Polymorphisms in Pneumocystis jirovecii strains in Spanish children with cystic fibrosis.
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Child↗
Biomedical subjects
F J Medrano
Publications and source records attributed to F J Medrano.
Pneumocystis jirovecii colonisation in patients with interstitial lung disease.
A prospective study was conducted to determine the prevalence of colonisation by Pneumocystis jirovecii in 80 consecutive patients who required bronchoscopy and bronchoalveolar lavage (BAL) following suspicion of interstitial lung disease (ILD). The mtLSU rRNA gene of P. jirovecii was identified by nested PCR in BAL samples. Patients with ILDs were divided into three groups: group A comprised those with idiopathic interstitial pneumonias; group B comprised those with sarcoidosis; and group C comprised those with other ILDs. The overall prevalence of P. jirovecii carriage was 33.8%, with colonisation rates of 37.8%, 18.8% and 37% in groups A, B and C, respectively (p not significant). There were more smokers among the carriers, but there were no other significant differences between carriers and non-carriers. The high prevalence of P. jirovecii carriers found among immunocompetent patients with ILDs in Spain suggests a possible role of P. jirovecii in the natural history of these diseases.
Adult↗
Prevalence of colonisation and genotypic characterisation of Pneumocystis jirovecii among cystic fibrosis patients in Spain.
Pneumocystis jirovecii colonisation may occur among cystic fibrosis (CF) patients because of their underlying pulmonary disease. A wide epidemiological analysis was performed among CF patients from Spain to assess the prevalence of P. jirovecii colonisation and the distribution of different genotypes. P. jirovecii was identified by nested PCR targeting the mitochondrial large-subunit rRNA gene from sputum samples or oropharyngeal washes. The genotype was determined by direct sequencing. The prevalence of P. jirovecii colonisation among 88 consecutive CF patients was 21.5%. The polymorphisms identified were 85C/248C (45.4%), 85T/248C (27.2%) and 85A/248C (18.1%); in one case, a mix of genotypes was found. Colonisation was more frequent in subjects aged < 18 years (25.5% vs. 15.1%). Among the patients studied, 20.8% received treatment with azithromycin; all of these patients were colonised with P. jirovecii, but none developed Pneumocystis pneumonia (PcP) during a 1-year follow-up period. Concordance in the colonisation status of siblings suggested a common source of infection or person-to-person transmission.
Adolescent↗
Purification of a 6.5 kDa protease inhibitor from Amazon Inga umbratica seeds effective against serine proteases of the boll weevil Anthonomus grandis.
A 6.5 kDa serine protease inhibitor was purified by anion-exchange chromatography from the crude extract of the Inga umbratica seeds, containing inhibitor isoforms ranging from 6.3 to 6.7 kDa and protease inhibitors of approximately 19 kDa. The purified protein was characterized as a potent inhibitor against trypsin and chymotrypsin and it was named I. umbratica trypsin and chymotrypsin inhibitor (IUTCI). MALDI-TOF spectra of the IUTCI, in the presence of DTT, showed six disulfide bonds content, suggesting that this inhibitor belongs to Bowman-Birk family. The circular dichroism spectroscopy indicates that IUTCI is predominantly formed by unordered and beta-sheet secondary structure. It was also characterized, by fluorescence spectroscopy, as a stable protein at range of pH from 5.0 to 7.0. Moreover, this inhibitor at concentration of 75 microM presented a remarkable inhibitory activity (60%) against digestive serine proteases from boll weevil Anthonomus grandis, an important economical cotton pest.
Chromatography, Ion Exchange↗
Crystallization, data collection and phasing of infestin 4, a factor XIIa inhibitor.
Infestin is a protein from Triatoma infestans (kissing bug) composed of seven Kazal-type domains that is further processed to yield several serine protease inhibitors with varying specificities. Infestins 3 and 4 are the last two domains of the infestin gene and are found in vivo in the insect's anterior midgut. The last domain, infestin 4, has been cloned, expressed and purified. Here, the crystallization of infestin 4 using the sitting-drop vapour-diffusion method with PEG 8000 as precipitant is described. Crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 25.89, b = 45.64, c = 57.41 A. X-ray diffraction data were collected to a maximum resolution of 1.8 A using a synchrotron-radiation source. Initial phases were calculated by molecular replacement using an edited rhodniin molecule as the search model. Structure refinement is in progress.
Animals↗
Pneumocystis jiroveci genotypes in the Spanish population.
This study describes the genotype distribution of Pneumocystis jiroveci in 79 respiratory samples obtained from 15 patients with acquired immunodeficiency syndrome (AIDS) with P. jiroveci pneumonia and 64 human immunodeficiency virus-negative subjects with different chronic pulmonary diseases. The genotyping was based in analysis of 2 independent genetic loci: the mitochondrial large subunit ribosomal RNA (mt LSU rRNA) fragment (assessed by direct sequencing) and the gene for dihydropteroate synthase (DHPS; assessed by restriction fragment-length polymorphism). The mt LSU rRNA analysis revealed the presence of 3 different polymorphisms for both populations. The major genotype, 85C/248C, was found to be significantly higher in patients with AIDS and P. jiroveci pneumonia than in patients with pulmonary disease. The rate of genotypes 85A/248C and 85T/248C was similar in both groups. The analysis of DHPS genotypes assesses the prevalence of its 4 possible genotypes, with 35.5% of genotypes related to sulfa resistance. The data suggest a common source of infection between both groups.
Gene Frequency↗
Pneumocystis jiroveci isolates with dihydropteroate synthase mutations in patients with chronic bronchitis.
Since mutations in the dihydropteroate synthase (DHPS) gene possibly associated with sulfonamide resistance have been reported in patients with Pneumocystis jiroveci (previously carinii) pneumonia, and since P. jiroveci colonization has been recently demonstrated in patients with chronic pulmonary diseases, the present study aimed to investigate the possible occurrence of P. jiroveci DHPS mutations in patients with chronic bronchitis. P. jiroveci colonization was detected in 15 of 37 non-selected patients with chronic bronchitis by amplifying the large subunit of the mitochondrial gene of the ribosomal RNA using nested PCR. DHPS mutations were demonstrated using touchdown PCR and restriction enzyme analysis in two of eight patients with chronic bronchitis and in two of six patients from the same region who had AIDS-associated Pneumocystis pneumonia. In all cases, mutations were observed in subjects with no prior exposure to sulfonamides. These data could have important implications for public health, since (i) P. jiroveci colonization could speed the progression of chronic bronchitis, and (ii) these patients, who are customary sputum producers, could represent a reservoir for sulfonamide-resistant strains with the potential ability to transmit them to immunocompromised hosts susceptible to Pneumocystis pneumonia.
Age Distribution↗
Association between human-Pneumocystis infection and small-cell lung carcinoma.
BACKGROUND: Tobacco smoking is the most important but not the only risk factor in lung carcinoma. There is evidence that certain infections, which cause chronic inflammatory reactions, can also induce tumour development. It has recently been shown that patients with chronic pulmonary diseases present a high rate of subclinical Pneumocystis infection, and that the latter is able to induce inflammatory responses and alveolar cell alterations. The possible role of Pneumocystis infection in the development of lung neoplasms thus deserves consideration. MATERIAL AND METHODS: Polymerase chain reaction has been used to analyze the presence of DNA of two independent loci of the Pneumocystis genome: the mitochondrial region (mtLSU rRNA) and the gene encoding for the dihydropteroate synthase enzyme, in paraffin-embedded tissue blocks of 10 cases of small cell lung carcinoma (SCLC) and 10 cases of nonsmall cell lung carcinoma (NSCLC) with similar demographic and clinical characteristics. Five cases without lung pathology, and two cases of Pneumocystis pneumonia were also analyzed as controls. RESULTS: DNA of the microorganism was found in all the cases of SCLC but in only two of the NSCLC, and in none of the controls without pulmonary disease - thus implying a statistically significant association (P < 0.0001) between subclinical Pneumocystis infection and SCLC. CONCLUSIONS: While the nature of this association is not clear, it nevertheless constitutes an important finding - either the infection is specifically facilitated by this tumour or induces the development of this type of neoplasm in combination with other factors. Eur J Clin Invest 2004; 34 (3): 229-335
Adult↗
Epidemiology of Pneumocystis carinii pneumonia in southern Spain.
In order to investigate the impact of Pneumocystis carinii infection in southern Spain following the introduction of highly active anti-retroviral therapy (HAART), all cases of pneumocystosis between 1998 and 1999 were identified from data compiled by the national surveillance system. In total, 498 cases of pneumocystosis were recorded, of which 87% involved HIV-positive patients. The mean age, length of hospital stay and mortality were higher for HIV-negative patients. There was a higher number of cases in winter. Despite HAART implementation, pneumocystosis remains a significant health problem for both HIV-positive and HIV-negative patients.
AIDS-Related Opportunistic Infections↗
Climatic factors and Pneumocystis jiroveci infection in southern Spain.
The modes of infection and transmission of Pneumocystis jiroveci remain unclear. This study explored the relationship between the incidence of infection and climatic factors. In total, 536 cases of P. jiroveci infection were identified in the period 1994-1998, with an inverse correlation between the incidence of Pneumocystis pneumonia and the minimum mean ambient temperature (Spearman correlation coefficient: r - 0.30; p 0.02; ARIMA model: r - 0.250, p 0.07). The highest number of cases occurred in winter (anova test, p < 0.05), and there was a clear season-related incidence of P. jiroveci infection.
Climate↗
High seroprevalence of Pneumocystis infection in Spanish children.
Pneumocystis infection occurs worldwide, and most individuals test seropositive for Pneumocystis early in childhood. Little is known about the epidemiology of this infection in western Europe. The seroprevalence of Pneumocystis infection in 233 Spanish children was determined in a community study by immunoblot analysis of sera. The overall seroprevalence was 73%, with an age-related increase from 52% at 6 years to 66% at 10 years and 80% at 13 years. The data indicated a high seroprevalence of Pneumocystis infection in healthy Spanish children, thereby demonstrating that this pathogen is widespread in southern Spain.
Adolescent↗
Crystallization, data collection and phasing of black-eyed pea trypsin/chymotrypsin inhibitor in complex with bovine beta-trypsin.
The black-eyed pea trypsin and chymotrypsin inhibitor (BTCI) is a Bowman-Birk-type inhibitor from Vigna unguiculata seeds. A complex of BTCI with bovine beta-trypsin was crystallized by the hanging-drop vapour-diffusion method with ammonium sulfate as precipitant. Crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 59.3, b = 61.8, c = 80.0 A. Diffraction data were collected to 2.36 A resolution and were processed to give an overall R(sym) of 0.137. The Matthews coefficient for one complex per asymmetric unit is 2.2 A(3) Da(-1), with a corresponding solvent content of 43%. After molecular replacement and initial refinement, the model gives an R(cryst) of 0.361 and an R(free) of 0.432.
Animals↗
The pathogenesis of Leishmania/HIV co-infection: cellular and immunological mechanisms.
The intracellular protozoan parasites of the genus Leishmania have been recognized as opportunistic pathogens in immunosuppressed individuals, including those infected with human immunodeficiency virus type-1 (HIV-1). Leishmaniasis and AIDS overlap in several sub-tropical and tropical regions around the world, including the Mediterranean area. In 1994, 3%-7% of HIV-1-infected individuals in southern Europe developed visceral leishmaniasis. In humans, interestingly, both HIV-1 and Leishmania interact with, invade, and multiply within cells of myeloid or lymphoid origin. The combined modulation of Leishmania - and HIV-1-related pathogenesis in the co-infected cases is therefore probably a realistic goal. In the light of the recent demonstration that L. donovani can up-regulate HIV-1 replication, both in monocytoid and lymphoid cells in vitro and in co-infected individuals, it is clear from the epidemiological data available that Leishmania can probably act as a powerful co-factor in the pathogenesis of HIV-1 infection. In those who are co-infected, complex mechanisms involving cytokine secretion and cellular-signalling events play pivotal roles in the Leishmania-mediated activation and pathogenesis of HIV-1. An overview of the recent findings concerning this Leishmania/HIV-1 interaction is presented here.
AIDS-Related Opportunistic Infections↗
Visceral leishmaniasis in those infected with HIV: clinical aspects and other opportunistic infections.
Cases of visceral leishmaniasis (VL) in HIV-positive individuals have been reported from most areas of the world where the geographical distributions of the two infections overlap. The majority of the co-infected cases that have been recorded, however, live around the Mediterranean basin. In these subjects, the length of the incubation period of VL is presumably very short, particularly in those who have severe immunodepression. At diagnosis, almost all cases of VL/HIV co-infection have been found to have fewer than 200 CD4+ cells/microl blood, and about 50% meet the AIDS-defining criteria during their first episode of VL. The clinical manifestations of VL in HIV-infected individuals may be similar to those seen in HIV-negative cases; fever, pancytopenia and hepato-splenomegaly, for example, are found in 75% of all the HIV-positive cases. Following the dissemination of the parasites, however, the HIV-positive cases may develop unusual, multi-organ pathology. Almost all the cases of co-infection are very prone to VL relapses, even after carefully managed antileishmanial treatment. The opportunistic infections that are often seen in HIV-positives frequently develop during VL episodes, the signs and symptoms of the leishmaniasis then confusingly overlapping with those of the other infections.
AIDS-Related Opportunistic Infections↗
Investigation of the functional role of active site loop II in a hypoxanthine phosphoribosyltransferase.
Hypoxanthine phosphoribosyltransferases (HPRTs) are of biomedical interest because defects in the enzyme from humans can result in gouty arthritis or Lesch-Nyhan syndrome, and in parasites these enzymes are potential targets for antiparasite chemotherapy. In HPRTs, a long flexible loop (active site loop II) closes over the active site during the enzyme catalyzed reaction. Functional roles for this loop have been proposed but have yet to be substantiated. For the present study, seven amino acids were deleted from loop II of the HPRT from Trypanosoma cruzi to probe the functional role of this active site loop in catalysis. The mutant enzyme (Deltaloop II) was expressed in bacteria, purified by affinity chromatography, and kinetic constants were determined for substrates of both forward (purine salvage) and reverse (pyrophosphorolysis) reactions catalyzed by the enzyme. Loop II deletion resulted in moderate (0.6-2.7-fold) changes in the Michaelis constants (K(m)s) for substrates other than pyrophosphate (PP(i)), for which there was a 5.8-fold increase. In contrast, k(cat) values were severely affected by loop deletion, with rates that were 240-840-fold below those for the wild-type enzyme. Together with previously reported structural data, these results are consistent with active site loop II participating in transition-state stabilization by precise positioning of the substrates for in line nucleophilic attack and in the liberation of PP(i) as a product of the salvage reaction.
Animals↗
Pneumocystis carinii pneumonia in heart transplant recipients.
OBJECTIVES: In spite of the high prevalence of Pneumocystis carinii (PC) pneumonia in immunocompromised patients, little is known about the epidemiological characteristics of this infection, and whether the cases of PC pneumonia in immunosuppressed patients are the result of a reactivation of a latent infection or a due to a recent infection is unknown. The aim of this study was to provide information about the epidemiological characteristics of PC pneumonia in a cohort of heart transplant (HT) recipients when compared with the epidemiology of PC infection in a cohort of chronic sputum producers (CSP) representative of the general population of the same geographical area. METHODS: We identified all the cases of PC pneumonia in the cohort of 72 subjects who underwent cardiac transplantation at our institution between January 1991 and December 1996 and compared them with the cases of PC infection identified in a non-selected cohort of 34 CSP. This second group was included to obtain an approximation of the frequency of PC carriers in the general population. Identification of PC was accomplished through customary stain techniques and immunofluorescence with monoclonal antibodies. RESULTS: Of the 72 HT recipients four (5.5%) developed PC pneumonia, but one had two episodes. Only one had received primary chemoprophylaxis, but developed PC pneumonia 2 months after discontinuing prophylactic therapy. PC pneumonia episodes were produced 53, 102, 230, 181 and 772 days after the moment of transplant, respectively. PC was identified in two (5.8%) of the 34 CSP. No significant differences were found when the accumulative incidences of PC pneumonia in HT patients and PC infection in CSP were compared (P=0.7). CONCLUSIONS: The frequency of PC pneumonia among HT patients is the same as the frequency of PC infection in the general population. This observation and the long interval between transplantation and the development of PC pneumonia observed in the study support the hypothesis that the occurrence of PC pneumonia in immunocompromised patients might be from a new infection rather than from the reactivation of latent organisms. Therefore, continuous prophylaxis might be indicated in areas with a high prevalence of PC for patients at highest risk.
Aged↗
Amino-terminal domain exchange redirects origin-specific interactions of adeno-associated virus rep78 in vitro.
The unique ability of adeno-associated virus type 2 (AAV) to site-specifically integrate its genome into a defined sequence on human chromosome 19 (AAVS1) makes it of particular interest for use in targeted gene delivery. The objective underlying this study is to provide evidence for the feasibility of retargeting site-specific integration into selected loci within the human genome. Current models postulate that AAV DNA integration is initiated through the interactions of the products of a single viral open reading frame, REP, with sequences present in AAVS1 that resemble the minimal origin for AAV DNA replication. Here, we present a cell-free system designed to dissect the Rep functions required to target site-specific integration using functional chimeric Rep proteins derived from AAV Rep78 and Rep1 of the closely related goose parvovirus. We show that amino-terminal domain exchange efficiently redirects the specificity of Rep to the minimal origin of DNA replication. Furthermore, we establish that the amino-terminal 208 amino acids of Rep78/68 constitute a catalytic domain of Rep sufficient to mediate site-specific endonuclease activity.
Base Sequence↗
Do sequence repeats play an equivalent role in the choline-binding module of pneumococcal LytA amidase?
LytA amidase breaks down the N-acetylmuramoyl-l-alanine bonds in the peptidoglycan backbone of Streptococcus pneumoniae. Its polypeptide chain has two modules: the NH(2)-terminal module, responsible for the catalytic activity, and the COOH-terminal module, constructed by six tandem repeats of 20 or 21 amino acids (p1-p6) and a short COOH-terminal tail. The polypeptide chain must contain at least four repeats to efficiently anchor the autolysin to the choline residues of the cell wall. Nevertheless, the catalytic efficiency decreases by 90% upon deletion of the final tail. The structural implications of deleting step by step the two last (p5 and p6) repeats and the final COOH-tail and their effects on choline-amidase interactions have been examined by comparing four truncated mutants with LytA amidase by means of different techniques. Removal of this region has minor effects on secondary structure content but significantly affects the stability of native conformations. The last 11 amino acids and the p5 repeat stabilize the COOH-terminal module; each increases the module transition temperature by about 6 degrees C. Moreover, the p5 motif also seems to participate, in a choline-dependent way, in the stabilization of the NH(2)-terminal module. The effects of choline binding on the thermal stability profile of the mutant lacking the p5 repeat might reflect a cooperative pathway providing molecular communication between the choline-binding module and the NH(2)-terminal region. The three sequence motives favor the choline-amidase interaction, but the tail is an essential factor in the monomer <--> dimer self-association equilibrium of LytA and its regulation by choline. The final tail is required for preferential interaction of choline with LytA dimers and for the existence of different sets of choline-binding sites. The p6 repeat scarcely affects the amidase stability but could provide the proper three-dimensional orientation of the final tail.
Choline↗