Biomedical subjects
Publications and source records attributed to J M Hopkin.
DNA amplification of specific sequences and subsequent oligonucleotide hybridization were used to search for Pneumocystis carinii in post-mortem lung samplings from non-immunosuppressed individuals ranging from 15 to 70 years of age. No P. carinii-specific DNA was detected in 45 DNA amplification reactions from 15 lungs.
We have developed a highly specific and sensitive technique for the detection of Pneumocystis carinii DNA using DNA amplification by the polymerase chain reaction (PCR). PCR products are detected by agarose gel electrophoresis and Southern hybridization to an oligonucleotide probe. Here we report the calibration of parasite numbers with amplification and hybridization signals and show that we can detect P. carinii to a lower limit of one to two organisms. The quantification of this diagnostic technique allows us to establish the number of organisms in a clinical sample which correspond to pneumocystis pneumonia or to sub-clinical pulmonary colonization.
Two studies were performed to compare the sensitivity of DNA amplification with immunofluorescence for the detection of Pneumocystis carinii in asymptomatic normal and immunosuppressed subjects receiving no anti-Pneumocystis chemoprophylaxis. In the first study, immunofluorescence and silver stains were used to examine 12 induced sputa and 12 bronchoalveolar lavage specimens from 24 normal control subjects; induced sputa from 20 renal transplant recipients; and induced sputa from 11 patients with fibrosing alveolitis. All specimens were negative for P carinii using both stains, apart from one renal patient in whom 2 P carinii cysts were seen by immunofluorescence alone. In the second study, DNA amplification and immunofluorescence were used to examine induced sputa from 3 groups of 10 control, renal, and heart/lung transplant recipients. All 30 specimens were negative for P carinii by immunofluorescence. However, 3 renal and 2 heart/lung patients were positive for P carinii by DNA amplification alone. One of these patients developed P carinii pneumonia 6 weeks after sputum induction. DNA amplification is a more sensitive technique than immunofluorescence for detecting P carinii. P carinii colonization occurs in asymptomatic organ transplant recipients, but not in normal individuals.
The results of testing for linkage between atopy and the chromosome 11 marker D11S97 is shown for all the 723 subjects genotyped by us up to January 1992. Lod score estimations were confounded by the high population prevalence of atopy, maternal inheritance of atopy at the 11q locus, genetic heterogeneity, and excess of atopy in families not ascertained through a single proband. Affected sib-pair analysis shows evidence for linkage which is not dependent on the definition of atopy or model specification. We suggest that presentation of sib-pair data will be suitable for meta-analysis of the different studies of genetic linkage and atopy.
We have undertaken a double blind placebo controlled study of the effect of nasal beclomethasone on the tendency to wheeze in 20 unselected hay fever sufferers, half with a history of previous seasonal wheezing. We found no difference between either bronchial hyperresponsiveness, as measured by methacholine challenge, home-monitored PEFR, nor recorded wheeze nor cough between treated and placebo groups although the numbers were small. All were allowed the antihistamine cetirizine hydrochloride 10 mg daily. Eighteen out of the 19 patients had either bronchial hyperresponsiveness (PD20 methacholine < 8 mumol or a > 2 doubling dose change in their PD20 during the pollen season). We have shown a significant positive correlation between a hay fever score (HFS) (created by taking the sum of the home scored; nasal discharge, nasal blockage, eye irritation, sneeze and antihistamine use) and peak seasonal specific IgE to mixed grass pollen (Spearman correlation coefficient 0.5 P < 0.02). There was also a positive correlation between the rise in specific IgE from pre to peak season and the HFS, correlation coefficient 0.6 P = 0.03).
Pairwise analysis of siblings from 21 families showed that house dust mite (HDM) sensitive children were exposed to higher concentrations of Der p I allergen in their mattress (P = 0.005) and bedding (P = 0.04), but not bedroom floor (P = 0.33), than their atopic sibling who was not sensitive to HDM antigens. There was no difference in the exposure to HDM numbers/100 mg of dust in the mattress (P = 0.61) or bedroom floors (P = 0.09). In contrast, pairwise analysis of siblings from 15 families showed that HDM sensitive children were not exposed to significantly different concentrations of Der p I in the mattress (P = 0.96), bedding (P = 0.11) or bedroom floor (P = 0.70) nor HDM numbers/100 mg of dust in the mattress (P = 0.12) and bedroom floor (P = 0.98) than their non-atopic siblings. These findings were identical when absolute allergen load was compared in these pairs. Genetic linkage studies in these families suggest the tendency to atopic IgE responses is conferred by a putative atopy locus on chromosome 11q. These results together suggest that differences in allergen levels in beds, among siblings with a comparable genetic tendency to atopy, play a significant role in determining the development of HDM allergy.
Pneumocystis carinii causes life-threatening pneumonia in T-lymphocyte-immunodeficient subjects in transplant and oncology units or with acquired immune deficiency syndrome (AIDS). Recent DNA homology studies show P. carinii to be a fungus. To investigate the biology and epidemiology of this parasite further, we elected to determine for it a more precise taxonomic assignment within the fungal kingdom. We screened a wide range of organisms representing the major orders of fungi using DNA amplification and subsequently sequenced a portion of the mitochondrial gene encoding the large subunit ribosomal RNA. Our data show that the opportunistic pulmonary pathogen P. carinii is closely related to the ustomycetous red yeast fungi, a group which includes organisms that are extensively distributed throughout the environment and which release many widely dispersed airborne spores.
Genetic linkage between atopic IgE responses and chromosome 11q13 (D11S97) has been previously reported in a limited number of extended families. Difficulties of phenotyping in the older family members, poor family structure in some families, and genetic heterogeneity were proposed as possible explanations for the variability in lod scores. To test this finding a second linkage study of 64 young nuclear families was undertaken and gave a two point lod score of 3.8 at theta = 0.07 (assuming theta m = theta f). A test of genetic heterogeneity in the nuclear families shows that atopic IgE responses are linked to this locus in 60 to 100% of families (approximate 95% confidence limits).
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DNA amplification and silver staining were used to identify Pneumocystis carinii in bronchoscopic lavage and induced sputum samples during 51 episodes of respiratory illness in 47 subjects with HIV infection. In 20 episodes, in which the clinical diagnosis was pneumocystis pneumonia (PCP), silver stain was positive in 14 lavage samples (70%) and 7 sputum samples (35%), whereas DNA amplification was positive in 19 lavage samples (95%) and 18 sputum samples (90%). DNA amplification was positive in only 1 of 31 patients without PCP (PCP developed in this patient within 10 weeks). DNA amplification on induced sputum offers a powerful technique for diagnosis of PCP.
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A 6.8 kilobase fragment of mitochondrial DNA from Pneumocystis carinii encodes for apocytochrome b, NADH dehydrogenase subunits 1, 2, 3, and 6, cytochrome oxidase subunit II, and the small subunit of ribosomal RNA. Comparative sequence analysis with a series of organisms representative of the fungal and protozoan groups shows that P. carinii has, consistently, an average similarity of 60% with the fungi but only 20% with the protozoa. The data indicate homology with the fungi for this opportunistic pathogen.
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Mycobacterial infections occurred in 11 of 633 (1.7 per cent) recipients of successful renal transplants. There were no cases of tuberculosis in patients receiving chemoprophylaxis, but amongst those who did not receive prophylaxis disease occurred in six of the 27 (22 per cent) high-risk patients. The major cause of morbidity during treatment was renal allograft rejection, largely due to reduction in immunosuppressive drug therapy.
Oligonucleotide primers and probes were used in the polymerase chain reaction to amplify Pneumocystis carinii specific DNA sequences from alveolar lavage samples from 47 diagnostic bronchoscopies. No P carinii DNA was found in lavage from 10 immunocompetent patients; only low levels were found in 3 of 13 samples from immunosuppressed individuals without P carinii pneumonia (PCP), and the highest levels, readily demonstrated by simple ethidium bromide staining, were found in all of 16 samples from immunosuppressed patients with PCP confirmed by means of standard silver staining and in 4 from patients with clinical PCP but negative silver staining. DNA amplification provides a highly sensitive and specific technique for the identification of P carinii that should be valuable in epidemiological studies on this parasitic infection and in diagnosis.
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Pneumocystis carinii specific DNA sequences have been cloned from the experimental rat model. The sequence of the gene coding for the large subunit of mitochondrial ribosomal RNA has been used to construct P. carinii specific oligonucleotide primers for the polymerase chain reaction. These oligonucleotides produced amplification of specific sequences from both P. carinii infected rat and human lung samplings, but none from a range of other organisms including potential pulmonary pathogens. Comparison of the sequence of amplified products from the infected rats and humans demonstrated limited but consistent differences between P. carinii from these two hosts and allowed for the construction of a human specific internal oligonucleotide. The application of the specific oligonucleotides for DNA amplification and subsequent Southern hybridisation affords extremely sensitive and specific detection of P. carinii in human samples, which may be applicable to both epidemiological research and clinical studies.
Twenty-five atopic children under 11 years of age were studied, using skin and RAST tests, for their specific IgE response to four species of pyroglyphid house dust mites, Dermatophagoides pteronyssinus, D. farinae, D. microceras and Euroglyphus maynei. All of the children were sensitive to D. pteronyssinus, 20 (80%) of these children were also sensitive to D. farinae and D. microceras, and 16 of the latter (64%) were also sensitive to E. maynei. Dust samples from various sites in the homes of the children revealed D. pteronyssinus in all homes studied but no D. farinae or D. microceras. E. maynei, although identified, was not present in significant numbers in any site. A control group of 20 atopic children of similar age who were not sensitive to house dust mite allergens had a similar exposure to the four mite species. These results suggest that factors in addition to mite exposure are important in the development of specific IgE responses to house dust mites.