Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0021051 (immunodeficiency)
 71,517 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We report on the development of a rapid nested PCR protocol for the detection of Pneumocystis carinii DNA in bronchoalveolar lavage (BAL) specimens in which the protocol included the use of a commercially available DNA extraction kit (GeneReleaser). GeneReleaser enabled us to obtain amplification-ready DNA within 20 min without requiring the purification of the DNA. The nested PCR was performed with the primers pAZ102-E, pAZ102-H, and pAZ102-L2 (A. E. Wakefield, F. J. Pixley, S. Banerji, K. Sinclair, R. F. Miller, E. R. Moxon, and J. M. Hopkin, Lancet 336:451-453, 1990.). Results were obtained in about 4 h with the adoption of denaturation, annealing, and extension steps shortened to 20 seconds. The sensitivity of the nested PCR was tested with a P. carinii cyst suspension and was found to be less than one cyst (one to eight nuclei). The detection limit was the same with the use of GeneReleaser or proteinase K-phenol chloroform for DNA extraction. The nested PCR assay was prospectively compared with staining with Giemsa and methenamine silver stains for the detection of P. carinii in 127 BAL samples from 105 human immunodeficiency virus-infected patients investigated for acute respiratory illness. Twenty-five BAL specimens (20%) were positive by staining and the nested PCR and 25 (20%) were negative by staining and positive by the nested PCR. These 25 BAL specimens with conflicting results were obtained from 23 patients, 82% of whom were receiving prophylactic therapy against P. carinii pneumonia (PCP). Only two patients were diagnosed with possible PCP. The final diagnosis was not PCP for 20 patients who were considered to be colonized or to have a low level of infection. This colonization is not of clinical importance but is of epidemiological importance. Our rapid, simple, and sensitive amplification protocol may be performed in clinical laboratories for the routine diagnosis of PCP with BAL specimens.
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PMID:Rapid detection of Pneumocystis carinii in bronchoalveolar lavage specimens from human immunodeficiency virus-infected patients: use of a simple DNA extraction procedure and nested PCR. 935 Jul 26

To study the initiation of human immunodeficiency virus type 1 reverse transcription, we have used the viral nucleocapsid protein (NC7) to anneal tRNA3Lys primer onto viral genomic RNA and have then eliminated NC7 from this primer-template complex by digestion with proteinase K and phenol-chloroform extraction of residual protein. Our data show that saturating concentrations of NC7 resulted in the formation of an active tRNA-template complex that yielded enhanced production of full-length negative-strand strong-stop DNA [(-)ssDNA] and that this complex remained active even after the elimination of NC7. While both of the two Zn finger motifs found within NC7 were essential for efficient elongation, NC protein that contained a point mutation in the first Zn finger or that was devoid of both Zn fingers yielded primer-template complexes that could still be initiated in 1-base-extension assays. In contrast, the use of heat annealing to produce primer-template complexes resulted in proportions of full-length (-)ssDNA lower than those seen with NC protein, and the addition of NC protein to such preformed primer-template complexes was able to reverse this defect only to a marginal extent.
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PMID:Roles of the human immunodeficiency virus type 1 nucleocapsid protein in annealing and initiation versus elongation in reverse transcription of viral negative-strand strong-stop DNA. 976 88

The detection of Pneumocystis carinii DNA in blood by PCR could be useful for studying the natural history of pneumocystosis and could also be a noninvasive diagnostic method. The results of previous studies are nevertheless conflicting. In our study, we compared three commercially available DNA extraction kits (GeneReleaser, QIAamp Tissue Kit, and ReadyAmp Genomic DNA Purification System) and proteinase K and proteinase K-phenol-chloroform treatments for the extraction of P. carinii DNA from dilutions of a P. carinii f. sp. hominis cyst suspension mixed with human whole blood. A rapid and simple nested PCR protocol which amplifies a portion of the mitochondrial large-subunit rRNA gene was applied to all the extraction products. The QIAmp Tissue Kit was the most effective kit for the isolation of amplification-ready P. carinii DNA and was used with nested PCR for the testing of whole-blood specimens from 35 immunocompetent control patients and 84 human immunodeficiency virus (HIV)-infected patients investigated for pulmonary disease and/or fever. In HIV-infected patients, P. carinii DNA was detected by nested PCR in blood samples from 3 of 14 patients with microscopically proven P. carinii pneumonia, 7 of 22 patients who were considered to be colonized with P. carinii, and 9 of 48 patients who were neither infected nor colonized with P. carinii. P. carinii DNA was not detected in blood specimens from the 35 immunocompetent patients. P. carinii DNA in blood might represent viable P. carinii organisms or DNA complexes released from pulmonary phagocytes. In conclusion, P. carinii DNA may be detected in whole blood from HIV-infected patients, but the nature and the meaning of the circulating form of P. carinii remain to be established.
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PMID:Detection of Pneumocystis carinii DNA in blood specimens from human immunodeficiency virus-infected patients by nested PCR. 985 76

A crude extract of Caulerpa taxifolia was tested for its antiviral activity. The chloroform-methanol residue showed an interesting inhibitor effect in vitro toward the feline immunodeficiency virus (FIV), a valid model for studying the acquired immunodeficiency syndrome. This extract reduced the virus-induced syncytia in the cultured cells, the viral reverse transcriptase activity and the viral capsid protein P24 expression.
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PMID:Antiviral properties of a crude extract from a green alga Caulerpa taxifolia (Vahl) C. Agardh. 1035 70

Polymerase chain reaction (PCR) techniques are used increasingly for the diagnosis of Mycobacterium tuberculosis infection and can be used on the DNA obtained from both frozen and formalin fixed, paraffin wax embedded tissues. However, the extraction of DNA by means of the conventional phenol/chloroform method is time consuming and requires the use of potentially dangerous chemical reagents. This paper describes a method based upon the use of magnetic beads for the extraction of M tuberculosis DNA from both routinely formalin fixed, paraffin wax embedded tissues and frozen tissues. Magnetic bead extracted DNA from brain, lymph node, and lung tissues collected from patients with human immunodeficiency virus and tuberculosis was compared with that extracted using the phenol/chloroform method. The magnetic bead extraction procedure requires less than two hours, including the time necessary to dewax the tissue sections. In all cases, the DNA extracted with both methods was amplified successfully by PCR for the M tuberculosis IS6110 sequence. Magnetic bead DNA extraction can be used on both frozen and archival tissues: the method is reliable, simple, sensitive, and rapid; in addition, it does not use hazardous procedures or specialised laboratory equipment and can be used for routine DNA isolation from various human tissues.
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PMID:Use of magnetic beads for tissue DNA extraction and IS6110 Mycobacterium tuberculosis PCR. 1062 38

The aim of this study was to validate the virus-inactivating/eliminating capacity of the manufacturing process of spongiosa cuboids. Both the sterilization step with peracetic acid (PAA)/ethanol and the defatting step of bones with chloroform/methanol (2:1, v/v) were investigated. Relevant enveloped, non-enveloped, and model viruses belonging to different virus families were included in the investigation: human immunodeficiency virus type 2 (HIV-2), hepatitis A virus (HAV), poliovirus (PV-1), pseudorabies virus (PRV), porcine parvovirus (PPV), and bovine virus diarrhoea virus (BVDV). Treatment of virus-spiked spongiosa cuboids for 4 hours at room temperature (RT) with 1% PAA/24% ethanol (PES) efficiently inactivated most viruses. Titres were reduced by more than 4 log(10)with the exception of HAV. The defatting step with chloroform/methanol reduced HAV titres by a factor of >/=7.0 log(10). From these results it can be concluded that the treatment of spongiosa cuboids with (i) chloroform/methanol and (ii) 1% PAA/24% ethanol solution leads to a virus-safe medicinal product.
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PMID:Virus safety of avital bone tissue transplants: evaluation of sterilization steps of spongiosa cuboids using a peracetic acid-methanol mixture. 1065 75

A sensitive bio-analytical assay for amprenavir, a human immunodeficiency virus protease inhibitor, based on reversed-phase liquid chromatography and fluorescence detection, is reported. The analyte is extracted from the matrix, plasma, cerebrospinal fluid (CSF) or semen, with chloroform using propyl-p-hydroxybenzoate as an internal standard. After centrifugation, evaporation of the organic phase and reconstitution in the eluent, the sample is injected into the chromatograph. The analyte is detected spectrofluorometrically at 270 and 340 nm for excitation and emission, respectively. The method has been validated in the 1-1000 ng/ml range for a 50-microl volume of plasma and in the 0.5-50 ng/ml range for a 100-microl volume of CSF and semen. The lower limit of quantification was 0.5 ng/ml in CSF and 1 ng/ml in both plasma and semen. Precision and accuracy both meet the current requirements for a bio-analytical assay and are <15% in the validated ranges. The assay was successfully used to obtain a concentration-time curve of amprenavir in plasma.
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PMID:Sensitive liquid chromatographic assay for amprenavir, a human immunodeficiency virus protease inhibitor, in human plasma, cerebrospinal fluid and semen. 1089 97

A low molecular weight (LMW) antigen of Eimeria tenella, initially identified using a murine monoclonal antibody (mAb C(3)4F(1)) raised against E. tenella sporozoites, was partially characterized using enzymatic degradation. solvent extraction, and immunization into various inbred lines of mice. The LMW antigen could be isolated using Folch extraction (methanol/chloroform/ water) and the epitope recognized by mAb C(3)4F(1) was resistant to degradation by alpha-amylase, pronase, and proteinase K, but was sensitive to sodium m-periodate treatment or digestion using mixed glycosidases (from Turbo cornutus). These observations suggest that the antigenic epitope recognized by mAb C(3)4F(1) is carbohydrate-dependent and, based on our ability to isolate the LMW antigen by Folch extraction, the epitope probably resides on a polar glycolipid. The inability of sporozoite-immunized nude mice to elicit a serum antibody response to this molecule indicates that it acts as a T-dependent antigen. Furthermore, sporozoite-immunized male CBA/N mice (with an X-linked immunodeficiency) also failed to elicit a serum antibody response to this molecule, which is consistent with a carbohydrate antigenic epitope. We propose that this antigenic molecule be designated ET-GL1 to reflect its origin and probable structure (E. tenella glycolipid 1).
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PMID:Partial characterization of a non-proteinaceous, low molecular weight antigen of Eimeria tenella. 1089 71

The accumulation of reports on the puzzling behavior of guanidinium-rich oligo/polymers in bilayer membranes, reaching from HIV-Tat-like (HIV Tat is the human immunodeficiency virus transactivator of transcription) translocation to selectivity and voltage-gating of ion channels, prompted us to investigate possible contributions from counteranions to these phenomena. We report that anion-mediated variability of charge and solubility makes guanidinium-rich oligo/polymers adaptable to many environments. For example, poly- and hexaarginine but not polylysine phase transferred from water into chloroform in the presence of amphiphilic anions such as monomeric sodium dodecyl sulfate (SDS), egg yolk phosphatidylglycerol (EYPG), cholesterol sulfate, pyrenebutyrate, and stearate. Hydrophilic anions with high affinity inhibited phase transfer of 5(6)-carboxyfluorescein (CF)-polyarginine complexes into bulk membranes (sulfate, adenosine 5'-triphosphate, adenosine 5'-monophosphate, heparin, and micellar SDS). At least binary anion cocktails were necessary to activate polyarginine as a carrier in bulk chloroform membranes. Refined combinations of EYPG, phosphate, and azide or TFA were found to maximize translocation of CF across bulk membranes by polyarginine. Polyarginine-mediated CF efflux from large unilamellar vesicles was best in the presence of EYPG in the bilayer as well as phosphate and TFA in the medium. Similar regulatory activities of several anions were in support of a common carrier mechanism for guanidinium-rich oligo/polymers in bulk and bilayer membranes. The identified activities of polyarginine in bulk and lipid membranes suggested that anion-mediated adaptability of the solubility of guanidinium-rich oligo/polymers cannot be ignored in studies on biological function. The infinite variability and dynamic nature of available regulatory anion cocktails may contribute to the elusive character of guanidinium-rich oligo/polymer function in biomembranes.
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PMID:Anion-mediated transfer of polyarginine across liquid and bilayer membranes. 1462 83

Human papillomavirus (HPV) has recently been associated with oral cancers. To prepare for a study of the natural history of oral HPV infection, the effect of the DNA purification method on HPV genomic DNA detection in Scope mouthwash oral rinse samples and the reproducibility of HPV detection in rinse samples collected 7 days apart were investigated. The study was conducted with a population at high risk for oral HPV infection: human immunodeficiency virus-infected men with CD4-cell counts <200. Five DNA purification methods were compared among equal aliquots of oral rinse samples collected from a subset of individuals. The purification methods included (i) proteinase K digestion (PKD) and heat inactivation; (ii) PKD and ethanol precipitation (EP); (iii) PKD, phenol-chloroform extraction, and EP; (iv) use of the Puregene DNA purification kit; and (v) use of the QIAamp DNA Blood Midi kit. HPV was detected by PCR amplification with PGMY09 and PGMY11 L1 primer pools and by use of a Roche linear array. Puregene-purified samples had higher human DNA yields and purities, and Puregene purification detected the greatest number of HPV-positive subjects and total HPV infections in comparison to the numbers detected by all other methods. The total number of HPV infections and HPV prevalence estimates were also higher for Puregene-processed oral rinse samples when a fixed volume (10 mul) rather than a fixed cell number ( approximately 50,000 cells) was used for PCR amplification. A good concordance was observed for oral HPV infection status (agreement, 80%; kappa value, = 0.60) and type-specific infection (agreement, 98%; kappa value, 0.57) in matched oral rinse samples. The method of DNA purification significantly affects the detection of HPV genomic DNA from oral rinse samples and may result in exposure misclassification that could contribute to the inconsistent associations reported in the literature.
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PMID:Analysis of the effect of DNA purification on detection of human papillomavirus in oral rinse samples by PCR. 1627 81


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