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Biomedical subjects

M Le

Publications and source records attributed to M Le.

At least 37 records · Page 2Linked to original sources
PubMed1998

Detection of reovirus RNA in hepatobiliary tissues from patients with extrahepatic biliary atresia and choledochal cysts.

Extrahepatic biliary atresia (EHBA) and choledochal cysts (CDC) are important causes of obstructive jaundice in pediatric patients. Viruses in general, and reoviruses in particular, have long been considered as possible etiologic agents responsible for inciting the inflammatory process that leads to these infantile obstructive cholangiopathies. In an effort to determine whether reovirus infection is associated with these disorders, we used a sensitive and specific reverse-transcriptase polymerase chain reaction (RT-PCR) technique designed to amplify a portion of the reovirus L1 gene segment from extracts of liver and/or biliary tissues. These tissues were obtained at the time of liver biopsy or surgical procedures from 23 patients with EHBA, 9 patients with CDC, and 33 patients with other hepatobiliary diseases. Hepatic and biliary tissues obtained at autopsy from 17 patients who died without known liver or biliary disease were also analyzed. Reovirus RNA was detected in hepatic and/or biliary tissues from 55% of patients with EHBA and 78% of patients with CDC. Reovirus RNA was found also in extracts of hepatic and/or biliary tissue from 21% of patients with other hepatobiliary diseases and in 12% of autopsy cases. The prevalence of reovirus RNA in tissues from patients with EHBA and CDC was significantly greater than that in patients with other hepatobiliary diseases (chi2 P = .012 EHBA vs. OTHER, P = .001 CDC vs. OTHER), or AUTOPSY cases (chi2 P = .006 EHBA vs. AUTOPSY, P < .001 CDC vs. AUTOPSY).

Biliary Atresia↗
PubMed1998

Differential effects of detergents on keratinocyte gene expression.

We have studied the effect of various detergents on keratinocyte gene expression in vitro, using an anionic detergent (sodium dodecyl sulfate), a cationic detergent cetyltrimethylammoniumbromide (CTAB), and two nonionic detergents, Nonidet P-40 and Tween-20. We measured the effect of these detergents on direct cellular toxicity (lactate dehydrogenase release), on the expression of markers for normal differentiation (cytokeratin 1 and involucrin expression), and on disturbed keratinocyte differentiation (SKALP) by northern blot analysis. As reported in other studies, large differences were noted in direct cellular toxicity. In a culture model that mimics normal epidermal differentiation we found that low concentrations of sodium dodecyl sulfate could induce the expression of SKALP, a proteinase inhibitor that is not normally expressed in human epidermis but is found in hyperproliferative skin. Sodium dodecyl sulfate caused upregulation of involucrin and downregulation of cytokeratin 1 expression, which is associated with the hyperproliferative/inflammatory epidermal phenotype found in psoriasis, wound healing, and skin irritation. These changes were not induced after treatment of cultures with CTAB, Triton X-100, and Nonidet-P40. This effect appeared to be specific for the class of anionic detergents because sodium dodecyl benzene sulfonate and sodium laurate also induced SKALP expression. These in vitro findings showed only a partial correlation with the potential of different detergents to induce clinical, biophysical, and cell biologic changes in vivo in human skin. Both sodium dodecyl sulfate and CTAB were found to cause induction and upregulation of SKALP and involucrin at low doses following a 24 h patch test, whereas high concentrations of Triton X-100 did not. Sodium dodecyl sulfate induced higher rates of transepidermal water loss, whereas CTAB treated skin showed more signs of cellular toxicity. We conclude that the action of anionic detergents on epidermal keratinocytes is qualitatively different from the other detergents tested, which might have implications for in vitro toxicology studies that use cell biologic parameters as a read-out. We would hypothesize that detergents cause skin injury by several mechanisms that include direct cellular toxicity, disruption of barrier function, and detergent specific effects on cellular differentiation, as demonstrated here for sodium dodecyl sulfate, sodium dodecyl benzene sulfonate, and sodium laurate.

Adult↗
PubMed1998

Changes in G protein expression account for impaired modulation of hepatic cAMP formation after BDL.

The regulation of cAMP synthesis by hormones and bile acids is altered in isolated hamster hepatocytes 2 days after bile duct ligation (BDL) [Y. Matsuzaki, B. Bouscarel, M. Le, S. Ceryak, T. W. Gettys, J. Shoda, and H. Fromm. Am. J. Physiol. 273 (Gastrointest. Liver Physiol. 36): G164-G174, 1997]. Therefore, studies were undertaken to elucidate the mechanism(s) responsible for this impaired modulation of cAMP formation. Hepatocytes were isolated 48 h after either a sham operation or BDL. Both preparations were equally devoid of cholangiocyte contamination. Although the basal cAMP level was not affected after BDL, the ability of glucagon to maximally stimulate cAMP synthesis was decreased by approximately 40%. This decreased glucagon effect after BDL was not due to alteration of the total glucagon receptor expression. However, this effect was associated with a parallel 50% decreased expression of the small stimulatory G protein alpha-subunit (GsalphaS). The expression of either the large subunit (GsalphaL) or the common beta-subunit remained unchanged. The expression of Gialpha2 and Gialpha3 was also decreased by 25 and 46%, respectively, and was associated with the failure of ANG II to inhibit stimulated cAMP formation. Therefore, alterations of the expression of GsalphaS and Galphai are, at least in part, responsible for the attenuated hormonal regulation of cAMP synthesis. Because cAMP has been reported to stimulate both bile acid uptake and secretion, impairment of cAMP synthesis and bile acid uptake may represent an initial hepatocellular defense mechanism during cholestasis.

Angiotensin II↗
PubMed1997

Therapeutic levels of functional human factor X in rats after retroviral-mediated hepatic gene therapy.

Factor X deficiency results in a rare but serious bleeding disorder that might be treated by expressing a normal factor X gene in patients. We generated an amphotropic retroviral vector with the human FX cDNA and delivered it to rat hepatocytes in vivo during liver regeneration. The human alpha1-antitrypsin promoter was chosen to direct expression because it was the most efficient of several tested in yielding expression of alpha1-antitrypsin protein from a retroviral vector in hepatocytes in vivo. We achieved expression of factor X in four rats at levels sufficient to maintain hemostasis in humans (10% to 43% of normal). The factor X was determined to be functional by using a chromogenic substrate assay after immunoprecipitation with human specific antibodies. Expression of factor X remained stable for more than 10 months in two rats. It is likely that expression will be maintained for the life of the animals, because retroviral vectors integrate into the chromosome and hepatocytes are long-lived. The high and stable levels of expression achieved using this liver-specific promoter overcomes one of the two major obstacles to successful human gene therapy for hemophilia.

3T3 Cells↗
PubMed1997

Effect of cholestasis on regulation of cAMP synthesis by glucagon and bile acids in isolated hepatocytes.

Previously, we have reported that bile acids can directly inhibit hormone-induced adenosine 3',5'-cyclic monophosphate (cAMP) formation through a protein kinase C (PKC)-dependent mechanism [Bouscarel, B., T.W. Gettys, H. Fromm, and H. Dubner. Am. J. Physiol. 268 (Gastrointest. Liver Physiol. 31): G300-G310, 1995]. Therefore, the regulation of cAMP synthesis by glucagon and bile acids was investigated in hepatocytes isolated after 2-day ligation of the common bile duct in Golden Syrian hamsters. The bile acid concentration was increased 30-fold in the serum, whereas it was not significantly different in the bile of duct-ligated vs. sham-operated hamsters. The glycine/taurine and cholate/chenodeoxycholate ratios were significantly increased fourfold and sevenfold, respectively, only in the serum of bile duct-ligated hamsters. Ligation of the bile duct decreased the efficacy of glucagon-stimulated cAMP synthesis by 40-50% without changing its potency. This attenuation of cAMP synthesis, which was also observed with forskolin, remained in the absence of any detectable amount of bile acids in the hepatocytes. The decrease in glucagon-stimulated cAMP production was also not attributable to changes in either the affinity or the number of receptors for this hormone. The potency and efficacy of the bile acids to inhibit glucagon-induced cAMP formation was also reduced in bile duct-ligated hamsters. The inhibitory regulation of cAMP synthesis through angiotensin II was similarly diminished after bile duct ligation. Although the total expression of PKC-alpha was not affected, an increased translocation by 60% from the cytosol to the membrane fraction was observed in hepatocytes isolated after bile duct ligation. Therefore, during cholestasis and prolonged exposure of the liver to bile acids, both the stimulatory and inhibitory regulatory, mechanisms of cAMP synthesis are compromised in an irreversible manner because the effects persist even after isolation of the hepatocytes. This decreased regulation of cAMP synthesis is possibly mediated through PKC-alpha activation.

Angiotensin II↗
PubMed1996

Changes in keratinocyte differentiation following mild irritation by sodium dodecyl sulphate.

Although the induction of acute irritant dermatitis by detergents has been studied extensively in recent years, our understanding of the cell biological events in the repair phase, and its relevance for the development of chronic irritant dermatitis is limited. Here we studied the reaction pattern of human skin to short-term application of sodium dodecyl sulphate (SDS) in a model that induced a minimal acute inflammatory reaction (absence of polymorphonuclear leucocytes, PMN) and did not have cytopathic effects on the epidermal keratinocytes as determined by histological investigation. All parameters were measured up to 14 days after exposure to SDS. Application of SDS caused disturbances of barrier function as measured by transepidermal water loss and had vascular effects as judged by erythema. Several cell biological markers for epidermal growth and differentiation were examined by immunohistochemistry. A rapid and strong induction of the cornified envelope precursor protein involucrin was seen in the stratum spinosum, with a peak at 24 h. Within 24 h a strong upregulation of epidermal fatty acid binding protein (E-FABP) was noted, with a peak at 7 days after injury. Cellular proliferation in the basal layer was increased fivefold as assessed by nuclear staining for the Ki-67 antigen, showing a peak at 48 h. Surprisingly, no significant induction of cytokeratin 16 and SKALP/elafin expression, two markers associated with epidermal hyper-proliferation and inflammation, was seen. These findings suggest that the cellular changes following exposure to detergent are distinct from those seen in other forms of skin injury. We would speculate that the epidermal response to detergent exposure is primarily directed at restoration of barrier function.

Adult↗
PubMed1996

The timSL mutant of the Drosophila rhythm gene timeless manifests allele-specific interactions with period gene mutants.

To identify new components of the Drosophila circadian clock, we screened chemically mutagenized flies for suppressors or enhancers of the long periods characteristic of the period (per) mutant allele perL. We isolated a novel mutant that maps to the rhythm gene timeless (tim). This novel allele, timSL, alters the temporal pattern of perL protein nuclear localization and restores temperature compensation to perL flies. timSL more generally manifests specific interactions with different per alleles. The identification of this first period-altering tim allele provides further evidence that TIM is a major component of the clock, and the allele-specific interactions with PER provide evidence that the PER/TIM heterodimer is a unit of circadian function. Although timSL fails to restore PER-L/TIM temperature insensitivity in yeast, it alters the TIM phosphorylation pattern during the late night. The effects on phosphorylation suggest that timSL functions as a partial bypass suppressor of perL and provide evidence that the TIM phosphorylation program contributes to the circadian timekeeping mechanism.

Alleles↗
PubMed1996

Generation and analysis of 280,000 human expressed sequence tags.

We report the generation of 319,311 single-pass sequencing reactions (known as expressed sequence tags, or ESTs) obtained from the 5' and 3' ends of 194,031 human cDNA clones. Our goal has been to obtain tag sequences from many different genes and to deposit these in the publicly accessible Data Base for Expressed Sequence Tags. Highly efficient automatic screening of the data allows deposition of the annotated sequences without delay. Sequences have been generated from 26 oligo(dT) primed directionally cloned libraries, of which 18 were normalized. The libraries were constructed using mRNA isolated from 17 different tissues representing three developmental states. Comparisons of a subset of our data with nonredundant human mRNA and protein data bases show that the ESTs represent many known sequences and contain many that are novel. Analysis of protein families using Hidden Markov Models confirms this observation and supports the contention that although normalization reduces significantly the relative abundance of redundant cDNA clones, it does not result in the complete removal of members of gene families.

Adult↗
PubMed1996

[Histopathological and immunohistochemical studies in hepatoid adenocarcinomas of the stomach].

To clarify the pathomorphology and the characteristics of clinical biological behaviors in hepatoid adenocarcinoma of the stomach (HAS), the clinical data of 24 HAS cases were reviewed and the specimens studied with light, electron microscopy and immunohistochemistry methods. The results show that HAS was more often found in middle aged and elderly people, and their serum alphafetoprotein (AFP) levels were significantly high. Among the 16 deceased cases, 12 died within one year and the mean survival period after operation was 10 months, only one of the 24 cases was early stage carcinoma, all others were of the advanced stage. Vessels were invaded in 18 cases (75%), metastasis to the regional or distant lymph nodes in 22 cases (91.7%) and liver metastasis in 14 cases (58.3%). HAS is a highly malignant tumor with poor prognosis. The tumors were composed of two different but closely related hepatoid and adenocarcinomatous areas. The tumor cells in the hepatoid region possess many features of hepatic cell carcinoma, such as abundant eosinophilic or clear cytoplasm, PAS positive hyaline globules in both intra- and extracellular spaces. AFP positive staining, intracytoplasmic bile, glycogen granules and intercarcinocyte bile canaliculus. Microvilli with intestinal epithelial type were found not only in adenocarcinoma areas but also in hepatoid areas. An indication that the HAS was differentiating into hepatic cell and intestinal epithelial types. We conclude that HAS possesses similar pathomorphologic pattern and clinicobiological behaviors of hepatic cell carcinoma and HAS should be classified as a special subtype of gastric carcinoma.

Adenocarcinoma↗
PubMed1995

A procedure for the determination of monothiols in the presence of dithiothreitol--an improved assay for the reduction of disulfides.

4,4'-Dipyridyl disulfide (4-PDS) and sodium arsenite have been used to develop a rapid and sensitive assay for monothiols in the presence of dithiothreitol (DTT). The procedure is similar to that of Zahler and Cleland (J. Biol. Chem. 243, 716-719, 1968) but involves the use of a lower pH and 4-PDS, which is more effective than 5,5'-dithiobis-(2-nitrobenzoic acid) at low pH. Background reactions that interfere with the determination of slow reacting thiol groups are much reduced in the new assay procedure. The reduction of oxidized glutathione by DTT at three different pH values and a partial and the complete reduction of bovine serum albumin by DTT have been characterized in order to demonstrate the use of this procedure. In the first two cases monothiol groups were readily detected without interference from DTT. Lower than expected numbers of thiol groups in the case of three reduced and denatured model proteins appeared to reflect their partial complexation by arsenite. Equilibrium and kinetic constants for the formation and dissociation of the DTT-arsenite complex from pH 5 to 8 are presented.

Animals↗
PubMed1994

Induction of systemic immunologic hyporesponsiveness to ovalbumin in neonatal rats by the enteric administration of peptic fragments of ovalbumin.

The orogastric administration of protein antigens can induce specific systemic immunologic hyporesponsiveness to the antigens. Some evidence has suggested that fragmention of the antigens by proteolytic enzymes in the gastrointestinal tract is a necessary step in this phenomenon. We administered either ovalbumin (OVA) or fragments OVA, produced by digestion with pepsin, into the stomach or jejunum of suckling rats and measured the serum IgG antibody response to a subsequent intraperitoneal challenge with OVA. Whereas OVA administered by gastric gavage caused virtually a complete unresponsiveness to the OVA challenge, administration into the jejunum had no significant effect. Administration of peptic fragments of OVA (about 8000 Da) into either the jejunum or stomach caused systemic unresponsiveness in 62% and 67% of animals, respectively (p < .05 vs buffer-fed controls). Smaller fragments of OVA (< 2000 Da) given into the stomach or jejunum had no effect on systemic IgG responses, nor did administration of the 8000 Da fragments intraperitoneally. We conclude the 1) systemic immunologic tolerance to OVA can be induced by enteric administration of critical-size fragments of OVA, and 2) encounter of the OVA fragments, like intact OVA, via the gut yields a distinctly different response from that which follows parenteral administration.

Animals↗
PubMed1993

Pili in microspheres protect rabbits from diarrhoea induced by E. coli strain RDEC-1.

We tested whether pilus proteins of rabbit diarrhoeagenic Escherichia coli (RDEC-1), incorporated into biodegradable microspheres, could function as safe and effective oral immunogens in the rabbit diarrhoea model. The RDEC-1 adhesin, AF/R1, incorporated into poly(D,L-lactide-co-glycolide) microspheres, was administered intraduodenally. Vaccinated and unvaccinated rabbits were challenged with RDEC-1 and killed 1 week later. Vaccination with AF/R1 in microspheres did not cause diarrhoea or weight loss. After challenge, rabbits given AF/R1 in microspheres, in contrast to unvaccinated animals, remained in good health. RDEC-1 attachment to caecal epithelium of vaccinated rabbits was reduced (p = 0.02), whereas numbers of RDEC-1 in intestinal fluids were little affected. Also, in vaccinated animals, biliary anti-AF/R1 IgA levels were increased, and AF/R1-induced blast-cell transformation was vigorous in spleen cell cultures. We conclude that vaccination with AF/R1 in microspheres was safe and protected rabbits against RDEC-1 disease, probably by interfering with adherence of the bacteria to the intestinal mucosa. The interference might have been due to the presence of specific antibodies secreted in bile.

Animals↗
PubMed1993

Trityl monitoring of automated DNA synthesizer operation by conductivity: a new method of real-time analysis.

AutoAnalysis is a new method for detecting and quantitating the trityl cation released each cycle on automated DNA synthesizers. The trityl (dimethoxytrityl) cation is removed from the growing oligonucleotide after each base addition and is a useful measure of synthesis efficiency. The traditional absorbance method of collecting each trityl effluent with a fraction collector, followed by dilution with an acid solution and careful quantitation by UV/VIS spectroscopy is costly, tedious and prone to error. The absorbance method for trityl cation analysis must usually wait until the synthesis is complete. Interruption of a failed operation, for a variety of reasons, such as an empty reagent reservoir, is thus not possible. Taking advantage of the conductive properties of the trityl cation, immediate and real-time quantitation is now possible by integrating the total conductance of the flowing stream during the detritylation step after each nucleoside addition in DNA synthesis. A conductivity cell is mounted downstream, past the synthesis column. The conductivity signal is processed and displayed as the current average stepwise yield and overall yield. If the yield drops below a pre-set threshold value because of a failure situation, the synthesizer will interrupt, preserving reagents. AutoAnalysis allows trityl monitoring with complete automation on the Applied Biosystems Models 392 and 394 DNA/RNA Synthesizers.

Base Sequence↗
PubMed1992

Mucosal immune response to RDEC-1 infection: study of lamina propria antibody-producing cells and biliary antibody.

Infection of rabbits with Escherichia coli RDEC-1 is a useful model for diarrheal disease caused by mucosally attaching E. coli. Understanding of the protective immunity induced by RDEC-1 infection in rabbits should provide information useful in the design of vaccines for protection against this infection and other mucosally attaching organisms as well. Thus, to define the time course and location of specific immunoglobulin A secretion in relation to bacterial colonization during primary RDEC-1 infection, we infected rabbits with RDEC-1, which express AF/R1 adherence pili, and compared sites of anti-AF/R1 antibody-containing cells in the intestinal mucosa with the sites of luminal colonization and mucosal attachment of RDEC-1. Also, anti-AF/R1 antibodies in intestinal fluids and bile were measured by enzyme-linked immunosorbent assay, and attachment sites of RDEC-1 to the intestinal epithelium were determined by immunohistochemical examination. Anti-AF/R1 pilus antibody-containing cells were most numerous in the proximal intestine (duodenum and jejunum). In contrast, both luminal colonization and attachment of RDEC-1 to epithelial cells were densest in the distal intestine (cecum and colon). Anti-AF/R1 antibodies were present in approximately equal amounts in fluids collected from all levels of the gut after week 1 postinfection. Anti-AF/R1 antibody levels in undiluted bile exceeded those in gut flushes by at least 2 orders of magnitude. Loss of RDEC-1 attachment to epithelial cells preceded resolution of diarrheal illness despite the presence of large numbers of organisms in the intestinal lumen. Our studies indicate that during RDEC-1 infection (i) sites of greatest mucosal anti-AF/R1 antibody secretion are proximal to sites of maximal RDEC-1 luminal colonization and attachment, (ii) bile is a major source of specific antibodies in the intestinal lumen, and (iii) interference with RDEC-1 attachment to epithelial cells may permit resolution of disease.

Animals↗
PubMed1992

[Immediate mammary reconstruction in the radical treatment of cancer of the breast].

Based on a series of 516 patients operated between 1976 and 1991, the authors present their experience of the various aspects of immediate breast reconstruction (IBR). A prosthesis was used in the majority of cases (80%) and the essential technical aspect was the creation of a complete muscular compartment, possibly by combining pectoralis major and serratus anterior, latissimus dorsi (5%) and by using an expander (8%) or even a musculocutaneous flap. The rectus abdominis musculocutaneous flap (TRAM, 10%) had the advantage of allowing IBR without a prosthesis. The mastectomy scar plays a major role in determining the final shape of the breast: the skin resection must be modulated according to the site of the tumour. The complications observed (40%), which required removal of the prosthesis in 12% of cases, were either immediate complications (20%), some of which (infections, necrosis, dehiscence) tended to delay adjuvant therapy, or secondary complications, principally grade III-IV contracture (20%). Evaluation of the cosmetic results of IBR in comparison with a population undergoing secondary reconstruction over the same period, did not reveal any significant difference between the two approaches. The authors discuss in detail the oncological and technical arguments for and against IBR and conclude on the psychological advantages of IBR for mastectomised patients.

Adult↗
PubMed1991

Long-term risk of sarcoma following radiation treatment for breast cancer.

Between 1954 and 1983, 7620 patients were treated for breast carcinoma at Institut Gustave Roussy (France). Of these patients, 6919 were followed for at least 1 year. Out of these, 11 presented with sarcomas thought to be induced by irradiation, 2 of which were Steward-Treves Syndrome, and 9 of which were sarcomas within the irradiated fields. All histological slides were reviewed and a comparison with those of breast cancer was done. The sites of these sarcomas were: parietal wall, 1 case; second costal cartilage, 1 case; infraclavicular region, 1 case; supraclavicular region, 2 cases; internal third of the clavicle, 2 cases; axillary region 2 cases; and the internal side of the upper arm (Stewart-Treves syndrome), 2 cases. The median age of these 11 patients at the diagnosis of sarcomas was 65.8 (49-83). The mean latent period was 9.5 years (4-24). Three patients underwent radical mastectomy and nine modified radical mastectomy. Only one patient received chemotherapy. The radiation doses received at the site of the sarcoma were 45 Gy/18 fr. for 10 cases and 90-100 Gy for 1 case (due to overlapping between two fields). The histology was as follows: malignant fibrous histiocytoma, 5 cases; fibrosarcoma, 3 cases; lymphangiosarcoma, 2 cases; and osteochondrosarcoma, 1 case. The median survival following diagnosis of sarcoma was 2.4 years (4 months-9 years). Two patients are still alive: one with recurrence of her breast cancer, the other in complete remission, with 7 and 3 years follow-up, respectively. All other patients died from their sarcomas. The cumulative incidence of sarcoma following irradiation of breast cancer was 0.2% (0.09-0.47) at 10 years. The standardized incidence ratio (SIR) of sarcoma (observed n# of cases (Obs)/expected n# of cases (Exp) computed from the Danish Cancer Registry for the same period) was 1.81 (CI 0.91-3.23). This is significantly higher than one, with a p = 0.03 (One Tailed Exact Test). The mean annual excess (Obs-Exp)/100.000 person-years at risk during the same period/(100,000) was 9.92. This study suggests that patients treated by radiation for breast cancer have a risk of subsequent sarcomas that is higher than the general population. However, the benefit from adjuvant radiation therapy in the treatment of breast cancer exceeds the risk of second cancer; therefore, the potential of radiation-induced sarcomas should not be a factor in the selection of treatment for patients with breast cancer.

Adult↗