural lining but not as a suspension in the effusions that they induce.


Subject(s)
Ascitic Fluid/chemistry , Endothelial Growth Factors/analysis , Lymphokines/analysis , Neoplasms/diagnosis , Adult , Aged , Aged, 80 and over , Animals , Ascites , Breast Neoplasms/diagnosis , Endothelial Growth Factors/blood , Endothelial Growth Factors/urine , Female , Fluoroimmunoassay , Guinea Pigs , Humans , Inflammation , Lung Neoplasms/diagnosis , Lymphokines/blood , Lymphokines/urine , Male , Middle Aged , Neoplasms/blood , Neoplasms/urine , Ovarian Neoplasms/diagnosis , Sensitivity and Specificity , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors
18.
Cancer Res ; 49(19): 5448-58, 1989 Oct 01.
Article in English | MEDLINE | ID: mdl-2475250

ABSTRACT

Fluorescein-labeled dextrans (FITC-D) from 3 to 5000 kDa (Stokes' radii from 1 to 40 nm) were used to study influx from the plasma into the peritoneum and efflux from the peritoneal cavity into the plasma in normal and ascites tumor-bearing mice and in mice whose peritoneal vessels had been rendered hyperpermeable by serotonin. Two syngeneic transplantable murine ascites tumors were studied: mouse ovarian tumor and the TA3/St breast adenocarcinoma. To control for effects of peritoneal fluid volume, influx and efflux were also analyzed in mice that had received 5 ml of 5% bovine serum albumin i.p. as "artificial ascites." Following i.v. or i.p. injection, levels of FITC-D in the plasma and peritoneal fluid were quantitated by fluorimetry at successive time intervals from 5 to 360 min posttracer injection. Influx and efflux data were analyzed with a model consisting of three compartments (plasma, peritoneal cavity, and the extravascular space of all other organs) to yield kinetic parameters that characterized macromolecular transport. Depending on the size of the FITC-D tracer, from 3- to 50-fold more FITC-D accumulated in mouse ovarian tumor or TA3/St tumor ascites fluid, and 3- to 10-fold more FITC-D accumulated in the peritoneum of serotonin-treated than normal mice, all of it intact by gel exclusion chromatography. Influx of the FITC-D from plasma into the peritoneum, as characterized by the rate constant k1, was 2- to 40-fold greater in ascites tumor-bearing animals and 2- to 10-fold greater in serotonin-treated animals than in controls. Control animals with artificial ascites showed at most a 4-fold increase in the value of k1. As judged by fluorescence microscopy, the permeability of peritoneal-lining vessels in ascites tumor-bearing animals was greatly increased to FITC-D of 70 to 5000 kDa. Efflux of FITC-D, characterized by the rate constant k2, was reduced from 5- to 50-fold in ascites tumor-bearing animals but was unchanged or actually somewhat enhanced following serotonin treatment. Efflux in animals that had received artificial ascites was reduced 2.5- to 12.5-fold, correlating increased peritoneal fluid volume with decreased efflux. We conclude that tracer accumulation in malignant ascites fluid results from both increased influx as well as impaired efflux. Influx, and to a lesser extent efflux, were significantly affected by tracer size. However, within the range of FITC-D tested, we found no absolute size barrier to macromolecular transport from plasma to the peritoneal cavity, or vice versa.(ABSTRACT TRUNCATED AT 400 WORDS)


Subject(s)
Ascites/metabolism , Dextrans/metabolism , Peritoneal Cavity/metabolism , Peritoneum/metabolism , Adenocarcinoma/blood supply , Adenocarcinoma/metabolism , Animals , Ascites/blood , Biological Transport , Breast Neoplasms/blood supply , Breast Neoplasms/metabolism , Capillary Permeability/drug effects , Dextrans/blood , Female , Fluorescein , Fluoresceins , Mice , Molecular Weight , Ovarian Neoplasms/blood supply , Ovarian Neoplasms/metabolism , Peritoneum/blood supply , Serotonin/pharmacology
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