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A E Wakefield

Publications and source records attributed to A E Wakefield.

At least 37 records · Page 2Linked to original sources
PubMed2000

Population structure of rat-derived Pneumocystis carinii in Danish wild rats.

The rat model of Pneumocystis carinii pneumonia is frequently used to study human P. carinii infection, but there are many differences between the rat and human infections. We studied naturally acquired P. carinii in wild rats to examine the relevance of the rat model for human infection. P. carinii DNA was detected in 47 of 51 wild rats and in 10 of 12 nonimmunosuppressed laboratory rats. Evidence for three novel formae speciales of rat-derived P. carinii was found, and these were provisionally named Pneumocystis carinii f. sp. rattus-secundi, Pneumocystis carinii f. sp. rattus-tertii, and Pneumocystis carinii f. sp. rattus-quarti. Our data suggest that low-level carriage of P. carinii in wild rats and nonimmunosuppressed laboratory rats is common and that wild rats are frequently coinfected with more than one forma specialis of P. carinii. We also examined the diversity in the internally transcribed spacer (ITS) regions of the nuclear rRNA operon of Pneumocystis carinii f. sp. carinii by using samples from wild rats and laboratory rats and spore trap samples. We report a lack of variation in the ITS1 and ITS2 regions that is consistent with an evolutionary bottleneck in the P. carinii f. sp. carinii population. This study shows that human- and rat-derived P. carinii organisms are very different, not only in genetic composition but also in population structure and natural history.

Animals↗
PubMed2000

The role of physiotherapy and clinical predictors of outcome after fracture of the distal radius.

The capacity for physiotherapy to improve the outcome after fracture of the distal radius is unproven. We carried out a randomised controlled trial on 96 patients, comparing conventional physiotherapy with a regime of home exercises. The function of the upper limb was assessed at the time of removal of the plaster cast and at three and six months after injury. Factors which may predict poor outcome in these patients were sought. Grip strength and hand function did not significantly differ between the two groups. Flexion and extension of the wrist were the only movements to improve with physiotherapy at six months (p = 0.001). Predictors of poor functional outcome were malunion and impaired function before the fracture. These patients presented with pain, decreased rotation of the forearm and low functional scores at six weeks. Our study has shown that home exercises are adequate rehabilitation after uncomplicated fracture of the distal radius, and routine referral for a course of physiotherapy should be discouraged. The role of physiotherapy in patients at high risk of a poor outcome requires further investigation.

Aged↗
PubMed1999

Discrimination of rat-derived Pneumocystis carinii f. sp. Carinii and Pneumocystis carinii f. sp. Ratti using the polymerase chain reaction.

The rat model of Pneumocystis carinii infection is widely used for the study of this non-culturable pathogen. Two genetically divergent < > of the organism have been detected in infected rat lungs, P. carinii formae specialis carinii and P. carinii formae specialis ratti, in some cases as a co-infection. We have developed a simple and rapid method to analyse rat-derived P. carinii samples, based on DNA amplification of a portion of the gene encoding the mitochondrial large subunit ribosomal RNA. A pair of oligonucleotide primers were designed for each special form of rat-derived P. carinii, the RC primer pair amplifying a 137 bp fragment from P. carinii f. sp. carinii DNA and the RR primer pair amplifying a 251 bp fragment from P. carinii f. sp. ratti DNA. The specificity of the primers was confirmed by sequencing the amplification products. The polymerase chain reaction (PCR) technique was consistent with, and more sensitive than, the electrophoretic karyotype method. The application of the specific PCR technique has implications for future studies on epidemiology, drug sensitivity, immunology and molecular biology of rat-derived P. carinii.

Animals↗
PubMed1999

Pneumocystis carinii infection in young non-immunosuppressed rabbits. Kinetics of infection and of the primary specific immune response.

The aim of this study was to determine the kinetics, the dissemination of the infection and the immunological response to Pneumocystis carinii primary infection in a non-immunosuppressed rabbit model. For this purpose, we developed a nested PCR that amplified a portion of the mitochondrial large-subunit rRNA gene of rabbit-derived P. carinii. The PCR detected P. carinii DNA in lung and bronchoalveolar lavage fluids from 14- to 45-day-old rabbits but not in their serum. No P. carinii DNA was detected in extrapulmonary organs from 28-day-old rabbits with P. carinii pneumonia. ELISA and immunoblotting analysis showed that 5-day-old pups had elevated specific IgG. The IgG concentration sharply decreased, reaching a trough on day 21, and from then onwards progressively increased as the infection cleared. Conversely, the specific IgM concentration increased during the infection and peaked on day 28. IgG mainly recognized a 50-kDa subunit of P. carinii organisms; IgM recognized first a 45-kDa subunit on day 21, whereas from day 28 onwards it also recognized the 50-kDa subunit. A P. carinii-specific splenocyte proliferative response was observed on day 45. These findings suggest that P. carinii primary infection is a time-limited and a lung-limited event and contribute new information on the relationship between the kinetics of primary P. carinii infection and the immunological response in a model that mimics the primary infections in humans.

Aging↗
PubMed1999

Cell surface protease PRT1 identified in the fungal pathogen Pneumocystis carinii.

The subtelomeric regions of the chromosomes of many organisms contain gene families that allow adaptation to a changing environment. In a number of parasites, these subtelomeric gene families encode cell surface proteins that undergo antigenic variation. Proteases are another important virulence determinant in pathogenic microorganisms. We report the localization of the PRT1 protease of the pathogenic fungus Pneumocystis carinii sp. f. carinii, encoded by a subtelomeric gene family, to the cell surface of both the trophozoite and the cyst phase of the organism. Using anti-PRT1 antiserum, we demonstrated specificity to P. carinii sp. f. carinii in sections of infected rat lungs and, using immunofluorescence, we showed that the PRT1 protease has the characteristic distribution of a surface protein. The anti-PRT1 antiserum showed cross-reactivity with a number of P. carinii sp. f. carinii proteins migrating between 185 kDa and 28 kDa, the majority migrating between 42 kDa and 52 kDa, a region that has been shown by serological studies to contain important immunodominant P. carinii proteins. Cross-reactivity was also observed with P. carinii sp. f. hominis proteins. We have also cloned a portion of the catalytic domain of PRT1 from P. carinii sp. f. hominis, P. carinii sp. f. muris and P. carinii sp. f. rattus. Our data suggest that the PRT1 protease plays an important role in the pathogenicity of P. carinii.

Amino Acid Sequence↗
PubMed1999

Association of primary Pneumocystis carinii infection and sudden infant death syndrome.

To delineate clinical and histological features of the first Pneumocystis carinii infection affecting the immunocompetent host, P. carinii-specific histological stains were performed on autopsy lung specimens from 534 consecutive pediatric patients (those with AIDS and malignancies were excluded) in Santiago, Chile. P. carinii clusters were found in 4 (25%) of 16 infants who died of no apparent cause at arrival to the emergency department, and in 10 (2.9%) of 342 infants who died of multiple conditions at the hospital (P=.002, Fisher's exact test). This prompted us to analyze additional series of infants with sudden infant death syndrome (SIDS). In 161 additional SIDS cases, 47 (35.1%) of 134 infants from Chile and 4 (14.8%) of 27 infants from Oxford, United Kingdom, were found to have P. carinii clusters in the lungs. The quantity of P. carinii cysts was small compared with the numbers seen in immunocompromised hosts with P. carinii pneumonitis. This study provides histological evidence that primary P. carinii infection is associated with SIDS.

Autopsy↗
PubMed1999

The affinity of lipid-coated microbubbles to maturing spinal cord injury sites.

OBJECTIVE: This laboratory has demonstrated that lipid-coated microbubbles (LCMs) effectively aggregate and deliver chemotherapeutic drugs into rat brain tumor cells and antigliosis agents into maturing rat brain injury sites. In this study, we report the affinity of tail vein-injected LCMs to the injured rat spinal cord by a compressive lesion to the upper thoracic region. METHODS: The accumulation of LCMs in the injured spinal cord was analyzed by labeling it with a lipid-soluble fluorescent dye, 3,3'-dioctadecyloxacarbocyanine perchlorate. Indices of glial fibrillary acidic protein were measured concomitantly with 3,3'-dioctadecyloxacarbocyanine perchlorate-labeled LCMs using confocal microscopy. RESULTS: There was no aggregation of LCMs accumulated 1 and 6 hours after injury; however, when given 2, 4, and 7 days after injury, LCMs showed a clear affinity for the injured region. LCM aggregation shifted from the central necrotic area of the injury on postinjury Day 2 and postinjury Day 4 to the white matter among glial fibrillary acidic protein-positive astrocytes by postinjury Day 7. CONCLUSION: Affinity of LCMs for spinal cord injury sites may be mediated in the early stages after injury by proliferating macrophages in the necrotic center, and then in later stages by glial fibrillary acidic protein-positive astrocytes in adjacent white matter. These findings suggest a potential for using LCMs as a delivery vehicle to concentrate lipid-soluble agents in spinal cord injury sites.

Animals↗
PubMed1998

Sequence polymorphisms in the Pneumocystis carinii cytochrome b gene and their association with atovaquone prophylaxis failure.

Atovaquone (Mepron, 566c80) is an effective agent against Pneumocystis carinii, which probably acts by binding to cytochrome b and inhibiting electron transport. To assess the possibility that atovaquone resistance might be developing, the genes for the cytochrome b from P. carinii sp. f. carinii and P. carinii sp. f. hominis were partially sequenced. Eight of 10 patient isolates had cytochrome b genes with the same amino acid sequence. The P. carinii cytochrome b genes from 2 of 4 patients who had atovaquone prophylaxis failure contained mutations resulting in amino acid changes in one of the ubiquinone (coenzyme Q) binding sites (Qo). These mutations are homologous to mutations in other microorganisms that confer resistance to similar inhibitors. Variations in the sequence of the P. carinii cytochrome b gene suggest but do not prove the development of drug resistance.

Amino Acid Sequence↗
PubMed1998

Clusters of Pneumocystis carinii pneumonia: analysis of person-to-person transmission by genotyping.

Genotyping at the internal transcribed spacer (ITS) regions of the nuclear rRNA operon was performed on isolates of P. carinii sp. f. hominis from three clusters of P. carinii pneumonia among eight patients with haematological malignancies and six with HIV infection. Nine different ITS sequence types of P. carinii sp. f. hominis were identified in the samples from the patients with haematological malignancies, suggesting that this cluster of cases of P. carinii pneumonia was unlikely to have resulted from nosocomial transmission. A common ITS sequence type was observed in two of the patients with haematological malignancies who shared a hospital room, and also in two of the patients with HIV infection who had prolonged close contact on the ward. In contrast, different ITS sequence types were detected in samples from an HIV-infected homosexual couple who shared the same household. These data suggest that person-to-person transmission of P. carinii sp. f. hominis may occur from infected to susceptible immunosuppressed patients with close contact within hospital environments. However direct transmission between patients did not account for the majority of cases within the clusters, suggesting that person-to-person transmission of P. carinii sp. f. hominis infection may be a relatively infrequent event and does not constitute the major route of transmission in man.

AIDS-Related Opportunistic Infections↗
PubMed1998

The use of lipid-coated microbubbles as a delivery agent of 7beta-hydroxycholesterol in a radiofrequency lesion in the rat brain.

OBJECTIVE: This laboratory has previously described the aggregation of intravenously administered lipid-coated microbubbles (LCM) around tumors and areas of injury. 7Beta-hydroxycholesterol has been used to inhibit astrocytic proliferation in nervous system injury models. The compound has been given by direct infusion, by epidural catheter, or in liposomes (delivered stereotactically to the injury site). In this article, we report the use of LCM to deliver 7beta-hydroxycholesterol to a radiofrequency injury site in the rat cerebrum. METHODS: First, the ability of LCM to target the thermal lesion in the rat brain was characterized using a lipid-soluble fluorescent dye 3,3-dioctadecyloxacarbocyanine perchlorate. Then, the effectiveness of this delivery system in suppression of glial proliferation was measured by glial fibrillary acidic protein immunoreactivity. RESULTS: Glial fibrillary acidic protein immunoreactivity was significantly reduced when 7beta-hydroxycholesterol was administered via LCM but not alone, suggesting that astrocytic proliferation would correspondingly be diminished. CONCLUSION: LCM were assessed as a delivery vehicle for 7beta-hydroxycholesterol in a rat brain radiofrequency lesion and found to be efficient in reducing astrogliosis, as measured by glial fibrillary acidic protein immunoreactivity.

Animals↗
PubMed1998

Genetic heterogeneity in Pneumocystis carinii: an introduction.

Molecular techniques have played an important role in demonstrating a high level of heterogeneity among the different types of organisms which are collectively known as Pneumocystis carinii. Genetic heterogeneity has been observed in Pneumocystis organisms isolated from different host species, suggesting that Pneumocystis infection is host-species specific. On the basis of this genetic divergence a provisional trinomial nomenclature has been adopted, in which different types of Pneumocystis organisms are designated a 'special form'. Multiple special forms of Pneumocystis have been detected in some host species, for example in infected rat and ferret lungs, but not in human lungs. Lower levels of genetic heterogeneity have been observed within each Pneumocystis special form, and in particular in human-derived Pneumocystis. Analysis of the genetic heterogeneity of populations of Pneumocystis is contributing to the understanding of the epidemiology and pathophysiology of this infection.

Animals↗
PubMed1998

Genetic heterogeneity in human-derived Pneumocystis carinii.

Genetic heterogeneity has been observed among isolates of human-derived Pneumocystis carinii (P. carinii sp. f. hominis). DNA sequence analysis has been shown to be informative in distinguishing between isolates of P. carinii sp. f. hominis. Single base polymorphisms have been observed in the genes encoding the mitochondrial large subunit ribosomal RNA, the mitochondrial small subunit ribosomal RNA and the AROM protein. The highest level of genetic variation has been found at the internal transcribed spacer (ITS) regions of the nuclear ribosomal RNA operon. Typing of isolates of P. carinii sp. f. hominis has enabled the examination of the frequency of different types of P. carinii sp. f. hominis in distinct populations. It has also facilitated studies on the acquisition and transmission of P. carinii sp. f. hominis infection.

Genetic Heterogeneity↗
PubMed1998

PCR for detecting Pneumocystis carinii in clinical or environmental samples.

Since Pneumocystis carinii cannot be cultured in vitro, the introduction of polymerase chain reaction (PCR) has been an enormous advantage for research purposes. It is now possible to detect P. carinii in specimens containing low numbers of organisms where conventional detection methods using microscopic examination of histochemical stains has been insufficient. PCR has been used to detect P. carinii in bronchoalveolar lavage, induced sputum, spontaneous expectorates, oropharyngeal gargles, nasopharyngeal aspirates, serum, blood and in environmental samples. The use of PCR will enable the study of the epidemiology of P. carinii infection by detecting the organism in environmental samples, permitting molecular typing and thereby the study of the transmission of the organism. Furthermore PCR will facilitate studies on the response to therapy, studies monitoring for the emergence of drug resistant strains of P. carinii and in the diagnosis of P. carinii pneumonia in noninvasive specimens, in patients unable to undergo more invasive diagnostic procedures.

Environmental Microbiology↗
PubMed1998

Pre-AIDS era isolates of Pneumocystis carinii f. sp. hominis: high genotype similarity with contemporary isolates.

Isolates of Pneumocystis carinii f. sp. hominis were examined from six individuals who died of P. carinii pneumonia between 1968 and 1981 and who had underlying immunodeficiencies which were not due to human immunodeficiency virus infection. DNA sequence variation was analyzed in the genes encoding the mitochondrial large subunit rRNA (mt LSU rRNA), the internal transcribed spacer (ITS) regions of the nuclear rRNA, the arom locus, and the mitochondrial small subunit rRNA. No major variations were observed when these isolates were compared to isolates from HIV-infected individuals. A small number of minor differences were detected. A new position at which variation occurred in the mt LSU rRNA was observed in one sample. Three new ITS sequence types were identified. A total of nine different ITS sequence types were found in the six samples. Mixed infection with different ITS sequence types of P. carinii f. sp. hominis was observed in four of the six samples. The ITS locus was the most informative of the four loci for distinguishing among the isolates of P. carinii f. sp. hominis. The data suggest that isolates of P. carinii f. sp. hominis from before the AIDS pandemic are genetically very similar to those currently found in HIV-infected individuals.

AIDS-Related Opportunistic Infections↗
PubMed1998

Genetics, metabolism and host specificity of Pneumocystis carinii.

Pneumocystis carinii is a major cause of severe pneumonia in immunosuppressed individuals, especially in those with human immunodeficiency virus (HIV) infection during their period of progression to acquired immunodeficiency syndrome (AIDS), and constitutes a worldwide problem to public health. Recently, significant advances in the development of experimental animal models of P. carinii infection, as well as in our knowledge of the genetic diversity and taxonomy of P. carinii, have been made. These advances may contribute to our understanding of the transmission of P. carinii pneumonia (PCP) and to the development of new prevention and control strategies. This paper addresses questions relating to the epidemiology of PCP including the detection of the parasite in the environment and in patients, the mechanism of genetic variation of the major surface glycoprotein (MSG) of P. carinii, and host-related genetic variation among isolates of this organism, emphasizing phenotypic expression and its impact on epidemiology and taxonomy.

AIDS-Related Opportunistic Infections↗
PubMed1997

Identification of porcine Pneumocystis carinii as a genetically distinct organism by DNA amplification.

DNA was amplified from lung samples from three piglets infected with Pneumocystis carinii, using oligonucleotide primers designed to the P. carinii mitochondrial large subunit ribosomal RNA gene. The nucleotide sequence of the amplification product was determined and indicated lack of sequence variation among these pig-derived P. carinii samples at this locus. The data showed that porcine P. carinii was genetically distinct from P. carinii isolated from other mammalian host species.

Animals↗
PubMed1996

Non specific PCR products using rat-derived Pneumocystis carinii dihydrofolate reductase gene-specific primers in DNA amplification of human respiratory samples.

DNA amplification using dihydrofolate reductase (DHFR) primers in bronchoalveolar lavage fluids (BALFs) from patients with Pneumocystis carinii (PC) pneumonia yielded low sensitivity and specificity. Amplified products of BALFs from an AIDS patient without PC pneumonia and five patients with PC pneumonia were cloned and sequenced. All samples showed the same sequence without any homology with DHFR cDNA of rat PC, or with any DHFR sequences in databases at the DNA or amino acid level. The data demonstrate that these DHFR primers amplify a non-specific region of DNA with a sequence unrelated to the human PC DHFR gene both in PC positive and in PC negative samples. This finding precludes the use of these DHFR primers for the diagnosis of PC pneumonia in respiratory specimens.

Base Sequence↗