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PubMed1972

Isolation and characterization of an isolate (HN) of Marek's disease virus with low pathogenicity.

A cytopathic agent was isolated and characterized as an isolate of Marek's disease herpesvirus (MDHV) with low pathogenicity, and referred to as the HN isolate. This isolate of MDHV did not cause clinical Marek's disease (MD) or death in a highly susceptible line of chickens within 5 weeks after exposure. Gross lesions of limited extent were noted in a few of the inoculated birds. Microscopic nerve lesions in the inoculated and contact-infected birds were invariably minimal, closely resembling C-type MD lesions.

Animals↗
PubMed1998

Lipopolysaccharide from nonvirulent Ara+ Burkholderia pseudomallei isolates is immunologically indistinguishable from lipopolysaccharide from virulent Ara- clinical isolates.

Different lines of evidence suggest that a discrepancy between the distribution of Burkholderia (Pseudomonas) pseudomallei in the environment and the distribution of the disease melioidosis is attributable, at least in part, to phenotypic differences between clinical and some environmental isolates. Two antigenically and biochemically distinct biotypes have been described, only one of which is virulent. In this study, lipopolysaccharides (LPSs) were extracted by the proteinase K digestion method from a total of 214 B. pseudomallei isolates, and their immunoreactivities with sera from patients with different clinical spectra and with other infections were evaluated. With the exception of4 isolates from a total of 214 tested, the sodium dodecyl sulfate-polyacrylamide gel electrophoresis silver-staining profiles of the LPSs from the two biotypes showed identical ladder patterns that were typical for smooth LPSs from other gram-negative bacteria. The 210 isolates with typical LPS patterns (119 Ara- clinical, 13 Ara- soil, 70 Ara+ soil, and 8 reference National Type Culture Collection strains) also exhibited similar immunoblot profiles against pooled sera from patients with melioidosis and hyperimmune mouse sera. Concordant findings were noted in the indirect enzyme-linked immunosorbent assay with Ara- and Ara+ LPSs to coat the microtiter plates. The LPSs of the different B. pseudomallei biotypes appear antigenically indistinguishable. It is, therefore, unlikely that this component is related to the virulence and pathogenicity of B. pseudomallei.

Animals↗
PubMed1999

Isolation of Enterococcus faecalis clinical isolates that efficiently adhere to human bladder carcinoma T24 cells and inhibition of adhesion by fibronectin and trypsin treatment.

The adherence of Enterococcus faecalis strains to human T24 cells was examined by scanning electron microscopy. Five highly adhesive strains were identified from 30 strains isolated from the urine of patients with urinary tract infections. No efficiently adhesive strains were found among the 30 strains isolated from the feces of healthy students. The five isolated strains also adhered efficiently to human bladder epithelial cells. Analysis of restriction endonuclease-digested plasmid DNAs and chromosome DNAs showed that the five strains were different strains isolated from different patients. The adhesiveness of these strains was inhibited by treatment with fibronectin or trypsin, implying that a specific protein (adhesin) on the bacterial cell surface mediates adherence to fibronectin on the host cell surfaces, and the adhesin differs from the reported adhesins.

Bacterial Adhesion↗
PubMed1989

Typing of urogenital, maternal, and neonatal isolates of Haemophilus influenzae and Haemophilus parainfluenzae in correlation with clinical source of isolation and evidence for a genital specificity of H. influenzae biotype IV.

Over a period of 6 years, 114 strains of Haemophilus influenzae and Haemophilus parainfluenzae were isolated from genital, mother-infant, or neonatal infections. Their serotypes, biotypes, antibiotic resistance phenotypes, and outer membrane protein (OMP) electrophoretic patterns were characterized and correlated with the various clinical outcomes. Genital H. influenzae and H. parainfluenzae appeared to behave mostly as opportunistic pathogens; for instance, 62% of the cases of endometritis or pelvic inflammatory disease were related to the presence of an intrauterine device. However, as seen clearly in one case, the strains may be sexually transmitted. The analysis of OMP patterns proved to be a very convenient method to seek evidence for the sexual origin of the infection. H. influenzae was more often involved in complicated genital infections than was H. parainfluenzae. Nontypeable and biotype II H. influenzae strains were the more frequent isolates, except in pelvic inflammatory diseases, in which biotype I prevailed, and in mother-infant infections, in which one-fourth of the cases were due to biotype IV. Characterization of H. influenzae isolates did not support a general concept of specific genital strains. However, strains of biotype IV clearly stood out with two characteristics: (i) a peritrichous fimbriation and (ii) a very peculiar homogeneous OMP pattern comprising an OMP of molecular weight approximately 18,000 unique to this biotype. These characteristics were also found in H. influenzae biotype IV strains isolated from genital infections in the United States and used as controls. H. influenzae biotype IV strains may thus correspond to a group somewhat adapted to the genital tract.

Female↗
PubMed1995

Identification and ribotypes of Staphylococcus caprae isolates isolated as human pathogens and from goat milk.

We report five cases of human infection with Staphylococcus caprae. Two were community acquired (one case each of endocarditis and urinary tract infection); the other three were acquired in a hospital (two cases of bacteremia associated with intravenous access and one case of urinary tract infection). Analysis of human isolates and goat isolates from eight herds showed that they could be misidentified by some commercial identification systems but were clearly identified as S. caprae by ribotyping, according to their species-specific ribotype. Phylogenetic methods applied to the ribotypes did not reveal two distinct lineages corresponding to the goat and human origins of the isolates, although human ribotypes were clearly distinguishable by the presence of a core of four specific bands. The latter observation may reflect some degree of evolutionary change within the species between human and goat isolates.

Adult↗
PubMed1996

A microsporidian isolated from an AIDS patient corresponds to Encephalitozoon cuniculi III, originally isolated from domestic dogs.

The ribosomal DNA internal transcribed spacer (ITS) region of a recently cultured human Encephalitozoon cuniculi isolate was analyzed by gene amplification and DNA sequencing. Restriction endonuclease digestion (FokI) and double-stranded DNA heteroduplex mobility shift analysis were performed to determine their utility for strain differentiation. The human E. cuniculi isolate was identical to E. cuniculi III, which had been isolated only from domestic dogs until now. The patient providing the isolate owned a pet dog, but no microsporidia were detected in the pet's urine.

AIDS-Related Opportunistic Infections↗
PubMed1999

Comparison of large restriction fragments of Mycobacterium avium isolates recovered from AIDS and non-AIDS patients with those of isolates from potable water.

We examined potable water in Los Angeles, California, as a possible source of infection in AIDS and non-AIDS patients. Nontuberculous mycobacteria were recovered from 12 (92%) of 13 reservoirs, 45 (82%) of 55 homes, 31 (100%) of 31 commercial buildings, and 15 (100%) of 15 hospitals. Large-restriction-fragment (LRF) pattern analyses were done with AseI. The LRF patterns of Mycobacterium avium isolates recovered from potable water in three homes, two commercial buildings, one reservoir, and eight hospitals had varying degrees of relatedness to 19 clinical isolates recovered from 17 patients. The high number of M. avium isolates recovered from hospital water and their close relationship with clinical isolates suggests the potential threat of nosocomial spread. This study supports the possibility that potable water is a source for the acquisition of M. avium infections.

AIDS-Related Opportunistic Infections↗
PubMed2005

In vitro susceptibilities of clinical isolates of Candida species, Cryptococcus neoformans, and Aspergillus species to itraconazole: global survey of 9,359 isolates tested by clinical and laboratory standards institute broth microdilution methods.

The in vitro activity of itraconazole was determined against 7,299 isolates of Candida spp., 1,615 isolates of Cryptococcus neoformans, and 445 isolates of Aspergillus spp. obtained from over 200 medical centers worldwide. Itraconazole was active against all Candida spp. (96% of MICs were < or =1 microg/ml) with the exception of C. glabrata (77% of MICs were < or =1 microg/ml). Itraconazole inhibited 94% of C. krusei and 84% of other fluconazole-resistant Candida species, exclusive of C. glabrata, at a MIC of < or =1 microg/ml. Itraconazole was not active against fluconazole-resistant isolates of C. glabrata. Only modest activity was seen against C. neoformans (80% of MICs were < or =1 microg/ml); however, itraconazole showed excellent activity against Aspergillus spp. (94% of MICs were < or =1 microg/ml). These results provide an update on the antifungal activity of itraconazole against major opportunistic fungal pathogens. In light of the new intravenous formulation of itraconazole these data suggest that this agent remains a viable systemically active antifungal agent.

Antifungal Agents↗
PubMed1977

Selective screening procedure for the isolation of heat- and cold-sensitive, DNA replication-deficient mutants of bacteriophage SPO1 and preliminary characterization of the mutants isolated.

A procedure is described for the selective isolation of temperature-sensitive replication-deficient mutants of Bacillus subtilis phage SPO1. A modification of the procedure permits the isolation of temperature-sensitive mutants in specific cistrons of interest. The applicability of these procedures to other viral systems is discussed. The mutations isolated were assigned to eight replication-deficient cistrons, with the cold-sensitive mutations showing a distribution strikingly different from that of the heat-sensitive mutations. As a preliminary to the identification of initiation-deficient mutants, the mutants were divided into three classes on the basis of their ability to synthesize DNA after a shift to nonpermissive temperature. We also report two incidental results: (i) the SPO1 dUMP hydroxymethylase, like the T4 dCMP hydroxymethylase, may be part of a multifunctional complex; and (ii) mutants were isolated that were replication positive but lysis deficient and failed to complement one of the replication-deficient mutants.

Bacillus subtilis↗
PubMed1992

Neutralization of multiple laboratory and clinical isolates of human immunodeficiency virus type 1 (HIV-1) by antisera raised against gp120 from the MN isolate of HIV-1.

Vaccines prepared from the envelope glycoprotein, gp120, of the common laboratory isolate of human immunodeficiency virus type 1 (HIV-1) (IIIB/LAV-1) elicit antibodies that neutralize the homologous virus but show little if any cross-neutralizing activity. This may be because the principal neutralizing determinant (PND) of gp120 is highly unusual in the IIIB/LAV-1 strain and is not representative of those found in the majority of field isolates. We have now examined the immunogenicity of recombinant gp120 prepared from the MN strain of HIV-1 (MN-rgp120), whose PND is thought to be representative of approximately 60% of the isolates in North America. Our results show that MN-rgp120 is a potent immunogen and elicits anti-gp120 titers comparable to those found in HIV-1-infected individuals. While both MN-rgp120 and IIIB-rgp120 induced antibodies able to block gp120 binding to CD4, strain-specific and type-common blocking antibodies were detected. Finally, antibodies to MN-rgp120 but not to IIIB-rgp120 were effective in neutralizing a broad range of laboratory and clinical isolates of HIV-1. These studies demonstrate that susceptibility or resistance to neutralization by antibodies to gp120 correlates with the PND sequence and suggest that the problem of antigenic variation may not be insurmountable in the development of an effective AIDS vaccine.

Amino Acid Sequence↗
PubMed1981

Isolation of Mallory bodies and an attempt to demonstrate cell mediated immunity to Mallory body isolate in patients with alcoholic liver disease.

Mallory bodies were isolated from necropsy livers from patients with alcoholic hepatitis with and without cirrhosis with a Ficoll viscosity barrier. The purity of Mallory bodies in the isolate varied between 70 and 90%, estimated by counting Mallory bodies and non-Mallory body structures in haematoxylin-eosin stained smears. Electron microscopy confirmed the presence of Mallory bodies in the isolates. The Mallory body isolate was used as antigen in the agarose leucocyte migration inhibition test in order to test the cell-mediated immunity. No significant difference in leucocyte migration was found between controls and patients with alcoholic hepatitis, alcoholic steatosis, alcoholic cirrhosis and miscellaneous liver diseases.

Adolescent↗
PubMed1987

Effects of dietary cod liver oil on fatty-acid composition and calcium transport in isolated adult rat ventricular myocytes and on the response of isolated hearts to ischemia and reperfusion.

Three-week-old male and female rats were placed either on standard rat chow or chow supplemented with 10% cod liver oil for 12 weeks. Animals fed cod liver oil demonstrated reduced body weights. Cod liver oil feeding produced a significant reduction in the ratio of (n - 6)/(n - 3) fatty acids in phospholipids of the isolated myocytes. The primary changes included a significant decrease in arachidonic acid (20:4, n - 6) and elevations in eicosapentaenoic acid (20:5, n - 3) and docosahexaenoic acid (22:6, n - 3). Furthermore, isolated myocytes from cod liver oil fed rats exhibited an enhanced 45Ca2+ uptake, although 45Ca2+ release was unaffected. Dietary cod liver oil had little effect on cardiac response to ischemia and reperfusion. Thus, neither developed force or resting tension was significantly affected by diet, although the latter tended to be elevated in hearts from cod liver oil fed animals. Release of creatine kinase was unaltered by diet. The release of 6-ketoprostaglandin F1 alpha from isolated hearts was significantly reduced by dietary cod liver oil, likely due to the reduced levels of arachidonic acid. Our study indicates that dietary cod liver oil and subsequent changes in phospholipid fatty-acid content are accompanied by changes in Ca2+ transport in isolated cardiac myocytes. However, this diet produces little effect on the cardiac response to acute ischemia and reperfusion.

6-Ketoprostaglandin F1 alpha↗
PubMed2003

Troglitazone inhibits isolated cell proliferation, and induces apoptosis in isolated rat mesangial cells.

BACKGROUND/AIMS: Troglitazone is one of thiazolidinedione derivatives as a high affinity ligand for peroxisome proliferator-activated receptor-gamma (PPAR-gamma). The in vivo studies demonstrated that troglitazone ameliorated microalbuminuria. There have been few reports about direct effect of thiazolidinedione derivatives on mesangial cell function. We determined the effect of troglitazone on isolated rat mesangial cell proliferation. METHODS: We determined PPAR-gamma mRNA expression in isolated rat mesangial cells. Chronic effects of 10(-6) to 10(-4) mol/l troglitazone on mesangial cell proliferation and mitogen-activated protein (MAP) kinase activity were also determined. The effects of troglitazone on apoptosis were investigated in rat mesangial cells. RESULTS: Rat PPAR-gamma mRNA was detected in isolated rat mesangial cells. Living cell number, assessed by colorimetric [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT) assay, was significantly decreased with 10(-4) mol/l troglitazone. The addition of 10(-6) to 10(-4) mol/l troglitazone dose-dependently inhibited 5-bromo-2'-deoxyuridine (BrdU) uptake into isolated rat mesangial cells. The addition of 10(-4) and 10(-5) mol/l troglitazone significantly reduced MAP kinase activity. Troglitazone at the concentrations of 10(-6) to 10(-4) mol/l dose-dependently increased DNA fragmentation rates, indicating that troglitazone may cause apoptosis in rat mesangial cells. Bax and Bcl-xL proteins were not changed, although Bcl-2 proteins increased with troglitazone. CONCLUSIONS: The present data demonstrated that troglitazone inhibits cell proliferation, and induces apoptosis in rat mesangial cells, raising a possibility that it directly affects renal function.

Animals↗
PubMed2003

Isolation of Plasmodium berghei ookinetes in culture using Nycodenz density gradient columns and magnetic isolation.

BACKGROUND: Large scale in vitro production of the mosquito stages of malaria parasites remains elusive, with only limited success for complete sporogonic development and only one report of development through to infective sporozoites. The initial step in this process is the production, in vitro, of ookinetes from gametocytaemic blood. Methods for isolation of these ookinetes from blood cells have been described; however, in addition to yield often being low, processing time and potential for contamination by erythrocytes remain high. METHODS: This study compares two procedures for retaining mature ookinetes from blood stage cultures, whilst removing red blood cells and other contaminants prior to further culture of the parasite. The well established method of isolation on Nycodenz cushions is compared with a novel method utilizing the innate magnetic properties of the haem pigment crystals found in the cytoplasm of ookinetes. RESULTS: Yield and viability of ookinetes were similar with both isolation methods. However, in our hands magnetic isolation produced a cleaner ookinete preparation much more quickly. Moreover, decreasing the flow rate through the magnetic column could further enhance the yield. CONCLUSION: We recommend the enrichment of an ookinete preparation prior to further culture being performed using the magnetic properties of Plasmodium berghei ookinetes as an alternative to their density. The former technique is faster, removes more erythrocytes, but day-to-day costs are greater.

Journal Article↗
PubMed2001

Current progress in isolation and characterization of toxins isolated from Pfiesteria piscicida.

The isolation and partial purification of toxic substances derived from Pfiesteria piscicida Steidinger & Burkholder extracts is described. Four distinct bioassay systems were used to monitor bioactivity of the P. piscicida extracts, including a high throughput cell cytotoxicity assay and a reporter gene assay as well as assays using brine shrimp and fish. Using these bioassays to guide fractionation, we have isolated two distinct, active fractions from Pfiesteria culture medium and cell mass extracts on the basis of their solubility characteristics. We have identified and characterized a bioactive lipophilic substance from Pfiesteria-derived extracts as di(2-ethylhexyl)phthalate, a commonly used plasticizer. The source of this typically man-made substance has been identified as originating from Instant Ocean (Aquarium Systems, Mentor, OH, USA), a commercially available seawater salt mixture used to prepare our mass culture growth medium. We have developed chromatographic methodology to isolate a bioactive polar compound isolated from extracts of Pfiesteria culture and presently report the characterization of the activity of this substance. The molecular structural analysis of the polar active component(s) using mass spectrometry and nuclear magnetic resonance spectroscopy is currently under way.

Animals↗
PubMed1975

Comparison of alpha- and beta-cell secretory responses in islets isolated with collagenase and in the isolated perfused pancreas of rats.

The inhibitory actions of somatostatin (100 ng./ml.) on insulin release, stimulated by high glucose (20 mM), and on glucagon release, stimulated by arginine (15 mM), were studied with two in vitro systems: the isolated perifused rat islets prepared by the collagenase procedure and the isolated perfused rat pancreas. Suppression of arginine-induced glucagon release by glucose (20 mM) and glyceraldehyde (5 mM) was also assessed in both systems. With the perfused pancreas, somatostatin caused 32 per cent inhibition of glucose-mediated insulin release and inhibited arginine-induced glucagon release by 72 per cent. In the same system, glucose and glyceraldehyde were similarly potent inhibitors of arginine-induced glucagon secretion. In contrast to the isolated perfused pancreas, there was no significant somatostation suppression of glucose-induced insulin release or arginine-induced glucagon release whether the inhibitor was present prior to or was added during stimulation by glucose or arginine. Furthermore, glucose was only minimally active and glyceraldehyde ineffective in inhibiting glucagon secretion due to arginine in the perifusion system. The most plausible explanation for the difference in the endocrine response of islet cells in the two types of widely used in vitro systems is that the alpha and beta cells have lost inhibitory receptors in the plasma membrane as a result of the collagenase isolation technic.

Animals↗
PubMed2003

Molecular relatedness between isolates Yersinia pseudotuberculosis from a patient and an isolate from mountain spring water.

A 40-yr-old buddhist monk was admitted to the hospital with abdominal pain, fever, and confusion. He had a history of drinking untreated mountain spring water in his temple, and experienced the above symptoms for several days before admission. In past medical history, he had suffered from hepatic cirrhosis. Yersinia pseudotuberculosis was isolated from his blood and ascitic fluid. The mountain spring water that he had ingested was cultivated and Y. pseudotuberculosis was also isolated. For identification of pathogenic Y. pseudotuberculosis, each isolate from the three sources (blood, ascitic fluid, and drinking water) was also analysed for the inv gene for Y. pseudotuberculosis and the virF gene for virulent plasmid by PCR. All strains were positive for both the virF and the inv genes and also positive for autoagglutination test. For relationship study, each isolate from the three sources was also analysed with serotyping and restriction endonuclease analysis of virulence plasmid DNA (REAP) using BamHI. All belonged to the serotype 4b and REAP pattern D. Thus, all these findings supported that the mountain spring water was the source of the Y. pseudotuberculosis infection in this case.

Adhesins, Bacterial↗
PubMed1993

Antigenic comparison of Ockelbo virus isolates from Sweden and Russia with Sindbis virus isolates from Europe, Africa, and Australia: further evidence for variation among alphaviruses.

The plaque-reduction neutralization test (PRNT) was used to compare 15 isolates of Ockelbo virus from Sweden, one isolate of Ockelbo virus from Russia, the Egyptian prototype Sindbis virus, and Sindbis-like viruses from Slovakia, South Africa, Cameroon, and Australia. Strains from northern Europe (Sweden and Russia) were indistinguishable by PRNT. We observed some antigenic variation between isolates of Sindbis virus from Europe, Africa, and Australia. An Australian strain (C-377) was shown to be distinct from prototype Sindbis virus, and the Acrocephalus strain from Slovakia was shown to be identical to prototype Sindbis virus. All other strains, including Ockelbo virus isolates, were shown to be subtypes of prototype Sindbis virus.

Alphavirus↗